Altered gut microbiota is associated with the formation of occult hepatitis B virus infection.

Hepatitis B virus (HBV), a common blood transmission pathogen worldwide, can lead to viral hepatitis, cirrhosis, liver cancer, and other liver diseases. In particular, occult hepatitis B virus infection (OBI) may be caused by an immune response leading to suppressed virus replication. Gut microbiota can change the immunity status of the human body and, therefore, affect the replication of HBV. Thus, to identify whether there are differences in gut microbiota between HBV carriers and OBI carriers, we collected fecal samples from 18 HBV carriers, 24 OBI blood donors, and also 20 healthy blood donors as negative control. After 16S sequencing, we found that the abundance of Faecalibacterium was significantly reduced in samples from OBI blood donors compared with those from healthy blood donors. Compared with samples from HBV carriers, the samples from OBI blood donors had a significantly increased abundance of Subdoligranulum, which might stimulate immune activation, thus inhibiting HBV replication and contributing to the formation of occult infection. Our findings revealed the potential role of gut microbiota in the formation of OBI and further provided a novel strategy for the treatment of HBV infection.IMPORTANCEOccult hepatitis B virus infection (OBI) is a special form of hepatitis B virus infection with hepatitis B surface antigen (HBsAg) positive and hepatitis B virus (HBV) DNA negative. Gut microbiota may contribute to the immune response leading to suppressed virus replication and, thus, participates in the development of OBI. The study on gut microbiota of OBI blood donors provides novel data considerably advancing our understanding of the immune mechanism for the determination of occult hepatitis B virus infection, which is helpful for improving the strategy of the treatment of HBV infection.

Lower peripheral blood CD4+ lymphocyte ratio is associated with severe bronchopulmonary dysplasia.

OBJECTIVE: To elucidate the characteristics of lymphocyte subsets in bronchopulmonary dysplasia (BPD) diagnosis following Jensen's criterion to understand the spectrum of lymphocytes in different degrees of BPD. STUDY DESIGN: This single-center retrospective cohort study included 120 neonates admitted to the neonatal intensive care unit between 1 July 2014 and 30 June 2021, who had undergone peripheral blood lymphocyte subpopulation detection. RESULTS: Thirty-one neonates were included in the control group, whereas 33 infants with BPD were included in the case group. In addition, we selected 56 infants with a gestational age (GA) <37 weeks without BPD who were receiving oxygen therapy. Among the three groups, the B cell and NK cell frequencies were significantly higher and the frequencies of T cells and CD4+ cells were significantly lower in the BPD group. In newborns without BPD, the distribution of T lymphocyte subsets was similar at different GAs. Comparing different degrees of BPD, the patients in the grades 2-3 BPD group had significantly lower percentages of T lymphocytes and CD4+ T cells than those in the other groups. Remarkably, the frequencies of NK cells were significantly higher in patients with grades 2-3 BPD, and the Treg cells slightly increased with BPD severity, although the differences were not significant. CONCLUSION: Healthy neonates had similar ratios of lymphocyte subsets among different GAs; although as the GAs increased, the percentage of lymphocytes increased slightly. Severe BPD was associated with lower CD4+ T cells and higher NK cells. However, whether such changes were the cause or the consequence of BPD has not been determined.

A reference interval study of ferritin and transferrin levels in donors from two blood centers.

Blood donors not only save the lives of patients but also play an important role in the development of medical and health services. Therefore, it is particularly important to pay attention to the blood health of blood donors who are at a high risk of iron deficiency. Detection of serum ferritin and transferrin is an important basis for the diagnosis of iron deficiency anemia. However, to the best of our knowledge, the levels of serum ferritin and transferrin, and the influencing factors, such as age and type of donation, in blood donors have not been clarified. In the present study, the serum ferritin and transferrin levels of donors from two blood centers were investigated. Demographic data were collected from the donors, and their serum ferritin and transferrin levels were tested. A total of 1,817 donors were enrolled and were eligible for evaluation. Reference intervals (RIs) for ferritin and transferrin were obtained from blood donors, and it was revealed that the ferritin and transferrin levels of blood donors were associated with age. Furthermore, serum transferrin levels were associated with the type of donation; the serum transferrin RI level was significantly higher in platelet-only donors compared with in whole blood donors. It was also demonstrated that ferritin levels were negatively associated with transferrin levels. The present study identified RIs for ferritin and transferrin levels in blood donors, and indicated that age and type of donation were important factors affecting ferritin and transferrin levels in blood donors. These findings may prove useful for blood donation recruitment and screening strategies in China, and could promote the health of blood donors.

Case Report: Identification of Two Variants of ALG13 in Families With or Without Seizure and Binocular Strabismus: Phenotypic Spectrum Analysis.

Background: Genetic causes in most affected children with intellectual disability and/or development delay remain unknown. Methods: To identify potential variants responsible for these disorders, we recruited 161 affected families and performed whole-exome sequencing and associated bioinformatics analysis. Results: In the present study, we report the identification of variants in the ALG13 gene in two of the families. In family 1, a known pathogenic missense variant (c.23T > C; p.V8A) of ALG13 was identified in a boy and his mother. In family 2, a novel missense variant (c.862C > G; p.L288V) of the same gene was identified in the affected boy and his phenotypically normal mother. Genotype-phenotype correlation analysis by comparing reported 28 different variants (HGMD) showed that three major phenotypes, including various seizures/epilepsy, intellectual disability, and development delay (such as growth, speech, motor, etc.), are present in most affected individuals. However, other phenotypes, such as strabismus and absence of seizure in our second patient, are not reported if any, which may represent a unique case of X-linked recessive nonsyndromic disorder caused by a mutation in ALG13. Conclusion: We identified two missense variants in ALG13 in a cohort of 161 families with affected individuals diagnosed as intellectual disability and/or development delay. A novel c.862C > G mutation may represent a case of X-linked recessive.

Molecular characteristics of the full-length genome of occult hepatitis B virus from blood donors in China.

The characteristics of a large sample size of the full-length genome of occult hepatitis B virus (HBV) infection (OBI) have not been extensively explored in China. Voluntary blood donors who were HBsAg-negative/HBV NAT-positive (HBsAg-/HBV NAT+) were identified by blood screening and recruited. Blood samples were tested for HBV serologic markers, viral loads, and PCR to identify OBI. HBV full-length genomes were obtained by amplifying two fragments using nested PCR. The characterization of OBI strains was based on sequence analyses compared with HBsAg+ strains obtained from the same donor population. Of the 50 full-length genomes of 172 identified OBI strains, 33 were classified as genotype B (OBIB) and 17 strains as genotype C (OBIC). Significantly higher nucleotide variabilities were observed in the Pre-S2/S promoter region (SP2) and core upstream regulatory sequence (CURS) in OBIB than in their HBsAg+ controls (P < 0.05). Both OBIB and OBIC showed higher amino acid (aa) variabilities in Pol and Pre-S/S regions than their controls (P < 0.05). In addition, 19 novel OBI-related mutations were found spanning the four open reading frames (ORFs) of the HBV genome. Four novel deletions and one novel insertion were also found in OBIC strains. Several novel OBI-related mutations spanning the four ORFs of the virus were identified by characterizing a large sample size of the full-length OBI genome, which may affect the production of HBsAg and contribute to the occult infection of HBV.

The evolutionary dynamics and epidemiological history of hepatitis C virus genotype 6, including unique strains from the Li community of Hainan Island, China.

Hepatitis C virus (HCV) is a highly diverse pathogen that frequently establishes a chronic long-term infection, but the origins and drivers of HCV diversity in the human population remain unclear. Previously unidentified strains of HCV genotype 6 (gt6) were recently discovered in chronically infected individuals of the Li ethnic group living in Baisha County, Hainan Island, China. The Li community, who were early settlers on Hainan Island, has a distinct host genetic background and cultural identity compared to other ethnic groups on the island and mainland China. In this report, we generated 33 whole virus genome sequences to conduct a comprehensive molecular epidemiological analysis of these novel gt6 strains in the context of gt6 isolates present in Southeast Asia. With the exception of one gt6a isolate, the Li gt6 sequences formed three novel clades from two lineages which constituted 3 newly assigned gt6 subtypes and 30 unassigned strains. Using Bayesian inference methods, we dated the most recent common ancestor for all available gt6 whole virus genome sequences to approximately 2767 bce (95 per cent highest posterior density (HPD) intervals, 3670-1397 bce), which is far earlier than previous estimates. The substitution rate was 1.20 × 10-4 substitutions/site/year (s/s/y), and this rate varied across the genome regions, from 1.02 × 10-5 s/s/y in the 5'untranslated region (UTR) region to 3.07 × 10-4 s/s/y in E2. Thus, our study on an isolated ethnic minority group within a small geographical area of Hainan Island has substantially increased the known diversity of HCV gt6, already acknowledged as the most diverse HCV genotype. The extant HCV gt6 sequences from this study were probably transmitted to the Li through at least three independent events dating perhaps from around 4,000 years ago. This analysis describes deeper insight into basic aspects of HCV gt6 molecular evolution including the extensive diversity of gt6 sequences in the isolated Li ethnic group.

Hepatitis B Virus-Specific Cellular Immunity Contributes to the Outcome of Occult Hepatitis B Virus Infection.

There is little known of immunologic factors leading to the occurrence of occult HBV infection (OBI). Specific cellular immune response to hepatitis B virus (HBV) core/pol peptides was compared between blood donor populations, including 37 OBIs, 53 chronic HBV infections (CHB), 47 resolved infections, and 56 non-infected controls, respectively. The rate of CD4+/CD8+ T cell proliferation in OBI or CHB carriers was higher than in HBV resolved and non-infected individuals (P < 0.05). The intensity of IFN-γ-secretion T-cell response of OBI carriers was highest, followed by CHB and resolved infections, and non-infected individuals (P < 0.05). The frequency of intracellular IFN-γ and IL-17A CD4+/CD8+ and IL-21 CD4+ T-cell responses was significantly higher in resolved infections than in OBI or CHB carriers (P < 0.05), while the level of extracellular IL-17A of peripheral blood mononuclear cells (PBMCs) was higher in OBI and CHB carriers than in resolved infections (P < 0.01). The frequency of intracellular IL-10 CD4+ T-cell response in CHB, OBI, and resolved infections was higher than in HBV non-infected individuals (P < 0.01). Intracellular IL-10 CD8+ T cell and extracellular IL-10 T-cell responses were higher in CHB than in OBI (P = 0.012) or HBV resolved infections (P < 0.01). In conclusion, the higher level of effective T-cell response with IFN-γ, IL-17A, and IL-21 contributes to resolved infection outcome, while higher levels of suppressive T-cell response with IL-10 result in HBV chronicity. OBI is an intermediary status between HBV resolved and chronic infections, in which IL-21 effector and IL-10 suppressor T-cell responses play an important role in directing the outcome of HBV infection.

Molecular Epidemiological Characteristics and Risk Factors for Acquiring HBV Among Li Ethnic in Baisha County, Hainan Island-Subgenotype D3 Was First Discovered in China.

The residents of Baisha, a county of Hainan Island, mainly composed of Li ethnic population and relatively closed living environment with its unique geographical location. Our previous study showed that Li ethnic population of Baisha is an endemic center for hepatitis C virus, with significantly higher rates than in other parts of China. However, the epidemiology of HBV in this region remains unclear. Therefore, we conducted a comprehensive epidemiological survey of HBV in Baisha County, including 1,682 Li ethnic residents. The total seropositive rate for HBsAg was 10.2% and was higher than other parts of China. HBV-positive status was associated with the 20-40-year-old group (OR = 1.27, 95%CI 1.04-1.39, P < 0.01) and alcohol consumption (OR = 2.17, 95%CI 1.58-2.99, P < 0.01). Phylogenetic analysis showed that HBV subgenotype D3 was predominant in Baisha County which was first discovered in China, followed by C5, C1, B2, and undetermined subgenotypes which were significantly different from other geographical distribution of main genotypes in China. The most recent common ancestor (tMRCA) of the HBV genotype C in the Li ethnic of Baisha County was 1846 (95%CI: 1739-1932), and Baisha-C5 was earlier than Baisha-C1 and Baisha-C2. Most Baisha-D3 sequences were concentrated in one bundle and unrelated to those D3 genome sequences elsewhere in the world. According to the phylogenetic tree, D3 was introduced into Baisha County in 1884 (95%CI: 1816-1993) and became a local endemic virus. In conclusion, HBV infection in the Li ethnic group is characterized by a high prevalence rate in 20-40-year-old individuals and a unique genotype distribution which were significantly different from other geographical distribution of main genotypes in China, and subgenotype D3 was first discovered in China.

Complete genome sequencing and evolutionary analysis of hepatitis C virus subtype 6a, including strains from Guangdong Province, China.

We performed an evolutionary analysis using whole genome sequence isolates of hepatitis C virus (HCV) 6a from Guangdong Province and reference sequences from various countries. Less than 5% of the HCV genome was found to be under positive selection. The E1 and E2 proteins had the highest proportion of positively selected sites both within and outside of CD8 T cell epitopes in all of the strains. Regions corresponding to CD8 T cell epitopes were under negative selection except in the isolates from Guangdong. Furthermore, we found evidence of three introductions of the virus into Guangdong from Vietnam and other Southeast Asian countries. Thus, this study provides information about the transmission of HCV 6a by comparison of full-length sequences, indicating the impact of selective constraints in Guangdong and across China.

E2 Site Mutations in S Protein Strongly Affect Hepatitis B Surface Antigen Detection in the Occult Hepatitis B Virus.

The mechanism of occult hepatitis B infection (OBI) has not yet been fully clarified. Our previous research found that novel OBI-related mutation within S protein, E2G, could cause the hepatitis B surface antigen (HBsAg) secretion impairment, which resulted in intracellular accumulation in OBI of genotype B. Here, to further explore the role of E2 site mutations in the occurrence of OBI, we analyzed these site mutations among 119 OBI strains identified from blood donors. Meanwhile, 109 wild-type HBV strains (HBsAg positive/HBV DNA positive) were used as control group. Furthermore, to verify the E2 site mutations, two conservative 1.3-fold full-gene expression vectors of HBV genotype B and C (pHBV1.3B and pHBV1.3C) were constructed. Then, the E2 mutant plasmids on the basis of pHBV1.3B or pHBV1.3C were constructed and transfected into HepG2 cells, respectively. The extracellular and intracellular HBsAg were analyzed by electrochemical luminescence and cellular immunohistochemistry. The structural characteristics of S proteins with or without E2 mutations were analyzed using relevant bioinformatics software. E2 mutations (E2G/A/V/D) existed in 21.8% (26/119) of OBIs, while no E2 mutations were found in the control group. E2G/A/V/D mutations could strongly affect extracellular and intracellular level of HBsAg (p < 0.05). Notably, unlike E2G in genotype B that could cause HBsAg intracellular accumulation and secretion decrease (p < 0.05), E2G in genotype C could lead to a very significant HBsAg decrease both extracellularly (0.46% vs. pHBV1.3C) and intracellularly (11.2% vs. pHBV1.3C) (p < 0.05). Meanwhile, for E2G/A mutations, the relative intracellular HBsAg (110.7-338.3% vs. extracellular) and its fluorescence intensity (1.5-2.4-fold vs. with genotype-matched pHBV1.3B/C) were significantly higher (p < 0.05). Furthermore, N-terminal signal peptides, with a typical cleavage site for peptidase at positions 27 and 28, were exclusively detected in S proteins with secretion-defective mutants (E2G/A). Our findings suggest that: (1) E2G/A/V/D mutations were confirmed to significantly influence the detection of HBsAg, (2) the underlying mechanism of OBI caused by E2G mutation is quite different between genotype B and genotype C, and (3) E2G/A could produce a N-terminal truncated S protein, which might attribute to the HBsAg secretion impairment in the OBIs.

miR-351-3p promotes rat amniotic fluid-derived mesenchymal stromal cell proliferation via targeting the coding sequence of Abca4.

Amniotic fluid-derived mesenchymal stromal cells (AFMSCs) present different features, depending on the isolation timing and culture conditions. The lack of uniform experimental standards hinders the comparison of results from different studies on AFMSCs. Moreover, understanding the molecular mechanisms that underlie the features of AFMSCs isolated at different embryonic developmental stages might allow the obtention of more viable and highly proliferative AFMSCs through genetic modification. We isolated AFMSCs from pregnant rats at embryonic day (E)12, E15, E18, and E21 and compared their cell proliferation capacity and transcriptome. The cell counting kit-8 assay and RNA sequencing revealed that E12 and E15 AFMSCs showed different characteristics from E18 and E21 AFMSCs. Therefore, AFMSCs were divided into two groups: early (E12 and E15) and late (E18 and E21) pregnancy-stage groups. Next, we screened the gene/microRNA pair Abca4/miR-351-3p that was related to cell proliferation. Abca4 knockdown/overexpression suggested that this gene represses the proliferation of AFMSCs, which is a newly discovered function of this gene. Finally, dual luciferase reporter gene assays confirmed that miR-351-3p targeted the coding sequence of Abca4 and regulated AFMSC proliferation. miR-351-3p promotes AFMSC proliferation via targeting the coding sequence of Abca4. Our findings provide a molecular foundation for further research for obtaining AFMSCs with a higher proliferation capacity.

Prevalence and evolutionary analyses of human T-cell lymphotropic virus in Guangdong province, China: Transcontinental and Japanese subtype lineages dominate the prevalence.

To systematically characterize the prevalence and evolution of human T-cell lymphotropic virus (HTLV) infection among voluntary blood donors (BDs) in Guangdong province, China. A three-year survey for HTLV epidemiology among BDs was performed in Guangdong during 2016-2018. Anti-HTLV-1/2 was screened by ELISA and ECLIA, and subsequently confirmed by western blot (WB) and nucleic acid testing (NAT). The prevalence of HTLV in donors from different cities was calculated. The identified HTLV-positive cases were phylogenetically genotyped and analyzed in a Bayesian phylogenetic framework. Among 3,262,271 BDs, 59 were confirmed positive for HTLV-1 (1.81 per 100,000) and no HTLV-2 infection was found. The prevalence of HTLV-1 varied significantly among 21 cities in Guangdong province, China. The highest prevalence was found in donors from Shanwei (13.94 per 100,000), which is a coastal city in eastern Guangdong. Viral genomic sequences genotyped from 55 HTLV-1 carriers showed that 39 were transcontinental subtype and 16 were Japanese subtype. Specially, 13 out of 39 transcontinental subtype sequences were characterized with L55P mutation and 21 out of 55 sequences were characterized with L19F mutation in viral gp46 protein. The L55P mutation seemed be specific to eastern Asia since it only presented in the sequences from Japan, mainland China, and Taiwan. Phylogenetic analysis of gp46 gene shows that HTLV-1a may have been introduced to Guangdong through four different introduction events and formed major transmission clusters: clades I(13,602 years ago), II(16, 010 years ago), III(15,639 years ago) and IV(16,517 years ago). In general, Guangdong is considered to be a low-prevalence region for HTLV-1 infection, but the prevalence is significantly higher in Shanwei city. Transcontinental and Japanese subtype lineages dominate the prevalence in Guangdong. In terms of blood safety, HTLV antibody screening for first-time blood donors can effectively reduce the risk of HTLV transmission.

Low prevalence of antibodies against SARS-CoV-2 among voluntary blood donors in Guangzhou, China.

Since the first case of COVID-19 reported in late December of 2019 in Wuhan, China, the SARS-CoV-2 virus has caused approximately 20 million infections and 732 thousand deaths around the world by 11 August 2020. Although the pathogen generally infects the respiratory system, whether it is present in the bloodstream and whether it poses a threat to the blood supply during the period of the outbreak is of serious public concern. In this study, we used enzyme-linked immunosorbent assay (ELISA) to screen total antibodies against SARS-CoV-2 in 2199 blood donors, who had donated blood at the Guangzhou Blood Center during the epidemic. The Ig-reactive samples were further characterized for IgA, IgG, and IgM subtypes by ELISA and viral nucleic acid by real-time polymerase chain reaction. Among the 2199 plasma samples, seven were reactive under total antibodies' screening. Further testing revealed that none of them had detectable viral nucleic acid or IgM antibody, but two samples contained IgA and IgG. The IgG antibody titers of both positive samples were 1:16 and 1:4, respectively. Our results indicated a low prevalence of past SARS-CoV-2 infection in our blood donors, as none of the tests were positive for viral nucleic acid and only 2 out of 2199 (0.09%) of samples were positive for IgG and IgA. There would be a limited necessity for the implementation of such testing in blood screening in a COVID-19 low-risk area.

Cytotoxicity Produced by Silicate Nanoplatelets: Study of Cell Death Mechanisms.

Nano-silicate platelets (NSP), an exfoliated product from natural clays, have been validated for biosafety and as an effective supplement to alleviate mycotoxicosis. Since NSP induced noticeable cell death, we therefore investigated further the mechanism of cytotoxicity caused by NSP. Exposure to NSP impaired membrane integrity and caused cell death in a dose-dependent manner. Reactive oxygen species (ROS) generation other than of NADH oxidase origin, and subcellular interactions by internalized NSP also contributed to NSP-induced cell death. NSP persistently provoked receptor-interacting protein 1 Ser/Thr (RIP1) kinase and caspase 6 and 3/7 activation without altering caspase 8 activity and induced evident chromatolysis of necrosis in the later stage. These events proceeded along with increased ER stress and mitochondrial permeability, to final Cyt-C (Cytochrome C) release and AIF (apoptosis inducing factor) translocation, a hallmark of cell necroptosis. Fluorescent probing further manifested NSP traffic, mostly adherence on the cell surfaces, or via internalization, being compartmentalized in the nuclei, cytosols, and mitochondria. Pharmacological approaches with specific inhibitors suggested that endocytosis and particularly RIP1 kinase provocation mediate NSP-induced cell death independent of caspase activation. In conclusion, the necroptotic process contributes to most of the cell death induced by NSP due to membrane interactions/impaired integrity, ROS generation, and subcellular interactions by internalized NSP.

Amniotic fluid mesenchymal stromal cells from early stages of embryonic development have higher self-renewal potential.

Amniotic fluid (AF) is a rich source of mesenchymal stromal cells (MSCs) that have the ability to differentiate into multiple lineages rendering them a promising and powerful tool for regenerative medicine. However, information regarding the differences among AFMSCs derived from different gestational stages is limited. In the present study, AFMSCs derived from 125 pregnant rats at four embryonic day (E) stages (E12, E15, E18, and E21) were isolated and cultured. The primary E15 cells were the smallest in size and the easiest to culture and usually grew in a spherical shape that resembled the growth morphology of embryonic stem cells (ESCs). Once adhered, the E12 and E15 AFMSCs grew faster and could be passaged more than 60 times while still maintaining a continuous proliferative state; however, AFMSCs derived from E18 and E21 could normally be maintained for only 10 passages. To identify the possible reasons for this difference, RT-qPCR was used to examine several genes associated with self-renewal ability and cell origin. The Sox2 expression levels indicated that AFMSCs from E12 and E15 possessed stronger self-renewal capability. The K19, Col2A1, FGF5, AFP, and SPC expression levels indicated there were mixed-population cells co-existing in the AFMSC culture. In conclusion, E15 cells were easier to culture than E12 cells, could be passaged more often, and had a higher Sox2 expression than E18 or E21 cells. The E15-derived AFMSCs had higher viability and proliferative capacity than cells from the later stages. Therefore, AF cells from the early stages could be a good choice for exploring potential treatments involving AFMSCs.

Complement factors and alpha-fetoprotein as biomarkers for noninvasive prenatal diagnosis of neural tube defects.

Neural tube defects (NTDs) are serious congenital malformations. In this study, we aimed to identify more specific and sensitive maternal serum biomarkers for noninvasive NTD screenings. We collected serum from 37 pregnant women carrying fetuses with NTDs and 38 pregnant women carrying normal fetuses. Isobaric tags for relative and absolute quantitation were conducted for differential proteomic analysis, and an enzyme-linked immunosorbent assay was used to validate the results. We then used a support vector machine (SVM) classifier to establish a disease prediction model for NTD diagnosis. We identified 113 differentially expressed proteins; of these, 23 were either up- or downregulated 1.5-fold or more, including five complement proteins (C1QA, C1S, C1R, C9, and C3); C3 and C9 were downregulated significantly in NTD groups. The accuracy rate of the SVM model of the complement factors (including C1QA, C1S, and C3) was 62.5%, with 60% sensitivity and 67% specificity, while the accuracy rate of the SVM model of alpha-fetoprotein (AFP, an established biomarker for NTDs) was 62.5%, with 75% sensitivity and 50% specificity. Combination of the complement factor and AFP data resulted in the SVM model accuracy of 75%, and receiver operating characteristic curve analysis showed 75% sensitivity and 75% specificity. These data suggest that a disease prediction model based on combined complement factor and AFP data could serve as a more accurate method of noninvasive prenatal NTD diagnosis.

The miR-532-3p/Chrdl1 axis regulates the proliferation and migration of amniotic fluid-derived mesenchymal stromal cells.

BACKGROUND: Amniotic fluid-derived mesenchymal stromal cells (AFMSCs) are promising stem cells for regeneration medicine. However, AFMSCs isolated at different stages of pregnancy have different biological characteristics, and the therapeutic effects can differ in vivo and in vitro. The mechanisms underlying these differences have not been defined. METHODS: Bioinformatics analysis of the AFMSC transcriptome identified Chrdl1 as one of the differentially expressed genes. We evaluated the effects of Chrdl1 overexpression or knockdown on the proliferation and migration of AFMSCs. Target prediction was performed using miRanda software to identify the upstream microRNA of Chrdl1. The interaction between Chrdl1 mRNA and its upstream microRNA was evaluated using a dual-luciferase reporter gene assay. RESULTS: Chrdl1 was expressed at lower levels in AFMSCs derived from the early stages of pregnancy. It could suppress AFMSC proliferation and migration. miR-532-3p promoted AFMSC proliferation and migration by targeting the 3' UTR of Chrdl1 and downregulating its expression.

Novel hepatitis B virus surface antigen mutations associated with occult genotype B hepatitis B virus infection affect HBsAg detection.

The causative factors of occult hepatitis B infection are complicated and not yet been fully elucidated. Mutations in hepatitis B virus (HBV) S gene are one of the factors may contributing to occult infection. In this study, 89 blood donors with genotype B occult HBV infection were investigated. Fifty-seven hepatitis B surface antigen (HBsAg)-positive/HBV DNA-positive blood donors served as control group for comparison. Occult HBV-related mutations with a high incidence (P < .05) in the S gene were identified. To further verify these occult infection-related mutations, a conservative full-gene expression vector of HBV B genotype (pHBV1.3B) was constructed. Then, the mutant plasmids on the basis of pHBV1.3B were constructed and transfected into HepG2 cells. Extracellular as well as intracellular HBsAg was analysed by electrochemical luminescence and cellular immunohistochemistry. Ten occult infection-related mutations (E2G, Q101R, K122R, M133T, D144E, G145R, V168A, S174N, L175S and I226S) were significantly more frequent in the occult infection group (P < .05). Five of the ten mutations (E2G, D144E, G145R, V168A and S174N) strongly decreased extracellular HBsAg level (P < .05) in the transfection system. Notably, the E2G mutation had the most significant impact on the ratio of extracellular HBsAg (3.8% vs pHBV1.3B) and intracellular HBsAg (239.3% vs pHBV1.3B) (P < .05), and the fluorescence density of E2G mutant HBsAg was significantly higher than that of pHBV1.3B (P < .0001). Hence, ten mutations were associated with genotype B occult HBV infection; E2G and V168A were novel mutations which we confirmed significantly affect HBsAg detection. E2G might cause HBsAg secretion impairment that results in intracellular accumulation and a decrease in HBsAg secretion.

miR-322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4.

AIMS: Failure of neural tube closure resulting from excessive apoptosis leads to neural tube defects (NTDs). NADPH oxidase 4 (NOX4) is a critical mediator of cell growth and death, yet its role in NTDs has never been characterized. NOX4 is a potential target of miR-322, and we have previously demonstrated that miR-322 was involved in high glucose-induced NTDs. In this study, we investigated the effect of NOX4 on the embryonic neuroepithelium in NTDs and reveal a new regulatory mechanism for miR-322 that disrupts neurulation by ameliorating cell apoptosis. METHODS: All-trans-retinoic acid (ATRA)-induced mouse model was utilized to study NTDs. RNA pull-down and dual-luciferase reporter assays were used to confirm the interaction between NOX4 and miR-322. In mouse neural stem cells and whole-embryo culture, Western blot and TUNEL were carried out to investigate the effects of miR-322 and NOX4 on neuroepithelium apoptosis in NTD formation. RESULTS: NOX4, as a novel target of miR-322, was upregulated in ATRA-induced mouse model of NTDs. In mouse neural stem cells, the expression of NOX4 was inhibited by miR-322; still further, NOX4-triggered apoptosis was also suppressed by miR-322. Moreover, in whole-embryo culture, injection of the miR-322 mimic into the amniotic cavity attenuated cell apoptosis in NTD formation by silencing NOX4. CONCLUSION: miR-322/NOX4 plays a crucial role in apoptosis-induced NTD formation, which may provide a new understanding of the mechanism of embryonic NTDs and a basis for potential therapeutic target against NTDs.