Lnc-AIFM2-1 promotes HBV immune escape by acting as a ceRNA for miR-330-3p to regulate CD244 expression.

Chronic hepatitis B (CHB) virus infection is a major risk factor for cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) immune escape is regulated by the exhaustion of virus-specific CD8+ T cells, which is associated with abnormal expression of negative regulatory molecule CD244. However, the underlying mechanisms are unclear. To investigate the important roles of non-coding RNAs play in CD244 regulating HBV immune escape, we performed microarray analysis to determine the differential expression profiles of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in patients with CHB and patients with spontaneous clearance of HBV. Competing endogenous RNA (ceRNA) was analyzed by bioinformatics methods and confirmed by the dual-luciferase reporter assay. Furthermore, gene silencing and overexpression experiments were used to further identify the roles of lncRNA and miRNA in HBV immune escape through CD244 regulation. The results showed that the expression of CD244 on the surface of CD8+ T cells was significantly increased in CHB patients and in the co-culture system of T cells and HBV-infected HepAD38 cells, which was accompanied by the reduction of miR-330-3p and the elevation of lnc-AIFM2-1. The down-regulated miR-330-3p induced the apoptosis of T cells by lifting the inhibition of CD244, which was reversed by miR-330-3p mimic or CD244-siRNA. Lnc-AIFM2-1 promotes the accumulation of CD244, which is mediated by decreased miR-330-3p, and then reduced the clearance ability of CD8+ T cells to HBV through regulated CD244 expression. And the injury in the ability of CD8+ T cells to clear HBV can be reversed by lnc-AIFM2-1-siRNA, miR-330-3p mimic, or CD244-siRNA. Collectively, our findings indicate that lnc-AIFM2-1 on CD244 by acting as a ceRNA of miR-330-3p contributes to HBV immune escape, which may provide novel insights into the roles of interaction networks among lncRNA, miRNA, and mRNA in HBV immune escape, highlighting potential applications of lnc-AIFM2-1 and CD244 for diagnosis and treatment in CHB.

Specific lateral flow detection of isothermal nucleic acid amplicons for accurate point-of-care testing.

For point-of-care testing (POCT), coupling isothermal nucleic acid amplification schemes (e.g., recombinase polymerase amplification, RPA) with lateral flow assay (LFA) readout is an ideal platform, since such integration offers both high sensitivity and deployability. However, isothermal schemes typically suffers from non-specific amplification, which is difficult to be differentiated by LFA and thus results in false-positives. Here, we proposed an accurate POCT platform by specific recognition of target amplicons with peptide nucleic acid (PNA, assisted by T7 Exonuclease), which could be directly plugged into the existing RPA kits and commercial LFA test strips. With SARS-CoV-2 as the model, the proposed method (RPA-TeaPNA-LFA) efficiently eliminated the false-positives, exhibiting a lowest detection concentration of 6.7 copies/µL of RNA and 90 copies/µL of virus. Using dual-gene (orf1ab and N genes of SARS-CoV-2) as the targets, RPA-TeaPNA-LFA offered a high specificity (100%) and sensitivity (RT-PCR Ct < 31, 100%; Ct < 40, 71.4%), and is valuable for on-site screening or self-testing during isolation. In addition, the dual test lines in the test strips were successfully explored for simultaneous detection of SARS-CoV-2 and H1N1, showing great potential in response to future pathogen-based pandemics.

A new droplet digital PCR assay: improving detection of paucibacillary smear-negative pulmonary tuberculosis.

OBJECTIVES: Smear-negative pulmonary TB (PTB) is difficult to diagnose. Current diagnosis and treatment monitoring methods have inherent limitations. Droplet digital PCR (ddPCR) is a new technique with high sensitivity. This study presents a novel ddPCR for rapid and sensitive identification of Mycobacterium tuberculosis (MTB). METHODS: MTB DNA was detected in respiratory specimens from suspected PTB cases using ddPCR assay, which was directed at two different locations within IS6110. We, for the first time, evaluated the clinical diagnostic ability of this ddPCR for paucibacillary smear-negative PTB. RESULTS: A total of 605 PTB suspects were recruited, including 263 patients with confirmed PTB (84.03% from smear-negative PTB) and 342 without PTB. The sensitivity and specificity of IS6110 ddPCR were 61.22% (95% confidence interval (CI) 55.00-67.10%) and 95.03% (95% CI 92.20-97.10%) for total PTB and 57.92% (95% CI 51.10-64.50%) and 94.57% (95% CI 91.20-96.90%) for smear-negative PTB. ddPCR assay outperformed Xpert MTB/RIF (53.08% vs 28.46%, P = 0.020) in smear-negative PTB detection. Furthermore, effective anti-TB treatment was linked to significantly lower IS6110 copies detected by ddPCR. CONCLUSION: Herein, we developed and validated a highly sensitive and robust ddPCR assay for MTB quantification in respiratory specimens, which improves diagnosis and therapeutic effect evaluation of smear-negative PTB.

Multiplex detection of meningitis and encephalitis pathogens: A study from laboratory to clinic.

Introduction: Infectious meningitis and encephalitis (ME) are life-threatening conditions are caused by various pathogens. Conventional laboratory tests with low sensitivity and specificity cannot help with early diagnosis. Methods: A prospective study using the novel multiplex PCR detection for 18 pathogens of ME (MME-18) was conducted to investigate the clinical utilization and the epidemiology characteristics of ME in southwestern China. Patients with suspected intracranial infection were recruited between May and October 2019 at West China Hospital of Sichuan University. The MME-18 was used to detect cerebrospinal fluid, and conventional experiments including cryptococcal capsular antigen detection, GeneXpert, real-time PCR, and clinical feedback were used to verify the result of MME-18. Results: Among 581 tested patients, 139 eligible individuals were enrolled in the study. Among them, Mycobacterium tuberculosis was the most common pathogen in mono-infection. Viruses and Cryptococcus neoformans were also frequently detected. Of 139 infected patients, 12 cases were diagnosed by MME-18 only, 57 patients by conventional testing only, and 70 cases by both comparator tests and MME-18. There were 96.3% (79/82) diagnoses made by MME-18 had a favorable outcome, and two of twelve diagnoses, made solely by MME-18, had a likely unclear clinical significance. Discussion: The MME-18 showed satisfactory consistency with expert clinical consensus for patients presenting with ME. Combined with conventional testing and clinical suspicion, MME-18 may help clinicians with the early identification of pathogens.

Epidemiological surveillance of common respiratory viruses in patients with suspected COVID-19 in Southwest China.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the peak season of common respiratory viral infections. However, the clinical symptoms of most SARS-CoV-2 infected patients are not significantly different from those of common respiratory viral infections. Therefore, knowing the epidemiological patterns of common respiratory viruses may be valuable to improve the diagnostic and therapeutic efficacy of patients with suspected COVID-19, especially in Southwest China (a mild epidemic area). METHODS: A total of 2188 patients with clinically suspected of COVID-19 in Southwest China were recruited from January 21 to February 29, 2020. Nasopharyngeal swabs, throat swabs and sputum specimens were collected to detect SARS-CoV-2 by using real-time reverse transcription-polymerase chain reaction (RT-PCR) and other 12 viruses via PCR fragment analysis combined with capillary electrophoresis. Clinical characteristics and laboratory test findings were acquired from electronic medical records. All data were analyzed to unravel the epidemiological patterns. RESULTS: Only 1.1% (24/2188) patients with suspected COVID-19 were eventually confirmed to have SARS-CoV-2 infection, and the most frequently observed symptoms were fever (75.0%, 18/24) and cough (20.8%, 5/24). The overall detection rate of other respiratory pathogens was 10.3% (226/2188). Among them, human rhinovirus (3.2%, 71/2188), human parainfluenza viruses (1.6%, 35/2188), influenza B virus (1.2%, 26/2188) and mycoplasma pneumonia (1.2%, 26/2188) were the predominantly detected pathogens in this study. Moreover, the co-infection was observed in 22 specimens. Notably, one COVID-19 case had a coexisting infection with human parainfluenza virus (4.2%, 1/24) and bocavirus was the most common virus tending to occur in co-infection with other respiratory pathogens. CONCLUSIONS: This study reveals the epidemiological features of common respiratory viruses and their clinical impact during the ongoing outbreak of COVID-19 in a mild epidemic area. The findings highlight the importance of understanding the transmission patterns of the common respiratory virus in COVID-19 regions, which can provide information support for the development of appropriate treatment plans and health policies, while eliminating unnecessary fear and tension.

Correlation of clinical features with inferior right hepatic vein incidence: a three-dimensional reconstruction-based study.

PURPOSE: The correlation between right hepatic vein (RHV) diameter and inferior RHV (IRHV) incidence and that between IRHV incidence and other clinical features remain unclear. We investigated factors correlated with IRHV incidence as well as provide a simple and reliable method for predicting IRHV presence preoperatively. METHODS: We obtained computed tomography (CT) imaging data of 1980 patients from the Department of Radiology, Qingdao Municipal Hospital, from July 1, 2016, to July 1, 2017. We excluded patients with heart disease, inferior vena cava (IVC) disease, history of liver surgery or trauma, space-occupying lesions in the liver, and other diseases, which can cause hepatic hemodynamic changes. CT images of patients were three-dimensionally reconstructed. We measured RHV and IRHV diameter as well as the angle between the RHV and the IVC. RESULTS: Data on 299 patients were included in this study; the incidence of IRHV was 34.44%. Sex, age, and the angle between the RHV and IVC did not correlate with IRHV incidence. RHV diameter negatively correlated with IRHV incidence (P < 0.05). The area under the receiver-operating characteristic curve for IRHV incidence was 0.878. The diagnostic threshold value of RHV diameter was 8.86 mm. CONCLUSION: A negative correlation was found between RHV diameter and IRHV incidence, suggesting that IRHV is absent with RHV diameter > 8.86 mm, but is present with RHV diameter < 8.86 mm. This suggests that measuring only RHV diameter can predict the presence of an IRHV when IRHV-related hepatectomy and IRHV preserved living donor liver transplantation are needed.

PCSK9 rs7552841 is associated with plasma lipids profiles in female Chinese adolescents without posttraumatic stress disorder.

To explain the inconsistent relationship between proprotein convertase subtilisin/kexin type 9 (PCSK9) rs7552841 and plasma lipids profiles, we hypothesized that interplays might occur among gender, PCSK9 rs7552841 and posttraumatic stress disorder (PTSD) on plasma lipids levels. To test this hypothesis, a population of 704 Chinese Han high school students was used, which had been recruited after the 2008 Wenchuan Earthquake. In this population, the plasma levels of glucose, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) had been measured by routine methods. PTSD had been assessed by the PTSD Checklist Civilian Version (PCL-C). PCSK9 rs7552841 was analyzed by polymerase chain reaction-restriction fragment length polymorphism analyses and verified by DNA sequencing. The T allele carriers had significantly higher levels of TG, TC, LDL-C, and glucose than the CC homozygotes of PCSK9 rs7552841 after the adjustment for age and BMI in the female students, but not in the male students. When PTSD was taken into consideration, the female T allele carriers had significantly higher TG, TC, LDL-C and glucose than the female CC homozygotes after the adjustment for age and BMI only in the subjects without PTSD, but not in the PTSD patients. No significant differences were observed in the male students regardless of PTSD and the adjustment for age and BMI. These results suggest that PCSK9 rs7552841 is associated with plasma lipids profiles only in female adolescents, but not in male students. This association can be modified and negated by PTSD.

Longitudinal interactions of estrogen receptor alpha gene rs9340799 with social-environmental factors on depression in adolescents after Wenchuan earthquake.

Inconsistent relationships were reported between rs9340799 on estrogen receptor alpha gene (ESR1) and depression in previous studies. The present study was to explore the longitudinal changes of prevalence and severity of depression in 439 Chinese Han adolescents with different genotypes of ESR1 rs9340799 at 6, 12 and 18months after the 2008 Wenchuan earthquake. Social-environmental factors were collected by questionnaires from 465 high school students. Variants of rs9340799 were genotyped by polymerase chain reaction-restriction fragment length polymorphism analyses and verified by DNA sequencing. Depression symptoms were assessed by Beck Depression Inventory (BDI). The results showed the female AA homozygotes had higher prevalence of depression at 12months and higher BDI scores at 18months than the female G allele carriers. Significantly decreased prevalence of depression was observed only in the female AA homozygotes at 18months when compared with that at 6 or 12 months. Consecutive decreases in BDI scores were observed only in the female AA homozygotes. The AA genotype was one of the risk factors at 12months and predictors of BDI scores at 18months. These results firstly suggest different interactions may occur in a gender and time dependent manner among rs9340799 and other potential factors of depression or predictors of its severity, and influence the development and natural rehabilitation of depression.

Posttraumatic stress disorder eliminates association of TrkB rs1187327 with HDL-C in Chinese Han adolescents.

Tropomyosin-related kinase receptor B (TrkB) has been observed to be a common player in posttraumatic stress disorder (PTSD) and the regulation of serum lipids levels. However, interplays of PTSD with TrkB on serum lipids levels have not been explored yet. This study was to investigate the interplays of PTSD and TrkB rs1187327 on serum lipid profiles. Variants of TrkB rs1187327 of 709 high school students were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses and verified by DNA sequencing. The PTSD Checklist Civilian Version (PCL-C) was used to assess PTSD. Colorimetric methods were used to determine the serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and glucose. The results show that the GG homozygotes had a significantly higher level of HDL-C than the A allele carriers of TrkB rs1187327 after the adjustment for gender, age and body mass index (BMI) (1.44 ± 0.299 mmol/L vs. 1.39 ± 0.266 mmol/L, p = 0.036). When PTSD was taken into account, the higher than the A allele carriers level of HDL-C of the GG homozygotes was observed significant after the adjustment for gender, age and BMI only in the subjects without PTSD (1.44 ± 0.293 mmol/ L vs. 1.39 ± 0.267 mmol/L, p = 0.030), but not in the subjects with PTSD. These results suggest that the A allele of TrkB rs1187327 may be associated with decreased levels of serum HDL-C in general healthy adolescents, but not in adolescents with PTSD.