[Automatic classification of immune-mediated glomerular diseases based on multi-modal multi-instance learning].

OBJECTIVE: To develop a multi-modal deep learning method for automatic classification of immune-mediated glomerular diseases based on images of optical microscopy (OM), immunofluorescence microscopy (IM), and transmission electron microscopy (TEM). METHODS: We retrospectively collected the pathological images from 273 patients and constructed a multi-modal multi- instance model for classification of 3 immune-mediated glomerular diseases, namely immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), and lupus nephritis (LN). This model adopts an instance-level multi-instance learning (I-MIL) method to select the TEM images for multi-modal feature fusion with the OM images and IM images of the same patient. By comparing this model with unimodal and bimodal models, we explored different combinations of the 3 modalities and the optimal methods for modal feature fusion. RESULTS: The multi-modal multi-instance model combining OM, IM, and TEM images had a disease classification accuracy of (88.34±2.12)%, superior to that of the optimal unimodal model [(87.08±4.25)%] and that of the optimal bimodal model [(87.92±3.06)%]. CONCLUSION: This multi- modal multi- instance model based on OM, IM, and TEM images can achieve automatic classification of immune-mediated glomerular diseases with a good classification accuracy.

[Survey of prevalence of hepatitis C in people aged 1-69 years in Henan Province, 2020].

Objective: To understand the infection status and epidemiological characteristics of hepatitis C in people aged 1-69 years in Henan Province in 2020. Methods: The estimated sample size was 5 827. From August to December 2020, multistage sampling was used to select 8 counties (districts) in Henan, and two survey sites were selected in each county (district), and a questionnaire survey was conducted in local people aged 1-69 years, blood samples were collected from them for anti-HCV, HCV RNA and genotype detections. Results: A total of 5 165 people aged 1-69 years completed the questionnaire survey. Men accounted for 44.76% (2 312/5 165), women accounted for 55.24% (2 853/5 165). In the people aged 1-69 years, the overall prevalence rates of anti-HCV and HCV RNA were 0.69% (95%CI: 0.68%-0.70%) and 0.20% (95%CI: 0.19%-0.21%) respectively. The prevalence rates of anti-HCV and HCV RNA were 0.48% (95%CI: 0.46%-0.50%), 0.09% (95%CI: 0.08%-0.10%) in men and 0.86% (95%CI: 0.85%-0.87%), 0.30% (95%CI: 0.28%-0.32%) in women. The prevalence rates of anti-HCV and HCV RNA increased with age. The prevalence rates of anti-HCV and HCV RNA were 0.87% (95%CI: 0.86%-0.88%), 0.28% (95%CI: 0.26%-0.30%) in urban residents and 0.53% (95%CI: 0.51%-0.55%), 0.14% (95%CI: 0.13%-0.15%) in rural residents. The genotyping results of 10 HCV RNA positive samples ware genotype 1b (4/10), genotype 2 (3/10), genotype 1b/3 (1/10), genotype 1b/3/6 (1/10) and genotype 2/6 (1/10). Conclusions: The prevalence of hepatitis C was low in Henan in 2020. It is necessary to strengthen hepatitis C surveillance in people aged 40 years and above. The major HCV genotypes were 1b and 2, and mixed genotype infection existed.

[A region-level contrastive learning-based deep model for glomerular ultrastructure segmentation on electron microscope images].

OBJECTIVE: We propose a novel region- level self-supervised contrastive learning method USRegCon (ultrastructural region contrast) based on the semantic similarity of ultrastructures to improve the performance of the model for glomerular ultrastructure segmentation on electron microscope images. METHODS: USRegCon used a large amount of unlabeled data for pre- training of the model in 3 steps: (1) The model encoded and decoded the ultrastructural information in the image and adaptively divided the image into multiple regions based on the semantic similarity of the ultrastructures; (2) Based on the divided regions, the first-order grayscale region representations and deep semantic region representations of each region were extracted by region pooling operation; (3) For the first-order grayscale region representations, a grayscale loss function was proposed to minimize the grayscale difference within regions and maximize the difference between regions. For deep semantic region representations, a semantic loss function was introduced to maximize the similarity of positive region pairs and the difference of negative region pairs in the representation space. These two loss functions were jointly used for pre-training of the model. RESULTS: In the segmentation task for 3 ultrastructures of the glomerular filtration barrier based on the private dataset GlomEM, USRegCon achieved promising segmentation results for basement membrane, endothelial cells, and podocytes, with Dice coefficients of (85.69 ± 0.13)%, (74.59 ± 0.13)%, and (78.57 ± 0.16)%, respectively, demonstrating a good performance of the model superior to many existing image-level, pixel-level, and region-level self-supervised contrastive learning methods and close to the fully- supervised pre-training method based on the large- scale labeled dataset ImageNet. CONCLUSION: USRegCon facilitates the model to learn beneficial region representations from large amounts of unlabeled data to overcome the scarcity of labeled data and improves the deep model performance for glomerular ultrastructure recognition and boundary segmentation.

Correlation between nocturnal intermittent hypoxemia and mild cognitive impairment in the older adult and the role of BDNF Val66Met polymorphism: a hospital-based cross-sectional study.

PURPOSE: To explore the prevalence of nocturnal intermittent hypoxemia (NIH) in a tertiary hospital geriatric department and the relationship between NIH and mild cognitive impairment (MCI) in older adults, and to examine the role of brain-derived neurotrophic factor (BDNF) Val66Met polymorphism. METHODS: Older adults aged ≥ 60 were enrolled. NIH and cognitive assessments were conducted. BDNF concentrations and BDNF Val66Met polymorphism were detected for a preliminary exploration of the possible mechanism of the process. RESULTS: Of 325 older adults enrolled, 157 (48%) had NIH and were further divided into mild, moderate, and severe NIH groups according to their oxygen desaturation of ≥ 4% per hour of sleep (ODI4). MCI detection rate in the four groups gradually increased, and the differences were statistically significant (chi-square = 4.457, P = 0.035). ODI4 was negatively correlated with MoCA score in all participants (r = - 0.115, P = 0.039) and patients with NIH (r = - 0.199, P = 0.012). After adjusting for sex, age, and cardiovascular risk factors, NIH and MCI remained independently associated (OR = 3.13, 95% CI 1.03-9.53, P = 0.045). BDNF levels were positively correlated with MoCA score (r = 0.169, P = 0.028) and negatively correlated with nocturnal average oxygen saturation in patients with NIH (r = - 0.288, P = 0.008). Older adults with different BDNF Val66Met genotypes did not show significant differences in MCI rate and BDNF levels (P > 0.05). CONCLUSION: The older adults with NIH have a higher MCI detection rate. BDNF levels may be a potential biomarker for cognitive dysfunction in patients with NIH.

[Serum levels of endothelin-1 and connective tissue growth factor are elevated in patients with atrial fibrillation and correlated with relapse following radiofrequency ablation].

OBJECTIVE: To investigate the changes in serum levels of endothelin-1 (ET-1) and connective tissue growth factor (CTGF) in patients with atrial fibrillation (AF) and their value for predicting recurrence of AF after radiofrequency ablation (RFCA). METHODS: Sixty-six patients with paroxysmal AF (PaAF) and 72 with persistent AF (PaAF) admitted in our hospital were recruited as AF group and 80 patients with sinus rhythm as the control group, and in all the participants, serum levels of ET-1 and CTGF were measured using ELISA and Western blotting. From 6 patients with AF and 6 with sinus rhythm undergoing cardiac surgery in our hospital, tissue samples of the right atrial appendage were taken intraoperatively for observation of structural changes of the cardiomyocytes, myocardial fibrosis and expression of ET-1 and CTGF protein. In AF group, the patients receiving RFCA were followed up for 6 months following the procedure for assessment of the outcomes. RESULTS: Compared with the control patients, the patients with AF showed obvious damages of the cardiomyocyte structure and myocardial fibrosis. Serum levels of ET-1 and CTGF levels were significantly higher in PaAF and PeAF groups than in the control group, and were higher in PeAF group than in PaAF group. In the patients with AF, serum ET-1 and CTGF levels were positively correlated with left atrial diameter (LAD) (P < 0.05), and ET-1 was positively correlated with CTGF levels (P < 0.05). In patients with postoperative AF recurrence, the serum levels of ET-1 and CTGF were significantly higher than those in patients without recurrence; serum ET-1 and CTGF levels before and after the operation were positively correlated with the recurrence of PeAF, and elevated serum levels of ET- 1 and CTGF were identified by logistic regression analysis as independent risk factors for postoperative recurrence of PeAF. CONCLUSION: Serum levels of ET-1 and CTGF are significantly elevated in AF patients in positive correlation with AF duration. ET-1 and CTGF levels are higher in AF patients with postoperative recurrence, and they both have predictive value for recurrence of PeAF following RFCA.

[Analysis of factors associated with the structure of the gut microbial community in HIV/AIDS patients in some areas of Henan province].

Objective: To investigate the related factors associated with the structure of the gut microbial community in HIV infection/AIDS cases (HIV/AIDS) in Henan province. Methods: The convenience sampling method was used to select 122 cases who were receiving Antiviral Treatment (ART) or ART-naive in Henan. Whole blood and stool specimens were collected. Genomic DNA of stool samples was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using Illumina NovaSeq 6000 high-throughput sequencing system. The analysis was performed mainly at the genus level, and the 30 genera with the highest abundance were selected as a measure of the gut microbial community structure. The correlation between community structure and related factors was analyzed using redundancy analysis and Envfit function. Results: 122 cases were finally completed sequencing and analysis, the average BMI was (23.62±2.78) kg/m2 and the average age was (47±13) years. Among them, male accounted for 66.39% (81/122), and heterosexual transmission route constituted the largest ratio, accounting for 51.64% (63/122). 36 cases were treatment naive (29.51%, 36/122). The top five dominant genera of the total population (122 cases) were Prevotella, Roseburia, Megamonas, Bacteroides and Faecalibacterium and the top five dominant genera of the ART population (86 cases) were Prevotella, Megamonas, Bacteroides, Roseburia and Faecalibacterium. The top five dominant genera of the ART-naive population (36 cases) appeared as Prevotella, Faecalibacterium, Roseburia, Bacteroides and Megamonas. In the total population, ART (P<0.001) was the most significant factors of community structure. Other significant factors were: duration of diagnosis (P=0.009), viral load (P=0.022) and anti-HCV (P=0.018). ART was positively correlated with Megamonas and negatively correlated with Prevotella, Roseburia and Faecalibacterium, while the other three factors of duration of diagnosis, viral load and anti-HCV were positively correlated with Prevotella, Roseburia and Faecalibacterium and negatively correlated with Megamonas. In the ART-naive population, duration of diagnosis (P=0.003) were the factors significantly associated with community structure. Duration of diagnosis was positively correlated with Roseburia, Faecalibacterium, Megamonas and Prevotella and negatively correlated with Bacteroides. Conclusion: ART and duration of diagnosis were factors significantly associated with gut microbial community structure and had a significant impact on multiple high-abundance genera.

Identification of Periosteal Osteogenic Progenitors in Jawbone.

Unlike long bones, jawbone development is mainly accomplished by intramembranous ossification resulting from the differentiation of periosteal progenitor cells. However, the spatiotemporal ontogeny of periosteal progenitor cells during jawbone development and repair remains elusive. In this study, we mapped the transcriptional landscape of the human jawbone periosteum at single-cell resolution and identified a cathepsin K (Ctsk)+ periosteal subset. Lineage tracing analysis indicated that Ctsk-Cre-labeled periosteal cells could make contributions to jawbone development. However, different from the periosteal-specific location of Ctsk+ cells in long bone, we also identified Ctsk+ stromal cells in jawbone marrow and implied the heterogeneity of jawbone Ctsk+ hierarchy. In further analysis of the periosteal progenitor cell subset of heterogeneous Ctsk+ hierarchy, we identified a unique Ctsk+Ly6a+ subset of cells. The additional marker Ly6a helped to further confine the progenitor subset to the jawbone periosteum and was nearly undetectable in the bone marrow. Defects in the jawbone could activate the migration and osteogenic differentiation of Ctsk+Ly6a+ cells. Local ablation of Ctsk+ cells by diphtheria reduced the number of Ctsk+Ly6a+ cells and delayed the repair of the bone defect. Taken together, we identify a novel periosteal osteogenic progenitor subset that is active in jawbone osteogenesis and healing.

CARBONATED BEVERAGES AFFECT LEVELS OF ANDROGEN RECEPTOR AND TESTOSTERONE SECRETION IN MICE.

Objectives: This work aimed to study the influences of carbonated beverages (CBs) on the testis growth and the expression levels of androgen receptor (AR) of mice. Methods: Two experimental groups of 30 mice each PEP-1 and PEP-2 drank 50% and 100% Pepsi-Cola, respectively for 15 days. Other 2 experimental groups of 30 mice each COC-1 and COC-2 drank 50% and 100% Coca-Cola, respectively for 15 days. The control group (CG) of 30 mice drank water. Bilateral testes were harvested aseptically on days 0, 5, 7, 10, 13 and 15. Real-time PCR and Western blot were implemented to detect levels of androgen receptor (AR) mRNA and protein in testis tissues. Results: Testes masses of PEP-2, COC-1 and COC-2 were greater than those of PEP-1 and CG (P < 0.05). On day 15, testis longitudinal diameter (TLD) of CBs-treated mice was increased as compared to CG. TLD, testes transverse diameters (TTD) and AR proteins levels of PEP-2 and COC-2 were increased in comparison with CG (P<0.05). Serum testosterone concentrations of PEP-2 were higher than that of COC-1 and CG (P < 0.05). Levels of AR mRNAs of four CBs-treated mice were increased by 60.18%, 67.26%, 65.93% and 78.76%. Conclusions: A high concentration of Coca-Cola and Pepsi-Cola could raise TLD and TDD, enhance testosterone secretion, and increase serum EGF concentrations.

[Correlation between the primary tumor size of endometrial carcinoma and lymph node metastasis and recurrence].

Objective: To investigate the clinical significance of the primary tumor size in patients with endometrial carcinoma (EC). Methods: A total of 385 patients with EC admitted to Peking University People's Hospital from January 2006 to December 2016 with complete follow up data were selected, whose tumor size data before biopsy were retrospectively studied. Results: (1) The mean diameter of the primary tumor was (3.6±1.8) cm (range: 1-15 cm). And 48 cases were 0-<2 cm, 78 cases were 2-<3 cm, 92 cases were 3-<4 cm, 73 cases were 4-<5 cm, 94 cases were ≥5 cm. The diameter of the tumor was associated with age <60 years old, premenopause, CA125≥35 kU/L, non-parturition, poor differentiation, stage Ⅲ-Ⅳ, depth of myometrial infiltration ≥1/2, cervical interstitial involvement, adnexal metastasis and lymph node metastasis (all P<0.05), but not associated with body mass index, hypertension, diabetes mellitus, pathology, lymph-vascular space invasion (all P>0.05). (2) Among the 334 patients underwent lymphadenectomy, 45 (13.5%, 45/334) cases with lymph node metastasis were observed. Stratified analysis showed that lymph node metastasis and recurrence rate of patients with EC gradually increased with the increase of tumor size (P<0.05). Adopting 2, 3, 4 and 5 cm as cut-off values of tumor size, there were significant differences in the rate of lymph node metastasis and recurrence among them observed (P<0.05), except for lymph node metastasis rate and recurrence rate when the cut-off value was 2 cm (P>0.05). (3) An receiver operating characteristic (ROC) curve analysis showed that a tumor diameter of 4.25 cm was the cut-off prognostic value to predict lymph node metastasis and recurrence of EC. Conclusions: Tumor diameter is significantly correlated with lymph node metastasis and recurrence in patients with EC. Tumor size should be considered in determining the scope of surgery and adjuvant therapy.

Expression and Clinical Significance of OX40 and OX40L mRNA in Hepatocellular Carcinoma.

In a retrospective study, the expression of mRNA of membrane receptor OX40 and its ligand OX40L in liver tissues was analyzed in 34 patients with hepatocellular carcinoma in order to assess their clinical implications and prognostic value. Expression of mRNA was analyzed by reverse transcription PCR and TaqMan probes. Expression of OX40 mRNA was significantly higher in tumor specimens in paired comparison with the samples of adjacent non-tumor tissue or normal liver tissue of control patients. In contrast, expression of OX40L mRNA was lower in tumor tissue in paired comparison with the samples of adjacent non-tumor tissue or normal liver tissue. The clinical and pathological analysis showed that expression of OX40 mRNA significantly correlated with the degree of tumor differentiation; there was an insignificant decreasing trend in the length of recurrence-free period. It was hypothesized that microenvironment of hepatocellular carcinoma can induce immunosuppression due to dysregulation of the expression of OX40 and OX40L in tumor tissue, which promotes tumor growth.

[Preliminary analysis on COVID-19 case spectrum and spread intensity in different provinces in China except Hubei province].

Objective: To analyze the characteristics of COVID-19 case spectrum and spread intensity in different provinces in China except Hubei province. Methods: The daily incidence data and case information of COVID-19 were collected from the official websites of provincial and municipal health commissions. The morbidity rate, severity rate, case-fatality rate, and spread ratio of COVID-19 were calculated. Results: As of 20 March, 2020, a total of 12 941 cases of COVID-19 had been conformed, including 116 deaths, and the average morbidity rate, severity rate and case-fatality rate were 0.97/100 000, 13.5% and 0.90%, respectively. The morbidity rates in Zhejiang (2.12/100 000), Jiangxi (2.01/100 000) and Beijing (1.93/100 000) ranked top three. The characteristics of COVID-19 case spectrum varied from province to province. The first three provinces (autonomous region, municipality) with high severity rates were Tianjin (45.6%), Xinjiang (35.5%) and Heilongjiang (29.5%). The case-fatality rate was highest in Xinjiang (3.95%), followed by Hainan (3.57%) and Heilongjiang (2.70%). The average spread ratio was 0.98 and the spread intensity varied from province to province. Tibet had the lowest spread ratio (0), followed by Qinghai (0.20) and Guangdong (0.23). Conclusion: The intervention measures were effective in preventing the spread of COVID-19 and improved treatment effect in China. However, there were significant differences among different regions in severity, case-fatality rate and spread ratio.

[Analysis on clinical characteristics, diagnosis and treatment of 13 cases of infant hairy polyp].

Objective: To explore and analyze the clinical characteristics, diagnosis and treatment of infant hairy polyp. Methods: A retrospective analysis was made on 13 cases of hairy polyp confirmed by pathology, who were admitted to the Children's Hospital of Hebei Province from January 2010 to September 2019, including 4 males and 9 females, with a male-female ratio of 1∶2.25. The age ranged from 3 hours to 1 year, and the median age was 1 month. Twelve of the 13 children were found to have difficulty breathing or feeding. All the children received coblation resection under general anesthesia. The root pedicle of the mass was found in the lateral nasopharyngeal wall in 8 cases, in the junction of palatine and palatopharyngeal arch of tonsil and the tongue and esophageal entrance in 1 case, respectively. Nasal septum was found in 2 cases, including 1 case located between two incisors. The wound at the root pedicle was ablated and the bleeding was stopped completely. Results: Postoperative follow-up lasted from 3 months to 2 years, and there was no recurrence in 12 cases. Fibrolaryngoscope showed a mass of the right eustachian tube and pharyngeal mouth in 1 case 2 years after the surgery, which was considered recurrence of hairy polyps and lost after that. Conclusion: Hairy polyps in infants is a rare clinical disease, and its main symptom is upper respiratory tract obstruction. Early diagnosis and radical surgery are the key to the treatment of the disease.

Triplex qRT-PCR with specific probe for synchronously detecting Bovine parvovirus, bovine coronavirus, bovine parainfluenza virus and its applications.

Bovine parvovirus (BPV), bovine coronavirus (BCoV) and bovine parainfluenza virus (BPIV) are common etiologies causing gastrointestinal and respiratory diseases in dairy herds. However, there are few reports on the synchronous detection of BPV, BCoV and BPIV. The present article aimed to develop a quick and accurate RT-PCR assay to synchronously detect BPV, BCoV and BPIV based on their specific probes. One pair universal primers, one pair specific primers and one specific probe was designed and synthesized. After the concentrations of primer and probe and annealing temperature were strictly optimized, the specificity, sensitivity and repeatability of the established triplex probe qRT-PCR were evaluated, respectively. The results showed the recombinant plasmids of pMD18-T-BPV, pMD18-T-BCoV and pMD18-T-BPIV were 554bp, 699bp and 704bp, respectively. The optimal annealing temperature was set at 45.0°C for triplex qRT-PCR. The triplex probe qRT-PCR can only synchronously detect BPV, BCoV and BPIV. Detection sensitivities were 2.0×102, 2.0×102 and 2.0×101 copies/µL for BPV, BCoV and BPIV, being 1000-fold greater than that in the conventional PCR. Detection of clinical samples demonstrated that triplex probe qRT-PCR had a higher sensitivity and specificity. The intra-assay and inter-assay coefficient of variation were lower than 2.0%. Clinical specimens verified that the triplex qRT-PCR had a higher sensitivity and specificity than universal PCR. In conclusion, this triplex probe qRT-PCR could detect only BPV, BCoV and BPIV. Minimum detection limits were 2.0×102 copies/µL for BPV and BCoV, and 2.0×101 copies/µL for BPIV. The sensitivity of this triplex probe qRT-PCR was 1000-fold greater than that in the conventional PCR. The newly qRT-PCR could be used to monitor or differentially diagnose virus infection.

[Consistency of ALK Ventana-D5F3 immunohistochemistry interpretation in lung adenocarcinoma among Chinese histopathologists].

Objective: To understand the consistency of ALK Ventana-D5F3 immunohistochemistry (IHC) interpretation in Chinese lung adenocarcinoma among histopathologists from different hospitals, and to recommend solution for the problems found during the interpretation of ALK IHC in real world, with the aim of the precise selection of patients who can benefit from ALK targeted therapy. Methods: This was a multicenter and retrospective study. A total of 109 lung adenocarcinoma cases with ALK Ventana-D5F3 IHC staining were collected from 31 lung cancer centers in RATICAL research group from January to June in 2018. All cases were scanned into digital imaging with Ventana iSCANcoreo Digital Slide Scanning System and scored by 31 histopathologists from different centers according to ALK binary (positive or negative) interpretation based on its manufacturer's protocol. The cases with high inconsistency rate were further analyzed using FISH/RT-PCR/NGS. Results: There were 49 ALK positive cases and 60 ALK negative cases, confirmed by re-evaluation by the specialist panel. Two cases (No. 2302 and No.2701) scored as positive by local hospitals were rescored as negative, and were confirmed to be negative by RT-PCR/FISH/NGS. The false interpretation rate of these two cases was 58.1% (18/31) and 48.4% (15/31), respectively. Six out of 31 (19.4%) pathologists got 100% accuracy. The minimum consistency between every two pathologists was 75.8%.At least one pathologist gave negative judgement (false negative) or positive judgement (false positive) in the 49 positive or 60 negative cases, accounted for 26.5% (13/49), 41.7% (25/60), respectively, with at least one uncertainty interpretation accounted for 31.2% (34/109). Conclusion: There are certain heterogeneities and misclassifications in the real world interpretation of ALK-D5F3 IHC test, which need to be guided by the oncoming expert consensus based on the real world data.

Structure and mode of action of a novel antibacterial peptide from the blood of Andrias davidianus.

Andrias davidianus is widely recognized in traditional medicine as a cure-all to treat a plethora of ailments. In a previous study, a novel antibacterial peptide named andricin B was isolated from A. davidianus blood. In this study, we investigated andricin B structure and its mode of action. Circular dichroism spectra suggested that andricin B adopts a random coil state in aqueous solution and a more rigid conformation in the presence of bacteria. Moreover propidium iodide/fluorescein diacetate double staining indicated that bacteria treated with andricin B were not immediately eliminated. Rather, there is a gradual bacterial death, followed by a sublethal stage. Scanning electronic microscope imaging indicates that andricin B might form pores on cell membranes, leading to the release of cytoplasmic contents. These results were consistent with flow cytometry analysis. Furthermore, Fourier transform infrared spectroscopy suggests that andricin B induces changes in the chemical properties in the areas surrounding these "pores" on the cell membranes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggested the new perspectives about the mode of action of antimicrobial peptide (AMP) active against sensitive bacteria. The AMP was able to be in a random coiled state in aqueous solution but to change to a more rigid one in the presence of sensitive bacteria. Exposure to AMP might not lead to immediate death of treated bacteria, rather bacteria concentration decreased gradually flattening at a sublethal stage. These findings will help people to understand better how the AMPs activate against sensitive bacteria.

TaqMan real-time PCR for detecting bovine viral diarrhea virus.

The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic protocol which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5' - and 3'-untranslated regions (5'UTR, 3'UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were transcribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 µmol/L primer, 7.5 µmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/µL for viral RNA. It was 10000-fold higher than conventional PCR with excellent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/µL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.

MiR-137-5p alleviates inflammation by upregulating IL-10R1 expression in rats with spinal cord injury.

OBJECTIVE: This study aims to explore the potential functions of miR-137-5p and interleukin-10R1 (IL-10R1) in mediating the immune inflammation after spinal cord injury (SCI). MATERIALS AND METHODS: Firstly, primary microglia were isolated from the spinal cord of newborn rats. Expression levels of miR-137-5p and IL-10R1 in LPS-induced microglia were determined by quantitative Real-time polymerase chain reaction (qRT-PCR). In addition, mRNA expressions of Janus kinase (Jak1) and signal transducer and activator of transcription 3 (STAT3) were also examined by qRT-PCR. SCI model in rats was established and randomly assigned to three different groups: Sham group, SCI group and miR-137-5p mimic group. Within one week of spinal injury, relative levels of miR-137-5p and IL-10R1 in rats of different groups were detected by qRT-PCR. The mRNA levels of JAK1, tyrosine kinase (Tyk2) and STAT3 in rats were also measured. Moreover, protein expression of IL-1ß, TNF-α and IL-6 in rats was measured by Western blotting. Finally, the improvement of locomotor function in three groups of rats within 4 weeks via BBB rating scale. RESULTS: Transfection of miR-137-5p mimics upregulated relative levels of IL-10R1, JAK1 and STAT3 in in vitro cultured microglia. Similarly, IL-10R1/JAK1/STAT3 pathway was activated in rats administrated with miR-137-5p mimics. Nevertheless, relative levels of classical inflammatory stimulators IL-1ß, TNF-α and IL-6 were downregulated accordingly by miR-137-5p overexpression. Moreover, miR-137-5p effectively improved the locomotor function of rats after SCI. CONCLUSIONS: MiR-137-5p exerts an anti-inflammatory response by upregulating IL-10R1, thus improving locomotor function and alleviating spinal cord injury.