Reverse electron transfer is activated during aging and contributes to aging and age-related disease.

Mechanisms underlying the depletion of NAD+ and accumulation of reactive oxygen species (ROS) in aging and age-related disorders remain poorly defined. We show that reverse electron transfer (RET) at mitochondrial complex I, which causes increased ROS production and NAD+ to NADH conversion and thus lowered NAD+ /NADH ratio, is active during aging. Genetic or pharmacological inhibition of RET decreases ROS production and increases NAD+ /NADH ratio, extending the lifespan of normal flies. The lifespan-extending effect of RET inhibition is dependent on NAD+ -dependent Sirtuin, highlighting the importance of NAD+ /NADH rebalance, and on longevity-associated Foxo and autophagy pathways. RET and RET-induced ROS and NAD+ /NADH ratio changes are prominent in human induced pluripotent stem cell (iPSC) model and fly models of Alzheimer's disease (AD). Genetic or pharmacological inhibition of RET prevents the accumulation of faulty translation products resulting from inadequate ribosome-mediated quality control, rescues relevant disease phenotypes, and extends the lifespan of Drosophila and mouse AD models. Deregulated RET is therefore a conserved feature of aging, and inhibition of RET may open new therapeutic opportunities in the context of aging and age-related diseases including AD.

The ubiquitin-specific protease USP36 SUMOylates EXOSC10 and promotes the nucleolar RNA exosome function in rRNA processing.

The RNA exosome is an essential 3' to 5' exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3' to 5' exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.

Histone methylation-mediated microRNA-32-5p down-regulation in sensory neurons regulates pain behaviors via targeting Cav3.2 channels.

microRNA (miRNA)­mediated gene regulation has been studied as a therapeutic approach, but its functional regulatory mechanism in neuropathic pain is not well understood. Here, we identify that miRNA-32-5p (miR-32-5p) is a functional RNA in regulating trigeminal-mediated neuropathic pain. High-throughput sequencing and qPCR analysis showed that miR-32-5p was the most down-regulated miRNA in the injured trigeminal ganglion (TG) of rats. Intra-TG injection of miR-32-5p agomir or overexpression of miR-32-5p by lentiviral delivery in neurons of the injured TG attenuated established trigeminal neuropathic pain. miR-32-5p overexpression did not affect acute physiological pain, while miR-32-5p down-regulation in intact rats was sufficient to cause pain-related behaviors. Nerve injury increased the methylated histone occupancy of binding sites for the transcription factor glucocorticoid receptor in the miR-32-5p promoter region. Inhibition of the enzymes that catalyze H3K9me2 and H3K27me3 restored the expression of miR-32-5p and markedly attenuated pain behaviors. Further, miR-32-5p­targeted Cav3.2 T-type Ca2+ channels and decreased miR-32-5p associated with neuropathic pain caused an increase in Cav3.2 protein expression and T-type channel currents. Conversely, miR-32-5p overexpression in injured TG suppressed the increased expression of Cav3.2 and reversed mechanical allodynia. Together, we conclude that histone methylation-mediated miR-32-5p down-regulation in TG neurons regulates trigeminal neuropathic pain by targeting Cav3.2 channels.

Perovskite Quantum Dots Lasing in Double-Heterostructure through Energy Transfer.

Planar double heterostructures were initially investigated and have been successfully applied in III-V semiconductor lasers due to their excellent roles in confining both the photons and carriers. Here, we design and fabricate a (PEA)2Csn-1PbnX3n+1 (quasi-2D)/CsPbBr3 QD/quasi-2D double-heterostructure sandwiched in a 3/2 λ DBR microcavity, and then demonstrate a single-mode pure-green lasing with a threshold of 53.7 µJ/cm2 under nanosecond-pulsed optical pumping. The thresholds of these heterostructure devices decrease statistically by about 50% compared to the control group with no energy donor layers, PMMA/QD/PMMA in an identical microcavity. We show that there is efficient energy transfer from the barrier regions of the quasi-2D phases to the QD layer by transient absorption and luminescence lifetime spectra and that such energy transfer leads to marked threshold reduction. This work indicates that the double-heterostructure configurations should play a significant role in the future perovskite electrically pumped laser.

Pirin Promotes the Progression of Non-Small-Cell Lung Cancer by Increasing ODC1 to Suppress Autophagy.

Non-small-cell lung cancer (NSCLC), a common malignant tumor, requires deeper pathogenesis investigation. Autophagy is an evolutionarily conserved lysosomal degradation process that is frequently blocked during cancer progression. It is an urgent need to determine the novel autophagy-associated regulators in NSCLC. Here, we found that pirin was upregulated in NSCLC, and its expression was positively correlated with poor prognosis. Overexpression of pirin inhibited autophagy and promoted NSCLC proliferation. We then performed data-independent acquisition-based quantitative proteomics to identify the differentially expressed proteins (DEPs) in pirin-overexpression (OE) or pirin-knockdown (KD) cells. Among the pirin-regulated DEPs, ornithine decarboxylase 1 (ODC1) was downregulated in pirin-KD cells while upregulated along with pirin overexpression. ODC1 depletion reversed the pirin-induced autophagy inhibition and pro-proliferation effect in A549 and H460 cells. Immunohistochemistry showed that ODC1 was highly expressed in NSCLC cancer tissues and positively related with pirin. Notably, NSCLC patients with pirinhigh/ODC1high had a higher risk in terms of overall survival. In summary, we identified pirin and ODC1 as a novel cluster of prognostic biomarkers for NSCLC and highlighted the potential oncogenic role of the pirin/ODC1/autophagy axis in this cancer type. Targeting this pathway represents a possible therapeutic approach to treat NSCLC.

Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSCs.

RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing. RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation, RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.

N-glycosylation of SCAP exacerbates hepatocellular inflammation and lipid accumulation via ACSS2-mediated histone H3K27 acetylation.

Sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) is a widely expressed membrane glycoprotein that acts as an important modulator of lipid metabolism and inflammatory stress. N-glycosylation of SCAP has been suggested to modulate cancer development, but its role in nonalcoholic steatohepatitis (NASH) is poorly understood. In this study, the N-glycosylation of SCAP was analyzed by using sequential trypsin proteolysis and glycosidase treatment. The liver cell lines expressing wild-type and N-glycosylation sites mutated SCAP were constructed to investigate the N-glycosylation role of SCAP in regulating inflammation and lipid accumulation as well as the underlying mechanisms. The hepatic SCAP protein levels were significantly increased in C57BL/6J mice fed with Western diet and sugar water (WD + SW) and diabetic db/db mice, which exhibited typical liver steatosis and inflammation accompanied with hyperglycemia. In vitro, the enhanced N-glycosylation by high glucose increased the protein stability of SCAP and hence increased its total protein levels, whereas the ablation of N-glycosylation significantly decreased SCAP protein stability and alleviated lipid accumulation and inflammation in hepatic cell lines. Mechanistically, SCAP N-glycosylation increased not only the SREBP-1-mediated acetyl-CoA synthetase 2 (ACSS2) transcription but also the AMPK-mediated S659 phosphorylation of ACCS2 protein, causing the enhanced ACSS2 levels in nucleus and hence increasing the histone H3K27 acetylation (H3K27ac), which is a key epigenetic modification associated with NASH. Modulating ACSS2 expression or its location in the nuclear abolished the effects of SCAP N-glycosylation on H3K27ac and lipid accumulation and inflammation. In conclusion, SCAP N-glycosylation aggravates inflammation and lipid accumulation through enhancing ACSS2-mediated H3K27ac in hepatocytes.NEW & NOTEWORTHY N-glycosylation of SCAP exacerbates inflammation and lipid accumulation in hepatocytes through ACSS2-mediated H3K27ac. Our data suggest that SCAP N-glycosylation plays a key role in regulating histone H3K27 acetylation and targeting SCAP N-glycosylation may be a new strategy for treating nonalcoholic steatohepatitis (NASH).

6-month consequences of COVID-19 in patients discharged from hospital: a cohort study.

BACKGROUND: The long-term health consequences of COVID-19 remain largely unclear. The aim of this study was to describe the long-term health consequences of patients with COVID-19 who have been discharged from hospital and investigate the associated risk factors, in particular disease severity. METHODS: We did an ambidirectional cohort study of patients with confirmed COVID-19 who had been discharged from Jin Yin-tan Hospital (Wuhan, China) between Jan 7 and May 29, 2020. Patients who died before follow-up; patients for whom follow-up would be difficult because of psychotic disorders, dementia, or readmission to hospital; those who were unable to move freely due to concomitant osteoarthropathy or immobile before or after discharge due to diseases such as stroke or pulmonary embolism; those who declined to participate; those who could not be contacted; and those living outside of Wuhan or in nursing or welfare homes were all excluded. All patients were interviewed with a series of questionnaires for evaluation of symptoms and health-related quality of life, underwent physical examinations and a 6-min walking test, and received blood tests. A stratified sampling procedure was used to sample patients according to their highest seven-category scale during their hospital stay as 3, 4, and 5-6, to receive pulmonary function test, high resolution CT of the chest, and ultrasonography. Enrolled patients who had participated in the Lopinavir Trial for Suppression of SARS-CoV-2 in China received SARS-CoV-2 antibody tests. Multivariable adjusted linear or logistic regression models were used to evaluate the association between disease severity and long-term health consequences. FINDINGS: In total, 1733 of 2469 discharged patients with COVID-19 were enrolled after 736 were excluded. Patients had a median age of 57·0 years (IQR 47·0-65·0) and 897 (52%) were male and 836 (48%) were female. The follow-up study was done from June 16 to Sept 3, 2020, and the median follow-up time after symptom onset was 186·0 days (175·0-199·0). Fatigue or muscle weakness (52%, 855 of 1654) and sleep difficulties (26%, 437 of 1655) were the most common symptoms. Anxiety or depression was reported among 23% (367 of 1616) of patients. The proportions of 6-min walking distance less than the lower limit of the normal range were 17% for those at severity scale 3, 13% for severity scale 4, and 28% for severity scale 5-6. The corresponding proportions of patients with diffusion impairment were 22% for severity scale 3, 29% for scale 4, and 56% for scale 5-6, and median CT scores were 3·0 (IQR 2·0-5·0) for severity scale 3, 4·0 (3·0-5·0) for scale 4, and 5·0 (4·0-6·0) for scale 5-6. After multivariable adjustment, patients showed an odds ratio (OR) of 1·61 (95% CI 0·80-3·25) for scale 4 versus scale 3 and 4·60 (1·85-11·48) for scale 5-6 versus scale 3 for diffusion impairment; OR 0·88 (0·66-1·17) for scale 4 versus scale 3 and OR 1·76 (1·05-2·96) for scale 5-6 versus scale 3 for anxiety or depression, and OR 0·87 (0·68-1·11) for scale 4 versus scale 3 and 2·75 (1·61-4·69) for scale 5-6 versus scale 3 for fatigue or muscle weakness. Of 94 patients with blood antibodies tested at follow-up, the seropositivity (96·2% vs 58·5%) and median titres (19·0 vs 10·0) of the neutralising antibodies were significantly lower compared with at the acute phase. 107 of 822 participants without acute kidney injury and with an estimated glomerular filtration rate (eGFR) of 90 mL/min per 1·73 m2 or more at acute phase had eGFR less than 90 mL/min per 1·73 m2 at follow-up. INTERPRETATION: At 6 months after acute infection, COVID-19 survivors were mainly troubled with fatigue or muscle weakness, sleep difficulties, and anxiety or depression. Patients who were more severely ill during their hospital stay had more severe impaired pulmonary diffusion capacities and abnormal chest imaging manifestations, and are the main target population for intervention of long-term recovery. FUNDING: National Natural Science Foundation of China, Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, National Key Research and Development Program of China, Major Projects of National Science and Technology on New Drug Creation and Development of Pulmonary Tuberculosis, and Peking Union Medical College Foundation.

Coplanar MoS2-MoTe2 Heterojunction With the Same Crystal Orientation.

Two-dimensional (2D) coplanar heterostructure enables high-performance optoelectronic devices, such as p-n heterojunctions. However, realizing site-controllable and shape-specific 2D coplanar heterojunctions composed of two semiconductors with the same crystal orientation still requires the development of new growth methods. Here, a route to fabricate MoS2-MoTe2 coplanar heterojunctions with the same crystal orientation is reported by exploiting the properties of phase transition and atomic rearrangement during the growth of 2H-MoTe2. Raman spectroscopy and electron microscopy techniques reveal the chemical composition and lattice structure of the heterostructure. Both MoS2 and MoTe2 in the heterojunction are single crystals and have the same lattice orientation, and their shapes can be arbitrarily defined by electron beam lithography. Electrical measurements show that the MoS2 and MoTe2 channels exhibit n-type and p-type transfer characteristics, respectively. The coplanar epitaxy technology can be used to prepare more coplanar heterostructures with novel device functions.

Large-Scale Vertically Interconnected Complementary Field-Effect Transistors Based on Thermal Evaporation.

With the rapid development of integrated circuits, there is an increasing need to boost transistor density. In addition to shrinking the device size to the atomic scale, vertically stacked interlayer interconnection technology is also an effective solution. However, realizing large-scale vertically interconnected complementary field-effect transistors (CFETs) has never been easy. Currently-used semiconductor channel synthesis and doping technologies often suffer from complex fabrication processes, poor vertical integration, low device yield, and inability to large-scale production. Here, a method to prepare large-scale vertically interconnected CFETs based on a thermal evaporation process is reported. Thermally-evaporated etching-free Te and Bi2S3 serve as p-type and n-type semiconductor channels and exhibit FET on-off ratios of 103 and 105, respectively. The vertically interconnected CFET inverter exhibits a clear switching behavior with a voltage gain of 17 at a 4 V supply voltage and a device yield of 100%. Based on the ability of thermal evaporation to prepare large-scale uniform semiconductor channels on arbitrary surfaces, repeated upward manufacturing can realize multi-level interlayer interconnection integrated circuits.

Expression and function of cytokine interleukin-22 gene in the tumor microenvironment of triple negative breast cancer.

BACKGROUND: The tumor microenvironment (TME) and interleukin-22 (IL-22) in cytokines have recently attracted much attention due to their potential impact on tumor biology. However, the role of IL-22 in triple negative breast cancer (TNBC) TME is still poorly understood. This article investigated the gene expression and function of IL-22 in TNBC TME. METHODS: Tumor samples from TNBC patients were collected, and adjacent noncancerous tissues were used as controls. A functional test was performed to evaluate the impact of IL-22 for TNBC cells, including proliferation, migration, and apoptosis. RESULTS: IL-22 gene expression in TNBC tumor samples was markedly higher relative to adjacent non-cancerous tissues (P < 0.05). In addition, it was also observed that IL-22facilitated proliferation and migration of TNBC cells, and inhibit apoptosis. This article reveals the role of IL-22 in the TME of TNBC. The up-regulation of IL-22 gene expression in TNBC tumors and its promoting effect on cancer cell invasiveness highlight its potential as a therapeutic target in TNBC treatment strategies. CONCLUSION: The findings suggested that targeting IL-22 and its related pathways can offer new insights for developing effective therapies for TNBC.

Red Meat Intake and the Risk of Cardiovascular Diseases: A Prospective Cohort Study in the Million Veteran Program.

BACKGROUND: Red meat consumption was associated with an increased risk of cardiovascular disease (CVD) in prospective cohort studies and a profile of biomarkers favoring high CVD risk in short-term controlled trials. However, several recent systematic reviews and meta-analyses concluded with no or weak evidence for limiting red meat intake. OBJECTIVES: To prospectively examine the associations between red meat intake and incident CVD in an ongoing cohort study with diverse socioeconomic and racial or ethnic backgrounds. METHODS: Our study included 148,506 participants [17,804 female (12.0%)] who were free of cancer, diabetes, and CVD at baseline from the Million Veteran Program. A food frequency questionnaire measured red meat intakes at baseline. Nonfatal myocardial infarction and acute ischemic stroke were identified through a high-throughput phenotyping algorithm, and fatal CVD events were identified by searching the National Death Index. RESULTS: Comparing the extreme categories of intake, the multivariate-adjusted relative risks of CVD was 1.18 (95% CI: 1.01, 1.38; P-trend < 0.0001) for total red meat, 1.14 (95% CI: 0.96, 1.36; P-trend = 0.01) for unprocessed red meat, and 1.29 (95% CI: 1.04, 1.60; P-trend = 0.003) for processed red meat. We observed a more pronounced positive association between red meat intake and CVD in African American participants than in White participants (P-interaction = 0.01). Replacing 0.5 servings/d of red meat with 0.5 servings/d of nuts, whole grains, and skimmed milk was associated with 14% (RR: 0.86; 95% CI: 0.83, 0.90), 7% (RR: 0.93; 95% CI: 0.89, 0.96), and 4% (RR: 0.96; 95% CI: 0.94, 0.99) lower risks of CVD, respectively. CONCLUSIONS: Red meat consumption is associated with an increased risk of CVD. Our findings support lowering red meat intake and replacing red meat with plant-based protein sources or low-fat dairy foods as a key dietary recommendation for the prevention of CVD.

Sequence-based design and construction of synthetic nanobody library.

Nanobody (Nb), the smallest antibody fragments known to bind antigens, is now widely applied to various studies, including protein structure analysis, bioassay, diagnosis, and biomedicine. The traditional approach to generating specific nanobodies involves animal immunization which is time-consuming and expensive. As the understanding of the antibody repertoire accumulation, the synthetic library, which is devoid of animals, has attracted attention widely in recent years. Here, we describe a synthetic phage display library (S-Library), designed based on the systematic analysis of the next-generation sequencing (NGS) of nanobody repertoire. The library consists of a single highly conserved scaffold (IGHV3S65*01-IGHJ4*01) and complementary determining regions of constrained diversity. The S-Library containing 2.19 × 108 independent clones was constructed by the one-step assembly and rapid electro-transformation. The S-Library was screened against various targets (Nb G8, fusion protein of Nb G8 and green fluorescent protein, bovine serum albumin, ovalbumin, and acetylcholinesterase). In comparison, a naïve library (N-Library) from the source of 13 healthy animals was constructed and screened against the same targets as the S-Library. Binders were isolated from both S-Library and N-Library. The dynamic affinity was evaluated by the biolayer interferometry. The data confirms that the feature of the Nb repertoire is conducive to reducing the complexity of library design, thus allowing the S-Library to be built on conventional reagents and primers.

Reactive Oxygen Species-Responsive Delivery of a Notch Inhibitor to Alleviate Nonalcoholic Steatohepatitis by Inhibiting Hepatic de Novo Lipogenesis and Inflammation.

With the increased prevalence of nonalcoholic steatohepatitis (NASH) in the world, effective pharmacotherapy in clinical practice is still lacking. Previous studies have shown that dibenzazepine (DBZ), a Notch inhibitor, could alleviate NASH development in a mouse model. However, low bioavailability, poor water solubility, and extrahepatic side effects restrict its clinical application. To overcome these barriers, we developed a reactive oxygen species (ROS)-sensitive nanoparticle based on the conjugation of bilirubin to poly(ethylene glycol) (PEG) chains, taking into account the overaccumulation of hepatic ROS in the pathologic state of nonalcoholic steatohepatitis (NASH). The PEGylated bilirubin can self-assemble into nanoparticles in an aqueous solution and encapsulate insoluble DBZ into its hydrophobic cavity. DBZ nanoparticles (DBZ Nps) had good stability, rapidly released DBZ in response to H2O2, and effectively scavenged intracellular ROS of hepatocytes. After systemic administration, DBZ Nps could accumulate in the liver of the NASH mice, extend persistence in circulation, and improve the bioavailability of DBZ. Furthermore, DBZ Nps significantly improved glucose intolerance, relieved hepatic lipid accumulation and inflammation, and ameliorated NASH-induced liver fibrosis. Additionally, DBZ Nps had no significant extrahepatic side effects. Taken together, our results highlight the potential of the ROS-sensitive DBZ nanoparticle as a promising therapeutic strategy for NASH.

Mechanism of HCG regulating epithelial-mesenchymal transition progression in endometrial cells through the FoxO1/miR223-5p/Wnt5α pathway.

RESEARCH QUESTION: Does human chorionic gonadotrophin (HCG) influence endometrial receptivity and epithelial-mesenchymal transition (EMT) via the FoxO1/miR223-5p/Wnt5α pathway? DESIGN: This study aimed to establish the co-culture system of human embryonic trophoblast cell line (HTR-8-Svneo) cells and human endometrial epithelial cell line (HEEC) cells. The expression of Wnt5α protein and EMT-related proteins in HTR-8-Svneo and HEEC cells treated in a gradient-dependent manner using HCG and exosome inhibitor GW4869 were detected in the co-culture system. RESULTS: In the HTR-8-Svneo/HEEC co-culture system, miR223-5p in HEEC cells increased significantly with induction of HTR-8-Svneo cells by 100 IU/ml HCG for 48 h (P = 0.046), and Wnt5α protein decreased significantly in HEEC cells (P = 0.021). Pretreatment of HTR-8-Svneo cells with GW4869, and knockdown of FoxO1 in HTR-8-Svneo cells, significantly inhibited the above effects of HCG on miR223-5p and Wnt5α expression in HEEC cells in the HTR-8-Svneo/HEEC co-culture system. HTR-8-Svneo cells induced with 100 IU/ml HCG for 48 h significantly enhanced the logarithmic phase proliferation activity of HEEC cells in the co-culture system (P < 0.001), while knockdown of FoxO1 in HTR-8-Svneo cells and inhibition of miR223-5p in HEEC cells suppressed proliferation of HEEC cells in the HTR-8-Svneo/HEEC co-culture system (P < 0.001). CONCLUSIONS: HCG exposure induces HTR-8-Svneo cells to up-regulate miR223-5p expression, which enters HEEC cells in the co-culture system through the exosomal pathway, and inhibits Wnt5α expression and the progress of EMT.

A novel homozygous nonsense variant of AK7 is associated with multiple morphological abnormalities of the sperm flagella.

RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.

Design, synthesis, and antiviral activity of 1-aryl-4-arylmethylpiperazine derivatives as Zika virus inhibitors with broad antiviral spectrum.

Zika virus (ZIKV) disease has been given attention due to the risk of congenital microcephaly and neurodevelopmental disorders after ZIKV infection in pregnancy, but no vaccine or antiviral drug is available. Based on a previously reported ZIKV inhibitor ZK22, a series of novel 1-aryl-4-arylmethylpiperazine derivatives was designed, synthesized, and investigated for antiviral activity by quantify cellular ZIKV RNA amount using RT-qPCR method in ZIKV-infected human venous endothelial cells (HUVECs) assay. Structure-activity relationship (SAR) analysis demonstrated that anti-ZIKV activity of 1-aryl-4-arylmethylpiperazine derivatives is not correlated with molecular hydrophobicity, multiple new derivatives with pyridine group to replace the benzonitrile moiety of ZK22 showed stronger antiviral activity, higher ligand lipophilicity efficiency as well as lower cytotoxicity. Two active compounds 13 and 33 were further identified as novel ZIKV entry inhibitors with the potential of oral available. Moreover, both ZK22 and newly active derivatives also possess of obvious inhibition on the viral replication of coronavirus and influenza A virus at low micromolar level. In summary, this work provided better candidates of ZIKV inhibitor for preclinical study and revealed the promise of 1-aryl-4-arylmethylpiperazine chemotype in the development of broad-spectrum antiviral agents.

New meroterpenoids and polyketides from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1 and their anti-inflammatory and SARS-CoV-2 Mpro inhibitory activities.

Seven new meroterpenoids, paraphaeones A-G (1-7), and two new polyketides, paraphaeones H-I (8-9), along with eight known compounds (10-17), were isolated from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1. The structures of 1-9 were identified through the analysis of 1H, 13C, and 2D NMR spectra, assisted by HR-ESI-MS data. Compounds 1 and 7 exhibited a dose-dependent decrease in lactate dehydrogenase levels, with IC50 values of 1.78 µM and 1.54 µM, respectively. Moreover, they inhibited the secretion of IL-1ß and CASP-1, resulting in a reduction in the activity levels of NLRP3 inflammasomes. Fluorescence microscopy results indicated that compound 7 concentration-dependently attenuated cell pyroptosis. Additionally, compounds 4 and 7 showed potential inhibitory effects on the severe acute respiratory syndrome coronavirus-2 main protease (SARS-CoV-2 Mpro), with IC50 values of 10.8 ± 0.9 µM and 12.9 ± 0.7 µM, respectively.

Exploration of costunolide derivatives as potential anti-inflammatory agents for topical treatment of atopic dermatitis by inhibiting MAPK/NF-κB pathways.

Atopic dermatitis (AD) is a common inflammatory disease and it is very difficult to treat. In the present work, a series of costunolide derivatives have been prepared, and in vitro and in vivo anti-inflammatory activities have evaluated. The results showed that most derivatives displayed good inhibition of NO generation with low cytotoxicity, and 7d could inhibit the phosphorylation of P38, P65 NF-κB and IκB-α in LPS-induced RAW264.7 model. The in vivo researches showed that 7d could improve skin injury symptoms, decrease Th2-type cytokine levels, inhibit HIS levels, alleviate scratching and repaire the damaged skin barrier through the inhibition of phosphorylation of MAPK and NF-κB signaling pathways on MC903-induced AD model. Therefore, costunolide derivatives may be new potent anti-AD agents for further study.

Primary care providers' preferences for pay-for-performance programs: a discrete choice experiment study in Shandong China.

BACKGROUND: Pay-for-performance (P4P) schemes are commonly used to incentivize primary healthcare (PHC) providers to improve the quality of care they deliver. However, the effectiveness of P4P schemes can vary depending on their design. In this study, we aimed to investigate the preferences of PHC providers for participating in P4P programs in a city in Shandong province, China. METHOD: We conducted a discrete choice experiment (DCE) with 882 PHC providers, using six attributes: type of incentive, whom to incentivize, frequency of incentive, size of incentive, the domain of performance measurement, and release of performance results. Mixed logit models and latent class models were used for the statistical analyses. RESULTS: Our results showed that PHC providers had a strong negative preference for fines compared to bonuses (- 1.91; 95%CI - 2.13 to - 1.69) and for annual incentive payments compared to monthly (- 1.37; 95%CI - 1.59 to - 1.14). Providers also showed negative preferences for incentive size of 60% of monthly income, group incentives, and non-release of performance results. On the other hand, an incentive size of 20% of monthly income and including quality of care in performance measures were preferred. We identified four distinct classes of providers with different preferences for P4P schemes. Class 2 and Class 3 valued most of the attributes differently, while Class 1 and Class 4 had a relatively small influence from most attributes. CONCLUSION: P4P schemes that offer bonuses rather than fines, monthly rather than annual payments, incentive size of 20% of monthly income, paid to individuals, including quality of care in performance measures, and release of performance results are likely to be more effective in improving PHC performance. Our findings also highlight the importance of considering preference heterogeneity when designing P4P schemes.