Viral infection can induce endoplasmic reticulum stress(ERS)and unfolded protein re-sponse(UPR)in host cells,resulting in perturbation of endoplasmic reticulum homeostasis.To e-lucidate the action mechanism of isochlorogenic acid A(IAA)in regulating peste des petits rumi-nant virus(PPRV)-induced ERS and UPR,MTT assay,indirect immunofluorescence assay and Western blot were used to evaluate the anti-PPRV activity of IAA,and the effects of IAA on PPRV-induced ERS and PERK signaling pathway were studied by Western blot and quantitative real-time PCR.The results showed that the PPRV replication and virus-induced cytopathic in LDG-2 cells were significantly inhibited,and the survival rate of virus-infected cells was significantly in-creased due to IAA treatment.Compared with the virus control group,the expression levels of GRP78 and p-eIF2α,the ratios of p-PERK/PERK and p-eIF2α/eIF2α in IAA treated PPRV-infec-ted cells were significantly decreased.The expression level of GADD153 significantly decreased at 24,36 h,and significantly increased at 48,60 h.Furthermore,treatment with ERS inhibitor 4-PBA could significantly suppress the expression levels of GRP78,PPRV-N protein and GADD153 in PPRV-infected cells,and the ratios of p-eIF2α/eIF2α and p-PERK/PERK in PPRV-infected cells were also significantly decreased caused by treatment with IAA or 4-PBA and IAA combination.These findings implicated that the PPRV-induced ERS could be alleviated by inhibiting activation of the PERK-eIF2α-GADD1 53 signaling pathway,which led to restriction of PPRV replication in host cells.

Viral infection can induce endoplasmic reticulum stress(ERS)and unfolded protein reac-tion(UPR)in host cells.This study aims to further explore the effects of ERS induced by pest des petits ruminants virus(PPRV)infection on UPR signaling pathway,virus replication and apopto-sis of host cells.MTT assay,indirect immunofluorescence assay(IFA)and Western blot were used to observe the proliferation of PPRV in goat kidney cells(LDG-2).Western blot and real-time flu-orescence quantitative PCR(qRT-PCR)were used to observe the effects of PPRV infection on the expression levels of GRP78,PERK and its downstream signal molecules,apoptosis-related proteins Bcl-2 and Bax.The result indicated that the cell survival rate was significantly declined with evident cytopathic effect at 36 h post-infection,and the expression level of PPRV-N protein tended to be elevated,and was significantly higher than that of cell control at 30 h post-infection.Meanwhile,the expression levels of GRP78,p-eIF2α and GADD153,the ratio of p-PERK/PERK and p-eIF2α/eIF2α were significantly increased.Moreover,the expression levels of PPRV-N protein,GRP78,p-eIF2α and GADD1 53,the ratio of p-eIF2α/eIF2α and p-PERK/PERK were significantly decreased in PPRV-infected cells due to 4-PBA treatment.The expression level of apoptosis-related Bcl-2 was down-regulated,Bax was up-regulated,and the ratio of Bcl-2/Bax was significantly decreased.Therefore,the activation of PERK/eIF2α/GADD153 signaling pathway could be induced by PPRV infection resulting in alleviating of virus-induced ERS,which is beneficial to viral replication.Bloc-king PPRV-induced ERS could inhibit the activation of PERK signaling pathway and virus replica-tion.PPRV infection and prolonged ERS can induce apoptosis of LDG-2 cells.

MTT assay,indirect immunofluorescence assay(IFA)and real-time fluorescence quanti-tative PCR(qRT-PCR)were used to observe the proliferation of PPRV in goat kidney cells(LDG-2).Western blot and qRT-PCR were used to evaluate the effects of PPRV infection on the expres-sion levels of TLR2,MyD88,NF-κB signaling pathway-related factors and their downstream in-flammatory factors.The results indicated that the significantly decreased cell survival rate and ob-vious cytopathic effect were observed at 36 h post PPRV infection,and the mRNA expression level of PPRV-N gene was significantly up-regulated.At the same time,the expression levels of TLR2,MyD88,p-p65 and p-IκBα,the ratio of p-p65/p65 and p-IκBα/IκBα and the mRNA expression levels of downstream inflammatory factors TNFα,IL-1β,IL-4 and IL-10 were significantly increased.Mo-reover,the expression levels of PPRV-N mRNA,TLR2,MyD88,p-p65 and p-IκBα,the ratio of p-p65/p65 and p-IκBα/IκBα and the mRNA expression levels of downstream inflammatory factors IL-1β and IL-4 in PPRV-infected cells were significantly decreased in the presence of the inhibitor C29 of TLR2.These findings implied that the TLR2/MyD88/NF-κBα signaling pathway can be activated by PPRV infection,which is beneficial to the replication and spread of the virus.Blocking down the activation of TLR2 can inhibit MyD88/NF-κB signaling pathway and viral replication in PPRV-infected cells.

Objective As primary Sj?gren's syndrome(pSS)primarily affects the salivary glands,saliva can serve as an indicator of the glands'pathophysiology and the disease's status.This study aims to illustrate the salivary proteomic profiles of pSS patients and identify potential candidate biomarkers for diagnosis. Methods The discovery set contained 49 samples(24 from pSS and 25 from age-and gender-matched healthy controls[HCs])and the validation set included 25 samples(12 from pSS and 13 from HCs).Totally 36 pSS patients and 38 HCs were centrally randomized into the discovery set or to the validation set at a 2:1 ratio.Unstimulated whole saliva samples from pSS patients and HCs were analyzed using a data-independent acquisition(DIA)strategy on a 2D LC-HRMS/MS platform to reveal differential proteins.The crucial proteins were verified using DIA analysis and annotated using gene ontology(GO)and International Pharmaceutical Abstracts(IPA)analysis.A prediction model for SS was established using random forests. Results A total of 1,963 proteins were discovered,and 136 proteins exhibited differential representation in pSS patients.The bioinformatic research indicated that these proteins were primarily linked to immunological functions,metabolism,and inflammation.A panel of 19 protein biomarkers was identified by ranking order based on P-value and random forest algorichm,and was validated as the predictive biomarkers exhibiting good performance with area under the curve(AUC)of 0.817 for discovery set and 0.882 for validation set. Conclusions The candidate protein panel discovered may aid in pSS diagnosis.Salivary proteomic analysis is a promising non-invasive method for prognostic evaluation and early and precise treatments for pSS patients.DIA offers the best time efficiency and data dependability and may be a suitable option for future research on the salivary proteome.

Objective:To investigate the possible mechanism of DiI labeled bone marrow mesenchymal stem cells (BMSCs) in contact co-cultured with neonatal rat cardiomyocytes (CMs) on polycaprolactone (PCL) film to make myocardial patch.Methods:BMSCs from Sprague Dawley rats (aged 5-6 weeks) were isolated, cultured, and characterized for surface marker expression using flow cytometry. CMs from 15 neonatal rats were isolated and cultured. After cultured for 3 generations, BMSCs were labeled with DiI dye. On PCL film, DiI labeled BMSCs were co-cultured with CMs as the experimental group, and CMs were replaced with the same amount of unlabeled BMSCs in the control group. After 24 h of co-culture, the cell growth was observed under fluorescence microscope and the co-culture was observed under scanning electron microscope. Immunofluorescence staining was performed after 7 days to detect myocardial markers, including cardiac troponin T (cTnT) and α-actinin. BMSC differentiation on the PCL film was observed under a fluorescence microscope. The differentiation efficiency of BMSCs into cardiomyoid cells was analyzed by flow cytometry on days 1 and 7 of co-culture. Intercellular dye transfer was observed by staining CMs with calcein and co-culturing them with DiI-labeled BMSCs on PCL film. The cells were stained with immunofluorescence to detect the expression of connexin 43 (Cx43) and observe the relationship between gap junction and contact co-culture.Results:Flow cytometry showed strong positivity for CD90 and CD44 and negativity for CD11b/c and CD45 on BMSCs. After 24 h of co-culture, DiI labeled BMSCs glowed red on the PCL film, while unlabeled CMs did not; the number of cells on PCL film was large and cell morphology appeared normal under scanning electron microscope. On the 7th day of co-culture, some DiI labeled BMSCs expressed cTnT and α-actinin. Flow cytometry showed a higher differentiation rate of stem cells in the experimental group on day 7 compared to the control group ((20.12±0.15)% vs. (3.49±0.20)%, P<0.05). From the second day of co-culture, some BMSCs exhibited green dot fluorescence in Cx43 immunofluorescence staining; and by the third day, dye transfer test showed green fluorescence emission from some BMSCs. Conclusion:Contact co-culture of DiI labeled BMSCs and CMs on PCL film can make myocardial patch. The mechanism of contact co-culture promoting the differentiation and formation of myocardial patch may be associated with gap junctions and intercellular signal pathways mediated by gap junctions.

Background: Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The activation of UPR signaling pathways and their impact on apoptosis and virus replication remains controversial. Objectives: To investigate the role of PPRV-induced ER stress and the IRE1-XBP1 and IRE1-JNK pathways and their impact on apoptosis and virus replication. Methods: The cell viability and virus replication were assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, immunofluorescence assay, and Western blot. The expression of ER stress biomarker GRP78, IRE1, and its downstream molecules, PPRV-N protein, and apoptosis-related proteins was detected by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. 4-Phenylbutyric acid (4-PBA) and STF-083010 were respectively used to inhibit ER stress and IRE1 signaling pathway. Results: The expression of GRP78, IRE1α, p-IRE1α, XBP1s, JNK, p-JNK, caspase-3, caspase-9, Bax and PPRV-N were significantly up-regulated in PPRV-infected cells, the expression of Bcl-2 was significantly down-regulated. Due to 4-PBA treatment, the expression of GRP78, p-IRE1α, XBP1s, p-JNK, caspase-3, caspase-9, Bax, and PPRV-N were significantly downregulated, the expression of Bcl-2 was significantly up-regulated. Moreover, in PPRV-infected cells, the expression of p-IRE1α, p-JNK, Bax, and PPRV-N was significantly decreased, and the expression of Bcl-2 was increased in the presence of STF-083010. Conclusions PPRV infection induces ER stress and IRE1 activation, resulting in apoptosis and enhancement of virus replication through IRE1-XBP1s and IRE1-JNK pathways.

Objective    To provide experimental data and theoretical support for further studying the maturity of cardiac patches in other in vitro experiments and the safety in other in vivo animal experiments, through standard chemically defined and small molecule-based induction protocol (CDM3) for promoting the differentiation of human induced pluripotent stem cells (hiPSCs) into myocardium, and preliminarily preparing cardiac patches. Methods    After resuscitation, culture and identification of hiPSCs, they were inoculated on the matrigel-coated polycaprolactone (PCL). After 24 hours, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and the stemness of hiPSCs was identified by OCT4 fluorescence. After fixation, electron microscope scanning was performed to observe the cell morphology on the surface of the patch. On the 1st, 3rd, 5th, and 7th days of culture, the cell viability was determined by CCK-8 method, and the growth curve was drawn to observe the cell growth and proliferation. After co-cultured with matrigel-coated PCL for 24 hours, hiPSCs were divided into a control group and a CDM3 group, and continued to culture for 6 days. On the 8th day, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and hiPSCs stemness was identified by OCT4 fluorescence, and cTnT and α-actin for cardiomyocyte marker identification. Results    Immunofluorescence of hiPSCs co-cultured with matrigel-coated PCL for 24 hours showed that OCT4 emitted green fluorescence, and hiPSCs remained stemness on matrigel-coated PCL scaffolds. DAPI emitted blue fluorescence: cells grew clonally with uniform cell morphology. Scanning electron microscope showed that hiPSCs adhered and grew on matrigel-coated PCL, the cell outline was clearly visible, and the morphology was normal. The cell viability assay by CCK-8 method showed that hiPSCs proliferated and grew on PCL scaffolds coated with matrigel. After 6 days of culture in the control group and the CDM3 group, immunofluorescence showed that the hiPSCs in the control group highly expressed the stem cell stemness marker OCT4, but did not express the cardiac markers cTnT and α-actin. The CDM3 group obviously expressed the cardiac markers cTnT and α-actin, but did not express the stem cell stemness marker OCT4. Conclusion    hiPSCs can proliferate and grow on matrigel-coated PCL. Under the influence of CDM3, hiPSCs can be differentiated into cardiomyocyte-like cells, and the preliminary preparation of cardiac patch can provide a better treatment method for further clinical treatment of cardiac infarction.

Objective:To study the clinical application of a new classification on location of hepatolithiasis in guiding treatment using percutaneous transhepatic choledochoscopic lithotomy (PTCSL).Methods:The clinical data of 85 consecutive patients with preoperatively diagnosed hepatolithiasis who underwent PTCSL at the First Affiliated Hospital of Nanjing Medical University from January 2017 to July 2021 were prospectively collected. There were 27 males and 58 females, aged from 15 to 86(62±14) years. Hepatolithiasis was classified into five types of stone location based on preoperative imagings: type Ⅰ ( n=12) , stones located in central bile duct, including hilar bile duct and common hepatic duct; type Ⅱ ( n=17) in unilateral hepatic duct with multiple branches; type Ⅲ ( n=24) in unilateral hepatic duct with multiple branches plus central bile duct; type Ⅳ ( n=31) in bilateral hepatic ducts with multiple branches; and type Ⅴ ( n=1) in unilateral hepatic duct with a single branch. Fistulation path, number of procedures, number of bile duct fistula, and complications were recorded. The residual stone rate and stone recurrence rate were compared among the five types. The follow-up was performed to analyse prognosis. Results:A total of 99 biliary fistulae were performed, with one single tract created in 74 patients, two tracts in 9 patients, three tracts in 1 patient, and four tracts in 1 patient. The fistulation path was B2 in 12 patients, B3 in 18 patients, B4 in 1 patient, B5 in 4 patients, B6 in 10 patients, B7 in 4 patients, and B8 in 50 patients. Altogether, 151 choledochoscopic lithotomy procedures were performed (1-3 times per patient, mean 1.78 times). For the 9 patients with residual stones (10.6%, 9/85), there were 3 patients with type Ⅱ and 6 patients with type Ⅳ. There were significant differences in the residual stone rates among the 5 types (χ 2=11.13, P=0.025). Stone recurrence developed in 33 (38.8%) patients, including 2 patients with type Ⅰ, 7 patients with type Ⅱ, 10 patients with type Ⅲ and 14 patients with type Ⅳ (χ 2=9.07, P=0.046). The total intraoperative and postoperative complications rates was 28.2% (24/85). The follow-up period was 4-58 months with the median follow-up time of 30 months. Twelve patients died during the follow-up period, including 1 patient who died from postoperative bleeding, 3 cholangiocarcinoma, 7 biliary cirrhosis-related liver failure, and 1 stone-unrelated disease. Conclusion:Type Ⅳ in the location classification of hepatolithiasis based on PTCSL had significantly higher rates of residual stones and stone recurrence. This new classification is helpful for clinicaians to determine the optimal path using a smaller number of fistulation tracts to clear stones. It improved the efficacy of PTCSL in treating hepatolithiasis.

Inflammation alters the neural stem cell (NSC) lineage from neuronal to astrogliogenesis. However, the underlying mechanism is elusive. Autophagy contributes to the decline in adult hippocampal neurogenesis under E. coli lipopolysaccharide (LPS) stimulation. SRY-box transcription Factor 2 (SOX2) is critical for NSC self-renewal and proliferation. In this study, we investigated the role of SOX2 in induced autophagy and hippocampal adult neurogenesis under LPS stimulation. LPS (5 ng•100 g -1 •hour -1 for 7 days) was intraperitoneally infused into male Sprague–Dawley rats (8 weeks old) to induce mild systemic inflammation. Beclin 1 and autophagy protein 12 (Atg12) were significantly upregulated concurrent with decreased numbers of Ki67- and doublecortin (DCX)-positive cells in the dentate gyrus. Synchronically, the levels of phospho(p)-mTOR, the p-mTOR/mTOR ratio, p-P85s6k, and the p-P85s6k/P85s6k ratio were suppressed. In contrast, SOX2 expression was increased. The fluorescence micrographs indicated that the colocalization of Beclin 1 and SOX2 was increased in the subgranular zone (SGZ) of the dentate gyrus. Moreover, increased S100β-positive astrocytes were colocalized with SOX2 in the SGZ. Intracerebroventricular infusion of 3-methyladenine (an autophagy inhibitor) effectively prevented the increases in Beclin 1, Atg12, and SOX2. The SOX2 + -Beclin 1 + and SOX2 + -S100β + cells were reduced. The levels of p-mTOR and p-P85s6k were enhanced. Most importantly, the number of DCX-positive cells was preserved. Altogether, these data suggest that LPS induced autophagy to inactivate the mTOR/P85s6k pathway, resulting in a decline in neural differentiation. SOX2 was upregulated to facilitate the NSC lineage, while the autophagy milieu could switch the SOX2-induced NSC lineage from neurogenesis to astrogliogenesis.

Objective To explore the somatotype characteristics and changing rules of Tajik adults. Methods The Heath-Carter bod)' type method was used to determine the body size of 280 (124 males and 156 females) Tajik adults. Results The average body size of Tajik males and females were 4. 3-3. 1-1. 8 and 7. 0-3. 1-1. 1, respectively, and both are mesomorphic endomorphy.The ectomorphy of Tajik nationality were negatively correlated with age, female endomorphy and mesomorphy were positively correlated with age, while endomorphy and mesomorphy were not correlated with age. With increasing age, the difference in body shape between female age groups was more obvious than that of males. Conclusion The Tajik have less skeletal muscle mass, and women have developed body fat, which is different from the Tibetan people and other people in the Altaic language family.