Category Archives: Utah Stem Cells

Introduction

Mechanical stress enhances bone metabolism and periodontal tissue remodeling.1 During orthodontic tooth movement (OTM), bone remodeling is initiated via the periodontal ligament.2 As the main mesenchymal stem cells (MSCs) in the periodontal ligament, periodontal ligament stem cells (PDLSCs) play an important role in mechanical signal transduction. Currently, a consensus has been reached that cyclic mechanical tension is a strong driver of the differentiation of PDLSCs into the osteoblast lineage.24 Mechanical tension activates calcium channels,5 which activate the ERK1/2 and P38 MAPK pathways through integrin-FAK or protein kinase (PKC)-SR signaling6 and induce the phosphorylation of Runt-associated transcription factor 2 (Runx2),7 promoting osteogenic precursor cell synthesis and the transcription of mineralizable proteins.8 At present, the TGF-, BMP, MAPK, Notch, Wnt, Hedgehog, FGF, and Hippo signaling pathways have been found to be involved in this process.

Force parameters (including magnitude, frequency, and duration) are crucial for well-regulated tissue remodeling. However, numerous in vitro studies performed to date show enormous heterogeneity in tensile force parameters.9 In different studies, cyclic tension was applied with magnitudes ranging from 1% to 24%, frequencies ranging from 0.1 Hz to 1.0 Hz, and stimuli duration ranging from 1 hour to 6 days, thereby reducing comparability between different studies.9 To establish strategies to optimize tensile force parameters, it is of particular importance to understand how different tensile force parameters affect the osteogenic differentiation of PDLSCs.

The effects of different tensile force magnitudes and durations have been investigated in some studies. Among the magnitudes, a magnitude of 10% generally led to a lower level of inflammation and a higher level of osteogenesis,10 whereas a magnitude of 12% was found to correlate well with strain conditions at the mid-root under physiological loading conditions11,12 and to induce optimal effects in both the proliferation and osteogenesis of PDLSCs.13 Cyclic tension alone at 3000 strain significantly enhanced SATB Homeobox 2 (Satb2) after 3 h of loading and significantly upregulated Runx2 after 6 h.14 The synthesis of BMP9 increased under 6-h continuously applied cyclic tension.15 In addition, 12% cyclic tensile force gradually upregulated the expression of Runx2, alkaline phosphatase (ALP), and osteocalcin (OCN) with force durations of 6 h, 12 h, and 24 h, respectively.16,17 The protein level of osterix increased stepwise following 3 h, 6 h, 12 h, and 24 h of exposure to tensile strain.14 Recently, temporal gene expression patterns were delineated.17

Tensile frequency varies largely among different studies. The ROCKTAZ pathway and its interaction with Cbf1 were found to be essential for the cyclic tension (12% elongation, 0.1 Hz)-induced osteogenic differentiation in PDLSCs.18 Cyclic tension (10% elongation, 0.5 Hz) stimulated the osteogenic differentiation of PDLSCs by inhibiting miR-129-5p expression and activating the BMP2/Smad pathway.17 LncRNAs-miRNAs-mRNAs networks in PDLSCs were depicted under cyclic tension (10% elongation, 1.0 Hz).19 However, there have been rare studies examining the impact of different cyclic tensile frequencies on osteogenesis of PDLSCs and the expression of relevant genes thus far. The low-magnitude high-frequency (LMHF) vibration approach was excluded because it is used to simulate a masticatory force, while cyclic tension is used to simulate an orthodontic force, and the two methods of force application are completely different.2,20 Previous animal studies on long bone distraction osteogenesis have shown that loading frequency affects the osteogenic response of bone tissue.21 The mechano-regulation of trabecular bone adaptation is logarithmically dependent on the loading frequency.22 Therefore, we hypothesized that tensile frequency would affect osteogenesis of PDLSCs, in which some tensile frequency-sensitive genes may play an important role. To test our hypotheses, human PDLSCs were subjected to cyclic mechanical tension at different frequencies of 0.10.7 Hz to examine the osteoblastic differentiation of PDLSCs, and high-throughput sequencing was performed to characterize the frequency-course expression patterns of mRNA during the osteogenic differentiation of PDLSCs. This study aimed to investigate the effects of tensile frequency on the osteogenic differentiation of PDLSCs as well as the relevant molecular mechanisms.

Healthy periodontal ligament tissues were scraped from the middle third of tooth roots, which were extracted for orthodontic reasons, with informed consents. All donors were aged from 14 to 16 years and had no systemic or oral diseases. The periodontal ligament tissues were cut into small pieces and enzymatically digested for 40 min at 37C with collagenase I (3 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) and dispase II (4 mg/mL; Sigma-Aldrich). The cells were seeded in 25 cm2 flasks (Falcon, BD Biosciences, Franklin Lakes, NJ, USA) with -minimal essential medium (-MEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies Co., Grand Island, NY, USA) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin, Hyclone, Logan, Utah, USA), and incubated in a humidified atmosphere (37C, 5% CO2). The medium was changed every 3 days. After reaching 80% of confluence, the cells were detached with 0.25% trypsin/EDTA (Gibco, Life Technologies Co., Grand Island, NY, USA), and single-cell suspensions were cloned with the limiting-dilution method to purify the stem cells.17 Cell clusters from the colony were trypsinized and serially sub-cultured.

The third-passage PDLSCs were sub-cultured into six-well plates until confluent. The culture medium was then removed, and cells were fixed with 4% formaldehyde (Zhonghuihecai, Xian, CN) for 20 min, permeabilized with 0.3% Triton X-100 (Zhonghuihecai) for 5 min, and incubated with primary antibodies (anti-pan-cytokeratin, 1:300, Abcam, Cambridge, MA, USA; anti-vimentin, 1:500, Abcam; anti-STRO-1, 1:200, Abcam; anti-CD146, 1:200, Abcam) overnight at 4C. The cells were then washed with PBS and incubated with CY3/FITC-conjugated secondary antibodies (1:500, Zhuangzhi, Xian, CN) in darkness for 30 min, and then washed with PBS. Finally, the nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, Zhuangzhi), and fluorescent images were captured with a fluorescence microscope (Olympus, Japan).

For osteogenic and adipogenic differentiation, PDLSCs were seeded into six-well plates at a density of 2105 cells/well, and after reaching 80% confluence, the medium was replaced with an osteogenic or adipogenic inductive medium (Osteogenesis or Adipogenesis Differentiation Kit, Cyagen, USA). Seven days after osteogenic induction, the cells were stained with ALP (ALP staining kit, Solarbio, CN), and 14 days after osteogenic incubation, the cells were stained with Alizarin Red (ARS staining kit, Cyagen, USA). After 21 days of adipogenic incubation, the cells were stained with Oil Red O (Cyagen, USA).

Flexcell FX-5000T Tension Plus System (Flexcell International Corporation, Hillsborough, NC, USA) was used to mimic the tensile force exerted on PDLSCs during OTM, according to previous studies.23 Cyclic tensile loading experiments were performed on the fourth-passage PDLSCs from four different healthy donors in triplicate. PDLSCs were seeded onto six-well type I collagen (COL-I)-coated silicone culture plates (Flexcell International Corporation) at a density of 2105 cells/well. Upon reaching 80% confluence, the cells were serum-starved overnight, and the medium was changed to osteogenic medium (Cyagen, USA). Cyclic tensile force (12% bottom membrane elongation) was applied to different plates at different frequencies of 0.1 Hz, 0.5 Hz, and 0.7 Hz. Control cells were cultured under identical culture condition but without mechanical stimulation.

After 10 h of cyclic tensile force, the cells were collected. Proteins were isolated, electrophoretically separated, and immunoblotted as previously described.23 Briefly, PDLSCs were lysed with RIPA buffer containing 1% phenylmethanesulfonyl fluoride (PMSF, proteinase inhibitor, Zhonghuihecai) and 1% phosphatase inhibitor (Zhonghuihecai). After centrifugation, the supernatant was collected and measured quantitatively using a BCA Protein Assay Kit (Absin, Shanghai, CN). Total protein from cell lysates (20 g/lane) was separated by SDS-PAGE gels (Beyotime, Hangzhou, CN) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk in tris-buffered saline tween-20 (TBST) for 2 h at room temperature, the membranes were incubated overnight at 4C with primary antibodies (runt-related transcription factor 2 (Runx2), 1:500, ImmunoWay, USA; COL-I, 1:1000, Proteintech, USA; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1:10000, Proteintech, USA), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, Proteintech, USA) for 2 h at room temperature. The protein expression was visualized using ChemiDocTM XRS+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with an enhanced chemiluminescence (ECL) kit (Millipore, Billerica, MA, USA). GAPDH was used as an internal control for normalization.

The fourth-passage PDLSCs from three donors were used for RNA sequencing after tension loading. Total RNA was extracted from the four groups of cells (normal PDLSCs and PDLSCs tensioned at frequencies of 0.1 Hz, 0.5 Hz, and 0.7 Hz for 6 h) using the Trizol (Sigma-Aldrich), according to the manufacturers protocols. After digestion with DNase, rRNA were depleted using a Ribo-Zero magnetic kit, and sequencing libraries were constructed as previously described.24 The sequencing of the cDNA library was carried out by Gene Denovo Biotechnology Co. (Guangzhou, China). The gene expression level was evaluated by reads per kilobase transcriptome per million mapped reads (RPKM). Requirements for filtering differentially expressed genes (DEGs) were as follows: (1) |log2 (fold-change)| 1; (2) p value < 0.05. DESeq2 (differential gene expression analysis based on the negative binomial distribution)25 was used to calculate p values and adjusted p (adj. p) values. Heatmaps and volcano plots analyses were used to visualize these DEGs using the Complex Heatmap package and ggplots2 package of R software. The online tool Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) was applied to identify the common DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for annotation visualization and integrated discovery. Raw data of the performed RNA sequencing (RNA-seq) were recorded in the SRA database with the SRA accession: PRJNA665587.

Gene expression pattern analysis is used to cluster genes of similar expression patterns for multiple samples in a tensile frequency order. To examine the expression pattern of DEGs in different frequencies, the expression data of each sample (in the order of treatment) were normalized to 0, log2 (v1/v0), and log2 (v2/v0), and then clustered using the Short Time-series Expression Miner software (STEM).26 The parameters were set as follows: (1) maximum unit change in model profiles between frequency points was 1; (2) maximum output profiles number was 20 (similar profiles will be merged; and (3) minimum ratio of fold change of DEGs was no less than 2.0. The clustered profiles with p value < 0.05 were considered as significant profiles. Then, the DEGs in all profiles or in each profile were subjected to GO and KEGG pathway enrichment analysis. STRING (https://string-db.org/) was used for PPI network analysis. The DEGs were mapped onto the PPI network with a minimum interaction score of 0.4. Cytoscape v3.7.1 software was used to visualize the PPI network. Gene network clustering analysis was performed to identify the key PPI network modules, using the MCODE and cytoHuba app from the Cytoscape software suite. Adj. p value < 0.05 was set as the significance threshold.

Total RNA was extracted using Trizol (Sigma-Aldrich) according to the manufacturers protocols. Quantitative real-time PCR (RTqPCR) was performed in triplicate using the Power SYBR Green PCR Mastermix (Applied Biosystems, Foster City, CA, USA). Sequences of the primers used are shown in Table S1. The mean expression values were calculated relative to GAPDH, which was used as an internal control for normalization.

For differential gene expression analysis, a likelihood ratio test was used assuming an underlying zero-inflated negative binomial distribution.25,27 FDR-corrected p-values were calculated using the Benjamini-Hochberg procedure.28 K-means cluster method29 was used for frequency cluster expression pattern analysis by the STEM software. P and Q values of GO/KEGG functional analysis and frequency cluster analysis were calculated based on a hypergeometric distribution.30 Values of the relative protein and mRNA expression were expressed as mean SD within each group. One-way ANOVA followed by SNK post hoc tests was used in the Western blotting assay and RTqPCR assay. The significance threshold was set at 0.05.

Immunofluorescent staining revealed that the isolated cells were positive for CD146, vimentin, and STRO-1 (Figure 1AC, respectively) but negative for pan-cytokeratin (Figure 1D), confirming that the cells were mesenchymal stem cells of mesodermal origin. The osteogenic potential of PDLSCs was determined by positive ALP staining after 7 days of osteogenic induction (Figure 1E and F) and red mineralized matrix nodules in Alizarin Red staining after 14 days of osteogenic induction (Figure 1G and H). The presence of red lipid droplets in Oil Red staining after 21 days of adipogenic induction (Figure 1I) indicated the adipogenic differentiation of PDLSCs.

Figure 1 Identification of PDLSCs. The Immunofluorescence showed that the cells were positive for CD146 (A, red), vimentin (B, green), and STRO-1 (C, red) but negative for pan-cytokeratin (D). Scale bar = 50 m. ALP staining was positive both visually (E, black) and under the microscope (F, black, Scale bar = 100 m), after 7 days of osteogenesis induction culture. After 14 days of osteogenesis induction, ARS staining was observed to be positive by the naked eye (G, red), and mineralized nodules were obvious under the microscope (H, red, Scale bar = 100 m). After 21 days of adipogenic induction, oil red O-positive lipid clusters were observed microscopically (I, red, Scale bar = 100 m).

PDLSCs were exposed to 12% cyclic tension, which contributed to the cellular reorientation, including an irregular arrangement at the central region and a parallel arrangement at the peripheral region of the plate (Figure 2A). As shown in Figure 2B and C, after consecutive 10-h cyclic tensile force loading, the protein expression levels of Runx2 and COLI increased with increasing tensile frequency from 0.1 Hz to 0.7 Hz and were remarkably higher than that in the group without tension application (p < 0.05). The result suggested that mechanical tension (12% deformation) upregulated the osteogenesis of PDLSCs in a frequency-dependent manner. Higher frequencies of cyclic tension were associated with higher osteogenic differentiation of PDLSCs.

Figure 2 Cyclic tension promoted the osteogenic differentiation of PDLSCs. Under cyclic equibiaxial tension, PDLSCs reoriented in parallel alignment at the peripheral region of the plate, while in random orientation at the center of the plate (A, Scale bar = 100 m). Western blotting was used to detect protein levels of osteogenesis-related genes, COL-I and Runx2, at different tensile frequencies (B and C). **p < 0.01, ***p < 0.001, vs control group.

It has been previously shown that cyclic mechanical tensile stress can improve osteogenesis of PDLSCs, and that consecutive 46 h of tension can significantly upregulate the mRNA expression of osteogenesis-related genes.16,17 To gain insight into the molecular mechanisms by which mechanical tension stimulates osteoblast differentiation of PDLSCs, total RNA was extracted from PDLSCs to conduct RNA-seq after 6 h of cyclic tension. The mRNA expression profiles of the PDLSCs at different tensile frequencies (0.1 Hz, 0.5 Hz, and 0.7 Hz) were detected. Comparative expression analyses were performed according to the different frequencies of the tensile stress (0.1 Hz vs control, 0.5 Hz vs control, and 0.7 Hz vs control). Heat maps of the top 40 DEGs (Figure 3AC) and volcano plots (Figure 3DF) were depicted. In total, 50 mRNAs were upregulated, and 261 mRNAs were downregulated at 0.1 Hz. At 0.5 Hz, 656 mRNAs were upregulated, and 1474 mRNAs were downregulated. At 0.7Hz, 139 mRNAs were upregulated, and 194 mRNAs were downregulated. A Venny analysis (Figure 3G and H) showed that 78 genes were simultaneously upregulated and 118 were simultaneously downregulated among the 0.1 Hz, 0.5 Hz, and 0.7 Hz groups. The GO analysis (Figure 4A) demonstrated that changes in biological processes (BPs) were mainly enriched in metabolic process, response to stimulus, biological regulation, signaling, and localization. Changes in Cellular Components (CCs) were mainly enriched in organelle, membrane, macromolecular complex, and membrane-enclosed lumen. Moreover, binding, catalytic activity, and nucleic acid binding transcription factor activity emerged as the highest-ranked Molecular Function (MF) groups. As shown in Figure 4B, DNA replication, cell cycle, and the TNF signaling pathway were significantly enriched in the KEGG pathway. Within the primary category Environmental Information Processing, Signal transduction, and Signaling molecules and interaction were strongly enriched (Figure 4C).

Figure 3 Identification of DEGs among different frequencies. (AC) Heatmaps of the top 40 DEGs between 0.1 Hz/0.5 Hz/0.7 Hz and static culture, respectively. Red rectangles represent high expression, and blue rectangles represent low expression. (DF) Volcano plot of DEGs between 0.1 Hz/0.5 Hz/0.7 Hz and static culture, respectively. The red plots represent upregulated genes, the blue plots represent downregulated genes, and the black plots represent nonsignificant genes. (G) Venn diagram of upregulated DEGs among 0.1 Hz, 0.5 Hz, and 0.7 Hz. (H) Venn diagram of downregulated DEGs among all the three frequencies.

Figure 4 Functional enrichment analysis of all DEGs. (A) GO enrichment analysis of all DEGs among different frequencies. (B) Top 20 pathways of the KEGG enrichment analysis of all DEGs among different frequencies, with the KEGG pathway annotation (C). The screening criteria for significance were p value < 0.05.

The sequencing data were normalized to the control, and trend analyses of DEGs were identified using STEM. In Figure 5A, within the 20 model profiles, eight mRNA trend profiles were statistically significant. The profile number assigned by STEM was on the top left corner of each profile box, p value was on the bottom left, and the number of the cardinality of each cluster was on the top right corner. As shown in Table 1, among different profiles, the top-ranked KEGG pathways were mainly in the metabolic pathways, PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, and MAPK signaling pathway. A continuous downregulation pattern was found in profile 0 (Figure 5B), in which the high-ranked BPs, CCs, and MFs in GO enrichment (Figure 5C) were similar to those in Figure 4A, and inflammatory pathways such as arachidonic acid metabolism, peroxisome, and cytokinecytokine receptor interaction were strongly enriched (Figure 5D).

Table 1 Top 10 of KEGG Enrichment Among Different Profiles

Figure 5 Frequency series clustering analysis on expression profiles of mRNAs by STEM. (A) Within the 20 model profiles, eight mRNA trend profiles were statistically significant. The number at the upper-left corner of each profile box was the profile number assigned by STEM, the number on the bottom left was the p value, and the number on the top-right corner was the number of genes within each cluster. (B) Persistently downregulated genes along frequency were clustered in profile 0. (C) GO enrichment of profile 0. (D) Top 10 pathways of the KEGG enrichment of profile 0.

The interactions of 194 DEGs in profile 0 were analyzed using the STRING online database, and the PPI network was obtained using the Cytoscape software (Figure 6A). The MCODE plugin was then used to investigate the key PPI network modules, and one key module with four genes (EYA1, SIX5, SALL1, FRAS1) was identified (Figure 6B). The cytoHubba plugin was then used to analyze hub genes with maximum correlation criterion (MCC)/Degree, and genes with the top 10 scores were respectively identified. The intersection (EYA1, SALL1) of hub genes according to the above three methods were selected for further RTqPCR validation (Figure 6C). The results of RTqPCR (Figure 6D and E) showed that the mRNA expression of EYA1 and SALL1 decreased with increasing frequency from 0.1 Hz to 0.7 Hz, which were highly consistent with our high-throughput sequencing.

Figure 6 Identification and validation of mechanofreqency-sensitive hub genes. (A) The interaction network between proteins coded by the DEGs in profile 0. The nodes represent genes, and the edges represent links between genes. Blue represents downregulated genes. (B) The highest scoring module was extracted by MCODE. (C) The intersection was obtained among modules measured by MCODE, and the top 10 highly connected genes were identified using MCC and Degree in cytoHubba. (D and E) Validation of the expression of the two intersection genes, EYA1 and SALL1, using RTqPCR. *p < 0.05, vs control group.

OTM is based on remodeling processes in the periodontal ligament and the alveolar bone. PDLSCs play an important role in mechano-transduction and in promoting periodontal tissue regeneration in OTM.31,32 It is evident that cyclic tensile force regulates the osteogenic differentiation of PDLSCs.33 A complex network of signaling molecules regulates the osteoblastic differentiation of PDLSCs under cyclic tension.5,17,34 The heterogeneity of mechanical force parameters (duration, magnitude, frequency, and others)9 led to the heterogeneity of the osteogenic phenotype and gene regulation in different studies. This study focused on the effect of tensile frequency on the osteogenic differentiation of PDLSCs and attempted to elucidate a potential mechanism.

In the current study, we successfully isolated and characterized human PDSLCs. The cells at the 4th to 6th passage were used, whose phenotype was generally believed to be maintained.35 Mechanical tension was applied using the Flexcell tension system, which has been widely used in PDLSCs studies.2,20 We observed that the cellar reorientation after force loading was similar to that of the previous study,23 and this could be attributed to the mechano-responsive stress fibersfocal adhesion system.36 The present study showed that cyclic mechanical tension (magnitude: 12% deformation, duration: 10 h) in the range of 0.10.7 Hz promoted osteogenic marker genes including Runx2 and COL-I in PDLSCs, and their protein expressions increased with the increasing tensile frequency. Runx2 is an osteogenic lineage commitment specific transcription factor, which binds to the specific cis-acting elements of osteoblasts to promote the transcription and translation of OCN, osteopontin (OPN), bone sialoprotein (BSP), and COL-I.8 COL-I, which acts as a template onto which minerals are deposited to form bone matrix,37 is the major constituent of extracellular matrix in the periodontal ligament and bone, and is confirmed to be essential for osteogenesis in response to tension during OTM.38 Consistently, upregulation of RUNX2 and COL-I in response to tension was reported in most studies.9 To the best of our knowledge, the present study is the first to reveal the frequency dependence during cyclic tension in enhancing the expression of osteogenic markers within the first 10 h of cyclic tension application, which may inform a new method of accelerating OTM.

Using RNA-seq, we observed that mRNAs in strained PDLSCs were mainly enriched in response to the stimulus process, signal transduction, and relative pathways such as mismatch repair, TNF signaling pathway, and FOXO signaling pathway, which were associated with cell survival and differentiation as well as immune and inflammatory responses.39,40 The STEM platform was also used to investigate how gene expression profiles change with tensile frequency during the osteoblast differentiation of PDLSCs under cyclic tension. Eight trend profiles were noted as significant. Genes in these profiles were mainly enriched in the metabolic pathways, PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, and MAPK signaling pathway. The PI3K-Akt signaling pathway has been reported to be involved in the mechanical force-induced osteoblast differentiation of PDLSCs.41,42 The MAPK signaling pathway also have been found to participate in the mechano-transduction of PDLSCs.43,44

Genes in profile 0 showed a continuous downward trend from 0.1 Hz to 0.7 Hz, an inverse trend of osteogenic genes, and were mainly enriched for pathways related to an inflammatory response, such as arachidonic acid metabolism and cytokinecytokine receptor interaction. Cyclooxygenase-2 (COX2) and prostaglandin E synthase (PTGES) participate in the arachidonic acid metabolism pathway. TNF Receptor Superfamily Member 14 (TNFRSF14) and Interleukin 9 (IL9) are involved in the cytokinecytokine receptor interaction. PTGES is induced by inflammatory mediators.45 COX2 is involved in the synthesis of prostaglandin E2 (PGE2), which is a potent pro-inflammatory cytokine, and participates in bone resorption.2 The expression of COX2 and PGE2 after mechanical stimulation was previously reported to be correlated with force duration and force magnitude,9 and it showed a negative correlation with tensile frequency in the present study. TNFRSF14 is a membrane-bound receptor leading to the induction of proinflammatory genes by activating the NF-B pathway.46 IL-9 plays a role in regulating inflammatory immunity, and it has demonstrated pro-inflammatory activity in several mouse models of inflammation.47 The response of periodontal ligament to mechanical stress generated by OTM is known as an aseptic transitory inflammatory process, which is regulated by various cytokines and chemokines.48 Proinflammatory cytokines activate matrix metalloproteinases (MMPs), degrade the ECM, and inhibit the expression of COL-I.49 Increased osteogenesis is usually accompanied by lower levels of inflammatory cytokines and chemokines.10,50,51 Accordingly, in the present study, with increasing frequency, the osteogenic commitment increased, and the suppression of pro-inflammatory genes and the relative inflammatory response pathway were observed.

Furthermore, through PPI network screening, we identified two candidate genes, EYA1 and SALL1, which were specifically sensitized to tensile frequency. The result was validated by RTqPCR, which confirmed the decreased expression of EYA1 and SALL1 with increasing frequency of tension stimulation. EYA1 is a conserved critical regulator of organ-specific stem cells.52 SALL1 is also considered a stem cell marker.53 The osteoblastic differentiation of PDLSCs increased with increasing tensile frequency; thus, stemness and related genes correspondingly reduced. In view of the high-throughput sequencing and validation after 6 h of tensile force exposure, further studies over a longer period are needed. Whether overexpression of EYA1 and SALL1 would reverse the frequency-dependent trend of the osteogenic differentiation of PDLSCs also deserves further study.

In the present study, the role of tensile frequency on osteogenic commitment of PDLSCs were identified, and the mRNA transcriptomes of PDLSCs during the osteogenic differentiation under cyclic tension with different frequencies were delineated. Frequency series clustering were defined using STEM, and tensile frequency-sensitive genes were identified. This study extends the knowledge about the role of tensile frequency in cyclic tension induced PDLSCs osteogenesis.

The osteoblastic differentiation of PDLSCs under mechanical tensile force is frequency dependent. EYA1 and SALL1 were identified as potential important tensile frequency-sensitive genes, which may contribute to the cyclic tension-induced osteogenic differentiation of PDLSCs in a frequency-dependent manner.

Raw data of the performed RNA sequencing (RNA-seq) have been recorded in the SRA database with the SRA accession: PRJNA665587 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA665587). Other data in this study are available from the corresponding author Xi Chen upon request.

The study was performed in accordance with the principles stated in the Declaration of Helsinki and approved by the Medical Ethics Committee of the First Affiliated Hospital of Medical College of Xi an Jiaotong University (No: XJTU1AF2019LSK-078). Informed consent was obtained from all donors and their legal guardians involved in the study. Written informed consent was also obtained from the donors and their legal guardians to publish this paper.

All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

This research was funded by the Key Research and Development project of Shaanxi Province under Grant 2018SF-037.

The authors declare that they have no competing interests.

1. Wang H, Sun W, Ma J, Pan Y, Wang L, Zhang W. Polycystin-1 mediates mechanical strain-induced osteoblastic mechanoresponses via potentiation of intracellular calcium and Akt/-catenin pathway. PLoS One. 2014;9(3):e91730. doi:10.1371/journal.pone.0091730

2. Li M, Zhang C, Yang Y. Effects of mechanical forces on osteogenesis and osteoclastogenesis in human periodontal ligament fibroblasts: a systematic review of in vitro studies. Bone Joint Res. 2019;8(1):1931. doi:10.1302/2046-3758.81.Bjr-2018-0060.R1

3. Chang M, Lin H, Fu H, Wang B, Han G, Fan M. MicroRNA-195-5p regulates osteogenic differentiation of periodontal ligament cells under mechanical loading. J Cell Physiol. 2017;232(12):37623774. doi:10.1002/jcp.25856

4. Yang Y, Wang BK, Chang ML, Wan ZQ, Han GL. Cyclic stretch enhances osteogenic differentiation of human periodontal ligament cells via YAP activation. Biomed Res Int. 2018;2018:2174824. doi:10.1155/2018/2174824

5. Jin SS, He DQ, Wang Y, et al. Mechanical force modulates periodontal ligament stem cell characteristics during bone remodelling via TRPV4. Cell Prolif. 2020;53(10):e12912. doi:10.1111/cpr.12912

6. Katz S, Boland R, Santilln G. Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation. Int J Biochem Cell Biol. 2006;38(12):20822091. doi:10.1016/j.biocel.2006.05.018

7. Ren D, Wei F, Hu L, Yang S, Wang C, Yuan X. Phosphorylation of Runx2, induced by cyclic mechanical tension via ERK1/2 pathway, contributes to osteodifferentiation of human periodontal ligament fibroblasts. J Cell Physiol. 2015;230(10):24262436. doi:10.1002/jcp.24972

8. Xu J, Li Z, Hou Y, Fang W. Potential mechanisms underlying the Runx2 induced osteogenesis of bone marrow mesenchymal stem cells. Am J Transl Res. 2015;7(12):25272535.

9. Sun C, Janjic Rankovic M, Folwaczny M, Otto S, Wichelhaus A, Baumert U. Effect of tension on human periodontal ligament cells: systematic review and network analysis. Front Bioeng Biotechnol. 2021;9:695053. doi:10.3389/fbioe.2021.695053

10. Sun C, Janjic Rankovic M, Folwaczny M, et al. Effect of different parameters of in vitro static tensile strain on human periodontal ligament cells simulating the tension side of orthodontic tooth movement. Int J Mol Sci. 2022;23(3). doi:10.3390/ijms23031525

11. Natali AN, Pavan PG, Scarpa C. Numerical analysis of tooth mobility: formulation of a non-linear constitutive law for the periodontal ligament. Dent Mater. 2004;20(7):623629. doi:10.1016/j.dental.2003.08.003

12. Pinkerton MN, Wescott DC, Gaffey BJ, Beggs KT, Milne TJ, Meikle MC. Cultured human periodontal ligament cells constitutively express multiple osteotropic cytokines and growth factors, several of which are responsive to mechanical deformation. J Periodontal Res. 2008;43(3):343351. doi:10.1111/j.1600-0765.2007.01040.x

13. Liu J, Li Q, Liu S, et al. Periodontal ligament stem cells in the periodontitis microenvironment are sensitive to static mechanical strain. Stem Cells Int. 2017;2017:1380851. doi:10.1155/2017/1380851

14. Tang N, Zhao Z, Zhang L, et al. Up-regulated osteogenic transcription factors during early response of human periodontal ligament stem cells to cyclic tensile strain. Arch Med Sci. 2012;8(3):422430. doi:10.5114/aoms.2012.28810

15. Tantilertanant Y, Niyompanich J, Everts V, Supaphol P, Pavasant P, Sanchavanakit N. Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells. J Cell Physiol. 2019;234(4):45284539. doi:10.1002/jcp.27257

16. Shen T, Qiu L, Chang H, et al. Cyclic tension promotes osteogenic differentiation in human periodontal ligament stem cells. Int J Clin Exp Pathol. 2014;7(11):78727880.

17. Wu X, Li Y, Cao Z, Xie Y, Fu C, Chen H. Mechanism of cyclic tensile stress in osteogenic differentiation of human periodontal ligament stem cells. Calcif Tissue Int. 2021;108(5):640653. doi:10.1007/s00223-020-00789-x

18. Wang Y, Hu B, Hu R, et al. TAZ contributes to osteogenic differentiation of periodontal ligament cells under tensile stress. J Periodontal Res. 2020;55(1):152160. doi:10.1111/jre.12698

19. Wang H, Feng C, Li M, Zhang Z, Liu J, Wei F. Analysis of lncRNAs-miRNAs-mRNAs networks in periodontal ligament stem cells under mechanical force. Oral Dis. 2021;27(2):325337. doi:10.1111/odi.13530

20. Yang L, Yang Y, Wang S, Li Y, Zhao Z. In vitro mechanical loading models for periodontal ligament cells: from two-dimensional to three-dimensional models. Arch Oral Biol. 2015;60(3):416424. doi:10.1016/j.archoralbio.2014.11.012

21. Hsieh YF, Turner CH. Effects of loading frequency on mechanically induced bone formation. J Bone Miner Res. 2001;16(5):918924. doi:10.1359/jbmr.2001.16.5.918

22. Scheuren AC, Vallaster P, Kuhn GA, et al. Mechano-regulation of trabecular bone adaptation is controlled by the local in vivo environment and logarithmically dependent on loading frequency. Front Bioeng Biotechnol. 2020;8:566346. doi:10.3389/fbioe.2020.566346

23. Yang SY, Wei FL, Hu LH, Wang CL. PERK-eIF2-ATF4 pathway mediated by endoplasmic reticulum stress response is involved in osteodifferentiation of human periodontal ligament cells under cyclic mechanical force. Cell Signal. 2016;28(8):880886. doi:10.1016/j.cellsig.2016.04.003

24. Borodina T, Adjaye J, Sultan M. A strand-specific library preparation protocol for RNA sequencing. Methods Enzymol. 2011;500:7998. doi:10.1016/b978-0-12-385118-5.00005-0

25. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. doi:10.1186/s13059-014-0550-8

26. Ernst J, Bar-Joseph Z. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinform. 2006;7:191. doi:10.1186/1471-2105-7-191

27. Brulois K, Rajaraman A, Szade A, et al. A molecular map of murine lymph node blood vascular endothelium at single cell resolution. Nat Commun. 2020;11(1):3798. doi:10.1038/s41467-020-17291-5

28. Stanton BZ, Hodges C, Calarco JP, et al. Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin. Nat Genet. 2017;49(2):282288. doi:10.1038/ng.3735

29. Beauchaine TP, Beauchaine RJ. A comparison of maximum covariance and K-means cluster analysis in classifying cases into known taxon groups. Psychol Methods. 2002;7(2):245261. doi:10.1037/1082-989x.7.2.245

30. Iacono G, Dubos A, Mziane H, et al. Increased H3K9 methylation and impaired expression of Protocadherins are associated with the cognitive dysfunctions of the Kleefstra syndrome. Nucleic Acids Res. 2018;46(10):49504965. doi:10.1093/nar/gky196

31. Tantilertanant Y, Niyompanich J, Everts V, Supaphol P, Pavasant P, Sanchavanakit N. Cyclic tensile force-upregulated IL6 increases MMP3 expression by human periodontal ligament cells. Arch Oral Biol. 2019;107:104495. doi:10.1016/j.archoralbio.2019.104495

32. Symmank J, Zimmermann S, Goldschmitt J, et al. Mechanically-induced GDF15 secretion by periodontal ligament fibroblasts regulates osteogenic transcription. Sci Rep. 2019;9(1):11516. doi:10.1038/s41598-019-47639-x

33. Yu N, Prodanov L, Te Riet J, et al. Regulation of periodontal ligament cell behavior by cyclic mechanical loading and substrate nanotexture. J Periodontol. 2013;84(10):15041513. doi:10.1902/jop.2012.120513

34. Wei FL, Wang JH, Ding G, et al. Mechanical force-induced specific MicroRNA expression in human periodontal ligament stem cells. Cells Tissues Organs. 2014;199(56):353363. doi:10.1159/000369613

35. Jnsson D, Nebel D, Bratthall G, Nilsson BO. The human periodontal ligament cell: a fibroblast-like cell acting as an immune cell. J Periodontal Res. 2011;46(2):153157. doi:10.1111/j.1600-0765.2010.01331.x

36. Livne A, Bouchbinder E, Geiger B. Cell reorientation under cyclic stretching. Nat Commun. 2014;5:3938. doi:10.1038/ncomms4938

37. Rather HA, Jhala D, Vasita R. Dual functional approaches for osteogenesis coupled angiogenesis in bone tissue engineering. Mater Sci Eng C Mater Biol Appl. 2019;103:109761. doi:10.1016/j.msec.2019.109761

38. Jacobs C, Grimm S, Ziebart T, Walter C, Wehrbein H. Osteogenic differentiation of periodontal fibroblasts is dependent on the strength of mechanical strain. Arch Oral Biol. 2013;58(7):896904. doi:10.1016/j.archoralbio.2013.01.009

39. Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science. 2002;296(5573):16341635. doi:10.1126/science.1071924

40. Arden KC. FoxO: linking new signaling pathways. Mol Cell. 2004;14(4):416418. doi:10.1016/s1097-2765(04)00213-8

41. Qi L, Zhang Y. The microRNA 132 regulates fluid shear stress-induced differentiation in periodontal ligament cells through mTOR signaling pathway. Cell Physiol Biochem. 2014;33(2):433445. doi:10.1159/000358624

42. Jiang N, He D, Ma Y, et al. Force-induced autophagy in periodontal ligament stem cells modulates M1 macrophage polarization via AKT signaling. Front Cell Dev Biol. 2021;9:666631. doi:10.3389/fcell.2021.666631

43. Ziegler N, Alonso A, Steinberg T, et al. Mechano-transduction in periodontal ligament cells identifies activated states of MAP-kinases p42/44 and p38-stress kinase as a mechanism for MMP-13 expression. BMC Cell Biol. 2010;11:10. doi:10.1186/1471-2121-11-10

44. Tang M, Peng Z, Mai Z, et al. Fluid shear stress stimulates osteogenic differentiation of human periodontal ligament cells via the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways. J Periodontol. 2014;85(12):18061813. doi:10.1902/jop.2014.140244

45. Blair PJ, Hwang SJ, Shonnard MC, et al. The role of prostaglandins in disrupted gastric motor activity associated with type 2 diabetes. Diabetes. 2019;68(3):637647. doi:10.2337/db18-1064

46. Shui JW, Steinberg MW, Kronenberg M. Regulation of inflammation, autoimmunity, and infection immunity by HVEM-BTLA signaling. J Leukoc Biol. 2011;89(4):517523. doi:10.1189/jlb.0910528

47. Goswami R, Kaplan MH. A brief history of IL-9. J Immunol. 2011;186(6):32833288. doi:10.4049/jimmunol.1003049

48. Lee SI, Park KH, Kim SJ, Kang YG, Lee YM, Kim EC. Mechanical stress-activated immune response genes via Sirtuin 1 expression in human periodontal ligament cells. Clin Exp Immunol. 2012;168(1):113124. doi:10.1111/j.1365-2249.2011.04549.x

49. Sun C, Liu F, Cen S, et al. Tensile strength suppresses the osteogenesis of periodontal ligament cells in inflammatory microenvironments. Mol Med Rep. 2017;16(1):666672. doi:10.3892/mmr.2017.6644

50. Loi F, Crdova LA, Pajarinen J, Lin TH, Yao Z, Goodman SB. Inflammation, fracture and bone repair. Bone. 2016;86:119130. doi:10.1016/j.bone.2016.02.020

51. Orapiriyakul W, Tsimbouri MP, Childs P, et al. Nanovibrational stimulation of mesenchymal stem cells induces therapeutic reactive oxygen species and inflammation for three-dimensional bone tissue engineering. ACS Nano. 2020;14(8):1002710044. doi:10.1021/acsnano.0c03130

52. Sun Y, Kaneko S, Li XK, The LX. PI3K/Akt signal hyperactivates Eya1 via the SUMOylation pathway. Oncogene. 2015;34(19):25272537. doi:10.1038/onc.2014.179

53. Griesche N, Luttmann W, Luttmann A, Stammermann T, Geiger H, Baer PC. A simple modification of the separation method reduces heterogeneity of adipose-derived stem cells. Cells Tissues Organs. 2010;192(2):106115. doi:10.1159/000289586

Read more:
Effect of tensile frequency on osteogenic differentiation | IJGM - Dove Medical Press

By Martin Neunzert They say life's what happens as we try to re-center from the little detours chance puts in our path. Here's a little story that encircles medicine, perseverance and evolving bicycle technology and chronicles three trips on the White Rim Trail in Southern Utah taken over the last 33 years.

CRAAARACK-ACK! The sound yerked me from half-sleep and echoed around the Wingate Sandstone cliffs, unseen above me in the darkness. What the? It sounded like a rifle shot! In the middle of the night? At Hardscrabble Bottom, deep in the heart of Canyonlands National Park? When I had gotten my permit to camp on the White Rim, the ranger had chuckled and said, Youre on your own! to my query about there being anyone else in the area. I shivered the rest of the night away, and at first light threw everything into the panniers and rode as fast as I could to the sun. Once I got a good look at the Green River, I could see what had made the noise: Long shelves of ice had formed along the banks. When the water level dropped during the night, the shelves snapped off violently.

I had never been so cold. I had stripped my equipment well past the point of comfortjust a summer-weight synthetic sleeping bag and a bivy sackin order to carry enough water. That night it was 14 degrees in Moab. The photo is of my feet wearing my heavy gloves in an attempt to get some feeling back into them.

A modern treatment for leukemia is to kill off the patients bone marrow, chemically, then rebuild it using stem cells from a suitable donor, my brother in my case. Although a miraculous procedure, the recipients body feverishly, literally, tries to reject the transplant. I ran a 104 degree fever for four days yet I felt intensely cold. Even under heated blankets, my uncontrollable shivering was so bad at times they gave me Demerol just so I could sleep for short periods.

PD and I stopped next to a woman standing at the edge of one of the uppermost switchbacks on the Shafer Trail. East of our toes, the road dropped 1,300 feet to the White Rim. A whooshing sound behind us caused us all to whip around. A guy on a mountain bike flew by. Nobody spoke for a few seconds, watching. Can you believe hes doing it in flats and tennis shoes? the woman asked the sky. He forgot his cleats this morning. I turned slightly and half-stepped toward my bike so she couldnt see my platform pedals and light hiking shoes. Then she was gone, too, and silence returned, in its enormity. I muttered Try that with partial vision loss, anemia (only two-thirds the amount of red blood cells of normal), osteopenia, chemo-brain, persistent left-side weakness, and, depending on who you talk to, sleep apnea, elevated creatinine level and residual PTSD.

I know who the real hero is, PD said quietly.

Two switchbacks lower, we stopped again as some vehicles were bunched up. Young Dude asked What kind of bike is that!? His tone of voice hovered between complete incomprehension and incredulousness, as if he had only seen a lugged-and-brazed frame used as a rusty bike rack, bolted to a sidewalk outside a bike shop.

Its an antique I said, politely leaving unsaid the part that it was state-of-the-art before he was born. It was bikes like these that blew open the whole mountain biking industry. Want to see it? Go to the parking area at the Slickrock Bike Trail above Moab and find the little interpretive sign at the south end that provides the history of the Trail. Mines a twin to the one in the photo, except silver.

We werent out to be the first or to break records. After all, websites for bragging about your accomplishments wouldnt go on-line for another twenty years. I wasnt even sure mountain biking in the desert would be practical: Too hot, too big, too sandy. But with each experience, we gained confidence and efficiency, always dreaming of future adventures.

Somewhere near Candlestick Butte, a movement and clattering of rocks caused me to skid to a halt. A mature desert bighorn ram had come partway down a shaly slope, our paths crossing by incomprehensible randomness. He intently studied me, perhaps confused by my bull moose handlebars. I was in awe, and a little jealous. Until then, I had felt smugly autonomous, carrying all my water for four days, yet he thrived in this environment, sculpted by evolution, sustained by the instinctive drive to survive.

Thank God for disc brakes! another woman said, grinning and letting gravity take her easily down the fun descent off Murphy Hogback. I wondered if she knew what center-pull brakes were. I was confident they were capable of locking up the wheels even when loaded with all my camping gear and nine quarts of water. Then as now, logistics are the real challenge of cycling the White Rim. My first time around, I chose to do it solo and self-contained. I had managed to cache a gallon at Potato Bottom, but failed to place another near Monument Basin. But when the opportunity came, I adapted by figuring out how to carry enough and going when it wasnt hot. Not once did it occur to me to mooch water the way modern bikepackers sometimes do, mostly because I truly value self-sufficiency, but partly because there was no one else.

Out by the Black Crack, I stopped to take in the expansive panorama, from Ekker Butte to the Buttes of the Cross. Youre riding a non-suspended bike? Mister More-Brand-Logos-Than-Spokes asked, pulling alongside. I laughed because I could see he was in too much of a hurry to listen to me explain why I wasnt on my full-suspension off-road recumbent, so I just answered, perhaps a little too flippantly, Oh, Ive been doing this for 30 years, I dont know what the big deal is.

Well, he replied, It makes a big difference to your forearms! and he flapped his hands as if he was shaking water from them. I flashed back to my initial days in rehab. After six weeks of immobilization from pneumonia and a stroke, my muscles were so weakened that my joints, particularly my knees and hips, would separate when I tried to relax or sleep. I dont mind telling you I had never felt such exquisite pain. It got so bad I asked for Oxycodone several times. That was kind of scary in itself. My physical therapists thought I was working extra hard in the gym to get back on my bike, I just wanted to build up my strength to stop the pain.

But in the present, I watched him sprint away, no doubt a drum-machine-and-synthesizer soundtrack playing in his mind like in the bike porn videos. I sincerely hoped he would someday learn the value of developing skills, tenacity and patience

When PD had invited me to ride the Rim again, I was terrified. It had been six months since I had tried to ride a diamond-frame bike, and that attempt had ended in a crash. But he refused to accede. I think my physical therapists had secretly gotten to him and persuaded him to find ways to get me back on the horse. I seriously considered moving him to the ex-friends category. Nevertheless, I was deeply curious to see if anything had changed in two-and-a-half decades. Fundamentally, no, nothing has. One still feels very insignificant out there in the vastness. But when I dragged into camp that evening at dusk, I sensed something was different.

Not just the breeze rustling the yellow cottonwood leaves, not the position of a cactus spine, definitely not the enduring sandstone that changes only imperceptibly during a humans lifespan. No, it was I who had changed.

Cancer (and its treatment and complications) had, for a time, taken away my freedom, my sanity, my dignity, my hair, my balance, my mobility, strength, even my appetite, leaving me only with my determination. Now everything I do is, in some way, therapy in the long fight to return to some level of normalcy.

Just last week one of my docs mentioned she was amazed at how well I handled 22 days in the ICU, horribly uncomfortably proned, with what felt like a garden-hose-sized ventilator tube jammed down my throat. Inspirational tenacity? Im not sure. I just did what it took to get through it. Perhaps there was an element of luck. One of the nurses in the ICU where they took me to after my stroke told me In the six years Ive worked here, youre the only one who has survived the combination of acute myeloid leukemia, a bone marrow transplant, pneumonia and stroke.

And I learned so much. Like genetics, the insane complexities of medicine or about being a professional patient. Stuff I never wanted to know that will now haunt me forever. Never again would I take for granted the profound compassion and intrinsic nonjudgmental nature of the myriad of people who helped me along the way. Or to be able to just jump on a bike and ride. Or to sleep under the rotating stars.

The White Rim Trail is a classic and deservedly popular 100-mile loop, mostly in Canyonlands National Park, mostly off-pavement.

Martin Neunzert is a long-time cyclist and tourer. He cycled the White Rim in 1989, 1990 and 2016 and has completed many other on- and off-road adventures along the way. He is now likely seen around Ogden, Utah, on his recumbent trike.

(Visited 97 times, 1 visits today)

See the original post here:
The White Rim - Around Again - Cycling West - Cycling Utah

Introduction

Gastric and gastroesophageal junction (G/GEJ) cancer is the fifth most common cancer and the third most deadly cancer worldwide, with an estimated 1 million new cases and 783,000 death in 2018.1 In China, the incidence and mortality rate of G/GEJ cancer is substantially higher than that of the rest of the world. According to the data from Cancer Registry in China, the country has about 403,000 new cases and 291,000 death each year. Moreover, about 54% of patients are diagnosed with locally advanced (stage II and III) G/GEJ cancers2 which are characterized by tumor infiltration of the serosa and the presence of regional lymph node metastasis. At this stage, surgery alone might not be sufficient, and even with R0 surgical resection, the prognosis remains dismal.3 Regarding the patients developing T3-4 and N2-3 cancer, the 5-year survival rate after surgery is lower than 15%.4

Multimodality regimens, such as perioperative chemotherapy and adjuvant or neoadjuvant concurrent chemoradiotherapy (cCRT), have been developed to improve the poor prognosis in advanced G/GEJ cancers. Clinical trials have demonstrated that perioperative chemotherapy has specific advantages in degrading tumor stage, increasing R0 resection rate, and improving pathological complete response (pCR) rate. Thus, patients with operable stage III to IVa G/GEJ cancers can obtain significant survival benefits.57 The therapeutic benefits of preoperative cCRT, on the other hand, have been confirmed by the long-term follow-up results from CROSS and POET trials.8,9 In addition, the multidisciplinary team (MDT) guided cCRT has dominated the treatment for stage IIIIV inoperable tumors in western countries, which provides a therapeutic window for radical surgery to extend the overall survival.

In addition to surgery, chemotherapy and radiation therapy, immunotherapy has become a new pillar of cancer therapy, improving the prognosis across a wide variety of solid tumors. One main driver behind this success is the development of immune checkpoint inhibitors (ICIs). Immunotherapy targeting PD-1/PD-L1 signalling is the most widely investigated regimen, and emerging clinical evidence has demonstrated its efficacy and safety. As a monotherapy regimen, anti-PD-(L)1 has conferred an approximately 12% objective response rate (ORR) in patients with advanced G/GEJ cancers, and an overall survival benefit was noted.1012 In particular, the ORR in PD-L1 positive tumors was reported to be 15.5% versus 5.5% in PD-L1 negative ones in the KEYNOTE-059 study. Based on such results, in 2017 the US Food and Drug Administration (FDA) approved the use of pembrolizumab in the third-line treatment for patients with recurrent or advanced gastric cancer whose tumors express PD-L1. Also, in 2017 the Japanese Ministry of Health, Labor and Welfare approved nivolumab for the treatment of unresectable recurrent or advanced gastric cancer, which has progressed after chemotherapy.

Enormous combination studies have been carried out to improve the anti-tumor activities of the anti-PD-(L)1 regimen. In addition to inhibiting tumor proliferation, chemotherapy can modulate the immune system by enhancing tumor antigenicity, disturbing immune-suppressive pathways, inducing immunogenic cell death, and increasing effector T-cell functions.13,14 Therefore, it is hypothesized that the addition of chemotherapy to anti-PD-(L)1 may render superior benefits in advanced G/GEJ cancer patients. The global Phase 3 trial CHECKMATE-649 demonstrated a significant improvement in PFS (7.7 months versus 6.0 months in chemotherapy alone cohort) and OS (14.4 months versus 11.1 months in chemotherapy alone cohort) by combining chemotherapy with anti-PD-(L)1.15,16 As a result of this study, in April 2021 FDA approved nivolumab in combination with chemotherapy as the first-line treatment for advanced or metastatic G/GEJ and esophageal adenocarcinoma. Likewise, in the ongoing ATTRACTION-4 trials, nivolumab combined with chemotherapy has shown encouraging clinical activity in first-line treatment in Asian patients with advanced G/GEJ, with improved PFS and ORR and favourable safety profile.17

On the other hand, irradiation activates host immunity by triggering immunogenic cell death to release the damage-associated molecular patterns (DAMPs), which leads to dendritic cell activation, tumor antigens presentation and antigen-specific T cells priming.18 Based on the insights gained, there is a strong rationale to support the use of PD-(L)1 inhibitors with cCRT to convert the cold tumors into hot tumors. The therapeutic efficacy of such regimen has been demonstrated in the Phase III PACIFIC trial of the PD-L1 inhibitor durvalumab in locally advanced, unresectable NSCLC following platinum-based cCRT.19 Compared to the placebo, an improved PFS (from 5.6 to 16.8 months) with a similar safety profile was noted with durvalumab, and its efficacy was irrespective of tumor PD-L1 status. Based on that, in 2018 FDA approved durvalumab for patients with unresectable stage III NSCLC whose disease has not progressed following platinum-based cCRT. Moreover, the phase 3 CHECKMATE-577 trial is the first study to evaluate a checkpoint inhibitor in the adjuvant setting after cCRT and has demonstrated a statistically significant improvement in disease-free survival (DFS) from 11.0 months to 22.4 months in patients with resected esophageal and GEJ cancers.20 Despite these advances, the ideal combination regimen of checkpoint inhibitors and cCRT is yet to be optimized in patients with advanced G/GEJ cancers.

Sintilimab is a highly selective, monoclonal IgG4 antibody that inhibits interactions between PD-1 and its ligands, with a robust anti-tumor response.21 In vitro, compared to nivolumab or pembrolizumab, sintilimab has a higher binding affinity and is able to bind with more PD-1 molecules on CD3+T cells, and has better T cell activating characteristics.21 In a phase 1b study (NCT03745170), sintilimab combined with oxaliplatin/capecitabine has shown promising efficacy in gastric cancer with an ORR of 85%22 which warranted a phase 3 ORIENT-16 study (NCT03745170) to further investigate the therapeutic potential of sintilimab in combination with chemotherapy in patients with advanced G/GEJ cancers.23 The interim results from ORIENT-16 study were orally presented on 2021 ESMO, in which sintilimab was demonstrated to be the first PD-1 inhibitor to show superior OS and PFS with an acceptable safety profile, in combination with chemo, in Chinese patients with G/GEJ cancer regardless of PD-L1 expression status.24

In addition, in Japan, D2 gastrectomy plus adjuvant S-1 is the standard treatment for locally advanced gastric cancer [18, 19], while nab-paclitaxel (nab-PTX) is an approved second-line gastric cancer treatment [23, 24]. The combination of S-1 with nab-PTX has proven to be an effective and safe first-line regimen in clinical [25, 26]. Furthermore, the interim analysis from the JACCRO GC-07 trial has demonstrated that the postoperative S-1 plus docetaxel is effective with few safety concerns in patients with stage III gastric cancer25 and similar benefits were also noted with S-1 plus nab-PTX in untreated patients with metastatic gastric cancer.26

Therefore, we propose to conduct the SHARED (Sintilimab plus chemoradiotherapy in gastric and GEJ adenocarcinoma) study, a phase 2 trial designed to evaluate perioperative sintilimab combined cCRT (S-1 plus nab-PTX) for patients with locally advanced G/GEJ adenocarcinoma.

The primary objective is to demonstrate whether adding sintilimab to cCRT (a combination of nab-PTX and S-1 with radiotherapy) improves pathological complete response (pCR) rate in patients with locally advanced G/GEJ adenocarcinoma. The secondary objectives include disease-free survival (DFS), major pathological response (MPR), R0 resection rate, surgical adverse events (AEs), overall survival (OS), event-free survival (EFS), and safety profile. Moreover, the prognostic value of tumor biomarkers and immune biomarkers will be evaluated.

This is a prospective, multicentre, single-arm phase II trial in China. Eligible patients are aged 18 or older, histological or cytological confirmed locally advanced gastric or GEJ adenocarcinoma as defined according to 8th edition of American Joint Committee on Cancer (AJCC) classification of malignant tumors, a clinical-stage of III to IVa (T3N2-3M0, T4aN+M0 or T4bNanyM0) according to the 8th edition of Tumor, Node, Metastasis (TNM) classification, an Eastern Cooperative Oncology Group performance (ECOG) score 1, no prior cancer treatment, and one or more measurable lesions based on Response Evaluation Criteria in Solid Tumors (RECIST v1.1).

Eligibility criteria on physiological parameters and organ function included adequate haematological function (absolute neutrophil count [ANC] 1.5109/L, platelet count [PLT] 100109/L, haemoglobin [Hb] 90 g/L, international normalized ratio (INR)/prothrombin time (PT) 1.5upper limit of normal [ULN], and activated partial thromboplastin time (aPTT) 1.5ULN), adequate hepatic function (plasma total bilirubin [PTB] 1.5ULN, alanine transaminase [ALT], aspartate transaminase [AST] and alkaline phosphatase [AKP] concentration 2.5ULN), and adequate renal function (creatinine concentration 1ULN, albumin concentration 30g/L).

Pregnant, breastfeeding women or those positive in baseline pregnancy tests are not eligible, and all female patients will be on contraception during the study. Other exclusion criteria include patients diagnosed with gastric neuroendocrine tumors; patients with distant metastasis according to computed CT/MRI or endoscopic ultrasound (EUS); history of chemotherapy, radiotherapy or immunotherapy; patients with active malignant tumor in recent 5 years, except for basal cell or squamous cell cancer, superficial bladder cancer, and in-situ cervical or breast carcinoma; uncontrollable pleural effusion, pericardial effusion or ascites; severe cardiovascular disease within 12 months before recruitment, including coronary artery disease, grade 2 congestive heart failure, uncontrollable arrhythmias and myocardial infarction; patients with upper GI tract obstruction/bleeding or functional abnormality or malabsorption syndrome, which can affect absorption of S-1; uncontrollable concurrent infection and other concomitant diseases systemic diseases, or moderate or severe renal injury; allergic to any drug in this study; use of steroids or any other systemic immunosuppressive agents within 14 days before recruitment; use of corticosteroids (dosage >10mg/d prednisone or equivalent dose of other glucocorticoids) or other immunosuppressive agents within 4 weeks before recruitment, except patient treated with hormone for preventing allergy to contrast agents; receiving treatment from other studies within 4 weeks before recruitment; patients with autoimmune diseases or primary immunodeficiency; vaccinated with attenuated vaccine within 4 weeks before recruitment or plan to vaccinate during the study; active infections, including TB, HIV, HBV and HCV; and history of allogeneic stem cell transplantation or organ transplantation.

The study protocol and the informed consent forms have been reviewed and approved by institutional review boards and ethics committees at each institution. The study will be done in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines, and the written informed consent will be obtained from all enrolled patients. The study has been registered (ChiCTR1900024428).

All patients will start with one cycle (3 weeks) of induction chemotherapy of S-1 (40mg/m2, PO, bid, D1 to D14) and nab-PTX (100120mg/m2, IV, D1 and D8) in combination with sintilimab (200mg, IV, D1), followed by radiation therapy (45Gy/1.8Gy in 25 factions) with concurrent nab-PTX (80100 mg/m2, IV, D1, D8, D15, and D22) and sintilimab (200mg, IV, D1 and D22) for 5 weeks. One to three weeks after the completion, patients will receive another cycle (3 weeks) of S-1 (40mg/m2, PO, bid, D1 to D14) and nab-PTX (100120mg/m2, IV, D1 and D8) in combination with sintilimab (200mg, IV, D1). All patients will be preferably operated on within 1 to 3 weeks later. In case of any treatment-related adverse event (TRAE) that cannot be resolved shortly is noted, the patient will be urged to tumor assessment followed by the surgery. The adjuvant therapy will start in 46 weeks after the operation with 3 cycles (3 weeks) of S-1 (40mg/m2, PO, bid, D1 to D14 of each cycle), nab-PTX (100120mg/m2, IV, D1 and D8 of each cycle) and sintilimab (200mg, IV, D1 of each cycle). Schematic diagram of SHARED regimen is in Figure 1.

Figure 1 Schematic diagram of SHARED regimen. All patients will receive one cycle of S-1 (40mg/m2, PO, Bid, D1 to D14) and nab-PTX (100120mg/m2, IV, D1 and D8) in combination with sintilimab (200mg, IV, D1) for 3 weeks, followed by radiation therapy (45Gy/1.8Gy in 25 factions) with nab-PTX (80100 mg/m2, IV, D1, D8, D15, and D22) and sintilimab (200mg, IV, D1 and D22) for 5 weeks. One to three weeks after the completion, patients will receive one cycle of S-1 (40mg/m2, PO, Bid, D1 to D14) and nab-PTX (100120mg/m2, IV, D1 and D8) in combination with sintilimab (200mg, IV, D1). All patients will be operated on within 1 to 3 weeks later. The adjuvant therapy will start in 46 weeks after the operation with 3 cycles (3 weeks) of S-1 (40mg/m2, PO, Bid, D1 to D14 of each cycle), nab-PTX (100120mg/m2, IV, D1 and D8 of each cycle) and sintilimab (200mg, IV, D1 of each cycle).

Dose modification of sintilimab is not allowed in this study. Sintilimab will be withheld to manage intolerable adverse event until toxicity resolves. Corticosteroids will be used to manage immune-related adverse events (irAEs) with Sintilimab discontinuation allowed for no more than 12 weeks. Sintilimab will be terminated upon completion of treatment, disease progression or intolerance. Guidance for sintilimab delay or discontinuation after adverse events is in Table 1.

Table 1 Guidance for Sintilimab Delay or Discontinuation

Before the operation, all patients will be assessed by the multidisciplinary committee, and surgical protocol will be decided according to the clinical judgment. A subject must be withdrawn from the study if a disease progression is noted. The surgical protocol includes transthoracic esophagectomy with resection of the proximal stomach and mediastinal and abdominal lymphadenectomy for type 1 GEJ cancers and gastrectomy with transhiatal distal oesophagectomy plus D2 lymphadenectomy for types 2 and 3 GEJ cancers. For the patients with gastric cancer, total or subtotal distal gastrectomy with D2 lymphadenectomy will be performed. For inoperable patients, the treatment options will be evaluated and tailored to the context of the patients situation and needs. Exploratory laparoscopy will be conducted to exclude peritoneal metastases, if necessary.

During the study, all patients will be assessed by physical examination, weight, ECOG performance status, vital signs, routine laboratory tests (blood routine, blood chemistry, blood coagulation routine, urine routine, stool routine for occult blood, and thyroid function), and 12-lead electrocardiogram (ECG) within 7 days before day one of the first cycle, one day before day one in subsequent cycles, one day before the surgery, one day after study completion or withdrawal, and at the first follow-up visit. Besides, all patients will be examined by echocardiography within 7 days before starting the study, one day before the surgery, one day before the first adjuvant cycle, and one day after study completion or withdrawal.

Tumor assessment by means of computed CT/MRI and EUS will be done at baseline (within 4 weeks before enrolment), every six weeks (7 days) perioperatively, every nine weeks (7 days) postoperatively, and every 3 months until the development of disease progression or the start of new anticancer treatment after treatment completion.

All patients will be followed up on a monthly basis in the first 3 months for safety, and telephone visits will be conducted after the first in-person hospital visit. After completing safety follow-up, monthly telephone visits for a maximum of 2 years will be implemented until death from any cause, lost to follow-up, consent withdrawal, or sponsors election to terminate the study prematurely.

The primary endpoint is the rate of pCR, which is defined as the absence of viable tumor cells assessed by histological evaluation criteria after preoperative therapy.

DFS is defined as the time to postoperative recurrence or death from any cause. OS is defined as the time interval from enrolment to all-caused death. EFS is defined as the time from enrolment to recurrence or death from any cause. Patients (including those lost to follow-up) still alive at the time of analysis (DFS, OS and EFS) will be censored at the last disease assessment date.

MPR is defined as tumor residual cells 10% in the surgical specimen. R0 resection rate is defined as the rate of the complete surgical removal of any residual cancer cells in the tumor bed.

Adverse events (AEs) will be monitored and graded according to the US National Cancer Institutes Common Terminology Criteria for Adverse Events (NCI CTCAE, version 4.03). All AEs will be recorded from the first dose of the study drug until 90 days after the last dose of the study drugs. Serious adverse events (SAEs) are defined as death, hospitalization or prolonged hospitalization, permanent or severe disability, teratogenesis, or other significant clinical sequels. Surgical AEs are defined as the complication which happens during or in 30 days after the operation.

Exploratory endpoints are assessments of potential tumor biomarkers and the relationship between immune biomarkers and clinical response.

Peripheral blood samples (10ml) will be collected at baseline and on the same day of sintilimab administration to evaluate serum levels of related tumor biomarkers (eg, CEA, AFP, CA19-9, CA72-4, and CA24-2).

Tumor tissue samples will be collected at baseline and intrasurgery. TMB and MSI will be measured by FoundationOne CDx assay (Foundation Medicine, Cambridge, MA, USA). TMB-H will be defined as 10mut/Mb, and MSI-H will be defined as more than 30% of markers show instability in marker panels. PD-L1 expression will be measured by 22C3 pharmaDx assay (Agilent Technologies, Carpinteria, CA, USA), and PD-L1+ will be defined as PD-L1 CPS 1%. HER2 immunohistochemistry was performed on the BenchMark XT platform (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturers recommendation. Normal gastric tissue will be collected concomitantly from each patient, and whole-exome sequencing (WES) will be performed to compare the transcriptome between tumor tissue and normal gastric tissue.

For the purpose of study design, a pCR rate at 15% (H0) in this setting will not be viewed as compelling for further study, while a pCR rate at 35% (Ha) or more will be considered of interest for further investigation. Based on this, a Simon optimal two-stage design will be employed with an H0=0.15 vs Ha=0.35, =0.05 (one-sided) and power of 80%. Using these operating characteristics, the study plan is as follows (Figure 2):

Figure 2 Simon optimal two stage design for SHARED study. At stage 1, nine patients who meet the inclusion criteria will be enrolled. If one or more patients demonstrate pCR, the study will advance to stage 2 to include 25 additional patients.

The sample size was calculated using the Power Analysis and Sample Size software program (PASS 16, NCSS Kaysville Utah USA).

EFS, DFS and OS will be analysed with the KaplanMeier method and the stratified Log rank test. The hazard ratio and its 95% confidence interval will be estimated with the Cox proportional hazards model. The incidences of the AEs and surgical AEs will be analysed using Fishers exact test. All comparisons are one-sided at the 0.05 level of significance. Data will be analysed with SAS version 9.4 statistical software (SAS Institute Inc., Cary, NC, USA).

A number of clinical studies in patients with locally advanced G/GEJ cancers have evaluated the feasibility and efficacy of multimodal treatment approaches that center on a tumor-removing surgery with preoperative or postoperative chemotherapy or radiotherapy.9,2731 Encouraged by the promising results from CHECKMATE-57720 the focus has been shifted towards the regimens that combine checkpoint inhibitors with chemotherapy in adjuvant or neoadjuvant settings (eg, ATTRACTION-5, KEYNOTE-585, MATTERHORN, and NCT04139135) for advanced G/GEJ cancers. The SHARED study is designed to begin with induction chemotherapy with anti-PD-1 to achieve maximum synergistic effects with subsequent cCRT. Given the potential of radiotherapy to revert the immune-suppressive tumor microenvironment, the addition of anti-PD-1 to cCRT is expected to provide a holistic solution for patients at a clinical-stage of T3N2-3M0, T4aN+ M0 or T4bNanyM0, a heterogenous population of both operable and inoperable patients.

Overall, there is a strong rationale warranting a phase 2 clinical trial to explore sintilimab combined with concurrent chemoradiotherapy in locally advanced G/GEJ cancers. The study aims are twofold, targeting to increase the pCR rate for operable patients to prolong the overall survival while ensuring that all patients, including inoperable patients, will gain more survival benefits from surgery. In addition, this study also has the capacity to prospectively evaluate the prognostic value of the status of TMB and MSI, PD-L1 expression level and tumor biomarkers, which can be applied to define eligibility criteria and stratification factors for further trials in locally advanced G/GEJ cancers.

The trial is ongoing, and no data is available.

The trial has been approved by the Ethics Committee of the Comprehensive Cancer Centre of Drum Tower Hospital. All patients are required to sign the written informed consent.

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

All investigators will not receive any remuneration. This research did not receive any grant from funding agencies in the public, commercial or not-for-profit sectors.

Innovent Biologics, Inc. provided the sintilimab, but had no role in study design, or writing of the protocol. The authors report no other conflicts of interest in this work.

1. Rawla P, Barsouk A. Epidemiology of gastric cancer: global trends, risk factors and prevention. Prz Gastroenterol. 2019;14(1):2638. doi:10.5114/pg.2018.80001

2. Zheng L, Wu C, Xi P, et al. The survival and the long-term trends of patients with gastric cancer in Shanghai, China. BMC Cancer. 2014;14(1):300. doi:10.1186/1471-2407-14-300

3. Ajani JA, Bentrem DJ, Besh S, et al. Gastric cancer, version 2.2013: featured updates to the NCCN guidelines. J Natl Compr Canc Netw. 2013;11(5):531546. doi:10.6004/jnccn.2013.0070

4. Sites A. SEER Cancer Statistics Review, 19752011. Bethesda, MD: National Cancer Institute; 2014.

5. Ji J, Shen L, Li Z, et al. Perioperative chemotherapy of oxaliplatin combined with S-1 (SOX) versus postoperative chemotherapy of SOX or oxaliplatin with capecitabine (XELOX) in locally advanced gastric adenocarcinoma with D2 gastrectomy: a randomized phase III trial (RESOLVE trial). Ann Oncol. 2019;30:v877. doi:10.1093/annonc/mdz394.033

6. Kang YK, Yook JH, Park YK, et al. Phase III randomized study of neoadjuvant chemotherapy (CT) with docetaxel (D), oxaliplatin (O) and S-1(S) (DOS) followed by surgery and adjuvant S-1, vs surgery and adjuvant S-1, for resectable advanced gastric cancer (GC) (PRODIGY). Ann Oncol. 2019;30:v876v877. doi:10.1093/annonc/mdz394.032

7. Al-Batran SE, Homann N, Pauligk C, et al. Perioperative chemotherapy with fluorouracil plus leucovorin, oxaliplatin, and docetaxel versus fluorouracil or capecitabine plus cisplatin and epirubicin for locally advanced, resectable gastric or gastro-oesophageal junction adenocarcinoma (FLOT4): a randomised, phase 2/3 trial. Lancet. 2019;393(10184):19481957. doi:10.1016/s0140-6736(18)32557-1

8. Stahl M, Walz MK, Riera-Knorrenschild J, et al. Preoperative chemotherapy versus chemoradiotherapy in locally advanced adenocarcinomas of the oesophagogastric junction (POET): long-term results of a controlled randomised trial. Eur J Cancer. 2017;81:183190. doi:10.1016/j.ejca.2017.04.027

9. Shapiro J, van Lanschot JJB, Hulshof M, et al. Neoadjuvant chemoradiotherapy plus surgery versus surgery alone for oesophageal or junctional cancer (CROSS): long-term results of a randomised controlled trial. Lancet Oncol. 2015;16(9):10901098. doi:10.1016/s1470-2045(15)00040-6

10. Muro K, Chung HC, Shankaran V, et al. Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trial. Lancet Oncol. 2016;17(6):717726. doi:10.1016/S1470-2045(16)00175-3

11. Fuchs CS, Doi T, Jang RW, et al. Safety and efficacy of pembrolizumab monotherapy in patients with previously treated advanced gastric and gastroesophageal junction cancer: phase 2 clinical KEYNOTE-059 trial. JAMA Oncol. 2018;4(5):e180013e180013. doi:10.1001/jamaoncol.2018.0013

12. Kang Y-K, Boku N, Satoh T, et al. Nivolumab in patients with advanced gastric or gastro-oesophageal junction cancer refractory to, or intolerant of, at least two previous chemotherapy regimens (ONO-4538-12, ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2017;390(10111):24612471. doi:10.1016/S0140-6736(17)31827-5

13. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of cancer chemotherapy. Nat Rev Immunol. 2008;8(1):5973. doi:10.1038/nri2216

14. Hato SV, Khong A, de Vries IJM, Lesterhuis WJ. Molecular pathways: the immunogenic effects of platinum-based chemotherapeutics. Clin Cancer Res. 2014;20(11):28312837. doi:10.1158/1078-0432.CCR-13-3141

15. Moehler M, Shitara K, Garrido M, et al. LBA6_PR Nivolumab (nivo) plus chemotherapy (chemo) versus chemo as first-line (1L) treatment for advanced gastric cancer/gastroesophageal junction cancer (GC/GEJC)/esophageal adenocarcinoma (EAC): first results of the CheckMate 649 study. Ann Oncol. 2020;31:S1191. doi:10.1016/j.annonc.2020.08.2296

16. Janjigian YY, Shitara K, Moehler M, et al. First-line nivolumab plus chemotherapy versus chemotherapy alone for advanced gastric, gastro-oesophageal junction, and oesophageal adenocarcinoma (CheckMate 649): a randomised, open-label, phase 3 trial. Lancet. 2021;398(10294):2740. doi:10.1016/S0140-6736(21)00797-2

17. Boku N, Ryu MH, Oh DY, et al. Nivolumab plus chemotherapy versus chemotherapy alone in patients with previously untreated advanced or recurrent gastric/gastroesophageal junction (G/GEJ) cancer: ATTRACTION-4 (ONO-4538-37) study. Ann Oncol. 2020;31:S1192. doi:10.1016/j.annonc.2020.08.2297

18. Golden EB, Frances D, Pellicciotta I, Demaria S, Helen Barcellos-Hoff M, Formenti SC. Radiation fosters dose-dependent and chemotherapy-induced immunogenic cell death. Oncoimmunology. 2014;3(4):e28518. doi:10.4161/onci.28518

19. Antonia SJ, Villegas A, Daniel D, et al. Durvalumab after chemoradiotherapy in stage III nonsmall-cell lung cancer. N Engl J Med. 2017;377(20):19191929. doi:10.1056/NEJMoa1709937

20. Kelly RJ, Ajani JA, Kuzdzal J, et al. Adjuvant nivolumab in resected esophageal or gastroesophageal junction cancer. N Engl J Med. 2021;384(13):11911203. doi:10.1056/NEJMoa2032125

21. Wang J, Fei K, Jing H, et al. Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit. INMABS. 2019;11(8):14431451.

22. Xu J, Jin Y, Liu Y, Zhou H, Wang Y. Abstract CT213: ORIENT-16: sintilimab plus XELOX vs placebo plus XELOX as 1< sup> st line treatment for unresectable advanced gastric and GEJ adenocarcinoma. Cancer Res. 2019;79(13 Supplement):CT213. doi:10.1158/1538-7445.AM2019-CT213

23. Jiang H, Zheng Y, Qian J, et al. Safety and efficacy of sintilimab combined with oxaliplatin/capecitabine as first-line treatment in patients with locally advanced or metastatic gastric/gastroesophageal junction adenocarcinoma in a phase Ib clinical trial. BMC Cancer. 2020;20(1):760. doi:10.1186/s12885-020-07251-z

24. Xu J, Jiang H, Pan Y, et al. LBA53 sintilimab plus chemotherapy (chemo) versus chemo as first-line treatment for advanced gastric or gastroesophageal junction (G/GEJ) adenocarcinoma (ORIENT-16): first results of a randomized, double-blind, phase III study. Ann Oncol. 2021;32:S1331. doi:10.1016/j.annonc.2021.08.2133

25. Yoshida K, Kodera Y, Kochi M, et al. Addition of docetaxel to oral fluoropyrimidine improves efficacy in patients with stage III gastric cancer: interim analysis of JACCRO GC-07, a randomized controlled trial. J Clin Oncol. 2019;37(15):12961304. doi:10.1200/jco.18.01138

26. He -M-M, Wang F, Jin Y, et al. Phase II clinical trial of S-1 plus nanoparticle albumin-bound paclitaxel in untreated patients with metastatic gastric cancer. Cancer Sci. 2018;109(11):35753582. doi:10.1111/cas.13813

27. Ajani JA, Winter K, Fau Okawara GS, et al. Phase II trial of preoperative chemoradiation in patients with localized gastric adenocarcinoma (RTOG 9904): quality of combined modality therapy and pathologic response. J Clin Oncol. 2006;24(24):39533958.

28. Cunningham D, Allum WH, Stenning SP, et al. Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer. N Engl J Med. 2006;355(1):1120. doi:10.1056/NEJMoa055531

29. De Paoli A, Navarria F, Torrisi E, et al. Neoadjuvant epirubicyn, oxaliplatin, capecitabine and radiation therapy (NEOX-RT) followed by surgery for locally advanced gastric cancer (LAGC): a phase II multicentric study. J Clin Oncol. 2019;37(15_suppl):4066. doi:10.1200/JCO.2019.37.15_suppl.4066

30. Leong T, Smithers BM, Haustermans K, et al. TOPGEAR: a randomized, phase III trial of perioperative ECF chemotherapy with or without preoperative chemoradiation for resectable gastric cancer: interim results from an international, intergroup trial of the AGITG, TROG, EORTC and CCTG. Ann Surg Oncol. 2017;24(8):22522258.

31. Macdonald JS, Smalley SR, Benedetti J. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med. 2001;345(10):725730. doi:10.1056/NEJMoa010187

View post:
Efficacy and Safety of Sintilimab in Combination with Concurrent Chemo | CMAR - Dove Medical Press

Democratic Sen. Ted Kennedy ( left) and Republican Sen. Orrin Hatch teamed up on a series of landmark legislative health care achievements, such as the Ryan White program on AIDS treatment, the Americans with Disabilities Act, and the first major federal child care law. John Duricka/AP hide caption

Democratic Sen. Ted Kennedy ( left) and Republican Sen. Orrin Hatch teamed up on a series of landmark legislative health care achievements, such as the Ryan White program on AIDS treatment, the Americans with Disabilities Act, and the first major federal child care law.

When it comes to health policy, former Utah Republican Sen. Orrin Hatch, who died Saturday at age 88, leaves a complex legacy of major legislative achievements, changing positions, compromises and fierce opposition. In many ways, though, Hatch's evolution and leadership on health policy during his four decades in the U.S. Senate mirror that of the Republican Party.

When he came to Washington as a neophyte politician after an upset victory in 1976, Hatch was a conservative firebrand, one of the early leaders of the "New Right" bent on dismantling the federal welfare state and banning abortion. A former trial lawyer, the new senator had never before held public office.

But the election of Ronald Reagan in 1980 and the Republican takeover of the Senate that made Hatch chairman of the powerful Labor and Human Resources Committee (now the Health, Education, Labor and Pensions Committee) turned him into something of a pragmatist. That pragmatism, it should be noted, was somewhat forced: Even though Hatch was technically the chair, there were enough moderate Republicans on the panel to give the ranking Democrat, Massachusetts' Edward Kennedy, effective control over what could be passed by the committee.

So Hatch learned to compromise and to legislate. In 1984, he negotiated with liberal Rep. Henry Waxman, D-Calif., what is still referred to as the "Hatch-Waxman Act." It's better known as the law that allowed, for the first time, approval of generic copies of brand-name drugs. Although far from a panacea, it is still the single-biggest advance in the fight to rein in high drug prices.

When the Democrats took back the Senate after the 1986 elections, Kennedy became chairman of the committee and Hatch, the ranking Republican. The two teamed up on a series of landmark legislative achievements, from the Ryan White program on AIDS treatment and the Americans with Disabilities Act to the first major federal child care law. And while Hatch was a strong foe of national health insurance, he and Kennedy ultimately pushed through Congress in 1997 the bill to create the Children's Health Insurance Program, which provides low-cost health insurance for low-income families who don't qualify for Medicaid.

The stridently anti-abortion Hatch was outspoken about his support for federal funding for research on embryonic stem cells derived from aborted fetuses. "I think it's the ultimate pro-life position, because I believe being pro-life is not just caring for the unborn but caring for those who are living," he told NPR in 2007.

But like much of the Republican Party in Congress, Hatch returned to his conservative roots after the election of President Barack Obama in 2008. A supporter of the so-called individual mandate requiring people to have health insurance when it was the quasi-official GOP position in the early 1990s, Hatch became an outspoken foe. "Congress has never crossed the line between regulating what people choose to do and ordering them to do it," he said in 2010.

After moderate Utah Republican Sen. Robert Bennett was ousted in a primary in 2010 and replaced by conservative favorite Mike Lee, Hatch grew more conservative to win reelection in 2012. His final term in the Senate was marked by efforts to overturn the Affordable Care Act and further restrict abortion access. The devout Mormon, who in his spare time wrote lyrics for best-selling Christian music, even called the ACA "the stupidest, dumb-a** bill that I've ever seen. Now some of you may have loved it; if you do, you are one of the stupidest dumb-a** people I've ever met." He later apologized for the statement.

A former Kennedy aide, Jim Manley, told The Salt Lake Tribune that "no one epitomizes the rightward lurch of the Republican Party more than Sen. Hatch."

In one final twist, however, Hatch pushed as his successor the 2012 GOP presidential nominee, Mitt Romney. In just his first few years, Romney has become one of the most moderate Republicans in the chamber. That may prove to be Orrin Hatch's final legacy.

KHN (Kaiser Health News) is a national newsroom that produces in-depth journalism about health issues. It is an editorially independent operating program of KFF (Kaiser Family Foundation).

Continue reading here:
Sen. Orrin Hatch's legacy tracks the GOP's evolution on health - NPR

Somatic stem cells (also called adult stem cells) exist naturally in the body. They are important for growth, healing, and replacing cells that are lost through daily wear and tear.

Stem cells from the blood and bone marrow are routinely used as a treatment for blood-related diseases. However, under natural circumstances somatic stem cells can become only a subset of related cell types. Bone marrow stem cells, for example, differentiate primarily into blood cells. This partial differentiation can be an advantage when you want to produce blood cells; but it is a disadvantage if you're interested in producing an unrelated cell type.

Most types of somatic stem cells are present in low abundance and are difficult to isolate and grow in culture. Isolation of some types could cause considerable tissue or organ damage, as in the heart or brain. Somatic stem cells can be transplanted from donor to patient, but without drugs that suppress the immune system, a patient's immune system will recognize transplanted cells as foreign and attack them.

Therapy involving somatic stem cells is not controversial; however, it is subject to the same ethical considerations that apply to all medical procedures.

Go here to read the rest:
Stem Cell Quick Reference - University of Utah

As people age, their intestinal stem cells begin to lose their ability to regenerate. These stem cells are the source for all new intestinal cells, so this decline can make it more difficult to recover from gastrointestinal infections or other conditions that affect the intestine.

This age-related loss of stem cell function can be reversed by a 24-hour fast, according to a new study from MIT biologists. The researchers found that fasting dramatically improves stem cells ability to regenerate, in both aged and young mice.

In fasting mice, cells begin breaking down fatty acids instead of glucose, a change that stimulates the stem cells to become more regenerative. The researchers found that they could also boost regeneration with a molecule that activates the same metabolic switch. Such an intervention could potentially help older people recovering from GI infections or cancer patients undergoing chemotherapy, the researchers say.

Fasting has many effects in the intestine, which include boosting regeneration as well as potential uses in any type of ailment that impinges on the intestine, such as infections or cancers, says Omer Yilmaz, an MIT assistant professor of biology, a member of the Koch Institute for Integrative Cancer Research, and one of the senior authors of the study. Understanding how fasting improves overall health, including the role of adult stem cells in intestinal regeneration, in repair, and in aging, is a fundamental interest of my laboratory.

David Sabatini, an MIT professor of biology and member of the Whitehead Institute for Biomedical Research and the Koch Institute, is also a senior author of the paper, which appears in the May 3 issue of Cell Stem Cell.

This study provided evidence that fasting induces a metabolic switch in the intestinal stem cells, from utilizing carbohydrates to burning fat, Sabatini says. Interestingly, switching these cells to fatty acid oxidation enhanced their function significantly. Pharmacological targeting of this pathway may provide a therapeutic opportunity to improve tissue homeostasis in age-associated pathologies.

The papers lead authors are Whitehead Institute postdoc Maria Mihaylova and Koch Institute postdoc Chia-Wei Cheng.

Boosting regeneration

For many decades, scientists have known that low caloric intake is linked with enhanced longevity in humans and other organisms. Yilmaz and his colleagues were interested in exploring how fasting exerts its effects at the molecular level, specifically in the intestine.

Intestinal stem cells are responsible for maintaining the lining of the intestine, which typically renews itself every five days. When an injury or infection occurs, stem cells are key to repairing any damage. As people age, the regenerative abilities of these intestinal stem cells decline, so it takes longer for the intestine to recover.

Intestinal stem cells are the workhorses of the intestine that give rise to more stem cells and to all of the various differentiated cell types of the intestine. Notably, during aging, intestinal stem function declines, which impairs the ability of the intestine to repair itself after damage, Yilmaz says. In this line of investigation, we focused on understanding how a 24-hour fast enhances the function of young and old intestinal stem cells.

After mice fasted for 24 hours, the researchers removed intestinal stem cells and grew them in a culture dish, allowing them to determine whether the cells can give rise to mini-intestines known as organoids.

The researchers found that stem cells from the fasting mice doubled their regenerative capacity.

It was very obvious that fasting had this really immense effect on the ability of intestinal crypts to form more organoids, which is stem-cell-driven, Mihaylova says. This was something that we saw in both the young mice and the aged mice, and we really wanted to understand the molecular mechanisms driving this.

Metabolic switch

Further studies, including sequencing the messenger RNA of stem cells from the mice that fasted, revealed that fasting induces cells to switch from their usual metabolism, which burns carbohydrates such as sugars, to metabolizing fatty acids. This switch occurs through the activation of transcription factors called PPARs, which turn on many genes that are involved in metabolizing fatty acids.

The researchers found that if they turned off this pathway, fasting could no longer boost regeneration. They now plan to study how this metabolic switch provokes stem cells to enhance their regenerative abilities.

They also found that they could reproduce the beneficial effects of fasting by treating mice with a molecule that mimics the effects of PPARs. That was also very surprising, Cheng says. Just activating one metabolic pathway is sufficient to reverse certain age phenotypes.

Jared Rutter, a professor of biochemistry at the University of Utah School of Medicine, described the findings as interesting and important.

This paper shows that fasting causes a metabolic change in the stem cells that reside in this organ and thereby changes their behavior to promote more cell division.In a beautiful set of experiments, the authors subvert the system by causing those metabolic changes without fasting and see similar effects, says Rutter, who was not involved in the research. This work fits into a rapidly growing field that is demonstrating that nutrition and metabolism has profound effects on the behavior of cells and this can predispose for human disease.

The findings suggest that drug treatment could stimulate regeneration without requiring patients to fast, which is difficult for most people. One group that could benefit from such treatment is cancer patients who are receiving chemotherapy, which often harms intestinal cells. It could also benefit older people who experience intestinal infections or other gastrointestinal disorders that can damage the lining of the intestine.

The researchers plan to explore the potential effectiveness of such treatments, and they also hope to study whether fasting affects regenerative abilities in stem cells in other types of tissue.

The research was funded by the National Institutes of Health, the V Foundation, a Sidney Kimmel Scholar Award, a Pew-Stewart Trust Scholar Award, the MIT Stem Cell Initiative through Fondation MIT, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the American Federation of Aging Research, the Damon Runyon Cancer Research Foundation, the Robert Black Charitable Foundation, a Koch Institute Ludwig Postdoctoral Fellowship, a Glenn/AFAR Breakthroughs in Gerontology Award, and the Howard Hughes Medical Institute.

Read the original:
Fasting boosts stem cells regenerative capacity | MIT ...

WASHINGTON -- Hundreds of clinics pushing unproven stem cell procedures caught a big break from the U.S. government in 2017: They would have three years to show that their questionable treatments were safe and worked before regulators started cracking down.

But when the Food and Drug Administration's grace period expired in late May extended six months due to the pandemic the consequences became clear: Hundreds more clinics were selling the unapproved treatments for arthritis, Alzheimers, COVID-19 and many other conditions.

It backfired, says Leigh Turner, a bioethicist at the University of California, Irvine. The scale of the problem is vastly larger for FDA today than it was at the start.

The continuing spread of for-profit clinics promoting stem cells and other so-called regenerative therapies including concentrated blood products illustrates how quickly experimental medicine can outpace government oversight. No clinic has yet won FDA approval for any stem cell offering and regulators now confront an enormous, uncooperative industry that contends it shouldn't be subject to regulation.

Although emerging research suggests stem cells could someday have broad use for a number of medical conditions, experts say they should not be used outside of well-controlled studies or a handful of established uses. For instance, stem cells collected from blood or bone marrow have long been used to treat leukemia and other blood diseases.

Many clinics use so-called adult stem cells collected from tissue like fat or bone marrow not the more versatile but controversial stem cells from embryos used in research.

Turner and other experts have tracked the growth of the clinics for nearly a decade. Clinics charge between $2,000 to $25,000 for adult stem cell injections and other infusions which they advertise for an assortment of diseases, including diabetes, autism, cancer, multiple sclerosis and vision problems. Some clinics use stem cells derived from fat, harvested via liposuction then reinjected into patients, aiming to repair joints or fight disease. Others use bone marrow or blood taken from umbilical cords after birth.

There is no government tally of how many clinics operate in the U.S. But Turner counted more than 1,200 of them in 2019, up from the 570 clinics he and a co-author identified in 2016. Hes working on an update but says the number has consistently grown.

The FDA has repeatedly warned Americans to steer clear of unapproved and unproven stem cell therapies, which have occasionally caused blindness, bacterial infections and tumors. During FDA's three-plus years of enforcement discretion, the agency sent formal warning letters to more than a dozen businesses performing the riskiest procedures. Regulators also prevailed in a Florida court case to shutdown a major clinic offering unproven treatments. Another case against a similar prominent company is pending in California.

Its time to actually get the data we need, to assess clinics' stem cell procedures, FDA's Dr. Peter Marks said at an industry conference in June. He pointed to a multiyear effort by FDA to help clinics through the review process.

Many stem cell doctors continue to argue that their in-office procedures are outside FDAs purview. But FDA has concluded that processing stem cells and giving them to patients with serious diseases amounts to creating a new drug, which the agency regulates.

The FDA hasnt disclosed how many clinics sought approval since 2017, but public comments suggest it was troublingly low.

We have been very disappointed in the number of clinics that have come in, FDAs Dr. Wilson Bryan said at the same conference.

Bryan, who directs FDA's cell therapies division, added that he is extremely concerned by how many stem cell and related offerings remain available.

Tracking injuries from the procedures is difficult. Drugmakers and hospitals are required to report drug-related complications to the FDA, but no such requirements exist for individual doctors. And patients often don't know where to report problems.

David Stringham of Provo, Utah, says undergoing a procedure for joint pain at a local clinic was the worst decision of my life.

In 2018, Stringham was looking for an alternative to surgery for chronic pain in his right shoulder and elbows after years of weightlifting. He paid $2,400 for injections of so-called platelet-rich plasma at a clinic. It doesn't involve stem cells but the procedure is similar: doctors take a blood sample, process it to concentrate the platelets and then reinject them into the patient's problem areas in an attempt to speed healing.

The procedure went smoothly, but within hours Stringham was wracked by pain in his back, shoulder and arms.

It was a crazy amount of pain and I kept calling them saying something is not right," said the 51-year-old. And to this day Im not right."

The clinic gave Stringham medication for the pain and told him to be patient. But things didn't improve, even after months of physical therapy. Since then, a neurologist has told Stringham he probably suffered nerve damage at the places where he was injected.

His case was included in a Pew Charitable Trusts review of 360 reported injuries from stem cell and other regenerative procedures between 2004 and 2020. Nearly all the reports came from medical journals, government publications, social media or news reports. Just five came from FDAs database for medical injuries.

There are a lot of holes in the safety system, said Liz Richardson of Pew, who led the project.

The FDA didn't clearly assert its authority over such clinics until 2017. The next year, it began sending form letters to some 400 clinics, warning that they may be violating FDA rules. But the names of the clinics havent been publicized, and such warnings are often ignored.

Traditional medical researchers welcome the FDA actions but say its impossible to gauge their effect.

The business model is this: We can keep offering these products until things get serious with the FDA and then we can just take down our website', said Laertis Ikonomou, a stem cell researcher at the University of Buffalo who also heads a task force on the topic for the International Society for Cell and Gene Therapy.

He and other specialists say the clinics have damaged the reputation of legitimate stem cell research while also siphoning off patients who might otherwise enroll in studies.

Lawyers representing stem cell clinics say they have no choice but to resist FDA regulation.

FDA is pushing them into this drug development pathway, which nobody is adopting because it requires a million dollars worth of toxicology and animal studies just to show something is safe for human use, said Marc Scheineson, a former FDA attorney.

For now, people on both sides are waiting to see what FDA does.

We shouldnt feel too confident that the FDA has this wrapped up said Turner, the bioethicist. They really have invested some resources and they are trying to do something here but I think theyre just outmatched and overwhelmed.

Follow Matthew Perrone on Twitter: @APFDAwriter

The Associated Press Health and Science Department receives support from the Howard Hughes Medical Institutes Department of Science Education. The AP is solely responsible for all content.

See the original post here:
US stem cell clinics boomed while FDA paused crackdown ...

Dr. Bill Cimikoski, Medical Director of Utah Stem Cellsjoined Surae on The Daily Dish to discuss the BodyTite and Facetite procedures. He tells Surae that these procedures are excellent for getting rid of unwanted fat, while at the same time shrink wrapping the skin so that any loose skin is simultaneously tightened at the same time.

For some individuals, there may only be minimal (or none at all) fat to extract and it might be only necessary to tighten the skin. Depending on the area Utah Stem Cells are treating, they often see that in some individuals, there isnt really any fat to speak of and their patients are just looking for skin tightening and this is an excellent way to achieve that goal!

Unfortunately, on the other hand, some patients do have a large amount of fat in certain areas and then this device is also accompanied by liposuction. This is where they can suck the fat in addition to tightening the loose skin at the same time.This procedure is called Radio Frequency assisted Liposuction. At Utah Stem Cells they also offer High Definition Radio Frequency assisted liposuction to sculpt abs.

They offer many different treatments for different areas of the body, including the following:

All procedures are in-office and with only small holes or needle punctures, which heal completely without scarring. There is no need for general anesthesia and all are completed with lidocaine fluid although they do offer nitrous oxide, ketamine, and other methods to keep people comfortable and less anxious.

As a special gift, anyone who calls in after viewing The Daily Dish today will be entitled to $200 off any procedure.

To find out more about how Dr. Bill Cimikoski and Utah Stem Cells can help you, visit their website or you can give them a call at Phone number: (801) 999-4860

This article contains sponsored content.

Continue reading here:
Learn how to remove unwanted fat with procedures beyond liposuction - ABC 4

This helmet measures changes in blood oxygenation levels.

Over the next few weeks, a company called Kernel will begin sending dozens of customers across the U.S. a $50,000 helmet that can, crudely speaking, read their mind. Weighing a couple of pounds each, the helmets contain nests of sensors and other electronics that measure and analyze a brain's electrical impulses and blood flow at the speed of thought, providing a window into how the organ responds to the world. The basic technology has been around for years, but it's usually found in room-size machines that can cost millions of dollars and require patients to sit still in a clinical setting.

The promise of a leagues-more-affordable technology that anyone can wear and walk around with is, well, mind-bending. Excited researchers anticipate using the helmets to gain insight into brain aging, mental disorders, concussions, strokes, and the mechanics behind previously metaphysical experiences such as meditation and psychedelic trips. "To make progress on all the fronts that we need to as a society, we have to bring the brain online," says Bryan Johnson, who's spent more than five years and raised about $110 million-half of it his own money-to develop the helmets.

Johnson with one of his helmets in a lab at Kernel's offices.

Johnson is the chief executive officer of Kernel, a startup that's trying to build and sell thousands, or even millions, of lightweight, relatively inexpensive helmets that have the oomph and precision needed for what neuroscientists, computer scientists, and electrical engineers have been trying to do for years: peer through the human skull outside of university or government labs. In what must be some kind of record for rejection, 228 investors passed on Johnson's sales pitch, and the CEO, who made a fortune from his previous company in the payments industry, almost zeroed out his bank account last year to keep Kernel running. "We were two weeks away from missing payroll," he says. Although Kernel's tech still has much to prove, successful demonstrations, conducted shortly before Covid-19 spilled across the globe, convinced some of Johnson's doubters that he has a shot at fulfilling his ambitions.

A core element of Johnson's pitch is "Know thyself," a phrase that harks back to ancient Greece, underscoring how little we've learned about our head since Plato. Scientists have built all manner of tests and machines to measure our heart, blood, and even DNA, but brain tests remain rare and expensive, sharply limiting our data on the organ that most defines us. "If you went to a cardiologist and they asked you how your heart feels, you would think they are crazy," Johnson says. "You would ask them to measure your blood pressure and your cholesterol and all of that."

The first Kernel helmets are headed to brain research institutions and, perhaps less nobly, companies that want to harness insights about how people think to shape their products. (Christof Koch, chief scientist at the Allen Institute for Brain Science in Seattle, calls Kernel's devices "revolutionary.") By 2030, Johnson says, he wants to bring down the price to the smartphone range and put a helmet in every American household-which starts to sound as if he's pitching a panacea. The helmets, he says, will allow people to finally take their mental health seriously, to get along better, to examine the mental effects of the pandemic and even the root causes of American political polarization. If the Biden administration wanted to fund such research, Johnson says, he'd be more than happy to sell the feds a million helmets and get started: "Let's do the largest brain study in history and try to unify ourselves and get back to a steady state."

Johnson is something of a measurement obsessive. He's at the forefront of what's known as the quantified-self movement. Just about every cell in his body has been repeatedly analyzed and attended to by a team of doctors, and their tests now cast him as a full decade younger than his 43 years. Along those lines, he wants to let everyone else analyze, modify, and perfect their minds. No one knows what the results will be, or even if this is a good idea, but Johnson has taken it upon himself to find out.

After selling his payments startup, Johnson radically changed his life.

Unlike many of his tech-millionaire peers, Johnson grew up relatively poor. Born in 1977, he was raised in Springville, Utah, the third of five children. "We had very little and lived a very simple life," says his mother, Ellen Huff. A devout Mormon, she stayed home with the kids as much as possible and earned a modest income from a rental unit on the other side of the family's duplex.

Johnson remembers his mother knitting his clothes and grinding wholesale batches of wheat to make bread. "We were not like my friends," he says. "They would buy things from stores, and we just did not do that." His dad, a trash collector turned lawyer, had a drug problem and an affair, which led to his divorce from Huff. Later, delinquent child support payments, missed pickups on the weekends, and legal troubles contributed to his disbarment. "After some time of challenge, my father successfully overhauled his life 20 years ago," Johnson says. "Throughout his struggles, we remained close and without conflict. He has been a unique source of wisdom, counsel, and stability in my life."

Johnson had little idea what to do with his life until he served a two-year church mission in Ecuador, where he interacted with people living in huts with dirt floors and walls made of mud and hay. "When I came back, the only thing I cared about was how to do the most good for the most people," he says. "Since I didn't have any skills, I decided to become an entrepreneur."

While at Brigham Young University, he started his own business selling cellphones and service plans, making enough money to hire a team of salespeople. After that, he invested in a real estate development company that collapsed and left him $250,000 in debt. To get out of the hole, he took a job selling credit card processing services to small businesses door to door. Soon he was the company's top salesman.

This was the mid-2000s, and Johnson's customers kept complaining about the hassle of setting up and maintaining credit card payment systems on their websites. In 2007 he started Braintree, a software company focused on easing the process with slick interfaces. It succeeded-and had good timing. After signing up a slew of restaurants, retailers, and other small businesses, Braintree became the middleman of choice for a profusion of startups premised on ordering services online, including Airbnb, OpenTable, and Uber. The company also made a great bet on mobile payments, acquiring Venmo for only $26 million in 2012. The next year, EBay bought Braintree for $800 million in cash, a little less than half of which went to Johnson.

Despite his newfound fortune, Johnson felt miserable. He was stressed out and overweight. He'd gotten married and had kids at a young age, but his marriage was falling apart, and he was questioning his life, religion, and identity. He says he entered a deep depressive spiral that included suicidal thoughts.

The decision to sell Braintree well before it peaked in value had been motivated in part by Johnson's need to change those patterns. "Once I had money, it was the first time in my life that I could eliminate all permission structures," he says. "I could do whatever I wanted." He broke with the Mormon church, got divorced, and moved from Chicago, where Braintree was headquartered, to Los Angeles to start over.

Arriving in California, Johnson consulted with all manner of doctors and mental health specialists. His bodily health improved with huge changes to his diet, exercise, and sleep routines. His mind proved a tougher puzzle. He meditated and studied cognitive science, particularly the ways people develop biases, in an effort to train himself to think more rationally. By late 2014 he was convinced his wealth would be best spent advancing humanity's understanding of the brain. He took a large portion of his windfall and started OS Fund, a venture firm that has invested in several artificial intelligence and biotech companies. These include Ginkgo Bioworks, Pivot Bio, Synthego, and Vicarious, some of the most promising startups trying to manipulate DNA and other molecules.

Mostly, though, Johnson staked his fortune on Kernel. When he founded the company, in 2015, his plan was to develop surgical implants that could send information back and forth between humans and computers, the way Keanu Reeves downloads kung fu into his brain in The Matrix. (In the early days, Johnson discussed a potential partnership with Elon Musk, whose company Neuralink Corp. has put implants in pigs and monkeys, but nothing came of it.) The idea was, in part, to transfer thoughts and feelings directly from one consciousness to another, to convey emotions and ideas to other people more richly than human language allows.

Perhaps more important, Johnson reckoned, AI technology was getting so powerful that for human intelligence to remain relevant, the brain's processing power would need to keep pace.

Johnson and I began discussing brains in mid-2018, when I was working on a story about the overlap between neuroscience and AI software. During an initial interview at his company's headquarters in L.A.'s Venice neighborhood, Johnson was cordial but somewhat vague about his aims. But at the end of the visit, I happened to mention the time I underwent a mental healing ritual that involved a Chilean shaman burning holes in my arm and pouring poisonous frog secretions into the wounds. (I do mention this a lot.) Excited, Johnson replied that he had a personal shaman in Mexico and doctors in California who guided him on drug-induced mind journeys. Based on this common ground, he decided to tell me more about Kernel's work and his own adventurous health practices.

By then, Johnson had abandoned neural implants in favor of helmets. The technology needed to make implants work is difficult to perfect-among other things, the human body tends to muddy the devices' signals over time, or to reject them outright-and the surgery seemed unlikely to go mainstream. With the helmets, the basic principle remained the same: put tiny electrodes and sensors as close as possible to someone's neurons, then use the electrodes to detect when neurons fire and relay that information to a computer. Watch enough of these neurons fire in enough people, and we may well begin to solve the mysteries of the brain's fine mechanics and how ideas and memories form.

A wider shot of the Flux helmet's enclosure at Kernel's lab. The booth shields against electromagnetic interference so the instrument can measure very sensitive brain output.

On and off for almost three years, I've watched as Kernel has brought its helmets into reality. During an early visit to the company's two-story headquarters in a residential part of Venice, I saw that Johnson's team had converted the garage into an optics lab full of mirrors and high-end lasers. Near the entryway sat a shed-size metallic cube designed to shield its contents from electromagnetic interference. On the second floor, dozens of the world's top neuroscientists, computer scientists, and materials experts were tinkering with early versions of the helmets alongside piles of other electrical instruments. At that point the helmets looked less like 21st century gadgets and more like something a medieval knight might wear into battle, if he had access to wires and duct tape.

Despite the caliber of his team, Johnson and his odd devices were considered toys by outsiders. "The usual Silicon Valley people and investors would not even talk to us or poke around at all," he says. "It became clear that we would have to spend the time, and I would have to spend the money, to show people something and demonstrate it working."

A hospital or research center will typically employ a range of instruments to analyze brains. The list is a smorgasbord of acronyms: fMRI (functional magnetic resonance imaging), fNIRS (functional near-infrared spectroscopy), EEG (electroencephalography), MEG (magnetoencephalography), PET (positron emission tomography), etc. (et cetera). These machines measure a variety of things, from electrical activity to blood flow, and they do their jobs quite well. They're also enormous, expensive, and not easily condensed into helmet form.

In some cases the machines' size owes in part to components that shield the patient's head from the cacophony of electrical interference present in the world. This allows the sensors to avoid distracting signals and capture only what's happening in the brain. Conversely, signals from the machines need to penetrate the human skull, which happens to be well-evolved to prevent penetration. That's part of the argument for implants: They nestle sensors right up against our neurons, where the signals come in loud and clear.

It's unlikely a helmet will ever gain the level of information an implant can, but Kernel has striven to close the gap by shrinking its sensors and finding artful ways to block electromagnetic interference. Among its breakthroughs, Johnson's team designed lasers and computer chips that were able to see and record more brain activity than any previous technology. Month after month, the helmet became more refined, polished, and lightweight as the team made and remade dozens of prototypes. The only trick was that, to suit the different applications Johnson envisioned for the helmet, Kernel wound up needing to develop two separate devices to mimic all the key functions of more traditional machines.

One of the devices, called Flow, looks like a high-tech bike helmet, with several brushed aluminum panels that wrap around the head and have small gaps between them. Flip it over, and you'll see a ring of sensors inside. A wire at the back can be connected to a computer system.

This helmet measures changes in blood oxygenation levels. As parts of the brain activate and neurons fire, blood rushes in to provide oxygen. The blood also carries proteins in the form of hemoglobin, which absorbs infrared light differently when transporting oxygen. (This is why veins are blue, but we bleed red.) Flow takes advantage of this phenomenon by firing laser pulses into the brain and measuring the reflected photons to identify where a change in blood oxygenation has occurred. Critically, the device also measures how long the pulse takes to come back. The longer the trip, the deeper the photons have gone into the brain. "It's a really nice way to distill out the photons that have gone into the brain vs. ones that only hit the skull or scalp and bounced away," says David Boas, a professor of biomechanical engineering and director of the Neurophotonics Center at Boston University.

The other Kernel helmet, Flux, measures electromagnetic activity. As neurons fire and alter their electrical potential, ions flow in and out of the cells. This process produces a magnetic field, if one that's very weak and changes its behavior in milliseconds, making it extremely difficult to detect. Kernel's technology can discover these fields all across the brain via tiny magnetometers, which gives it another way to see what parts of the organ light up during different activities.

The helmets are not only smaller than the devices they seek to replace, but they also have better bandwidth, meaning researchers will receive more data about the brain's functions. According to the best current research, the Flow device should help quantify tasks related to attention, problem-solving, and emotional states, while Flux should be better suited to evaluating brain performance, learning, and information flow. Perhaps the No.1 thing that has scientists gushing about Kernel's machines is their mobility-patients' ability to move around wearing them in day-to-day settings. "This unlocks a whole new universe of research," Boas says. "What makes us human is how we interact with the world around us." The helmets also give a picture of the whole brain, as opposed to implants, which look solely at particular areas to answer more specific questions, according to Boas.

Once their Kernel helmets arrive, Boas and his colleagues plan to observe the brains of people who've had strokes or suffer from diseases such as Parkinson's. They want to watch what the brain does as individuals try to relearn how to walk and speak and cope with their conditions. The hope is that this type of research could improve therapy techniques. Instead of performing one brain scan before the therapy sessions start and another only after months of work, as is the practice today, researchers could scan the brain each day and see which exercises make the most difference.

Devices are also going out to Harvard Medical School, the University of Texas, and the Institute for Advanced Consciousness Studies (a California lab focused on researching altered states) to study such things as Alzheimer's and the effect of obesity on brain aging, and to refine meditation techniques. Cybin Inc., a startup aiming to develop therapeutic mental health treatments based on psychedelics, will use the helmets to measure what happens when people trip.

All of this thrills Johnson, who continues to harbor the grandest of ambitions for Kernel. He may have given up on computer-interfacing implants, but he still wants his company to help people become something more than human.

A couple years ago, Johnson and I boarded his private jet and flew from California to Golden, Colo. Johnson, who has a pilot's license, handled the takeoffs and landings but left the rest to a pro. We were in Colorado to visit a health and wellness clinic run by physician-guru Terry Grossman and have a few procedures done to improve our bodies and minds.

The Grossman Wellness Center looked like a cross between a medical clinic and the set of Cocoon. Most of the other guests were elderly. In a large central room, about 10 black leather chairs and matching footrests were arranged in a loose circle. Each chair held a couple of fluffy white pillows, with a metal pole on the side for our IV drips. A few of the ceiling tiles had been replaced and fitted with pictures of clouds and palm trees. In rooms off to the side, medical personnel performed consultations and procedures.

Our morning began with an IV infusion of two anti-aging fluids: Myers' Cocktail-a blend of magnesium, calcium, B vitamins, vitamin C, and other good stuff-followed by a helping of nicotinamide adenine dinucleotide. Some of the IV fluids can trigger nausea, but Johnson set the drip to maximum and complemented the IV by having a fiber-optic cable fed into his veins to pepper his blood with red, green, blue, and yellow wavelengths of light for added rejuvenation. "I have to experience pain when I exercise or work," he said, adding that the suffering makes him feel alive.

A few hours later, Johnson went into one of the treatment rooms with Grossman to get a stem cell injection straight into his brain. Earlier he'd provided 5 ounces of his blood, which had then been spun in a centrifuge so Grossman could separate out the plasma and put it through a secret process to "activate the stem cells." Now, Johnson hopped onto a reclined exam table, lying on his back with his head angled toward the floor. Grossman pulled out a liquid-filled syringe. Instead of a needle at the end, it had a 4inchlong, curved plastic tube, which the doctor coated with some lubricating jelly. He pushed the tube into one of Johnson's nostrils, told the patient to take a big sniff, then pinched Johnson's nose shut. They repeated the process for the other nostril. The procedure looked incredibly uncomfortable, but again, Johnson was unfazed, pulling in the stem cells with determination and excitement.

This snorting procedure-designed to improve mood, energy, and memory-was just a small part of Johnson's overall health regimen. Each morning the CEO took 40 pills to boost his glands, cell membranes, and microbiome. He also used protein patches and nasal sprays for other jobs. After all this, he did 30 minutes of cardio and 15 minutes of weights. At lunch he'd have some bone broth and vegetables foraged by his chef from the yards of houses in Venice. He might have a light dinner later, but he never consumed anything after 5 p.m. He went to bed early and measured his sleep performance overnight. Every now and then, a shaman or doctor would juice him up with some drugs such as ketamine or psilocybin. He'd taken strongly enough to these practices to tattoo his arm with "5-MeO-DMT," the molecular formula for the psychoactive compound famously secreted by the Sonoran Desert toad.

To make sure all his efforts were doing some good, Johnson had a lab measure his telomeres. These are the protective bits at the end of DNA strands, which some Nobel Prize-winning science has shown can be good indicators of how your body is aging. The longer the telomeres, the better you're doing. Johnson used to register as 0.4 years older internally than his chronological age, but a couple of years into his regimen under Grossman, when he was in his early 40s, his doctors were telling him he was testing like a man in his late 30s.

During one of our most recent conversations, Johnson tells me he's stopped snorting stem cells and experimenting with hallucinogens. "I got what I wanted from that and don't need to mess with it right now," he says. After many tests and much analysis, he's discovered he operates best if he wakes up at 4 a.m., consumes 2,250 calories of carefully selected food over the course of 90 minutes, and then doesn't eat again for the rest of the day. Every 90 days he goes through another battery of tests and adjusts his diet to counteract any signs of inflammation in his body. He goes to bed each night between 8 and 8:30 p.m. and continues to measure his sleep metrics. "I have done tremendous amounts of trial and error to figure out what works best for my health," he says. "I have worked very hard to figure these algorithms out."

Johnson eats once a day, first thing. Yes, including the wine.

In terms of what our birth certificates say, Johnson and I are the same age. He'll turn 44 in August, a month before I do. To someone like me, who prizes late nights with friends, food, and drink, Johnson's rigid lifestyle doesn't exactly sound romantic. But it does seem to be paying off: When he last got tested, he had the exercise capacity of someone in his late teens or early 20s, and a set of DNA and other health markers pegged his age at somewhere around 30. As for me, I lack the courage to ask science what it makes of my innards and will go on celebrating my dad bod.

As Johnson sees it, had he not changed his lifestyle, he'd have remained depressed and possibly died far too young. Now he does what the data say and nothing else. "I did a lot of damage to myself working 18-hour days and sleeping under a desk," he says. "You might earn the praise of your peers, but I think that sort of lifestyle will very quickly be viewed as primitive." He says he's at war with his brain and its tendencies to lead him astray. "I used to binge-eat at night and could not stop myself," he says. "It filled me with shame and guilt and wrecked my sleep, which crushed my willpower. My mind was a terrible actor for all those years. I wanted to remove my mind from the decision-making process."

The nuance in his perspective can be tricky to navigate. Johnson wants to both master the mind and push it to the side. He maintains, however, that our brain is flawed only because we don't understand how it works. Put enough Kernel devices on enough people, and we'll find out why our brain allows us to pursue addictive, debilitating behaviors-to make reckless decisions and to deceive ourselves. "When you start quantifying the mind, you make thought and emotion an engineering discipline," he says. "These abstract thoughts can be reduced to numbers. As you measure, you move forward in a positive way, and the quantification leads to interventions."

Of course, not everyone will want to make decisions based on what a helmet says their brain activity means. Taking the decisions out of thought patterns-or analyzing them for the purposes of market research and product design-poses its own, perhaps scarier, questions about the future of human agency. And that's if the Kernel devices can fulfill the company's broader ambitions. While the big, expensive machines in hospitals have been teaching us about the brain for decades, our understanding of our most prized organ has remained, in many ways, pretty basic. It's possible Kernel's mountain of fresh data won't be of the kind that translates into major breakthroughs. The brain researchers who are more skeptical of efforts such as Johnson's generally argue that novel insights about how the brain works-and, eventually, major leaps in brain-machine interfaces-will require implants.

Yet scientists who have watched Kernel's journey remark on how the company has evolved alongside Johnson, a complete outsider to the field. "Everybody he's recruited to Kernel is amazing, and he's been able to listen to them and motivate them," says MIT neuroscientist Edward Boyden. "He didn't have scientific training, but he asked really good questions." The test now will be to see how the company's devices perform in the field and if they really can create a whole new market where consumers buy Flow and Flux helmets alongside their Fitbits and Oura rings. "There's a lot of opportunity here," Boyden says. "It's a high-risk, high-payoff situation."

If Johnson's theories are correct and the Kernel devices prove to be as powerful as he hopes, he'll be, in a sense, the first person to spark a broader sort of enlightened data awakening. He recently started a program meant to quantify the performance of his organs to an unprecedented degree. Meanwhile, he's taking part in several experiments with the Kernel helmets and is still looking for ways to merge AI with flesh. "We are the first generation in the history of Homo sapiens who could look out over our lifetimes and imagine evolving into an entirely novel form of conscious existence," Johnson says. "The things I am doing can create a bridge for humans to use where our technology will become part of our self."

(Except for the headline, this story has not been edited by NDTV staff and is published from a syndicated feed.)

Waiting for response to load...

See the original post here:
A $50,000 Helmet Can Read User's Mind. And It's Ready - NDTV

When a kids ask is the tooth fairy real or what does the tooth fairy look like, one might be tempted to give a simple yes or no answer and then makeup something that sounds like what one was told when they were a kid. Nevertheless, there actually is an origin story to the myth, and its a convoluted one that reaches further back in time than one might think.

This press release was orginally distributed by ReleaseWire

Bountiful, UT (ReleaseWire) 06/22/2021 If one has kids near the ages of four to six years old then the topic of the tooth fairy has probably come up a time or two (or on a daily basis if they're excited about a loose tooth).

When a kids ask "is the tooth fairy real" or "what does the tooth fairy look like", one might be tempted to give a simple yes or no answer and then makeup something that sounds like what one was told when they were a kid. Nevertheless, there actually is an origin story to the myth, and it's a convoluted one that reaches further back in time than one might think: nearly a millennium. Moreover, research is showing that saving those baby teeth could have health benefits later on in life. First, we'll go back in time, and then we'll talk about the future.

Baby Teeth and MythologyYes, baby teeth were a talisman of sorts in the old Norse and Northern European cultures as far back as the tenth century. In the earliest writings of these cultures, the Eddas, references to a tradition of exchanging money for a baby tooth can be found. Baby teeth were believed to bring luck to a warrior in battle, and some cultures even made necklaces of them. This superstitious tradition was known as the "tand-fe" which translates to tooth fee. Interestingly and perhaps completely benign, the German word for fairy is "Fee". Moreover, one Norse myth involves the god Frey who received an entire fairy kingdom as a "tooth gift".

In the Middle Ages in England, children were instructed to burn their baby teeth to save themselves hardship in the afterlife. Children who refused to do so were warned they would search for their baby teeth forever in the afterlife. Other cultures have children bury their teeth, or throw them into the air. Nevertheless, these baby teeth mythologies and superstitions didn't actually involve a tooth fairy until much more recent times: 1908 to be exact.

Where did the Tooth Fairy Come From?In 1908, the Chicago Daily Tribune Household Hints section featured a helpful tip from reader Lillian Brown and the tooth fairy was born. In response to the concern that children didn't want to have their loose teeth pulled, Ms. Brown wrote,

"Tooth Fairy.Many a refractory child will allow a loose tooth to be removed if he knows about the Tooth Fairy. If he takes his little tooth and puts it under the pillow when he goes to bed the Tooth Fairy will come in the night and take it away, and in its place will leave some little gift. It is a nice plan for mothers to visit the 5-cent counter and lay in a supply of articles to be used on such occasions."

Of course, it's entirely possible that this was a long-standing tradition in Ms. Brown's family going back generations, or maybe her family ran the 5-cent counter and she was cleverly thinking to drum up more business. Regardless, that is the first written mention of the tooth fairy as we know it.

Should One Pull a Loose Tooth?Just because it seems parents were looking for ways to pull loose teeth in 1908 does not mean one should be doing that in 2021. We should take special note that we do not believe in pulling a loose tooth unless absolutely necessary. We prefer to allow a loose tooth to fall out, on its own time and without force. A child's body, including the workings of their mouth, is a powerful and intentional force and we should trust that the tooth will fall out when it's good and ready.

If the gum around the loose tooth is inflamed, give us a call. There are certain circumstances in which we may decide pulling a tooth is best.

What Does the Tooth Fairy Do with Teeth?This one can be a stumper for parents. Because really, their little but very rational and inquisitive minds are investigating and asking questions about everything as they learn about the world around them. Why would anyone want to collect a bunch of fallen-out teeth since our warriors don't wear teeth necklaces anymore?

Save Those Baby Teeth for the FutureWell, there's some really interesting research out that shows one should collect and store a kids' baby teeth for later on in life. The advancements being made today in both medicine and science are astonishing and baby teeth may be an integral part of the future.

Baby teeth, and wisdom teeth, have adult stem cells that are referred to as "mesenchymal stem cells". These stem cells are important because they can be applied to bone and tissue regeneration treatments. As of today, more than 2,000 clinical trials have been completed or are in the process to study how these mesenchymal stem cells can help with treatments for a wide range of diseases including (but not limited to) the following:

Type 1 diabetes Stroke Parkinson's Alzheimer's Muscular dystrophy Bone loss Multiple sclerosis Cardiovascular disease Neural injuries Cancers (Leukemia, Lymphoma)

In order for a child's baby teeth to be viable in the future, they require proper storage. There are a few services already established for exactly this purpose such as Store-A-Tooth, orThe Tooth Bank. If one is interested in this idea for the future, do a little research about the companies to decide which one feels right.

About Utah Pediatric DentistsOur offices are open and we want to see happy healthy smiles in the community. Contact us today at 801-948-8880 to schedule an appointment at one of our four convenient locations.

Our mission is to provide comprehensive dental services to children of all ages from infant to adolescence, of all needs including special needs, in a compassionate and caring manner. Our pediatric dental professionals are educated, experienced, and specially trained to provide dental hygiene education and dental services in a fun and inviting environment that will ensure a child has a positive experience in our offices.

For more information on this press release visit: http://www.releasewire.com/press-releases/is-the-tooth-fairy-real-the-origin-story-to-the-tooth-fairy-and-why-it-is-a-story-commonly-told-today-1342142.htm

Follow this link:
Is the Tooth Fairy Real? the Origin Story to the Tooth Fairy, and Why It Is a Story Commonly Told Today - Digital Journal