MicroRNA‐146b‐5p overexpression attenuates premature ovarian failure in mice by inhibiting the Dab2ip/Ask1/p38‐Mapk pathway and γH2A.X phosphorylation

ObjectiveTo examine the role of high‐fat and high‐sugar (HFHS) diet‐induced oxidative stress, which is a risk factor for various diseases, in premature ovarian failure (POF).Materials and methodsOvarian granulosa cells (OGCs) were isolated from mice and cultured in medium supplemented with HFHS and poly (lactic‐co‐glycolic acid) (PLGA)‐cross‐linked miR‐146b‐5p nanoparticles (miR‐146@PLGA). RNA and protein expression levels were examined using quantitative real‐time polymerase chain reaction and Western blotting, respectively. HFHS diet‐induced POF model mice were administered miR‐146@PLGA.ResultsThe ovarian tissue of mice fed a HFHS diet exhibited the typical pathological characteristics of POF. HFHS supplementation induced oxidative stress injury in the mouse OGCs, activation of the Dab2ip/Ask1/p38‐Mapk signalling pathway and phosphorylation of γH2A.X in vitro and in vivo. The results of the luciferase reporter assay revealed that miR‐146 specifically downregulated p38‐Mapk14 expression. Meanwhile, co‐immunoprecipitation and Western blot analyses revealed that HFHS supplementation upregulated nuclear p38‐Mapk14 expression and consequently enhanced γH2A.X (Ser139) phosphorylation. The HFHS diet‐induced POF mouse model treated with miR‐146@PLGA exhibited downregulated p38‐Mapk14 expression in the OGCs, mitigated OGC ageing and alleviated the symptoms of POF.ConclusionsThis study demonstrated that HFHS supplementation activates the Dab2ip/Ask1/p38‐Mapk signalling pathway and promotes γH2A.X phosphorylation by inhibiting the expression of endogenous miR‐146b‐5p, which results in OGC ageing and POF development.     

Tumour microenvironment‐based molecular profiling reveals ideal candidates for high‐grade serous ovarian cancer immunotherapy

ObjectiveDue to limited immunological profiles of high‐grade serous ovarian cancer (HGSOC), we aimed to characterize its molecular features to determine whether a specific subset that can respond to immunotherapy exists.Materials and MethodsA training cohort of 418 HGSOC samples from TCGA was analysed by consensus non‐negative matrix factorization. We correlated the expression patterns with the presence of immune cell infiltrates, immune regulatory molecules and other genomic or epigenetic features. Two independent cohorts containing 482 HGSOCs and in vitro experiments were used for validation.ResultsWe identified immune and non‐immune groups where the former was enriched in signatures that reflect immune cells, infiltration and PD‐1 signalling (all, P < 0.001), and presented with a lower chromosomal aberrations but increased neoantigens, tumour mutation burden, and microsatellite instability (all, P < 0.05); this group was further refined into two microenvironment‐based subtypes characterized by either immunoactivation or carcinoma‐associated fibroblasts (CAFs) and distinct prognosis. CAFs‐immune subtype was enriched for factors that mediate immunosuppression and promote tumour progression, including highly expressed stromal signature, TGF‐β signalling, epithelial‐mesenchymal transition and tumour‐associated M2‐polarized macrophages (all, P < 0.001). Robustness of these immune‐specific subtypes was verified in validation cohorts, and in vitro experiments indicated that activated‐immune subtype may benefit from anti‐PD1 antibody therapy (P < 0.05).ConclusionOur findings revealed two immune subtypes with different responses to immunotherapy and indicated that some HGSOCs may be susceptible to immunotherapies or combination therapies.     

Vertical distribution of bacterial community diversity in the Greater Khingan Mountain permafrost region

Soil microorganisms are crucial contributors to the function of permafrost ecosystems, as well as the regulation of biogeochemical cycles. However, little is known about the distribution patterns and drivers of high‐latitude permafrost microbial communities subject to climate change and human activities. In this study, the vertical distribution patterns of soil bacterial communities in the Greater Khingan Mountain permafrost region were systematically analyzed via Illumina Miseq high‐throughput sequencing. Bacterial diversity in the active layer was significantly higher than in the permafrost layer. Principal coordinate analysis (PCoA) indicated that the bacterial community structure in the active layer and the permafrost layer was completely separated. Permutational multivariate analysis of variance (PERMANOVA) detected statistically significant differentiation across the different depths. The relative abundance of the dominant phyla Chloroflexi (17.92%–52.79%) and Actinobacteria (6.34%–34.52%) was significantly higher in the permafrost layer than in the active layer, whereas that of Acidobacteria (4.98%–38.82%) exhibited the opposite trend, and the abundance of Proteobacteria (2.49%–22.51%) generally decreased with depth. More importantly, the abundance of bacteria linked to human infectious diseases was significantly higher in the permafrost layer according to Tax4Fun prediction analysis. Redundancy analysis (RDA) showed that ammonium nitrogen (NH₄ ⁺‐N), total organic carbon (TOC), and total phosphorus (TP) were major factors affecting the bacterial community composition. Collectively, our findings provide insights into the soil bacterial vertical distribution patterns and major environmental drivers in high‐latitude permafrost regions, which is key to grasping the response of cold region ecosystem processes to global climate changes.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

CFTR interactome mapping using the mammalian membrane two‐hybrid high‐throughput screening system

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high‐throughput screening variant of the Mammalian Membrane Two‐Hybrid (MaMTH‐HTS) to map the protein–protein interactions of wild‐type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient‐specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient‐derived intestinal organoids has a significant effect on CFTR functional expression.     

FastD: Fast detection of insecticide target‐site mutations and overexpressed detoxification genes in insect populations from RNA‐Seq data

Target‐site mutations and detoxification gene overexpression are two major mechanisms conferring insecticide resistance. Molecular assays applied to detect these resistance genetic markers are time‐consuming and with high false‐positive rates. RNA‐Seq data contains information on the variations within expressed genomic regions and expression of detoxification genes. However, there is no corresponding method to detect resistance markers at present. Here, we collected 66 reported resistance mutations of four insecticide targets (AChE, VGSC, RyR, and nAChR) from 82 insect species. Next, we obtained 403 sequences of the four target genes and 12,665 sequences of three kinds of detoxification genes including P450s, GSTs, and CCEs. Then, we developed a Perl program, FastD, to detect target‐site mutations and overexpressed detoxification genes from RNA‐Seq data and constructed a web server for FastD (http://www.insect-genome.com/fastd). The estimation of FastD on simulated RNA‐Seq data showed high sensitivity and specificity. We applied FastD to detect resistant markers in 15 populations of six insects, Plutella xylostella, Aphis gossypii, Anopheles arabiensis, Musca domestica, Leptinotarsa decemlineata and Apis mellifera. Results showed that 11 RyR mutations in P. xylostella, one nAChR mutation in A. gossypii, one VGSC mutation in A. arabiensis and five VGSC mutations in M. domestica were found to be with frequency difference >40% between resistant and susceptible populations including previously confirmed mutations G4946E in RyR, R81T in nAChR and L1014F in VGSC. And 49 detoxification genes were found to be overexpressed in resistant populations compared with susceptible populations including previously confirmed detoxification genes CYP6BG1, CYP6CY22, CYP6CY13, CYP6P3, CYP6M2, CYP6P4 and CYP4G16. The candidate target‐site mutations and detoxification genes were worth further validation. Resistance estimates according to confirmed markers were consistent with population phenotypes, confirming the reliability of this program in predicting population resistance at omics‐level.     

Molecular evidence suggesting the persistence of residual SARS‐CoV‐2 and immune responses in the placentas of pregnant patients recovered from COVID‐19

ObjectivesRecent studies have shown the presence of SARS‐CoV‐2 in the tissues of clinically recovered patients and persistent immune symptoms in discharged patients for up to several months. Pregnant patients were shown to be a high‐risk group for COVID‐19. Based on these findings, we assessed SARS‐CoV‐2 nucleic acid and protein retention in the placentas of pregnant women who had fully recovered from COVID‐19 and cytokine fluctuations in maternal and foetal tissues.Materials and MethodsRemnant SARS‐CoV‐2 in the term placenta was detected using nucleic acid amplification and immunohistochemical staining of the SARS‐CoV‐2 protein. The infiltration of CD14+ macrophages into the placental villi was detected by immunostaining. The cytokines in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens at delivery were profiled using the Luminex assay.ResultsResidual SARS‐CoV‐2 nucleic acid and protein were detected in the term placentas of recovered pregnant women. The infiltration of CD14+ macrophages into the placental villi of the recovered pregnant women was higher than that in the controls. Furthermore, the cytokine levels in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens fluctuated significantly.ConclusionsOur study showed that SARS‐CoV‐2 nucleic acid (in one patient) and protein (in five patients) were present in the placentas of clinically recovered pregnant patients for more than 3 months after diagnosis. The immune responses induced by the virus may lead to prolonged and persistent symptoms in the maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid.     

Human breast milk‐derived exosomes through inhibiting AT II cell apoptosis to prevent bronchopulmonary dysplasia in rat lung

Human breast milk (HBM) effectively prevents and cures neonatal bronchopulmonary dysplasia (BPD). Exosomes are abundant in breast milk, but the function of HBM‐derived exosomes (HBM‐Exo) in BPD is still unclear. This study was to investigate the role and mechanism of HBM‐Exo in BPD. Overall lung tissue photography and H&E staining showed that HBM‐Exo improved the lung tissue structure collapse, alveolar structure disorder, alveolar septum width, alveolar number reduction and other injuries caused by high oxygen exposure. Immunohistochemical results showed that HBM‐Exo improved the inhibition of cell proliferation and increased apoptosis caused by hyperoxia. qPCR and Western blot results also showed that HBM‐Exo improved the expression of Type II alveolar epithelium (AT II) surface marker SPC. In vivo study, CCK8 and flow cytometry showed that HBM‐Exo improved the proliferation inhibition and apoptosis of AT II cells induced by hyperoxia, qPCR and immunofluorescence also showed that HBM‐Exo improved the down‐regulation of SPC. Further RNA‐Seq results in AT II cells showed that a total of 88 genes were significantly different between the hyperoxia and HBM‐Exo with hyperoxia groups, including 24 up‐regulated genes and 64 down‐regulated genes. KEGG pathway analysis showed the enrichment of IL‐17 signalling pathway was the most significant. Further rescue experiments showed that HBM‐Exo improved AT II cell damage induced by hyperoxia through inhibiting downstream of IL‐17 signalling pathway (FADD), which may be an important mechanism of HBM‐Exo in the prevention and treatment of BPD. This study may provide new approach in the treatment of BPD.     

Nonproductive exposure of PBMCs to SARS‐CoV‐2 induces cell‐intrinsic innate immune responses

Cell‐intrinsic responses mounted in PBMCs during mild and severe COVID‐19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS‐CoV and SARS‐CoV‐2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT–PCR experiments and single‐cell RNA sequencing revealed JAK/STAT‐dependent induction of interferon‐stimulated genes (ISGs) but not proinflammatory cytokines. This SARS‐CoV‐2‐specific response was most pronounced in monocytes. SARS‐CoV‐2‐RNA‐positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS‐CoV‐2‐specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS‐CoV‐2‐ and, to a much lesser extent, SARS‐CoV particles stimulate JAK/STAT‐dependent, monocyte‐accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID‐19.     

Chloride channel accessory 1 integrates chloride channel activity and mTORC1 in aging‐related kidney injury

The mechanism of kidney injury in aging are not well understood. In order to identify hitherto unknown pathways of aging‐related kidney injury, we performed RNA‐Seq on kidney extracts of young and aged mice. Expression of chloride (Cl) channel accessory 1 (CLCA1) mRNA and protein was increased in the kidneys of aged mice. Immunostaining showed a marked increase in CLCLA1 expression in the proximal tubules of the kidney from aged mice. Increased kidney CLCA1 gene expression also correlated with aging in marmosets and in a human cohort. In aging mice, increased renal cortical CLCA1 content was associated with hydrogen sulfide (H₂S) deficiency, which was ameliorated by administering sodium hydrosulfide (NaHS), a source of H₂S. In order to study whether increased CLCA1 expression leads to injury phenotype and the mechanisms involved, stable transfection of proximal tubule epithelial cells overexpressing human CLCA1 (hCLCA1) was performed. Overexpression of hCLCA1 augmented Cl⁻ current via the Ca⁺⁺‐dependent Cl⁻ channel TMEM16A (anoctamin‐1) by patch‐clamp studies. hCLCA1 overexpression also increased the expression of fibronectin, a matrix protein, and induced the senescence‐associated secretory phenotype (SASP). Mechanistic studies underlying these changes showed that hCLCA1 overexpression leads to inhibition of AMPK activity and stimulation of mTORC1 as cellular signaling determinants of injury. Both TMEM16A inhibitor and NaHS reversed these signaling events and prevented changes in fibronectin and SASP. We conclude that CLCA1‐TMEM16A‐Cl⁻ current pathway is a novel mediator of kidney injury in aging that is regulated by endogenous H₂S.     

Predictable fold switching by the SARS‐CoV‐2 protein ORF9b

Extant fold‐switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are associated with a number of diseases, including tuberculosis, cancer, Alzheimer''s, and autoimmune disorders. Thus, predictive methods are needed to identify more fold‐switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high‐throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS‐CoV‐1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high‐throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.     

RNA‐binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral activity

The type I interferon (IFN‐I, IFN‐α/β)‐mediated immune response is the first line of host defense against invading viruses. IFN‐α/β binds to IFN‐α/β receptors (IFNARs) and triggers the expression of IFN‐stimulated genes (ISGs). Thus, stabilization of IFNARs is important for prolonging antiviral activity. Here, we report the induction of an RNA‐binding motif‐containing protein, RBM47, upon viral infection or interferon stimulation. Using multiple virus infection models, we demonstrate that RBM47 has broad‐spectrum antiviral activity in vitro and in vivo. RBM47 has no noticeable impact on IFN production, but significantly activates the IFN‐stimulated response element (ISRE) and enhances the expression of interferon‐stimulated genes (ISGs). Mechanistically, RBM47 binds to the 3''UTR of IFNAR1 mRNA, increases mRNA stability, and retards the degradation of IFNAR1. In summary, this study suggests that RBM47 is an interferon‐inducible RNA‐binding protein that plays an essential role in enhancing host IFN downstream signaling.