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Supplementary Table 2. 10 OTUs with smallest P-values at genus and species levels. Comparisons are made between diarrhea and non-diarrhea patients

DDMS diagnostics for adjacent thyroid tissues. The table contains cancer risk scores according to DDMS, diagnosis according to DDMS and status of nodule to which tissue was adjacent.

Supplementary Appendix 2. Spike-in experiment with Salmonella typhimurium

Supplementary Table 1. 10 OTUs with smallest P-values at genus and species levels. Comparisons are made between the 2 clusters using PAM clustering with JSD distance

Supplementary Figure 2. Time on VEGF-TKI as it relates to (a) bacterial diversity as estimated by Shannon Index, (b) relative abundance of Bacteroides, and (c) relative abundance of Prevotella

Purpose: Diarrhea occurs in approximately half of patients with metastatic renal cell carcinoma (mRCC) receiving vascular endothelial growth factor–tyrosine kinase inhibitors (VEGF-TKI). We evaluated the relationship between VEGF-TKI–related diarrhea and stool microbiota.Experimental Design: Stool samples were collected from 20 mRCC patients receiving VEGF-TKIs. 16S rRNA sequencing was used to characterize the stool bacteriomic profiling of patients. Assay validation with Salmonella typhimurium spike-in experiments suggested greatest speciation with use of the V5 region.Results: Higher levels of Bacteroides spp. and lower levels of Prevotella spp. were found in patients with diarrhea. In addition, patients receiving VEGF-TKIs with mRCC appeared to have less relative abundance of Bifidobacterium spp. as compared with previous reports based on healthy subjects.Conclusions: We have thus demonstrated interplay between microbiota and VEGF-TKI–induced diarrhea. Further studies are warranted to evaluate the potential causative role of preexisting dysbiosis in VEGF-TKI–related diarrhea. Clin Cancer Res; 21(23); 5286–93. ©2015 AACR.

Supplementary Figure 3. PC1 and PC2 from PCoA plotted against age, diarrhea status, first line therapy vs others and sunitinib vs other drugs

DDMS locations are frequently co-localized with active enhancers. A. Representative snap-shot of H3K27Ac pattern in two analyzed adjacent thyroid tissues (7N and 10N). The snap-shot indicates the normalized number of reads. Transcriptional start sites and transcript directions are shown. B. Representative snap-shot of co-localization DDMS site with H3K27Ac enrichment at the NFIC gene body in two analyzed adjacent thyroid tissues (7N and 10N). The snap-shot indicates the normalized number of reads. The red dotted line indicates the position of the DDMS location. Transcriptional start site and transcript directions are shown. C. Representative snap-shot of co-localization DDMS site with H3K27Ac enrichment at the EPHA2 gene body in two analyzed adjacent thyroid tissues (7N and 10N). The snap-shot indicates the normalized number of reads. The red dotted line indicates the position of the DDMS location. Transcriptional start site and transcript direction are shown.

Supplementary Figure 1. Simpson index plotted against (a) age, (b) diarrhea status, (c) first line therapy vs others and (d) sunitinib vs other systemic treatment

Purpose:Thyroid cancer is frequently difficult to diagnose due to an overlap of cytologic features between malignant and benign nodules. This overlap leads to unnecessary removal of the thyroid in patients without cancer. While providing some improvement over cytopathologic diagnostics, molecular methods frequently fail to provide a correct diagnosis for thyroid nodules. These approaches are based on the difference between cancer and adjacent thyroid tissue and assume that adjacent tissues are the same as benign nodules. However, in contrast to adjacent tissues, benign thyroid nodules can contain genetic alterations that can be found in cancer.Experimental Design: For the development of a new molecular diagnostic test for thyroid cancer, we evaluated DNA methylation in 109 thyroid tissues by using genome-wide single-base resolution DNA methylation analysis. The test was validated in a retrospective cohort containing 65 thyroid nodules.Results:By conducting reduced representation bisulfite sequencing in 109 thyroid specimens, we found significant differences between adjacent tissue, benign nodules, and cancer. These tissue-specific signatures are strongly linked to active enhancers and cancer-associated genes. Based on these signatures, we developed a new epigenetic approach for thyroid diagnostics. According to the validation cohort, our test has an estimated specificity of 97% [95% confidence interval (CI), 81–100], sensitivity of 100% (95% CI, 87–100), positive predictive value of 97% (95% CI, 83–100), and negative predictive value of 100% (95% CI, 86–100).Conclusions:These data show that epigenetic testing can provide outstanding diagnostic accuracy for thyroid nodules.See related commentary by Mitmaker et al., p. 457

Supplementary Figure 4. Species belongs to genera Bacteroides and Prevotella are the driving factors for clustering. Species "Bacteroides, s__" is an unclassified species under genus Bacteroides. Species Copri belongs to genus Prevotella

Although the overall survival rate of B cell acute lymphoblastic leukemia (B-ALL) in childhood is more than 80%, it is merely 30% in refractory/relapsed and adult patients with B-ALL. This demonstrates a need for improved therapy targeting this subgroup of B-ALL. Here, we show that the ten-eleven translocation 1 (TET1) protein, a dioxygenase involved in DNA demethylation, is overexpressed and plays a crucial oncogenic role independent of its catalytic activity in B-ALL. Consistent with its oncogenic role in B-ALL, overexpression of TET1 alone in normal precursor B cells is sufficient to transform the cells and cause B-ALL in mice within 3 to 4 months. We found that TET1 protein is stabilized and overexpressed because of its phosphorylation mediated by protein kinase C epsilon (PRKCE) and ATM serine/threonine kinase (ATM), which are also overexpressed in B-ALL. Mechanistically, TET1 recruits STAT5B to the promoters of CD72 and JCHAIN and promotes their transcription, which in turn promotes B-ALL development. Destabilization of TET1 protein by treatment with PKC or ATM inhibitors (staurosporine or AZD0156; both tested in clinical trials), or by pharmacological targeting of STAT5B, greatly decreases B-ALL cell viability and inhibits B-ALL progression in vitro and in vivo. The combination of AZD0156 with staurosporine or vincristine exhibits a synergistic effect on inhibition of refractory/relapsed B-ALL cell survival and leukemia progression in PDX models. Collectively, our study reveals an oncogenic role of the phosphorylated TET1 protein in B-ALL independent of its catalytic activity and highlights the therapeutic potential of targeting TET1 signaling for the treatment of refractory/relapsed B-ALL.

METTL16 has recently been identified as an RNA methyltransferase responsible for deposition of N(6)-methyladenosine (m(6)A) in a few transcripts. Whether METTL16 methylates a large set of transcripts, similar to METTL3 and METTL14, remains unclear. Here we show that METTL16 exerts both methyltransferase activity-dependent and -independent functions in gene regulation. In cell nucleus, METTL16 functions as an m(6)A writer to deposit m(6)A into hundreds of its specific mRNA targets. In cytosol, METTL16 promotes translation in an m(6)A-independent manner. More specifically, METTL16 directly interacts with eukaryotic initiation factor 3a/b (eIF3a/b) and ribosomal RNAs (rRNAs) through its Mtase domain, thereby facilitating the assembly of the translation initiation complex (TIC) and promoting translation of over 4,000 mRNA transcripts. Moreover, we demonstrate that METTL16 is critical for the tumorigenesis of hepatocellular carcinoma. Collectively, our studies reveal previously unappreciated dual functions of METTL16 as an m(6)A writer and a translation initiation facilitator, which together contribute to its essential function in tumorigenesis.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome. CXCR5-PD-1hi peripheral T-helper (Tph) cells have an important pathogenic role in autoimmune diseases, but the role of Tph cells in cGVHD remains unknown. We show that in patients with cGVHD, expansion of Tph cells among blood CD4+ T cells was associated with cGVHD severity. These cells augmented memory B-cell differentiation and production of immunoglobulin G via interleukin 21 (IL-21). Tph cell expansion was also observed in a murine model of cGVHD. This Tph cell expansion in the blood is associated with the expansion of pathogenic tissue-resident T-helper (Trh) cells that form lymphoid aggregates surrounded by collagen in graft-versus-host disease (GVHD) target tissues. Adoptive transfer experiments showed that Trh cells from GVHD target tissues give rise to Tph cells in the blood, and conversely, Tph cells from the blood give rise to Trh cells in GVHD target tissues. Tph cells in the blood and Trh cells in GVHD target tissues had highly overlapping T-cell receptor α and β repertoires. Deficiency of IL-21R, B-cell lymphoma 6 (BCL6), or T-bet in donor T cells markedly reduced the proportions of Tph cells in the blood and Trh cells in GVHD target tissues and reduced T-B interaction in the lymphoid aggregates. These results indicate that clonally related pathogenic Tph cells and Trh cells traffic between the blood and cGVHD target tissues, and that IL-21R-BCL6 signaling and T-bet are required for the development and expansion of Tph and Trh cells in the pathogenesis of cGVHD.

Abnormal bone remodeling of subchondral bone is associated with osteoclast formation during early osteoarthritis (OA) while senescent phenotypes are detected during osteoclast differentiation. However, the associations between cellular senescence and osteoclastogenesis remain unclear in early OA. The present study found that Nox4 positively regulated the senescence of OCPs to promote multinucleated osteoclastogenesis. Overexpression of Nox4 induced overproduction of ROS, thereby promoting senescence of OCPs. Senescent OCPs secreted SASP factors to spread the senescence in OCPs clusters, over-activating the TRAF6/MAPK/c-Fos/NFATc1 axis to accelerate the fusion and osteoclast differentiation. Interestingly, the senescence of OCPs mediated by Nox4/ROS may be an essential step in initiating osteoclast differentiation. Compared to WT mice, subchondral bone loss was reduced in early OA and cartilage degeneration alleviated throughout the course of OA in Nox4-deficient mice. In addition, pharmacological inhibition of Nox4 significantly delayed OA development. In conclusion, we have identified Nox4-induced cellular senescence as an important regulator of osteoclast differentiation, suggesting that it might be a potential target to retard or prevent OA.

The pathogenesis of the osteoarthritis (OA) is complex. Abnormal subchondral bone metabolism is an important cause of this disease. Further understanding on the pathology of the subchondral bone in OA may provide a new therapy. This research is about to investigate the role of SDF-1 in the subchondral bone during the pathological process of OA. In vitro, Transwell was used to test the migratory ability of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs). Western blot presented the protein level after SDF-1 treatment in BMSCs and HUVESs. Alizarin red was used to assess the ability of osteogenic differentiation. To inhibit SDF-1 signaling pathway in vivo, AMD3100 (SDF-1 receptor blocker) was continuously delivered via miniosmotic pump for 4 weeks in mice after performing anterior cruciate ligament transaction surgery. Micro-CT, histology staining, immunofluorescence, immunohistochemistry, and TRAP staining were used to assess the role of SDF-1 on osteogenesis and angiogenesis in the subchondral bone. Our results showed that SDF-1 could recruit BMSCs, activate the p-ERK pathway, and enhance osteogenic differentiation. SDF-1 promoted the ability of proliferation, migration and tube formation of HUVECs by activating the ERK and AKT signaling pathways. In an animal study, inhibition of SDF-1/CXCR4 axis could significantly reduce subchondral osteogenesis differentiation and H-type vessel formation. Furthermore, the AMD3100-treated group showed less cartilage destruction and bone resorption. Our research shows that SDF-1 alters the microenvironment of the subchondral bone by promoting osteoid islet formation and abnormal H-type angiogenesis in the subchondral bone, resulting in articular cartilage degeneration.

Donor T cells mediate both graft-versus-leukemia (GVL) activity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Development of methods that preserve GVL activity while preventing GVHD remains a long-sought goal. Tolerogenic anti–interleukin-2 (IL-2) monoclonal antibody (JES6-1) forms anti–IL-2/IL-2 complexes that block IL-2 binding to IL-2Rβ and IL-2Rγ on conventional T cells that have low expression of IL-2Rα. Here, we show that administration of JES6 early after allo-HCT in mice markedly attenuates acute GVHD while preserving GVL activity that is dramatically stronger than observed with tacrolimus (TAC) treatment. The anti–IL-2 treatment downregulated activation of the IL-2-Stat5 pathway and reduced production of granulocyte-macrophage colony-stimulating factor (GM-CSF). In GVHD target tissues, enhanced T-cell programmed cell death protein 1 (PD-1) interaction with tissue–programmed cell death-ligand 1 (PD-L1) led to reduced activation of protein kinase–mammalian target of rapamycin pathway and increased expression of eomesodermin and B-lymphocyte-induced maturation protein-1, increased T-cell anergy/exhaustion, expansion of Foxp3–IL-10–producing type 1 regulatory (Tr1) cells, and depletion of GM-CSF–producing T helper type 1 (Th1)/cytotoxic T cell type 1 (Tc1) cells. In recipient lymphoid tissues, lack of donor T-cell PD-1 interaction with tissue PD-L1 preserved donor PD-1+TCF-1+Ly108+CD8+ T memory progenitors and functional effectors that have strong GVL activity. Anti–IL-2 and TAC treatments have qualitatively distinct effects on donor T cells in the lymphoid tissues, and CD8+ T memory progenitor cells are enriched with anti–IL-2 treatment compared with TAC treatment. We conclude that administration of tolerogenic anti–IL-2 monoclonal antibody early after allo-HCT represents a novel approach for preventing acute GVHD while preserving GVL activity.

Objective The antibacterial activity of copper (Cu)-alloy biomaterials has shown a great potential in clinical application. Here, we evaluated the osteogenesis and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy in an in vivo model of infected bone defects and determine their responsible proteins and pathways using proteomics. Methods After bone defects were filled with Ti6Al4V and Ti6Al4V-6.5wt%Cu implants for 6 week, the tissue and bone samples around the implants were harvested for radiographic, micro-CT, histological, and bone-related gene expression analyses. An iTRAQ-based protein identification/quantification approach was used to analyze the osteogenic and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy. Results Imaging and histological results showed Ti6Al4V alloy induced a stronger inflammatory response than Ti6Al4V-6.5wt%Cu alloy; imaging results and osteogenic protein levels showed Ti6Al4V-6.5wt%Cu alloy exerted a stronger osteogenic effect. In vitro experiment, we found the Ti6Al4V-6.5wt%Cu had significant antibacterial effects and inhibited the activity of Staphylococcus aureus in the early stage. In addition, the bacterial biofilm formed in Ti6Al4V-6.5wt%Cu group was significantly lower than that in Ti6Al4V group. Proteomic screening of 4279 proteins resulted in 35 differentially expressed proteins for further examination which were mainly associated with the cellular process, metabolic process, stimulus response, and cellular component organization. In further exploration of the mechanism of osteogenic mineralization of Ti6Al4V-6.5wt%Cu alloy, we found out SDC4 and AGRN were the top two target proteins associated with osteogenic differentiation and bone mineralization. Conclusion Ti6Al4V-6.5wt%Cu alloy shows a great potential as a bone implant material due to its positive effects against bacterial infection and on bone formation. The Translational potential of this article At present, titanium alloys and other non-antibacterial metal materials are used in orthopedic internal fixation operations. Our study demonstrates that Ti6Al4V-6.5wt%Cu alloy has good antibacterial and osteogenic effects in vivo and in vitro. This means that Ti6Al4V-6.5wt%Cu alloy may become a new kind of antimicrobial metallic material as internal fixation material to continuously exert its antimicrobial effects and reduce the infection rate after clinical internal fixation., Graphical abstract Image 1

MicroRNAs (miRNAs) have been shown to hold prognostic value in acute myeloid leukemia (AML); however, the temporal dynamics of miRNA expression in AML are poorly understood. Using serial samples from a mouse model of AML to generate time-series miRNA sequencing data, we are the first to show that the miRNA transcriptome undergoes state-transition during AML initiation and progression. We modeled AML state-transition as a particle undergoing Brownian motion in a quasi-potential and validated the AML state-space and state-transition model to accurately predict time to AML in an independent cohort of mice. The critical points of the model provided a framework to align samples from mice that developed AML at different rates. Our mathematical approach allowed discovery of dynamic processes involved during AML development and, if translated to humans, has the potential to predict an individual’s disease trajectory.

Our laboratory originally synthesized strontium(Sr)‐containing α‐calcium sulphate hemihydrate/nano‐hydroxyapatite composite (Sr‐α‐CSH/n‐HA) and demonstrated its ability to repair critical bone defects. This study attempted to incorporate aspirin into it to produce a better bone graft material for critical bone defects. After 5% Sr‐α‐CSH was prepared by coprecipitation and hydrothermal methods, it was mixed with aspirin solution of different concentrations (50 μg/ml, 200 μg/ml, 800 μg/ml and 3200 μg/ml) at a fixed liquid‐solid ratio (0.54 v/w) to obtain aspirin‐loaded Sr‐α‐CSH/n‐HA composite. In vitro experiments were performed on the composite extracts. The tibial defects (3 mm*5 mm) in SD rat model were filled with the composite for 4 weeks and 12 weeks to evaluate its osteogenic capacity in vivo. Our results showed its capability of proliferation, migration and osteogenesis of BMSCs in vitro got improved. In vivo treatment with 800 μg/ml aspirin–loaded Sr‐α‐CSH/n‐HA composite led to significantly more new bone formation in the defects compared with Sr‐α‐CSH/n‐HA composite and significantly promoted the expression of osteogenic‐related genes and inhibited osteoclast activity. In general, our research suggests that aspirin‐loaded Sr‐α‐CSH/n‐HA composite may have a greater capacity of repairing tibial defects in SD rats than simple Sr‐α‐CSH/n‐HA composite.

Summary Background Abnormal bone formation in subchondral bone resulting from uncoupled bone remodeling is considered a central feature in osteoarthritis (OA) pathogenesis. H-type vessels can couple angiogenesis and osteogenesis. We previously revealed that elevated H-type vessels in subchondral bone were correlated with OA and focal adhesion kinase (FAK) in MSCs is critical for H-type vessel formation in osteoporosis. The aim of this study was to explore the correlation between H-type vessels and MSCs in OA pathogenesis through regulation of H-type vessel formation using defactinib (an FAK inhibitor). Methods In vivo: 3-month-old male C57BL/6J (WT) mice were randomly divided into three groups: sham controls, vehicle-treated ACLT mice, and defactinib-treated ACLT mice (25 mg/kg, intraperitoneally weekly). In vitro: we explored the role of conditioned medium (CM) of MSCs from subchondral bone of different groups on the angiogenesis of endothelial cells (ECs). Flow cytometry, Western blotting, ELISA, real time (RT)-PCR, immunostaining, CT-based microangiography, and bone micro-CT (μCT) were used to detect changes in relative cells and tissues. Results This study demonstrated that inhibition of H-type vessels with defactinib alleviated OA by inhibiting H-type vessel-linked MSCs in subchondral bone. During OA pathogenesis, H-type vessels and MSCs formed a positive feedback loop contributing to abnormal bone formation in subchondral bone. Elevated H-type vessels provided indispensable MSCs for abnormal bone formation in subchondral bone. Flow cytometry and immunostaining results confirmed that the amount of MSCs in subchondral bone was obviously higher in vehicle-treated ACLT mice than that in sham controls and defactinib-treated ACLT mice. In vitro, p-FAK in MSCs from subchondral bone of vehicle-treated ALCT mice increased significantly relative to other groups. Further, the CM from MSCs of vehicle-treated ACLT mice enhanced angiogenesis of ECs through FAK-Grb2-MAPK-linked VEGF expression. Conclusions Our results demonstrate that defactinib inhibits OA by suppressing the positive feedback loop between H-type vessels and MSCs in subchondral bone. The translational potential of this article Our results provide a mechanistic rationale for the use of defactinib as an effective candidate for OA treatment.

Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.

Clonal hematopoiesis (CH), characterized by the accumulation of acquired somatic mutations in the blood, is associated with an elevated risk of aging-related diseases and premature mortality in non-cancer populations. Patients who undergo autologous hematopoietic cell transplantation (HCT) are also at high risk of premature onset of aging-related conditions. Therefore, we examined the association between pretreatment CH and late-occurring (≥1 year) nonrelapse mortality (NRM) after HCT. We evaluated pathogenic and likely pathogenic CH variants (PVs) in 10 patients who developed NRM after HCT and in 29 HCT recipient controls matched by age at HCT ± 2 years (median, 64.6 years; range, 38.5 to 74.7 years), sex (79.5% male), diagnosis (61.5% with non-Hodgkin lymphoma, 18.0% with Hodgkin lymphoma, and 20.5% with multiple myeloma), and duration of follow-up. We analyzed mobilized hematopoietic stem cell DNA in samples collected before HCT using a custom panel of amplicons covering the coding exons of 79 myeloid-related genes associated with CH. PVs with allele fractions >2% were used for analyses. Cases were significantly more likely than controls to have CH (70% versus 24.1%; P = .002), to have ≥2 unique PVs (60% versus 6.9%; P < .001), and to have PVs with allelic fractions ≥10% (40% versus 3.4%; P = .003). Here we provide preliminary evidence of an association between pre-HCT CH and NRM after HCT independent of chronologic age. Integration of CH analyses may improve the accuracy of existing pre-HCT risk prediction models, setting the stage for personalized risk assessment strategies and targeted treatments to optimally prevent or manage late complications associated with HCT.

Simple Summary Transformed mycosis fungoides (LCT-MF) is a histopathological marker of poor prognosis and associated with worse survival. We compared miRNA and mRNA expression profiles of LCT-MF with classic MF and found a distinct miRNA regulatory network modulated immunosuppressive tumor microenvironment in LCT-MF. Our findings provide novel insights and therapeutic targets for LCT-MF. Abstract Large cell transformation of mycosis fungoides (LCT-MF) occurs in 20–50% of advanced MF and is generally associated with poor response and dismal prognosis. Although different mechanisms have been proposed to explain the pathogenesis, little is known about the role of microRNAs (miRs) in transcriptional regulation of LCT-MF. Here, we investigated the miR and mRNA expression profile in lesional skin samples of patients with LCT-MF and non-LCT MF using RNA-seq analysis. We found miR-146a and miR-21 to be significantly upregulated, and miR-708 the most significantly downregulated miR in LCT-MF. Integration of miR and mRNA expression profiles revealed the miR-regulated networks in LCT-MF. Ingenuity pathway analysis (IPA) demonstrated the involvement of genes for ICOS-ICOSL, PD1-PDL1, NF-κB, E2F transcription, and molecular mechanisms of cancer signaling pathways. Quantitative real time (qRT)-PCR results of target genes were consistent with the RNA-seq data. We further identified the immunosuppressive tumor microenvironment (TME) in LCT-MF. Moreover, our data indicated that miR-146a, -21 and -708 are associated with the immunosuppressive TME in LCT-MF. Collectively, our results suggest that the key LCT-MF associated miRs and their regulated networks may provide insights into its pathogenesis and identify promising targets for novel therapeutic strategies.

The average fat mass in adults increases dramatically with age, and older people often suffer from visceral obesity and related adverse metabolic disorders. Unfortunately, how aging leads to fat accumulation is poorly understood. It is known that fat cell (adipocyte) turnover is very low in young mice, similar to that in young humans. Here, we find that mice mimic age-related fat expansion in humans. In vivo lineage tracing shows that massive adipogenesis (the generation of new adipocytes), especially in the visceral fat, is triggered during aging. Thus, in contrast to most types of adult stem cells that exhibit a reduced ability to proliferate and differentiate, the adipogenic potential of adipocyte progenitor cells (APCs) is unlocked by aging. In vivo transplantation and 3D imaging of transplants show that APCs in aged mice cell-autonomously gain high adipogenic capacity. Single-cell RNA sequencing analyses reveal that aging globally remodels APCs. Herein, we identify a novel committed preadipocyte population that is age-specific (CP-A), existing both in mice and humans, with a global activation of proliferation and adipogenesis pathways. CP-A cells display high proliferation and adipogenesis activity, both in vivo and in vitro. Macrophages may regulate the remodeling of APCs and the generation of CP-A cells during aging. Together, these findings define a new fundamental mechanism involved in fat tissue aging and offer prospects for preventing and treating age-related metabolic disorders.

Paralysis or paraplegia caused by transient or permanent spinal cord ischemia–reperfusion injury (SCIRI) remains one of the most devastating post-operative complications after thoracoabdominal aortic surgery, even though perioperative strategies and surgical techniques continue to improve. Uncovering the molecular and cellular pathophysiological processes in SCIRI has become a top priority. Recently, the expression, function, and mechanism of non-coding RNAs (ncRNAs) in various diseases have drawn wide attention. Non-coding RNAs contain a variety of biological functions but do not code for proteins. Previous studies have shown that ncRNAs play a critical role in SCIRI. However, the character of ncRNAs in attenuating SCIRI has not been systematically summarized. This review article will be the first time to assemble the knowledge of ncRNAs regulating apoptosis, inflammation, autophagy, and oxidative stress to attenuate SCIRI. A better understanding of the functional significance of ncRNAs following SCIRI could help us to identify novel therapeutic targets and develop potential therapeutic strategies. All the current research about the function of nRNAs in SCIRI will be summarized one by one in this review.

Background Administration of Magnoliae Cortex (MC) could induce remission of cisplatin-induced sarcopenia in mice, however, whether it is effective on sarcopenia patients and the underlying mechanisms remain unclear. Methods Sarcopenia related differentially expressed genes were analysed based on three Gene Expression Omnibus (GEO) transcriptome profiling datasets, which was merged and de duplicated with disease databases to obtain sarcopenia related pathogenic genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were than performed to analyse the role of proteins encoded by sarcopenia related pathogenic genes and the signal regulatory pathways involved in. The main active components and target proteins of MC were obtained by searching traditional Chinese medicine network databases (TCMSP and BATMAN-TCM). MC and sarcopenia related pathogenic genes shared target proteins were identified by matching the two. A protein–protein interaction network was constructed subsequently, and the core proteins were filtered according to the topological structure. GO and KEGG analysis were performed again to analyse the key target proteins and pathways of MC in the treatment of sarcopenia, and build the herbs-components-targets network, as well as core targets-signal pathways network. Molecular docking technology was used to verify the main compounds-targets. Results Sarcopenia related gene products primarily involve in aging and inflammation related signal pathways. Seven main active components (Anonaine, Eucalyptol, Neohesperidin, Obovatol, Honokiol, Magnolol, and beta-Eudesmol) and 26 target proteins of MC-sarcopenia, of which 4 were core proteins (AKT1, EGFR, INS, and PIK3CA), were identified. The therapeutic effect of MC on sarcopenia may associate with PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, longevity regulating pathway, and other cellular and innate immune signaling pathways. Conclusion MC contains potential anti-sarcopenia active compounds. These compounds play a role by regulating the proteins implicated in regulating aging and inflammation related signaling pathways, which are crucial in pathogenesis of sarcopenia. Our study provides new insights into the development of a natural therapy for the prevention and treatment of sarcopenia.

Background: Substantial evidence shows that crosstalk between cartilage and subchondral bone may play an important role in cartilage repair. Animal models have shown that hydroxyapatite-grafted-chitosan implant (HA-g-CS) and moderate-intensity exercise promote regeneration of osteochondral defects. However, no in vivo studies have demonstrated that these two factors may have a synergistic activity to facilitate subchondral bone remodeling in mice, thus supporting bone-cartilage repair. Questions: This study was to clarify whether HA-g-CS and moderate-intensity exercise might have a synergistic effect on facilitating (1) regeneration of osteochondral defects and (2) subchondral bone remodeling in a mouse model of osteochondral defects. Methods: Mouse models of osteochondral defects were created and divided into four groups. BC Group was subjected to no treatment, HC Group to HA-g-CS implantation into osteochondral defects, ME group to moderate-intensity treadmill running exercise, and HC+ME group to both HA-g-CS implantation and moderate-intensity exercise until sacrifice. Extent of subchondral bone remodeling at the injury site and subsequent cartilage repair were assessed at 4 weeks after surgery. Results: Compared with BC group, HC, ME and HC+ME groups showed more cartilage repair and thicker articular cartilage layers and HC+ME group acquired the best results. The extent of cartilage repair was correlated positively to bone formation activity at the injured site as verified by microCT and correlation analysis. Histology and immunofluorescence staining confirmed that bone remodeling activity was increased in HC and ME groups, and especially in HC+ME group. This bone formation process was accompanied by an increase in osteogenesis and chondrogenesis factors at the injury site which promoted cartilage repair. Conclusions: In a mouse model of osteochondral repair, HA-g-CS implant and moderate-intensity exercise may have a synergistic effect on improving osteochondral repair potentially through promotion of subchondral bone remodeling and generation of osteogenesis and chondrogenesis factors. Clinical Relevance: Combination of HA-g-CS implantation and moderate-intensity exercise may be considered potentially in clinic to promote osteochondral defect repair. Also, cartilage and subchondral bone forms a functional unit in an articular joint and subchondral bone may regulate cartilage repair by secreting growth factors in its remodeling process. However, a deeper insight into the exact role of HA-g-CS implantation and moderate-intensity exercise in promoting osteochondral repair in other animal models should be explored before they can be applied in clinic in the future.

Prevention of Epstein-Barr virus (EBV) infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). In this study, we generated 23 hybridomas producing gp350-specific antibodies. We compared the candidate gp350-specific antibodies to the well-characterized nAb 72A1 by: (1) testing their ability to detect gp350 using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using competitive cell and linear peptide binding assays. We performed sequence analysis to identify 15 mAbs with CDR regions unique from those of murine 72A1 (m72A1). We observed antigen binding competition between biotinylated m72A1, serially diluted unlabeled gp350 nAbs (HB1, HB5, HB11, HB20), and our recently humanized 72A1, but not gp350 non-nAb (HB17) or anti-KSHV gH/gL antibody.

So far, there has been no effective cure for osteoporotic cortical bone, the most significant change in long bone structure during aging and the main cause of bone fragility fractures, because its underlying molecular and cellular mechanisms remain largely unknown. We used 3- and 15-mo-old mice as well as 15-mo-old mice treated with vehicle and gefitinib to evaluate structural, cellular, and molecular changes in cortical bone. We found that the senescence of osteoprogenitors was increased, whereas the expression of phosphorylated epidermal growth factor receptor (EGFR) on the endosteal surface of cortical bone down-regulated in middle-aged 15-mo-old mice compared with young 3-mo-old mice. Further decreasing EGFR signaling by gefitinib treatment in middle-aged mice resulted in promoted senescence of osteoprogenitors and accelerated cortical bone degeneration. Moreover, inhibiting EGFR signaling suppressed the expression of enhancer of zeste homolog 2 (Ezh2), the repressor of cell senescence-inducer genes, through ERK1/2 pathway, thereby promoting senescence in osteoprogenitors. Down-regulated EGFR signaling plays a physiologically significant role during aging by reducing Ezh2 expression, leading to the senescence of osteoprogenitors and the decline in bone formation on the endosteal surface of cortical bone.-Liu, G., Xie, Y., Su, J., Qin, H., Wu, H., Li, K., Yu, B., Zhang, X. The role of EGFR signaling in age-related osteoporosis in mouse cortical bone.

Bone loss in Staphylococcus aureus (S. aureus) osteomyelitis poses a serious challenge to orthopedic treatment. The present study aimed to elucidate how S. aureus infection in bone might induce bone loss. The C57BL/6 mice were injected with S. aureus (106 CFU/ml, 100 μl) or with the same amount of vehicle (control) via the tail vein. Microcomputed tomography (microCT) analysis showed bone loss progressing from week 1 to week 5 after infection, accompanied by a decreased number of osteocalcin-positive stained osteoblasts and the suppressed mRNA expression of Runx2 and osteocalcin. Transcriptome profiles of GSE30119 were downloaded and analyzed to determine the differences in expression of inflammatory factors between patients with S. aureus infected osteomyelitis and healthy controls, the data showed significantly higher mRNA expression of granulocyte colony-stimulating factor (G-CSF) in the whole blood from patients with S. aureus infection. Enzyme-linked immunosorbent assay (ELISA) analysis confirmed an increased level of G-CSF in the bone marrow and serum from S. aureus infected mice, which might have been due to the increased amount of F4/80+ macrophages. Interestingly, G-CSF neutralizing antibody treatment significantly rescued the bone loss after S. aureus infection, as evidenced by its roles in improving BV/TV and preserving osteocalcin- and osterix-positive stained cells. Importantly, we found that G-CSF level was significantly up-regulated in the serum from osteomyelitis patients infected by S. aureus. Together, S. aureus infection might suppress the function of osteoblastic cells and induce progressive bone loss by up-regulating the level G-CSF, suggesting a therapeutic potential for G-CSF neutralization in combating bone loss in S. aureus osteomyelitis.