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Cancer cachexia is a lethal metabolic syndrome featuring muscle wasting with preferential loss of fast-twitching muscle mass through an undefined mechanism. Here, we show that cancer induces muscle wasting by selectively degrading myosin heavy chain (MHC) subtypes IIb and IIx through E3 ligase UBR2-mediated ubiquitylation. Induction of MHC loss and atrophy in C2C12 myotubes and mouse tibialis anterior (TA) by murine cancer cells required UBR2 up-regulation by cancer. Genetic gain or loss of UBR2 function inversely altered MHC level and muscle mass in TA of tumor-free mice. UBR2 selectively interacted with and ubiquitylated MHC-IIb and MHC-IIx through its substrate recognition and catalytic domain, respectively, in C2C12 myotubes. Elevation of UBR2 in muscle of tumor-bearing or free mice caused loss of MHC-IIb and MHC-IIx but not MHC-I and MHC-IIa or other myofibrillar proteins, including α-actin, troponin, tropomyosin, and tropomodulin. Muscle-specific knockout of UBR2 spared KPC tumor-bearing mice from losing MHC-IIb and MHC-IIx, fast-twitching muscle mass, cross-sectional area, and contractile force. The rectus abdominis (RA) muscle of patients with cachexia-prone cancers displayed a selective reduction of MHC-IIx in correlation with higher UBR2 levels. These data suggest that UBR2 is a regulator of MHC-IIb/IIx essential for cancer-induced muscle wasting, and that therapeutic interventions can be designed by blocking UBR2 up-regulation by cancer.

Supplementary table 1 showed the clinic-pathological data of patients with pancreatic adenocarcinoma.

Purpose: ZIP4 is overexpressed in human pancreatic cancer and promotes tumor growth. However, little is known about the role of ZIP4 in advanced stages of this dismal neoplasm. Our goal is to study the underlying mechanism and define a novel signaling pathway controlled by ZIP4-modulating pancreatic tumor metastasis.Experimental Design: The expression of ZIP4, ZO-1, claudin-1, and ZEB1 in human pancreatic cancer tissues, genetically engineered mouse model, xenograft tumor model, and pancreatic cancer cell lines were examined, and the correlations between ZIP4 and those markers were also analyzed. Functional analysis of ZO-1, claudin-1, and ZEB1 was investigated in pancreatic cancer cell lines and orthotopic xenografts.Results: Genetic inactivation of ZIP4 inhibited migration and invasion in pancreatic cancer and increased the expression of ZO-1 and claudin-1. Conversely, overexpression of ZIP4 promoted migration and invasion and increased the expression of ZEB1 and downregulation of the aforementioned epithelial genes. ZIP4 downregulation of ZO-1 and claudin-1 requires the transcriptional repressor ZEB1. Further analysis demonstrated that ZIP4-mediated repression of ZO-1 and claudin-1 leads to upregulation of their targets FAK and Paxillin. Silencing of ZIP4 caused reduced phosphorylation of FAK and Paxillin, which was rescued by simultaneous blocking of ZO-1 or claudin-1. Clinically, we demonstrated that ZIP4 positively correlates with the levels of ZEB1 and inversely associates with the expression of ZO-1 and claudin-1.Conclusions: These findings suggest a novel pathway activated by ZIP4-controlling pancreatic cancer invasiveness and metastasis, which could serve as a new therapeutic target for this devastating disease. Clin Cancer Res; 24(13); 3186–96. ©2018 AACR.

C/EBPβ is a key mediator of cancer-induced skeletal muscle wasting. However, the signaling mechanisms that activate C/EBPβ in the cancer milieu are poorly defined. Here, we report cancer-induced muscle wasting requires the transcriptional cofactor p300, which is critical for the activation of C/EBPβ. Conditioned media from diverse types of tumor cells as well as recombinant HSP70 and HSP90 provoked rapid acetylation of C/EBPβ in myotubes, particularly at its Lys39 residue. Overexpression of C/EBPβ with mutated Lys39 impaired Lewis lung carcinoma (LLC)–induced activation of the C/EBPβ-dependent catabolic response, which included upregulation of E3 ligases UBR2 and atrogin1/MAFbx, increased LC3-II, and loss of muscle proteins both in myotubes and mouse muscle. Silencing p300 in myotubes or overexpressing a dominant negative p300 mutant lacking acetyltransferase activity in mouse muscle attenuated LLC tumor–induced muscle catabolism. Administration of pharmacologic p300 inhibitor C646, but not PCAF/GCN5 inhibitor CPTH6, spared LLC tumor–bearing mice from muscle wasting. Furthermore, mice with muscle-specific p300 knockout were resistant to LLC tumor–induced muscle wasting. These data suggest that p300 is a key mediator of LLC tumor–induced muscle wasting whose acetyltransferase activity may be targeted for therapeutic benefit in this disease.Significance:These findings demonstrate that tumor-induced muscle wasting in mice is abrogated by knockout, mutation of Lys39 or Asp1399, and pharmacologic inhibition of p300.

Supplementary Figure 1. ZIP4 is correlated with EMT markers in pancreatic cancer tissue of KPC mice. Supplementary Figure 2. Knocking down ZIP4 upregulated ZO-1 and claudin-1 expression in pancreatic cancer. Supplementary Figure 3. Knocking down ZIP4 regulated mRNA of EMT markers in pancreatic cancer cell lines. Supplementary Figure 4. ZIP4 repressed ZO-1 and claudin-1 is independent with CREB. Supplementary Figure 5. Knocking down efficiency of ZO-1 and claudin-1 in AsPC-shZIP4 cells.

Figure S1. Genotyping p300 mKO Figure S2. Lys39 mutation effect on Thr188 phosphorylation Figure S3. Lys39 acetylation is required for muscle mass loss Figure S4. Acetyltransferase activity of p300 is required for muscle mass loss Figure S5. p300 is not required for activation of p38 and NF-kB

Cancer-associated cachexia, characterized by muscle wasting, is a lethal metabolic syndrome without defined etiology or established treatment. We previously found that p300 mediates cancer-induced muscle wasting by activating C/EBPβ, which then upregulates key catabolic genes. However, the signaling mechanism that activates p300 in response to cancer is unknown. Here, we show that upon cancer-induced activation of Toll-like receptor 4 in skeletal muscle, p38β MAPK phosphorylates Ser-12 on p300 to stimulate C/EBPβ acetylation, which is necessary and sufficient to cause muscle wasting. Thus, p38β MAPK is a central mediator and therapeutic target of cancer-induced muscle wasting. In addition, nilotinib, an FDA-approved kinase inhibitor that preferentially binds p38β MAPK, inhibited p300 activation 20-fold more potently than the p38α/β MAPK inhibitor, SB202190, and abrogated cancer cell–induced muscle protein loss in C2C12 myotubes without suppressing p38α MAPK–dependent myogenesis. Systemic administration of nilotinib at a low dose (0.5 mg/kg/day, i.p.) in tumor-bearing mice not only alleviated muscle wasting, but also prolonged survival. Therefore, nilotinib appears to be a promising treatment for human cancer cachexia due to its selective inhibition of p38β MAPK.Significance:These findings demonstrate that prevention of p38β MAPK–mediated activation of p300 by the FDA-approved kinase inhibitor, nilotinib, ameliorates cancer cachexia, representing a potential therapeutic strategy against this syndrome.

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NLRC5 has been reported to be involved in antiviral immunity; however, the underlying mechanism remains poorly understood. Here, we investigated the functional role of NLRC5 in the infection of a flavivirus, dengue virus (DENV). We found that the expression of NLRC5 was strongly induced by virus infection and IFNB or IFNG stimulation in different cell lines. Overexpression of NLRC5 remarkably suppressed DENV infection, whereas knockout of

SummaryCD4+ T follicular helper (TFH) cells are required for high-quality antibody generation and maintenance. However, the longevity and functional role of these cells are poorly defined in COVID-19 convalescents and vaccine recipients. Here, we longitudinally investigated the dynamics and functional roles of spike-specific circulating TFH cells and their subsets in convalescents at the 2nd, 5th, 8th, 12th and 24th months after COVID-19 symptom onset and in vaccinees after two and three doses of inactivated vaccine. SARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, of which spike-specific CXCR3+ TFH cells but not spike-specific CXCR3− TFH cells and neutralizing antibodies were persistent for at least two years in more than 80% of convalescents who experienced symptomatic COVID-19, which was well coordinated between spike-specific TFH cell and antibody responses at the 5th month after infection. Inactivated vaccine immunization also induced spike-specific TFH cell and antibody responses; however, these responses rapidly declined after six months with a two-dose standard administration, and a third dose significantly promoted antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells exhibited better responsiveness than spike-specific CXCR3− TFH cells upon spike protein stimulation in vitro and showed superior capacity in supporting spike-specific antibody secreting cell (ASC) differentiation and antibody production than spike-specific CXCR3− TFH cells cocultured with autologous memory B cells. In conclusion, spike-specific CXCR3+ TFH cells played a dominant functional role in antibody elicitation and maintenance in SARS-CoV-2 infection and vaccination, suggesting that induction of CXCR3-biased spike-specific TFH cell differentiation will benefit SARS-CoV-2 vaccine development aiming to induce long-term protective immune memory.HighlightsSARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, which persisted for at least two years in the majority of symptomatic COVID-19 convalescent patients.Inactivated vaccine immunization also elicited spike-specific TFH cell and antibody responses, which rapidly declined over time, and a third dose significantly promoted antibody maturation and potency.Spike-specific CXCR3+ TFH cells exhibited more durable responses than spike-specific CXCR3− TFH cells, correlated with antibody responses and showed superior capacity in supporting ASC differentiation and antibody production than spike-specific CXCR3− TFH cells.

Medicinal plants are one of the most important sources of antiviral agents and lead compounds. Lignans are a large class of natural compounds comprising two phenyl propane units. Many of them have demonstrated biological activities, and some of them have even been developed as therapeutic drugs. In this review, 630 lignans, including those obtained from medicinal plants and their chemical derivatives, were systematically reviewed for their antiviral activity and mechanism of action. The compounds discussed herein were published in articles between 1998 and 2020. The articles were identified using both database searches (e.g., Web of Science, Pub Med and Scifinder) using key words such as: antiviral activity, antiviral effects, lignans, HBV, HCV, HIV, HPV, HSV, JEV, SARS-CoV, RSV and influenza A virus, and directed searches of scholarly publisher's websites including ACS, Elsevier, Springer, Thieme, and Wiley. The compounds were classified on their structural characteristics as 1) arylnaphthalene lignans, 2) aryltetralin lignans, 3) dibenzylbutyrolactone lignans, 4) dibenzylbutane lignans, 5) tetrahydrofuranoid and tetrahydrofurofuranoid lignans, 6) benzofuran lignans, 7) neolignans, 8) dibenzocyclooctadiene lignans and homolignans, and 9) norlignans and other lignoids. Details on isolation and antiviral activities of the most active compounds within each class of lignan are discussed in detail, as are studies of synthetic lignans that provide structure-activity relationship information.

Early intervention services (EIS; in the United States, Coordinated Specialty Care) can lead to substantial improvements in psychiatric symptoms and social functioning for individuals with first-episode psychosis who engage in treatment. Nevertheless, stigma associated with early intervention services can limit their full potential benefits by preventing or reducing participation. Drawing from Corrigan's "why try" model positing relationships between public and self-stigma, engagement in treatment services, and the EIS treatment model, this article proposes a framework that delineates how distinct forms of stigma are linked to given stages of treatment engagement in first-episode psychosis. We identify three phases of engagement: (1) community outreach, which has associations with public stigma; (2) the referral and evaluation process, which primarily has associations with self-stigma; and (3) EIS, which have associations with self-stigma and its psychosocial consequences. For each phase, we describe evidence-based strategies typically provided by EIS programs, using OnTrackNY as an exemplary model, to illustrate potential linkages in our conceptual framework. By specifying how distinct forms of stigma are associated with EIS treatment stages, this framework is intended to guide EIS programs in explicitly addressing stigma to optimize recovery of individuals with first-episode psychosis.

Cancer-associated cachexia, characterized by muscle wasting, is a lethal metabolic syndrome without defined etiology or established treatment. We previously found that p300 mediates cancer-induced muscle wasting by activating C/EBPβ, which then upregulates key catabolic genes. However, the signaling mechanism that activates p300 in response to cancer is unknown. Here, we show that upon cancer-induced activation of Toll-like receptor 4 in skeletal muscle, p38β MAPK phosphorylates Ser-12 on p300 to stimulate C/EBPβ acetylation, which is necessary and sufficient to cause muscle wasting. Thus, p38β MAPK is a central mediator and therapeutic target of cancer-induced muscle wasting. In addition, nilotinib, an FDA-approved kinase inhibitor that preferentially binds p38β MAPK, inhibited p300 activation 20-fold more potently than the p38α/β MAPK inhibitor, SB202190, and abrogated cancer cell–induced muscle protein loss in C2C12 myotubes without suppressing p38α MAPK–dependent myogenesis. Systemic administration of nilotinib at a low dose (0.5 mg/kg/day, i.p.) in tumor-bearing mice not only alleviated muscle wasting, but also prolonged survival. Therefore, nilotinib appears to be a promising treatment for human cancer cachexia due to its selective inhibition of p38β MAPK. Significance: These findings demonstrate that prevention of p38β MAPK–mediated activation of p300 by the FDA-approved kinase inhibitor, nilotinib, ameliorates cancer cachexia, representing a potential therapeutic strategy against this syndrome.

Trio is a unique member of the Rho-GEF family that has three catalytic domains and is vital for various cellular processes in both physiological and developmental settings. TRIO mutations in humans are involved in craniofacial abnormalities, in which patients present with mandibular retrusion. However, little is known about the molecular mechanisms of Trio in neural crest cell (NCC)-derived craniofacial development, and there is still a lack of direct evidence to assign a functional role to Trio in NCC-induced craniofacial abnormalities. Methods: In vivo, we used zebrafish and NCC-specific knockout mouse models to investigate the phenotype and dynamics of NCC development in Trio morphants. In vitro, iTRAQ, GST pull-down assays, and proximity ligation assay (PLA) were used to explore the role of Trio and its potential downstream mediators in NCC migration and differentiation. Results: In zebrafish and mouse models, disruption of Trio elicited a migration deficit and impaired the differentiation of NCC derivatives, leading to craniofacial growth deficiency and mandibular retrusion. Moreover, Trio positively regulated Myh9 expression and directly interacted with Myh9 to coregulate downstream cellular signaling in NCCs. We further demonstrated that disruption of Trio or Myh9 inhibited Rac1 and Cdc42 activity, specifically affecting the nuclear export of β-catenin and NCC polarization. Remarkably, craniofacial abnormalities caused by trio deficiency in zebrafish could be partially rescued by the injection of mRNA encoding myh9, ca-Rac1, or ca-Cdc42. Conclusions: Here, we identified that Trio, interacting mostly with Myh9, acts as a key regulator of NCC migration and differentiation during craniofacial development. Our results indicate that trio morphant zebrafish and Wnt1-cre;Triofl/fl mice offer potential model systems to facilitate the study of the pathogenic mechanisms of Trio mutations causing craniofacial abnormalities.

Excess bone loss due to increased osteoclastogenesis is a significant clinical problem. Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation. The role of IFT80, an IFT complex B protein, in osteoclasts (OCs) is completely unknown. Here, we demonstrate that deletion of IFT80 in the myeloid lineage led to increased OC formation and activity accompanied by severe bone loss in mice. IFT80 regulated OC formation by associating with Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) to promote protein stabilization and proteasomal degradation of tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6). IFT80 knockdown resulted in increased ubiquitination of Cbl-b and higher TRAF6 levels, thereby hyperactivating the receptor activator of nuclear factor-κβ (NF-κβ) ligand (RANKL) signaling axis and increased OC formation. Ectopic overexpression of IFT80 rescued osteolysis in a calvarial model of bone loss. We have thus identified a negative function of IFT80 in OCs.

Rainfall runoff is one of the important sources of urban river pollution. In order to understand the pollution characteristics of rainfall runoff, the synchronous sampling and monitoring of rainfall runoff in the old urban area of Nanning were carried out; the pollution condition, initial scouring effect, and pollution contribution ratio of different underlying surfaces under different rainfall conditions were analyzed; and the calculation method and influence factor analysis of initial scouring of runoff pollution were discussed. According to the underlying surface of the vegetable market in the old urban area, the selection standard and necessity of the underlying surface of rainfall runoff were discussed. The results showed that the average concentration (EMC) of COD and TSS in roads and vegetable markets were greater than those in green spaces and roofs in the runoff pollution of the old urban area of Nanning, and the EMC values of nutrient pollutants in field rainfall runoff were ranked in decreasing order as vegetable markets, green spaces, roofs, and roads. Under the condition of heavy rain, each underlying surface had an obvious initial scouring effect, the average value of initial scouring coefficient (

Introduction: Knowledge about the distribution of hepatitis C virus (HCV) genotype and its correlation with viral load are important for the decision of treatment and the prediction of disease progression, however such information is very limited in Nepal. Here, we investigated the distribution of HCV genotypes and viral load for HCV-infected patients from Kathmandu, Nepal. Methodology: Ninety-six patients with HCV infection and not on antiviral therapy were enrolled from three different medical centers in Kathmandu valley, Nepal. Demographics were recorded and blood samples were collected. Plasma was separated and HCV RNA was extracted. Reverse transcriptase PCR (RT-PCR) was performed to measure the viral load, and virus genotype was determined. Results: Genotype 3a (n = 53, 55.2%) was the most prevalent, followed by 1b (n = 19, 19.8%), 1a (n = 18, 18.8%), 5a (n = 3, 3.1%), and mix types (n = 3, 3.1%). The median viral load for HCV genotype 1a was 770,942 IU/mL (IQR, 215,268-3,720,075), 1b was 700,000 IU/mL (IQR, 431,560-919,000), 3a was 1,060,000 IU/mL (IQR, 641,050-6,063,500), 5a was 673,400 IU/mL, and mixed was 6,428,000 IU/mL. A correlation between genotype and viral load was observed (p = 0.02), of which genotype 3a showed a high viral load. Conclusions: HCV genotypes 1a, 1b, 3a, and 5a were identified in Kathmandu, Nepal, and mixed genotype patients were observed in the patients studied. HCV genotype showed a correlation with viral load in patient plasma. This finding may contribute to the treatment and prevention of hepatitis C in Kathmandu, Nepal.

Despite years of research investigating osteoblast differentiation, the mechanisms by which transcription factors regulate osteoblast maturation, bone formation, and bone homeostasis is still unclear. It has been reported that runt-related transcription factor 1 (Runx1) is expressed in osteoblast progenitors, pre-osteoblasts, and mature osteoblasts; yet, surprisingly, the exact function of RUNX1 in osteoblast maturation and bone formation remains unknown. Here, we generated and characterized a pre-osteoblast and differentiating chondrocyte-specific Runx1 conditional knockout mouse model to study RUNX1's function in bone formation. Runx1 ablation in osteoblast precursors and differentiating chondrocytes via osterix-Cre (Osx-Cre) resulted in an osteoporotic phenotype and decreased bone density in the long bones and skulls of Runx1(f/f)Osx-Cre mice compared with Runx1(f/f) and Osx-Cre mice. RUNX1 deficiency reduced the expression of SRY-box transcription factor 9 (SOX9), Indian hedgehog signaling molecule (IHH), Patched (PTC), and cyclin D1 in the growth plate, and also reduced the expression of osteocalcin (OCN), OSX, activating transcription factor 4 (ATF4), and RUNX2 in osteoblasts. ChIP assays and promoter activity mapping revealed that RUNX1 directly associates with the Runx2 gene promoter and up-regulates Runx2 expression. Furthermore, the ChIP data also showed that RUNX1 associates with the Ocn promoter. In conclusion, RUNX1 up-regulates the expression of Runx2 and multiple bone-specific genes, and plays an indispensable role in bone formation and homeostasis in both trabecular and cortical bone. We propose that stimulating Runx1 activity may be useful in therapeutic approaches for managing some bone diseases such as osteoporosis.

BACKGROUND Geographic variation in male response to sex pheromone lures has been studied in the field in a number of moth species. However, only a few studies have investigated geographic variation in female calling and sex pheromone under field conditions. For an effective field implementation of sex pheromone lures, it is essential to know the local sex pheromone blend and local timing of sexual communication. We investigated the level and extent of geographic variation in the sexual communication of the important agricultural pest Helicoverpa armigera (Lepidoptera, Noctuidae) in three continents. RESULTS We found there is no genetic variation in the calling behavior of H. armigera. In the female sex pheromone, we found more between‐population variation than within‐population variation. In male response experiments, we found geographic variation as well. Strikingly, when adding the antagonistic compound Z11‐16:OAc to the pheromone blend of H. armigera, significantly fewer males were caught in Australia and China, but not in Spain. This variation is likely not only due to local environmental conditions, such as photoperiod and temperature, but also to the presence of other closely related species with which communication interference may occur. Conclusion Finding geographic variation in both the female sexual signal and the male response in this pest calls for region‐specific pheromone lures. Our study shows that the analysis of geographic variation in moth female sex pheromones as well as male responses is important for effectively monitoring pest species that occur around the globe. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., In this paper we found geographic variation in the female sex pheromone and the male response of the cotton bollworm, Helicoverpa armigera. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Aiming to solve the poor compactibility of the alcoholic extract of Zingiberis Rhizoma(ZR), this study explored the feasibility of its physical modification using co-spray drying with a small amount of hydroxypropyl methyl cellulose(HPMC). Based on the univariate analysis, the influence of two independent variables(the HPMC content in the product and the solid content of spray material) on the powder properties and tablet properties of the dried product was investigated by the central composite design. With the tensile strength and disintegration time of the tablets as the evaluation indexes, the optimal prescription was determined as follows: the HPMC content was 15% and the solid content of spray material was 25.6%. The accuracy of the regression model established for predicting tensile strength and disintegration time of tablets was verified, and the results revealed that the measured values were close to the predicted ones with deviations of 0.47% and-8.2%, indicating good prediction and reproducibility of the model. The tensile strength(4.24 MPa) of tablets prepared with the optimal prescription was 3.59 times that(1.18 MPa, far lower than the baseline of 2 MPa for qualified tablets) with the spray-dried powder of the ZR. On the other hand, due to the addition of HPMC, the disintegration time of tablets increased from 7.3 min to 24.6 min. On the whole, this study provided a new strategy to solve the common problem of poor compactibility of raw Chinese medicinal materials, which facilitated the successful preparation of Chinese medicinal tablets with high drug loads.

Cathepsins play a role in regulation of cell function through their presence in the cell nucleus. However, the role of Cathepsin K (Ctsk) as an epigenetic regulator in osteoclasts remains unknown. Our data demonstrated that

The alternating aerobic/anaerobic biofilm system had been applied for phosphorus (P) enrichment and recovery because of the advantage of low energy consumption and high efficiency. The metal ions and N-acyl-L-homoserine lactones (AHLs) in system were studied to better clarify the mechanism of P uptake/release under metal ion stress. The results indicated that the increase of metal ions stimulated the release of AHLs, and AHLs-guided quorum sensing (QS) enhanced P uptake. Moreover, biomineralization could stimulate the increase of P content in biofilm (P

G-protein-coupled receptors (GPCRs) signaling is critical to cell differentiation and activation. However, the function of GPCRs in osteoclast differentiation and activation remains unclear. We found that the G-protein coupled receptor 125 (GPCR 125) gene (Gpr125) gene was highly expressed in osteoclasts through RNA-sequencing technology, qRT-PCR, and Western blot analysis. We characterized the role of GPCR125 in osteoclast differentiation and activation by loss-of-function and gain-of-function methods in osteoclasts. Osteoclasts with lentivirus-mediated GPR125 silencing demonstrated a dramatic reduction in differentiation and impaired bone resorption function. In contrast, overexpression of Gpr125 in osteoclasts increased NFATC1 expression and enhanced osteoclast differentiation and enhanced osteoclast-mediated bone resorption. These results indicated that GPCR125 positively regulates osteoclast formation and function. Following receptor activator of nuclear factor kappa-Β ligand (RANKL) stimulation, the expression levels of MAPK signaling pathway proteins phosphorylated-ERK (p-ERK) and phosphorylated-p38 (p-p38) were significantly decreased in the Gpr125 knockdown (sh-GPR125) group compared to its control group. We also found that phosphorylated AKT (p-AKT) expression was downregulated, as well as nuclear factor kappa-B (NF-κB) signaling pathway protein phosphorylated-IKB alpha (p-IKBα). Our results demonstrated that GPCR125 positively regulates osteoclasts via RANKL-stimulated MAPK and AKT-NF-κB signaling pathways, and GPCR125 could potentially be utilized as a novel therapeutic target in bone related diseases including osteoporosis.

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5′UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3′UTR by constructing an intragenotypic recombinant using 5′UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3′UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5′UTR-NS5A and NS5B-3′UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml−1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Background: Hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis C, end-stage liver diseases, and hepatocellular carcinoma. The development of infectious HCV cell culture systems primarily relied on the replication enhancement effect of adaptive mutations. Although the mode of action may vary, those adaptive mutations could direct the study of virus-host interactions required for efficient virus infection. We previously identified a substitution D559G in NS5B (RNA dependent RNA polymerase) critical for the replication of HCV genomes. In this study, we set out to study whether D559G-NS5B specifically interacted with some host factors crucial for HCV infection.Methods: Through mass spectrometry analysis of immunoprecipitation mixture of ectopically expressed wild-type and D559G-mutated NS5B, we identified candidate factors showing potential interactions with NS5B and D559G-NS5B. The requirement of selected host factor in HCV infection in vitro was demonstrated by gene knockout, overexpression, virus infection, and co-immunoprecipitation approaches.Results: From the results of immunoprecipitation and mass spectrometry analysis, we selected protein phosphatase 2 regulatory subunit B’delta (PPP2R5D) for further characterization. Co-immunoprecipitation confirmed that both wild-type and D559G NS5B proteins interacted with PPP2R5D, but the interaction between D559G-NS5B and PPP2R5D was more efficient. Silencing of PPP2R5D decreased HCV infection, and knockout of PPP2R5D nearly eliminated HCV infection in Huh7.5 cells. Transient and stably overexpression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that seen for wild-type Huh7.5 cells. Conclusions: PPP2R5D is required for HCV infection in cultured hepatoma cells, and PPP2R5D may function through binding to HCV NS5B. The underlying mechanism of PPP2R5D in the complete HCV life cycle requires further investigation.

Background Hepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection. Methods Wild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc. Results Mass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B’delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B. Conclusions PPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

Altered adipose tissue (AT) metabolism in cancer-associated weight loss via inflammation, lipolysis, and white adipose tissue (WAT) browning is primarily implicated from rodent models; their contribution to AT wasting in cancer patients is unclear.Energy expenditure (EE), plasma, and abdominal subcutaneous WAT were obtained from men (aged 65 ± 8 years) with cancer, with (CWL, n = 27) or without (CWS, n = 47) weight loss, and weight-stable non-cancer patients (CON, n = 26). Clinical images were assessed for adipose and muscle area while plasma and WAT were assessed for inflammatory, lipolytic, and browning markers.CWL displayed smaller subcutaneous AT (SAT; P = 0.05) and visceral AT (VAT; P = 0.034) than CWS, and displayed higher circulating interleukin (IL)-6 (P = 0.01) and WAT transcript levels of IL-6 (P = 0.029), IL-1β (P = 0.042), adipose triglyceride lipase (P = 0.026), and browning markers (Dio2, P = 0.03; PGC-1a, P = 0.016) than CWS and CON. There was no difference across groups in absolute REE (P = 0.061), %predicted REE (P = 0.18), circulating free fatty acids (FFA, P = 0.13) or parathyroid hormone-related peptide (PTHrP; P = 0.88), or WAT protein expression of inflammation (IL-6, P = 0.51; IL-1β, P = 0.29; monocyte chemoattractant protein-1, P = 0.23) or WAT protein or gene expression of browning (uncoupling protein-1, UCP-1; P = 0.13, UCP-1, P = 0.14). In patients with cancer, FFA was moderately correlated with WAT hormone-sensitive lipase transcript (r = 0.38, P = 0.018, n = 39); circulating cytokines were not correlated with expression of WAT inflammatory markers and circulating PTHrP was not correlated with expression of WAT browning markers. In multivariate regression using cancer patients only, body mass index (BMI) directly predicted SAT (N = 25, RCancer-related weight loss was associated with elevated circulating IL-6 and elevations in some WAT inflammatory, lipolytic and browning marker transcripts. BMI, not weight loss, was associated with increased energy expenditure. The contribution of inflammation and lipolysis, and lack thereof for WAT browning, will need to be clarified in other tumour types to increase generalizability. Future studies should consider variability in fat mass when exploring the relationship between cancer and adipose metabolism and should observe the trajectory of lipolysis and energy expenditure over time to establish the clinical significance of these associations and to inform more mechanistic interpretation of causation.