Multi-Lineage Human Endometrial Organoids on Acellular Amniotic Membrane for Endometrium Regeneration.

Asherman's syndrome is an endometrial regeneration disorder resulting from injury to the endometrial basal layer, causing the formation of scar tissue in the uterus and cervix. This usually leads to uterine infertility, menstrual disorders, and placental abnormalities. While stem cell therapy has shown extensive progress in repairing the damaged endometrium and preventing intrauterine adhesion, issues of low engraftment rates, rapid senescence, and the risk of tumorigenesis remain to be resolved for efficient and effective application of this technology in endometrial repair. This study addressed these challenges by developing a co-culture system to generate multi-lineage endometrial organoids (MLEOs) comprising endometrial epithelium organoids (EEOs) and endometrial mesenchymal stem cells (eMSCs). The efficacy of these MLEOs was investigated by seeding them on a biocompatible scaffold, the human acellular amniotic membrane (HAAM), to create a biological graft patch, which was subsequently transplanted into an injury model of the endometrium in rats. The results indicated that the MLEOs on the HAAM patch facilitated endometrial angiogenesis, regeneration, and improved pregnancy outcomes. The MLEOs on the HAAM patch could serve as a promising strategy for treating endometrial injury and preventing Asherman's syndrome.

Patterns of HIV or AIDS Mortality Among Older People From 1990 to 2019 in China: Age-Period-Cohort Analysis.

BACKGROUND: With the increasing effectiveness of antiretroviral therapy and shifting demographics, the problem of older people with HIV or AIDS is increasingly grim in China, and neglecting infection among them may cause more serious social problems, exacerbate the difficulty of controlling HIV or AIDS transmission, and increase the risk of death. OBJECTIVE: We investigated the variations in the trends of Chinese mortality by age, period, and cohort, from 1990 to 2019, to reveal the relationship between age, period, cohort, and HIV burden, as well as providing guidance for resource allocation to prevent HIV-related deaths in vulnerable target populations. METHODS: We extracted the HIV or AIDS mortality data from the Global Burden of Disease. The joinpoint regression model was applied to detect changes in HIV or AIDS trends. The age-period-cohort model was used to explore the age, period, and cohort effects. RESULTS: The trends in age-standardized mortality rates in HIV or AIDS were increased in both genders, from 0.50 to 4.54/105 individuals for males, and from 0.19 to 1.43/105 individuals for females. Joinpoint regression model showed the average annual percentage change of age-standardized mortality rates was 7.0 for male and 6.4 for female individuals, showing an increasing trend. The age effect of male HIV or AIDS mortality showed a net increase of 0.59 (-0.21 to 0.38) from the ages 50-79 years. There is a gradual upward trend in the change in risk of death from HIV or AIDS for the period effect among the older population, lowest at ages 50-54 years (-0.80 for male and -0.78 for female individuals) and highest at ages 75-79 years (0.86 for male and 0.69 for female individuals). The variation of cohort effects was complex, but both genders had a nearly consistent tendency; people born in 1920-1929 had the lowest cohort effect, and those born in 1950-1954 had the highest values. CONCLUSIONS: Our study showed a marked rise in HIV mortality for both genders in China from 1990 to 2019. Aging is an important issue in current HIV prevention and control. There is an urgent need to promote HIV testing and health education. Our findings will help predict future HIV or AIDS mortality changes and identify age-specific priority populations for intervention.

The Second Human Pegivirus, a Non-Pathogenic RNA Virus with Low Prevalence and Minimal Genetic Diversity.

The second human pegivirus (HPgV-2) is a virus discovered in the plasma of a hepatitis C virus (HCV)-infected patient in 2015 belonging to the pegiviruses of the family Flaviviridae. HPgV-2 has been proved to be epidemiologically associated with and structurally similar to HCV but unrelated to HCV disease and non-pathogenic, but its natural history and tissue tropism remain unclear. HPgV-2 is a unique RNA virus sharing the features of HCV and the first human pegivirus (HPgV-1 or GBV-C). Moreover, distinct from most RNA viruses such as HCV, HPgV-1 and human immunodeficiency virus (HIV), HPgV-2 exhibits much lower genomic diversity, with a high global sequence identity ranging from 93.5 to 97.5% and significantly lower intra-host variation than HCV. The mechanisms underlying the conservation of the HPgV-2 genome are not clear but may include efficient innate immune responses, low immune selection pressure and, possibly, the unique features of the viral RNA-dependent RNA polymerase (RdRP). In this review, we summarize the prevalence, pathogenicity and genetic diversity of HPgV-2 and discuss the possible reasons for the uniformity of its genome sequence, which should elucidate the implications of RNA virus fidelity for attenuated viral vaccines.

Reinfection rate of hepatitis C in HIV-1 positive men who have sex with men: A systematic review and meta-analysis.

Purpose: A reduction of 80% in new Hepatitis C virus (HCV) infection is expected by 2030. However, high HCV reinfection rates have been reported among the high-risk population. This meta-analysis aimed to assess the HCV reinfection rate after successful treatment of HIV-1 coinfected MSM populations. Methods: Bibliographic databases were searched and a random-effect model was utilized to calculate the pooled HCV reinfection rate. Sub-group and meta-regression were used to explore heterogeneity among selected studies. A funnel plot and Egger's regression test were performed to estimate the publication bias. Results: Sixteen studies with 9,017.2 person-years (PY) follow-up were included. The overall HCV reinfection rate following successful treatment among HIV-1-infected MSM was 5.27/100 PY (95% CI, 3.98, 6.96). Lower reinfection rates were observed in developed parts of Europe (5.28/100 PY; 95% CI, 3.73, 6.84) and North America (3.92/100 PY; 95% CI, 1.67, 6.17). Reinfection rates among people with HCV test intervals of fewer than 6 months (7.59/100 PY; 95% CI: 5.15, 10.03) were significantly higher than those with more than 6 months test interval (2.88/100 PY; 95% CI: 2.26, 3.50), with an adjusted RR of 1.86 (95% CI, 1.06, 3.13). The adjusted study factors explained 91.03% the of studies' heterogeneity. Conclusion: HCV reinfection rate was high in successfully treated MSM who were coinfected with HIV-1. A shorter HCV test interval may help to explore more HCV reinfections. HCV reinfection rate studies from HIV-1 coinfected MSM in underdeveloped countries are urgently needed. Meta registration: PROSPERO: CRD42021285206, URL: https://www.crd.york.ac.uk/prospero/.

RNA-Dependent RNA Polymerase of the Second Human Pegivirus Exhibits a High-Fidelity Feature.

The virus-encoded RNA-dependent RNA polymerase (RdRp) is responsible for viral replication, and its fidelity is closely related to viral diversity, pathogenesis, virulence, and fitness. Hepatitis C virus (HCV) and the second human pegivirus (HPgV-2) belong to the family Flaviviridae and share some features, including similar viral genome structure. Unlike HCV, HPgV-2 preserves a highly conserved genome sequence and low intrahost variation. However, the underlying mechanism remains to be elucidated. In this study, we evaluated the fidelity of HPgV-2 and HCV RdRp in an in vitro RNA polymerase reaction system. The results showed higher fidelity of HPgV-2 RdRp than HCV NS5B with respect to the misincorporation rate due to their difference in recognizing nucleoside triphosphate (NTP) substrates. Furthermore, HPgV-2 RdRp showed lower sensitivity than HCV to sofosbuvir, a nucleotide inhibitor against HCV RdRp, which explained the insusceptibility of HPgV-2 to direct-acting antiviral (DAA) therapy against HCV infection. Our results indicate that HPgV-2 could be an excellent model for studying the mechanisms involved in viral polymerase fidelity as well as RNA virus diversity and evolution. IMPORTANCE RNA viruses represent the most important pathogens for humans and animals and exhibit rapid evolution and high adaptive capacity, which is due to the high mutation rates for using the error-prone RNA-dependent RNA polymerase (RdRp) during replication. The fidelity of RdRp is closely associated with viral diversity, fitness, and pathogenesis. Previous studies have shown that the second human pegivirus (HPgV-2) exhibits a highly conserved genome sequence and low intrahost variation, which might be due to the fidelity of HPgV-2 RdRp. In this work, we used a series of in vitro RNA polymerase assays to evaluate the in vitro fidelity of HPgV-2 RdRp and compared it with that of HCV RdRp. The results indicated that HPgV-2 RdRp preserves significantly higher fidelity than HCV RdRp, which might contribute to the conservation of the HPgV-2 genome. The unique feature of HPgV-2 RdRp fidelity provides a new model for investigation of viral RdRp fidelity.

Enhanced Transmissibility and Decreased Virulence of HIV-1 CRF07_BC May Explain Its Rapid Expansion in China.

HIV-1 CRF07_BC is one of the most common circulating recombinant forms (CRFs) in China and is becoming increasingly prevalent especially in HIV-infected men who have sex with men (MSM). The reason why this strain expanded so quickly in China remains to be defined. We previously observed that individuals infected with HIV-1 CRF07_BC showed slower disease progression than those infected with HIV-1 subtype B or CRF01_AE. CRF07_BC viruses carry two unique mutations in the p6Gag protein: insertion of PTAPPE sequences downstream of the original Tsg101 binding domain, and deletion of a seven-amino-acid sequence (30PIDKELY36) that partially overlaps with the Alix binding domain. In this study, we confirmed the enhanced transmission capability of CRF07_BC over HIV-1 subtype B or CRF01_AE by constructing HIV-1 transmission networks to quantitatively evaluate the growth rate of transmission clusters of different HIV-1 genotypes. We further determined lower virus infectivity and slower replication of CRF07_BC with aforementioned PTAPPE insertion (insPTAP) and/or PIDKELY deletion (Δ7) in the p6Gag protein, which in turn may increase the pool of people infected with CRF07_BC and the risk of HIV-1 transmission. These new features of CRF07_BC may explain its quick spread and will help adjust prevention strategy of HIV-1 epidemic. IMPORTANCE HIV-1 CRF07_BC is one of the most common circulating recombinant forms (CRFs) in China. The question is why and how CRF07_BC expanded so rapidly remains unknown. To address the question, we explored the transmission capability of CRF07_BC by constructing HIV-1 transmission networks to quantitatively evaluate the growth rate of transmission clusters of different HIV-1 genotypes. We further characterized the role of two unique mutations in CRF07_BC, PTAPPE insertion (insPTAP) and/or PIDKELY deletion (Δ7) in the p6Gag in virus replication. Our results help define the molecular mechanism regarding the association between the unique mutations and the slower disease progression of CRF07_BC as well as the quick spread of CRF07_BC in China.

Influence of Combination Antiretroviral Therapy on HIV-1 Serological Responses and Their Implications: A Systematic Review and Meta-Analysis.

We aimed to analyze HIV-1 seroreversion caused by combination antiretroviral therapy (cART) and to explore antibody levels of anti-HIV-1 as an alternative biomarker of HIV-1 reservoir. We searched PubMed, Embase, the Cochrane Library, and Web of Science up to August 2021 for publications about the performance of HIV-1 serological assays or the association between antibody responses against HIV-1 and HIV-1 reservoirs. Potential sources of heterogeneity were explored by meta-regression analysis, including the year of publication, country, pretreatment viral load, sample size, the timing of treatment, time on cART, and principle or type of serological assay. Twenty-eight eligible studies with a total population of 1,883 were included in the meta-analysis. The pooled frequency of HIV-1 seronegativity is 38.0% (95% CI: 28.0%-49.0%) among children with vertical HIV-1 infection and cART initiation at the age of less than 6 months, while the percentage of HIV-1 seronegativity declined to 1.0% (95% CI: 0%-3.0%) when cART was initiated at the age of >6 months. For adult patients, 16.0% (95% CI: 9.0%-24.0%) of them were serologically negative when cART was initiated at acute/early infection of HIV-1, but the seronegative reaction was rarely detected when cART was started at chronic HIV-1 infection. Substantial heterogeneity was observed among the studies to estimate the frequency of HIV-1 seronegativity in the early-cART population (I2 ≥ 70%, p < 0.05 and all), while mild heterogeneity existed for the deferred-cART subjects. Moreover, anti-HIV-1 antibody response positively correlates with HIV-1 reservoir size with a pooled rho of 0.43 (95% CI: 0.28-0.55), suggesting that anti-HIV antibody level may be a feasible biomarker of HIV-1 reservoir size.

Mechanisms of non-coding RNA-modulated alternative splicing in cancer.

Alternative splicing (AS) is a common and pivotal process for eukaryotic gene expression regulation, which enables a precursor RNA to produce multiple transcript variants with diverse cellular functions. Aberrant AS represents a hallmark of cancer, engaged in all stages of tumorigenesis from initiation to metastasis. Accumulating pieces of evidence have revealed the involvement of non-coding RNAs (ncRNAs) in regulating AS in human cancers. In this review, we overview the underlying mechanisms of non-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) modulated AS at diverse levels in human cancers, and summarize their regulatory functions in tumorigenesis.

Chlamydia trachomatis Stimulation Enhances HIV-1 Susceptibility through the Modulation of a Member of the Macrophage Inflammatory Proteins.

Sexually transmitted infections such as Chlamydia trachomatis can enhance HIV-1 infection. However, the molecular mechanisms modulating the enhancement of HIV-1 infectivity and replication during HIV-1/sexually transmitted infections coinfection remain elusive. In this study, we performed an ex vivo infection of HIV-1 in PBMCs of C. trachomatis‒infected patients and observed a significant increase in HIV-1 p24 levels compared with those in cells from healthy donors. Similarly, C. trachomatis‒stimulated PBMCs from healthy donors showed enhanced susceptibility to HIV-1. C. trachomatis‒stimulated CD4 T cells also harbored more HIV-1 copy numbers. RNA sequencing data revealed the upregulation of CCL3L1/CCL3L3, a paralog of CCL3 in C. trachomatis‒stimulated CD4 T cells infected with HIV-1. Furthermore, an increase in CCL3L1/CCL3L3 expression levels correlated with HIV-1 replication in C. trachomatis‒stimulated cells. However, the addition of exogenous CCL3L1 reduces HIV-1 infection of healthy cells, indicating a dual role of CCL3L1 in HIV-1 infection. Further investigation revealed that a knockout of CCL3L1/CCL3L3 in Jurkat T cells rescued the increased susceptibility of C. trachomatis‒stimulated cells to HIV-1 infection. These results reveal a role for CCL3L1/CCL3L3 in enhancing HIV-1 replication and production and highlight a mechanism for the enhanced susceptibility to HIV-1 among C. trachomatis‒infected patients.

Structural Analysis on the Severe Acute Respiratory Syndrome Coronavirus 2 Non-structural Protein 13 Mutants Revealed Altered Bonding Network With TANK Binding Kinase 1 to Evade Host Immune System.

Mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have made this virus more infectious. Previous studies have confirmed that non-structural protein 13 (NSP13) plays an important role in immune evasion by physically interacting with TANK binding kinase 1 (TBK1) to inhibit IFNß production. Mutations have been reported in NSP13; hence, in the current study, biophysical and structural modeling methodologies were adapted to dissect the influence of major mutations in NSP13, i.e., P77L, Q88H, D260Y, E341D, and M429I, on its binding to the TBK1 and to escape the human immune system. The results revealed that these mutations significantly affected the binding of NSP13 and TBK1 by altering the hydrogen bonding network and dynamic structural features. The stability, flexibility, and compactness of these mutants displayed different dynamic features, which are the basis for immune evasion. Moreover, the binding was further validated using the MM/GBSA approach, revealing that these mutations have higher binding energies than the wild-type (WT) NSP13 protein. These findings thus justify the basis of stronger interactions and evasion for these NSP13 mutants. In conclusion, the current findings explored the key features of the NSP13 WT and its mutant complexes, which can be used to design structure-based inhibitors against the SARS-CoV-2 new variants to rescue the host immune system.

Mutations in SARS-CoV-2 ORF8 Altered the Bonding Network With Interferon Regulatory Factor 3 to Evade Host Immune System.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been continuously mutating since its first emergence in early 2020. These alterations have led this virus to gain significant difference in infectivity, pathogenicity, and host immune evasion. We previously found that the open-reading frame 8 (ORF8) of SARS-CoV-2 can inhibit interferon production by decreasing the nuclear translocation of interferon regulatory factor 3 (IRF3). Since several mutations in ORF8 have been observed, therefore, in the present study, we adapted structural and biophysical analysis approaches to explore the impact of various mutations of ORF8, such as S24L, L84S, V62L, and W45L, the recently circulating mutant in Pakistan, on its ability to bind IRF3 and to evade the host immune system. We found that mutations in ORF8 could affect the binding efficiency with IRF3 based on molecular docking analysis, which was further supported by molecular dynamics simulations. Among all the reported mutations, W45L was found to bind most stringently to IRF3. Our analysis revealed that mutations in ORF8 may help the virus evade the immune system by changing its binding affinity with IRF3.

A point-of-care rapid HIV-1 test using an isothermal recombinase-aided amplification and CRISPR Cas12a-mediated detection.

Human immunodeficiency virus type one (HIV-1) infection is one of the major public health problems worldwide. Effective control of HIV-1 epidemic relies on early diagnosis of HIV-1 infection by using simple, rapid point-of-care test (POCT). An integrated assay was developed and evaluated in this study to combine a real-time isothermal reverse-transcription recombinase-aided amplification (rRT-RAA) and CRISPR Cas12a-mediated detection for HIV-1. The testing results could be directly observed with naked eye using a blue light imager, making it a suitable on-site testing assay. Our preliminary data indicated that the assay was capable of detecting 20 copies of purified HIV-1 DNA or RNA per reaction or as low as 123 copies/ml of HIV-1 viral load in clinical samples. When screening 155 clinical samples with or without HIV-1 infection, the sensitivity and specificity of the rRT-RAA assay were 98.95% (94/95) and 100% (60/60), respectively. The coefficient value was 0.986 when compared with the Chinese FDA approved HIV-1 RT-qPCR assay. Furthermore, the newly developed HIV-1 rRT-RAA assay could detect the major HIV-1 genotypes CRF01_AE, CRF07_BC, CRF08_BC, CRF08_BC and subtype B in China. Our preliminary results indicated that the rRT-RAA assay or its combination with CRISPR Cas12a-mediated detection could serve as a rapid, convenient, and robust assay for HIV-1 detection.

The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta.

The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.

Host cell response and distinct gene expression profiles at different stages of Chlamydia trachomatis infection reveals stage-specific biomarkers of infection.

BACKGROUND: Chlamydia trachomatis is the most common sexually transmitted infection and the bacterial agent of trachoma globally. C. trachomatis undergoes a biphasic developmental cycle involving an infectious elementary body and a replicative reticulate body. Little is currently known about the gene expression dynamics of host cell mRNAs, lncRNAs, and miRNAs at different stages of C. trachomatis development. RESULTS: Here, we performed RNA-seq and miR-seq on HeLa cells infected with C. trachomatis serovar E at 20 h post-infection (hpi) and 44 hpi with or without IFN-γ treatment. Our study identified and validated differentially expressed host cell mRNAs, lncRNAs, and miRNAs during infection. Host cells at 20 hpi showed the most differential upregulation of both coding and non-coding genes while at 44 hpi in the presence of IFN-γ resulted in a dramatic downregulation of a large proportion of host genes. Using RT-qPCR, we validated the top 5 upregulated mRNAs and miRNAs, which are specific for different stages of C. trachomatis development. One of the commonly expressed miRNAs at all three stages of C. trachomatis development, miR-193b-5p, showed significant expression in clinical serum samples of C. trachomatis-infected patients as compared to sera from healthy controls and HIV-1-infected patients. Furthermore, we observed significant upregulation of antigen processing and presentation, and T helper cell differentiation pathways at 20 hpi whereas T cell receptor, mTOR, and Rap1 pathways were modulated at 44 hpi. Treatment with IFN-γ at 44 hpi showed the upregulation of cytokine-cytokine receptor interaction, FoxO signaling, and Ras signaling pathways. CONCLUSIONS: Our study documented transcriptional manipulation of the host cell genomes and the upregulation of stage-specific signaling pathways necessary for the survival of the pathogen and could serve as potential biomarkers in the diagnosis and management of the disease.

A sensitive and rapid chemiluminescence immunoassay for point-of-care testing (POCT) of copeptin in serum based on high-affinity monoclonal antibodies via cytokine-assisted immunization.

Purpose: Antibodies are key reagents in the development of immunoassay. We attempted to develop high-performance CPP immunoassays using high-affinity monoclonal antibodies prepared via cytokine-assisted immunization. Methods: We used fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to assist traditional subcutaneous immunization of preparing high-affinity monoclonal antibodies, and further to develop high-performance immunoassay methods for CPP. Results: This novel immune strategy significantly enhanced immune response against CPP. Six anti-CPP monoclonal antibodies (mAbs) with high affinity were successfully screened and selected for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect CPP in the range of 1.2-1250 pmol L-1 with a detection limit of 6.25 pmol L-1. Significantly, the whole incubation process can be completed in 30 min as compared to about 4.5 hr for the control ELISA kit. Furthermore, this assay exhibited high sensitivity and specificity, low intra-assay and inter-assay coefficients of variation (CVs < 15%). The developed assay was applied in the detection of CPP in 115 random serum samples and results showed a high correlation with data obtained using a commercially available ELISA kit (correlation coefficient, 0.9737). Conclusion: Our assay could be applied in the point-of-care testing of CPP in the serum samples, and also the method developed in this study could be adopted to explore the detection and diagnosis of other biomarkers for various diseases.

Development of anti-Müllerian hormone immunoassay based on biolayer interferometry technology.

Anti-Müllerian hormone (AMH) is a biomarker for the assessment of female fertility. The accurate measurement of the concentration of AMH is relevant for the success of assisted reproductive therapies and diagnosis of clinical cases. In this study, we show that cytokines such as fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), granulocyte-macrophage colony-stimulating factor (GM-CSF), and ß2-microglobulin (ß2M) significantly enhance the immune response against AMH. Two anti-AMH monoclonal antibodies (mAbs) with high affinity were selected by biolayer interferometry (BLI) technology for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect AMH in the range of 0.125~20 ng mL-1 with a detection limit of 0.099 ng mL-1. This immunoassay showed high specificity with no cross-reaction with structurally related proteins and some of the other members of the TGF-ß super family, such as inhibin A, activin A, follicle-stimulating hormone, and luteinizing hormone. The average recovery rates of three different batches were 100.19%, 102.72%, and 103.59%, respectively, with coefficients of variation of less than 12%. The developed assay was applied in the detection of AMH in 69 serum samples from randomly selected patients. Our data showed a high correlation with those obtained using commercially available ELISA kits (correlation coefficient, 0.9831). Hence, we suggest that this immunoassay could find application in the development of POCT for the diagnosis of AMH in clinical samples. Graphical abstract.

Systematic evaluation of C. elegans lincRNAs with CRISPR knockout mutants.

BACKGROUND: Long intergenic RNAs (lincRNAs) play critical roles in eukaryotic cells, but systematic analyses of the lincRNAs of an animal for phenotypes are lacking. We generate CRISPR knockout strains for Caenorhabditis elegans lincRNAs and evaluate their phenotypes. RESULTS: C. elegans lincRNAs demonstrate global features such as shorter length and fewer exons than mRNAs. For the systematic evaluation of C. elegans lincRNAs, we produce CRISPR knockout strains for 155 of the total 170 C. elegans lincRNAs. Mutants of 23 lincRNAs show phenotypes in 6 analyzed traits. We investigate these lincRNAs by phenotype for their gene expression patterns and potential functional mechanisms. Some C. elegans lincRNAs play cis roles to modulate the expression of their neighboring genes, and several lincRNAs play trans roles as ceRNAs against microRNAs. We also examine the regulation of lincRNA expression by transcription factors, and we dissect the pathway by which two transcription factors, UNC-30 and UNC-55, together control the expression of linc-73. Furthermore, linc-73 possesses a cis function to modulate the expression of its neighboring kinesin gene unc-104 and thus plays roles in C. elegans locomotion. CONCLUSIONS: By using CRISPR/cas9 technology, we generate knockout strains of 155 C. elegans lincRNAs as valuable resources for studies in noncoding RNAs, and we provide biological insights for 23 lincRNAs with the phenotypes identified in this study.

Targetable long non-coding RNAs in cancer treatments.

Aberrant expression of many long non-coding RNAs has been observed in various types of cancer, implicating their crucial roles in tumorigenesis and cancer progression. Emerging knowledge with regard to the critical physiological and pathological roles of long non-coding RNAs in cancers makes them potential targets in cancer treatments. In this review, we present a summary of the relatively well studied long non-coding RNAs that are involved in oncogenesis and outline their functions and functional mechanisms. Recent findings that may be utilized in therapeutic intervention are also highlighted. With the fast development in nucleic acid-based therapeutic reagents that can target disease associated RNAs, lncRNAs should be explored as potential targets in cancer treatments.

Loss of miR-83 extends lifespan and affects target gene expression in an age-dependent manner in Caenorhabditis elegans.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the post-transcriptional regulation of protein-coding genes. miRNAs modulate lifespan and the aging process in a variety of organisms. In this study, we identified a role of miR-83 in regulating lifespan of Caenorhabditis elegans. mir-83 mutants exhibited extended lifespan, and the overexpression of miR-83 was sufficient to decrease the prolonged lifespan of the mutants. We observed upregulation of the expression levels of a set of miR-83 target genes in young mir-83 mutant adults; while different sets of genes were upregulated in older mir-83 mutant adults. In vivo assays showed that miR-83 regulated expression of target genes including din-1, spp-9 and col-178, and we demonstrated that daf-16 and din-1 were required for the extension of lifespan in the mir-83 mutants. The regulation of din-1 by miR-83 during aging resulted in the differential expression of din-1 targets such as gst-4 and gst-10. In daf-2 mutants, the expression level of miR-83 was significantly reduced compared to wild-type animals. We identified a role for miR-83 in modulating lifespan in C. elegans and provided molecular insights into its functional mechanism.

Convergent Transcriptional Programs Regulate cAMP Levels in C. elegans GABAergic Motor Neurons.

Both transcriptional regulation and signaling pathways play crucial roles in neuronal differentiation and plasticity. Caenorhabditis elegans possesses 19 GABAergic motor neurons (MNs) called D MNs, which are divided into two subgroups: DD and VD. DD, but not VD, MNs reverse their cellular polarity in a developmental process called respecification. UNC-30 and UNC-55 are two critical transcription factors in D MNs. By using chromatin immunoprecipitation with CRISPR/Cas9 knockin of GFP fusion, we uncovered the global targets of UNC-30 and UNC-55. UNC-30 and UNC-55 are largely converged to regulate over 1,300 noncoding and coding genes, and genes in multiple biological processes, including cAMP metabolism, are co-regulated. Increase in cAMP levels may serve as a timing signal for respecification, whereas UNC-55 regulates genes such as pde-4 to keep the cAMP levels low in VD. Other genes modulating DD respecification such as lin-14, irx-1, and oig-1 are also found to affect cAMP levels.