Knockdown of miR-150-5p reduces hypoxia-induced autophagy and epithelial-mesenchymal transition of endometriotic cells via regulating the PDCD4/NF-κB signaling pathway.

BACKGROUND: Hypoxia is an important microenvironmental factor that induces Endometriosis (EMs), but its mechanism remains unclear. Our study aims to investigate the mechanisms of miR-150-5p on hypoxia-induced EMs. METHODS: Ovarian endometriosis cyst wall stromal cell lines CRL-7566 cells were treated with hypoxia. Cell migration ability was measured by Transwell assay. qRT-PCR was performed to detect miR-150-5p and PDCD4 expression. The autophagy-related proteins (LC3-I, LC3-II, Beclin-1, and p62), epithelial-mesenchymal transition (EMT) related proteins (E-cadherin, N-cadherin, and Vimentin) and NF-κB signaling pathway related proteins p65 expression were measured by western blot. Dual-luciferase reporter gene assay verified the binding relationship between miR-150-5p and PDCD4. RESULTS: After hypoxia treatment, the miR-150-5p expression was up-regulated in CRL-7566 cells, while the expression of PDCD4 was down-regulated. In CRL-7566 cells, autophagy, migration and EMT were increased after hypoxia treatment. The autophagy inhibitor 3-MA inhibited hypoxia-induced the autophagy, migration and EMT of CRL-7566 cells. Hypoxia-induced autophagy and EMT of CRL-7566 cells were inhibited after knocking down miR-150-5p. Then miR-150-5p negatively regulated PDCD4 expression. PDCD4 knockdown reversed the inhibitory effect of miR-150-5p silencing on hypoxia-induced autophagy and EMT of CRL-7566 cells. Inhibiting the NF-κB signaling pathway weakened the effect of PDCD4 knockdown on hypoxia-induced autophagy and EMT of CRL-7566 cells. CONCLUSION: MiR-150-5p silencing inhibited hypoxia-induced autophagy and EMT of endometriotic cells by regulating the PDCD4/NF-κB signaling pathway.

lncRNA EGFR-AS1 facilitates leiomyosarcoma progression and immune escape via the EGFR-MYC-PD-L1 axis.

AIM: this study aimed to investigate the role of long non-coding RNA (lncRNA) epidermal growth factor receptor antisense RNA 1 (EGFR-AS1), an antisense transcript of EGFR, in leiomyosarcoma (LMS) and the underlying mechanisms. METHODS: levels of EGFR-AS1 and programmed death ligand 1 (PD-L1) were measured in LMS tissues and cell lines using quantitative real-time PCR (qRT-PCR), as well as western blotting and/or immunohistochemical staining; flow cytometry was employed to validate the role of EGFR-AS1 in altering the activity of CD8+ T cells; interaction of EGFR-AS1 and EGFR was determined by fluorescent in situ hybridization (FISH) and RNA pull-down; regulation of MYC on the PD-L1 promoter was assessed by chromatin immunoprecipitation (ChIP); a xenograft in vivo tumor growth assay was applied to verify the EGFR-AS1/EGFR/MYC/PD-L1 axis in vivo. RESULTS: up-regulation of EGFR-AS1 and PD-L1 in LMS tissues was negatively correlated with CD8+ T-cell infiltration; EGFR-AS1 positively regulated PD-L1, thereby strengthening interaction of LMS cells and CD8+ T cells and triggering CD8+ T cell apoptosis via the PD-1/PD-L1 checkpoint; EGFR-AS1 co-localized and interacted with EGFR to promote MYC activity; MYC was identified as a transcriptional activator of PD-L1. CONCLUSION: lncRNA EGFR-AS1 was demonstrated to increase PD-L1 expression through the EGFR/MYC pathway in LMS cells, thereby repressing T-cell infiltration and contributing to immune escape.

Interrelation of Chronic Lung Disease and Cardiovascular Disease Based on Two National Prospective Cohort Studies.

BACKGROUND: Chronic lung diseases (CLDs) and cardiovascular diseases (CVDs) are the main chronic diseases responsible for a considerable burden of disease. This study aimed to estimate the interrelation of CLDs and CVDs using two Chinese national longitudinal cohort studies. METHODS: The China Health and Retirement Longitudinal Study (CHARLS) and Chinese Longitudinal Healthy Longevity Survey (CLHLS) were used in this study with 15,052 and 9,765 participants, respectively. The Cox proportional risk model was used to estimate the interrelation between CLDs and CVDs. Mediating effects were performed to detect possible influencing pathways between CLDs and CVDs. RESULTS: The association of CLDs with CVDs was identified in 1,647 participants (10.9%) with newly diagnosed CVDs in CHARLS and 332 participants (11.6%) in CLHLS. The Cox proportional risk model showed that CLDs were a significant predictor of CVDs (HR 1.49, 95% CI 1.27-1.76) after adjusting for covariates, and the hazard ratios of stroke and CVDs excluding stroke were (HR 1.02, 95% CI 0.79-1.31) and (HR 1.76, 95% CI 1.46-2.13), respectively. These association were mediated by body mass index (BMI) and Center for Epidemiological Studies Depression Scale (CES-D-10) scores. No significant association was found in CHARLS and CLHLS regarding CVDs with CLDs. In CHARLS, CVDs was a significant predictor of CLDs (HR 1.40, 95% CI 1.09-1.79). CONCLUSIONS: Chronic lung disease was associated with increased incidence of CVDs in middle-aged and older people in the community population and vice versa. Body mass index and depressive symptoms might be mediated by the effect of CLD on CVD.

Higher baseline alanine aminotransferase level is associated with lower live birth rate after freeze-thawed embryo transfer.

OBJECTIVE: The aim of this retrospective analysis was to explore whether an elevated ALT level before pregnancy is associated with a reduction in live birth rate after IVF-FET. DESIGN: Retrospective observational study. SETTING: Shiyan People's Hospital, China between January 2019 and December 2019. PATIENTS: Women aged ≤ 40 years. INTERVENTION(S): Freeze-thawed embryo transfer (FET). MAIN OUTCOME MEASURE(S): The live birth rate, which was defined as the delivery of a live baby after 24 weeks of gestation. RESULTS: The analysis included 365 FET cycles. There was a significant difference between groups in the live birth rate (p < .05), which was highest for the low ALT tertile and lowest for the high ALT tertile. Multiple regression analysis with adjustment for multiple potential confounders revealed that the odds of live birth were decreased for each one standard deviation increase in ALT (OR = 0.56, 95%CI = 0.42-0.75, p < .0001) and lower for the high ALT tertile than for the low ALT tertile (OR = 0.38, 95%CI = 0.19-0.75, p = .0055). Smooth curve fitting showed an inverse relationship between ALT and live birth rate. CONCLUSION: Our findings indicate that relatively small elevations in baseline serum ALT level can have a clinically relevant impact on the success of FET.

Asymmetric Total Synthesis of Four Stereoisomers of the Sex Pheromone of the Western Corn Rootworm.

A convergent synthesis of four stereoisomers of the sex pheromone of the western corn rootworm (8-methyldecan-2-yl propionate, 1) from commercially available chiral starting materials is reported. The key step was Julia-Kocienski olefination between chiral BT-sulfone and chiral aldehyde. This synthetic route provided the four stereoisomers of 1 in 24-29% total yield via a six-step sequence. The simple scale-up strategy provides a new way to achieve the asymmetric synthesis of the sex pheromone.

Synthesis of the Sex Pheromone of the Tea Tussock Moth Based on a Resource Chemistry Strategy.

Synthesis of the sex pheromone of the tea tussock moth in 33% overall yield over 10 steps was achieved. Moreover, the chiral pool concept was applied in the asymmetric synthesis. The synthesis used a chemical available on a large-scale from recycling of wastewater from the steroid industry. The carbon skeleton was constructed using the C4+C5+C8 strategy. Based on this strategy, the original chiral center was totally retained.

In vitro and genomic mining studies of anti-Clostridium perfringens Compounds Derived from Bacillus amyloliquefaciens.

Clostridium perfringens is an important opportunistic microorganism in commercial poultry production that is implicated in necrotic enteritis (NE) outbreaks. This disease poses a severe financial burden on the global poultry industry, causing estimated annual losses of $6 billion globally. The ban on in-feed antibiotic growth promoters has spurred investigations into approaches of alternatives to antibiotics, among which Bacillus probiotics have demonstrated varying degrees of effectiveness against NE. However, the precise mechanisms underlying Bacillus-mediated beneficial effects on host responses in NE remain to be further elucidated. In this manuscript, we conducted in vitro and genomic mining analysis to investigate anti-C. perfringens activity observed in the supernatants derived from 2 Bacillus amyloliquefaciens strains (FS1092 and BaD747). Both strains demonstrated potent anti-C. perfringens activities in in vitro studies. An analysis of genomes from 15 B. amyloliquefaciens, 11 B. velezensis, and 2 B. subtilis strains has revealed an intriguing clustering pattern among strains known to possess anti-C. perfringens activities. Furthermore, our investigation has identified 7 potential antimicrobial compounds, predicted as secondary metabolites through antiSMASH genomic mining within the published genomes of B. amyloliquefaciens species. Based on in vitro analysis, BaD747 may have the potential as a probiotic in the control of NE. These findings not only enhance our understanding of B. amyloliquefaciens's action against C. perfringens but also provide a scientific rationale for the development of novel antimicrobial therapeutic agents against NE.

Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.

Intrarectal infection between men who have sex with men represents a predominant form of human immunodeficiency virus (HIV) transmission in developed countries. Currently there are no adequate small animal models that recapitulate intrarectal HIV transmission. Here we demonstrate that human lymphocytes generated in situ from hematopoietic stem cells reconstitute the gastrointestinal tract of humanized mice with human CD4(+) T cells rendering them susceptible to intrarectal HIV transmission. HIV infection after a single intrarectal inoculation results in systemic infection with depletion of CD4(+) T cells in gut-associated lymphoid tissue and other pathologic sequela that closely mimics those observed in HIV infected humans. This novel model provides the basis for the development and evaluation of novel approaches aimed at immune reconstitution of human gut-associated lymphoid tissue and for the development, testing, and implementation of microbicides to prevent intrarectal HIV-1 transmission.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) regulates CTL activation and memory programming.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the major carcinogens in tobacco. NNK has been associated with various cancers in tobacco users, especially lung cancer. However, the effects of NNK on cytotoxic T lymphocytes (CTLs), the cells responsible for destrcution of maligant and pathogen-infected cells, has not been elucidated. Using transgenic CTLs in vitro and in vivo, we show that NNK can directly affect CTL activation. NNK can enhance the expression of adhesion molecule CD62L in CTLs during their activation in vitro, but has no effects on their expansion and production of effector molecules such as IFN and granzyme B. After transferred into recipient mice, however, the NNK pretreated CTLs suffer an early loss in expansion. The percentage of memory precursors is higher in NNK pretreated CTLs, but the total amount of memory precursors is similar to controls. The final memory CTL population from NNK pretreated CTLs is reduced, but sustains a more central memory phenotype. In conclusion, NNK can affect CTL activation by modulating adhension molecule expression and reducing memory programming.

Characterization of Collagen Binding Activity of Clostridium perfringens Strains Isolated from Broiler Chickens.

Clostridium perfringens is the etiological agent for necrotic enteritis (NE) in broiler chickens, which causes a substantial economic loss of an estimated USD 6 billion annually in the global poultry industry. Collagen adhesion is involved in the NE pathogenesis in poultry. In this study, the binding capabilities of chicken C. perfringens isolates of various genetic backgrounds (netB-tpeL-, netB+tpeL-, netB+tpeL+) to collagen types I-V and gelatin were examined, and the putative adhesin protein cnaA gene was investigated at the genomic level. In total, 28 C. perfringens strains from healthy and NE-inflicted sick chickens were examined. The results on collagen adhesin-encoding gene cnaA by the quantitative-PCR results indicated that netB-tpeL- isolates had much lower copies of the detectable cnaA gene than netB+ isolates (10 netB+tpeL- isolates, 5 netB+tpeL+ isolates). Most of the virulent C. perfringens isolates demonstrated collagen-binding abilities to types I-II and IV-V, while some strains showed weak or no binding to collagen type III and gelatin. However, the netB+tpeL+ isolates showed significantly higher binding capabilities to collagen III than netB-tpeL- and netB+tpeL- isolates. The data in this study suggest that the collagen-binding capability of clinical C. perfringens isolates correlates well with their NE pathogenicity levels, especially for C. perfringens isolates carrying genes encoding crucial virulence factors and virulence-associated factors such as netB, cnaA, and tpeL. These results indicate that the presence of the cnaA gene may be correlated with C. perfringens virulence (particularly for netB+ isolates).

MicroRNA-223 Inhibits Soybean Glycinin- and ß-Conglycinin-Induced Apoptosis of IPEC-J2 Cells by Targeting NLRP-3 in the IEL/IPEC-J2 Co-culture System.

The apoptosis of intestinal porcine epithelial cells induced by soybean antigen protein allergy is one of the most important mechanisms responsible for enteritis. MicroRNAs (miRNAs) affect the cellular and physiological functions of all multicellular organisms. We hypothesize that microRNA-223 inhibits soybean glycinin- and ß-conglycinin-induced apoptosis of intestinal porcine enterocytes (IPEC-J2) by targeting the NLR family pyrin domain containing 3 (NLRP-3). Using the intestinal interepithelial lymphocyte (IEL)/IPEC-J2 co-culture system as an in vitro model, we investigate the role of microRNA-223 in the regulation of soybean glycinin- and ß-conglycinin-induced apoptosis. In co-cultured IEL/IPEC-J2 cells incubated with glycinin or ß-conglycinin, microRNA-223 decreased NLRP-3, ASC, caspase-1, caspase-3, FAS, BCL-2, and APAF-1 expressions in IPEC-J2 cells; decreased cytokine and cyclooxygenase-2 levels; significantly increased cell activity; and inhibited apoptosis. These data supported a novel antiallergic mechanism to mitigate the sensitization of soybean antigenic protein, which involves the upregulation of microRNA-223-targeting NLRP-3.

Repetitive peptide boosting progressively enhances functional memory CTLs.

Cytotoxic T lymphocytes (CTLs) play a critical role in controlling intracellular pathogens and cancer cells, and induction of memory CTLs holds promise for developing effective vaccines against critical virus infections. However, generating memory CTLs remains a major challenge for conventional vector-based, prime-boost vaccinations. Thus, it is imperative that we explore nonconventional alternatives, such as boosting without vectors. We show here that repetitive intravenous boosting with peptide and adjuvant generates memory CD8 T cells of sufficient quality and quantity to protect against infection in mice. The resulting memory CTLs possess a unique and long-lasting effector memory phenotype, characterized by decreased interferon-γ but increased granzyme B production. These results are observed in both transgenic and endogenous models. Overall, our findings have important implications for future vaccine development, as they suggest that intravenous peptide boosting with adjuvant following priming can induce long-term functional memory CTLs.

The Effects of City Lockdown Policy on Frozen Embryo Transfer Outcome during COVID-19 Epidemic in Hubei Province, China.

Objective: To evaluate whether there is a difference in clinical pregnancy rate and live birth rate between the corresponding period in 2019 and COVID-19 city lockdown period in 2020 in frozen embryo transfer (FET). Methods: In one single in vitro fertilization (IVF) center (Shiyan, Hubei province, China), a retrospective cohort analysis was conducted, with a sample size of 59 patients in the lockdown period (2020.1.23-2020.2.23, 2020 group) and 34 patients in the corresponding 2019 period (2019.1.23-2019.2.23, 2019 group). Implantation, biochemical and clinical pregnancy, miscarriage, and live birth rates were all measured. Results: Age, basal serum follicle-stimulating hormone (FSH), basal serum luteinizing hormone (LH), basal serum E2, and serum total T were all comparable between the two groups. On the day of progesterone administration, endometrial thickness was similar (8.5 ± 1.3 vs. 8.2 ± 1.4, P = 0.356). The number of transferred blastocysts was not significantly different. The two groups had similar clinical pregnancy rate (61.8% vs. 61.0%, P > 0.05) and live birth rate (47.1% vs. 49.2%, P > 0.05), which did not significantly differ. Nonetheless, there was a significant difference in the cancelled cycle rate between the two groups (0% vs. 28.0%, P = 0.043). Conclusions: Lockdown period FET versus corresponding period FET outcome did not show any significant difference in terms of pregnancy rate and live birth rate between two groups of patients. Although there was no significant difference, in the 2020 group, the live birth rate was higher compared with that in the 2019 group. There was a significant difference in the rate of cancelled cycles due to the seal off control. In summary, artificial endometrial preparation is an appropriate protocol for special periods.

Retraction Notice to: LINC00355 Promotes Tumor Progression in HNSCC by Hindering MicroRNA-195-Mediated Suppression of HOXA10 Expression.

[This retracts the article DOI: 10.1016/j.omtn.2019.11.002.].

Research Note: Development and characterization of monoclonal antibodies specific for chicken interleukin-7 receptor α (CD127).

CD127, also named interleukin-7 receptor (IL-7R), is expressed on various cell types including naive and memory T cells, and plays a critical role in the differentiation and activation of T lymphocytes. The availability of poultry-specific immune reagents to identify and measure chicken CD127 response will enhance fundamental and applied research in poultry immunology. Mouse monoclonal antibodies (MAbs) against chicken CD127 (chCD127) were developed and characterized. More specifically, a 678 bp ectodomain of chCD127 gene was cloned in the pET28a (+) vector and expressed in BL21-AI E. coli competent cells. The recombinant chCD127 protein with a size of 30 KDa which was also recognized by a mouse anti-human CD127 MAb (Clone G-11) was used to immunize mice, and 6 new mouse MAbs which specifically detected chicken CD127 were developed and characterized. Availability of these new sets of chCD127-specific MAbs will facilitate the immunological studies on CD127 in poultry, especially in understanding effector and memory T immune cell responses in normal and diseased states.

11S Glycinin Up-Regulated NLRP-3-Induced Pyroptosis by Triggering Reactive Oxygen Species in Porcine Intestinal Epithelial Cells.

11S glycinin is a major soybean antigenic protein, which induces human and animal allergies. It has been reported to induce intestinal porcine epithelial (IPEC-J2) cell apoptosis, but the role of pyroptosis in 11S glycinin allergies remains unknown. In this study, IPEC-J2 cells were used as an in vitro physiological model to explore the mechanism of 11S glycinin-induced pyroptosis. The cells were incubated with 0, 1, 5, and 10 mg·ml-1 11S glycinin for 24 h. Our results revealed that 11S glycinin significantly inhibited cell proliferation, induced DNA damage, generated active oxygen, decreased mitochondrial membrane potential, and increased the NOD-like receptor protein 3 (NLRP-3) expression of IPEC-J2 cells in a dose-dependent manner. Further, IPEC-J2 cells were transfected with designed sh-NLRP-3 lentivirus to silence NLRP-3. The results showed that 11S glycinin up-regulated the silenced NLRP-3 gene and increased the expression levels of apoptosis-related spot-like protein (ASC), caspase-1, the cleaved gasdermin D, and interleukin-1ß. The IPEC-J2 cells showed pyrolysis morphology. Moreover, we revealed that N-acetyl-L-cysteine can significantly inhibit the production of reactive oxygen species and reduce the expression levels of NLRP-3 and the cleaved gasdermin D. Taken together, 11S glycinin up-regulated NLRP-3-induced pyroptosis by triggering reactive oxygen species in IPEC-J2 cells.

Immunization with Pooled Antigens for Clostridium perfringens Conferred Partial Protection against Experimental Necrotic Enteritis in Broiler Chickens.

Necrotic enteritis (NE) is a multifactorial and important enteric infectious disease etiologically caused by pathogenic C. perfringens infection, accounting for the estimated loss of around USD 6 billion in the global poultry industry. The increasing incidence of NE was found to be associated with the voluntary reduction or withdrawal of antibiotic growth promoters from animal feed during recent years. Therefore, the development of effective vaccines specific to NE assumes a priority for the poultry industry. This study aimed to identify the potential C. perfringens proteins as vaccine targets for NE. Three recombinant C. perfringens proteins targeting five antigens were prepared: two chimeric proteins (alpha-toxin and NetB, fructose-1,6-bisphosphate aldolase (FBA) and a zinc metalloprotease (Zm)), and one single collagen adhesion protein (Cna). Their protection efficacies were evaluated with a potent challenge model of Eimeria maxima/C. perfringens dual infections using a netB+tpeL+ C. perfringens strain. Young chicks were immunized twice subcutaneously with adjuvanted C. perfringens proteins on Days 4 and 15. At six days after the second immunization, the chickens immunized with Cna, FBA, and Zm antigens, and alpha-toxin had much higher serum antibody titers than unvaccinated controls prior to the challenge. Following the challenge, the pooled antigen-immunized group demonstrated no mortality and the least lesion scores against virulent challenge. The results indicate that the immunization with multicomponent antigens, including C. perfringens housekeeping protein Cna, may confer partial protection.

Occupational risk factors and breast cancer in Beijing, China: a hospital-based case-control study.

INTRODUCTION: Studies on the association between breast cancer and occupational hazards are limited, especially in China. This is the first study to explore the relationship between breast cancer and occupational hazards in Beijing, China. DESIGN: A hospital-based case-control study. SETTING: Eight local hospitals in Beijing, China. PARTICIPANTS: A total of 973 female participants, comprising 495 cases and 478 controls, were recruited in our study. We identified patients who underwent diagnosis for breast cancer at one of the eight local hospitals in Beijing between 1 January 2015 and 31 December 2019; controls were individuals randomly matched from the same hospital where the cases were confirmed. MAIN OUTCOME AND MEASURE: Least absolute shrinkage and selection operator (LASSO) regression was used to estimate the occupational risk factors associated with breast cancer, including night shift work history and work posture. RESULTS: In the case group, the breast cancer type was mainly invasive, which accounted for 85.66% of all the breast cancer patients. Five risk factors were included in the final LASSO model, including body mass index (BMI), marital status, menopause, night shift work history and work posture. Furthermore, these risk factors were considered for multivariate logistic regression, and the analyses suggested that the risk of breast cancer was significantly associated with higher BMI (≥28.0 kg/m2, OR: 2.85, 95% CI: 1.29 to 6.30); married status: married (OR: 2.67, 95% CI: 1.28 to 5.56) or divorced (OR: 4.51, 95% CI: 1.84 to 11.07); menopause (OR: 6.89, 95% CI: 5.07 to 9.36); night shift work (OR: 1.53, 95% CI: 1.11 to 2.11); and maximum standing or walking, and minimal sitting (OR: 1.80, 95% CI: 1.19 to 2.73). CONCLUSION: Breast cancer is associated with occupational risk factors. Night shift work, especially in a standing posture, can increase the incidence of breast cancer in women in Beijing, China.

Small molecules facilitate single factor-mediated sweat gland cell reprogramming.

BACKGROUND: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands (SG), thus impairing the skin's physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin. METHODS: The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcino-embryonic antigen (CEA), aquaporin 5 (AQP5) and α-smooth muscle actin (α-SMA) was assessed with quantitative PCR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs. BALB/c nude mice were randomly divided into a normal group, SGM treatment group and iSGC transplantation group. Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs. RESULTS: EDA overexpression drove HDF conversion into iSGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18 and CEA in iSGCs, and flow cytometry data demonstrated (4.18 ± 0.04)% of iSGCs were CK5 positive and (4.36 ± 0.25)% of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails, (23.05 ± 2.49)% of iSGCs expressed CK5+ and (55.79 ± 3.18)% of iSGCs expressed CK18+, respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79 ± 7.71)% vs. (70.59 ± 0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2 ± 1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice. CONCLUSION: We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.

Sulfotyrosines of the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor promote tumorigenesis through autocrine activation.

The Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) is a bona fide signaling molecule that is implicated in KSHV-associated malignancies. Whereas vGPCR activates specific cellular signaling pathways in a chemokine-independent fashion, vGPCR binds a broad spectrum of CC and CXC chemokines, and the roles of chemokines in vGPCR tumorigenesis remain poorly understood. We report here that vGPCR is posttranslationally modified by sulfate groups at tyrosine residues within its N-terminal extracellular domain. A chemokine-binding assay demonstrated that the tyrosine sulfate moieties were critical for vGPCR association with GRO-alpha (an agonist) but not with IP-10 (an inverse agonist). A sulfated peptide corresponding to residues 12 through 33 of vGPCR, but not the unsulfated equivalent, partially inhibited vGPCR association with GRO-alpha. Although the vGPCR variant lacking sulfotyrosines activated downstream signaling pathways, the ability of the unsulfated vGPCR variant to induce tumor growth in nude mice was significantly diminished. Furthermore, the unsulfated vGPCR variant was unable to induce the secretion of proliferative cytokines, some of which serve as vGPCR agonists. This implies that autocrine activation by agonist chemokines is critical for vGPCR tumorigenesis. Indeed, GRO-alpha increased vGPCR-mediated AKT phosphorylation and vGPCR tumorigenesis in a sulfotyrosine-dependent manner. Our findings support the conclusion that autocrine activation triggered by chemokine agonists via sulfotyrosines is necessary for vGPCR tumorigenesis, thereby providing a rationale for future therapeutic design targeting the tumorigenic vGPCR.