Cascaded momentum-space polarization filters enabled label-free black-field microscopy for single nanoparticles analysis.

Label-free optical imaging of single-nanometer-scale matter is extremely important for a variety of biomedical, physical, and chemical investigations. One central challenge is that the background intensity is much stronger than the intensity of the scattering light from single nano-objects. Here, we propose an optical module comprising cascaded momentum-space polarization filters that can perform vector field modulation to block most of the background field and result in an almost black background; in contrast, only a small proportion of the scattering field is blocked, leading to obvious imaging contrast enhancement. This module can be installed in various optical microscopies to realize a black-field microscopy. Various single nano-objects with dimensions smaller than 20 nm appear distinctly in the black-field images. The chemical reactions occurring on single nanocrystals with edge lengths of approximately 10 nm are in situ real-time monitored by using the black-field microscopy. This label-free black-field microscopy is highly promising for a wide range of future multidisciplinary science applications.

Comparative analysis of in-silico tools in identifying pathogenic variants in dominant inherited retinal diseases.

Inherited retinal diseases (IRDs) are a group of rare genetic eye conditions that cause blindness. Despite progress in identifying genes associated with IRDs, improvements are necessary for classifying rare autosomal dominant (AD) disorders. AD diseases are highly heterogenous, with causal variants being restricted to specific amino acid changes within certain protein domains, making AD conditions difficult to classify. Here, we aim to determine the top-performing in-silico tools for predicting the pathogenicity of AD IRD variants. We annotated variants from ClinVar and benchmarked 39 variant classifier tools on IRD genes, split by inheritance pattern. Using area-under-the-curve (AUC) analysis, we determined the top-performing tools and defined thresholds for variant pathogenicity. Top-performing tools were assessed using genome sequencing on a cohort of participants with IRDs of unknown etiology. MutScore achieved the highest accuracy within AD genes, yielding an AUC of 0.969. When filtering for AD gain-of-function and dominant negative variants, BayesDel had the highest accuracy with an AUC of 0.997. Five participants with variants in NR2E3, RHO, GUCA1A, and GUCY2D were confirmed to have dominantly inherited disease based on pedigree, phenotype, and segregation analysis. We identified two uncharacterized variants in GUCA1A (c.428T>A, p.Ile143Thr) and RHO (c.631C>G, p.His211Asp) in three participants. Our findings support using a multi-classifier approach comprised of new missense classifier tools to identify pathogenic variants in participants with AD IRDs. Our results provide a foundation for improved genetic diagnosis for people with IRDs.

Mechanoredox/Nickel Co-Catalyzed Cross Electrophile Coupling of Benzotriazinones with Alkyl (Pseudo)halides.

An air-tolerant mechanoredox/nickel cocatalyzed cross electrophile coupling of benzotriazinones with alkyl (pseudo)halides is developed by liquid-assisting grinding in the presence of manganese powders and strontium titanate as a reductant and a cocatalyst, respectively. Mechanical activation of metal surfaces via ball milling eliminates the chemical activator for manganese, while mechanoredox cocatalysis of strontium titanate remarkably improves the aryl/alkyl cross electrophile coupling via piezoelectricity-mediated radical generation from alkyl halides. Both benzotriazinones and alkyl (pseudo)halides display reactivities in the mechanoredox/nickel cocatalysis different from those of conventional thermal chemistry in solution. The scope of the reaction is demonstrated with 26 examples, showing a high chemoselectivity of bromides vs chlorides.

Chromosomal abnormalities in recurrent pregnancy loss and its association with clinical characteristics.

OBJECTIVE: To evaluate the distribution of chromosomal abnormalities in a recurrent pregnancy loss (RPL) cohort and explore the associations between chromosomal abnormalities and clinical characteristics. METHOD: Over a 5-year period, fresh products of conception (POC) from women with RPL were analyzed by single-nucleotide polymorphism (SNP) array at our hospital. After obtaining the information on clinical characteristics, we investigated the associations between the causative chromosomal abnormalities and clinical characteristics by the chi-squared test or Fisher's exact test and logistic regression. RESULTS: A total of 2383 cases were enrolled. Overall, 56.9% (1355/2383) were identified with causative chromosomal abnormalities, of which 92.1% (1248/1355) were numerical abnormalities, 7.5% (102/1355) were structural variants, and 0.4% (5/1355) were loss of heterozygosity (LOH). The risk of numerical abnormalities was increased in women with maternal age ≥ 35 years (OR, 1.71; 95% CI, 1.41-2.07), gestational age at pregnancy loss ≤ 12 weeks (OR, 2.78; 95% CI, 1.79-4.33), less number of previous pregnancy losses (twice: OR, 2.32; 95% CI, 1.84-2.94; 3 times: OR, 1.59; 95% CI, 1.23-2.05, respectively), and pregnancy with a female fetus (OR, 1.37; 95% CI, 1.15-1.62). The OR of pregnancy loss with recurrent abnormal CMA was 4.00 (95% CI: 1.87-8.58, P < 0.001) and the adjusted OR was 5.05 (95% CI: 2.00-12.72, P = 0.001). However, the mode of conception was not associated with the incidence of numerical abnormality. No association was noted between structural variants and clinical characteristics. CONCLUSION: Chromosomal abnormality was the leading cause of RPL. Numerical chromosome abnormality was more likely to occur in cases with advanced maternal age, an earlier gestational age, fewer previous pregnancy losses, and pregnancy with a female fetus.

Nickel-Catalyzed Suzuki Coupling of Phenols Enabled by SuFEx of Tosyl Fluoride.

A practical and efficient Suzuki coupling of phenols has been developed by using trans-NiCl(o-Tol)(PCy3)2/2PCy3 as a catalyst in the presence of tosyl fluoride as an activator. The key for the direct use of phenols lies in the compatibility of the nickel catalyst with tosyl fluoride (TsF) and its sulfur(VI) fluoride exchange (SuFEx) with CAr-OH. Water has been found to improve the one-pot process remarkably. The steric and electronic effects and the functional group compatibility of the one-pot Suzuki coupling of phenols appear to be comparable to the conventional one of pre-prepared aryl tosylates. A series of electronically and sterically various biaryls could be obtained in good to excellent yields by using 3-10 mol% loading of the nickel catalyst. The applications of this one-pot procedure in chemoselective derivatization of complex molecules have been demonstrated in 3-phenylation of estradiol and estrone.

Pen-2 Negatively Regulates the Differentiation of Oligodendrocyte Precursor Cells into Astrocytes in the Central Nervous System.

Mutations on γ-secretase subunits are associated with neurologic diseases. Whereas the role of γ-secretase in neurogenesis has been intensively studied, little is known about its role in astrogliogenesis. Recent evidence has demonstrated that astrocytes can be generated from oligodendrocyte precursor cells (OPCs). However, it is not well understood what mechanism may control OPCs to differentiate into astrocytes. To address the above questions, we generated two independent lines of oligodendrocyte lineage-specific presenilin enhancer 2 (Pen-2) conditional KO mice. Both male and female mice were used. Here we demonstrate that conditional inactivation of Pen-2 mediated by Olig1-Cre or NG2-CreERT2 causes enhanced generation of astrocytes. Lineage-tracing experiments indicate that abnormally generated astrocytes are derived from Cre-expressing OPCs in the CNS in Pen-2 conditional KO mice. Mechanistic analysis reveals that deletion of Pen-2 inhibits the Notch signaling to upregulate signal transducer and activator of transcription 3, which triggers activation of GFAP to promote astrocyte differentiation. Together, these novel findings indicate that Pen-2 regulates the specification of astrocytes from OPCs through the signal transducer and activator of transcription 3 signaling.SIGNIFICANCE STATEMENT Astrocytes and oligodendrocyte (OLs) play critical roles in the brain. Recent evidence has demonstrated that astrocytes can be generated from OL precursor cells (OPCs). However, it remains poorly understood what mechanism governs the differentiation of OPCs into astrocytes. In this study, we took advantage of OL lineage cells specific presenilin enhancer 2 (Pen-2) conditional KO mice. We show that deletion of Pen-2 leads to dramatically enhanced astrocyte differentiation from OPCs in the CNS. Mechanistic analysis reveals that deletion of Pen-2 inhibits Hes1 and activates signal transducer and activator of transcription 3 to trigger GFAP activation which promotes astrocyte differentiation. Overall, this study identifies a novel function of Pen-2 in astrogliogenesis from OPCs.

Single polymeric microfiber waveguide platform for sensitive detection and discrimination of DNA methylation.

The development of a rapid and sensitive detection platform for DNA and DNA methylation in complex biological environments has attracted considerable attention. Herein, we describe a detection platform for p16 and p16 methylation in buffer and serum based on a single polymeric fluorescent microfiber waveguide with sandwich-structured hybridization designs. The target p16 could be captured by oligonucleotides conjugated on the surface of polymeric microfibers and oligonucleotides conjugated with gold nanoparticles, resulting in quenching the out-coupled tip emission of the microfiber waveguide. Then the restriction digestion enzyme HpaII was applied to specifically recognize the unmethylated 5'-CCGG-3' site and cut the formed sandwich structure. The gold nanoparticles could be removed from the surface of chitosan fiber so that the out-coupled tip emission of the polymeric fluorescent microfiber would be partially recovered. It is noteworthy that the proposed polymeric microfiber waveguide platform exhibited selective and sensitive detection of p16 with a low limit of 2 pM and excellent analytical performance of methylation as low as 5% difference. This strategy avoids the use of traditional PCR-based amplification and tedious operative processes, and we envisage that this technique could be extended to various DNA methylation analyses, which is meaningful for early clinical diagnosis of diseases.

Prenatal diagnosis for fetuses with isolated and non-isolated congenital heart defects using chromosomal microarray and exome sequencing.

OBJECTIVE: To investigate the use of chromosomal microarray (CMA) and Exome sequencing (ES) in fetuses with congenital heart disease (CHD). METHODS: The Fetal Medicine Unit of Shanghai First Maternity and Infant Hospital records were reviewed to ascertain all cases diagnosed with CHD by level 2 ultrasound examination between 2016 and 2019. Cases were categorized as isolated or associated with other abnormalities or fetal growth restriction. CMA was offered to all cases as a first-line genetic test followed by ES when CMA was non-diagnostic. RESULTS: Of the 586 ascertained, 84 (14.3%) had causative CMA abnormality, of which 8.8% (35/400) were in fetuses with isolated CHD and 26.3% (49/186) in those with other abnormalities. ES was performed in 47 cases with a negative CMA. Causative variants were identified in two (10.5%, 2/19) isolated cases and four(14.3%, 4/28) with other abnormalities. CONCLUSION: Invasive procedures with CMA should be offered in pregnancies complicated by both non-isolated and isolated cardiac abnormalities. When CMA is not diagnostic, ES can add diagnostic value in both groups and should be considered even for fetuses with an isolated CHD.

Pregnancy Loss After Amniocentesis with Double-Needle Insertions in Twin Pregnancies.

The aim of this study was to determine the pregnancy loss rate of amniocentesis with double-needle insertions in twin pregnancies. This was a retrospective study of twin pregnancies who underwent amniocentesis with double-needle insertion between 2010 and 2019 at a single center. The pregnancy loss rates were recorded as single or double fetal loss before 24 weeks' gestation and within 4 weeks after the procedure. Risk factors for pregnancy loss after amniocentesis were also assessed. A total of 678 twin pregnancies with amniocentesis were finally included. The pregnancy loss rates before 24 weeks' gestation and within 4 weeks after the procedure were 0.9% and 1.9%, respectively. Only one fetal loss was presumed to be a direct result of the procedure. All other cases were complicated by structural or chromosomal anomalies. Twin pregnancies with abnormal ultrasound findings had a significantly higher rate of pregnancy loss with a relative risk of 4.81 (95% CI [1.03, 22.2]). Our study showed a low pregnancy loss rate after amniocentesis in twin pregnancies with double-needle insertions technique of sampling, which can help decision making in prenatal screening and diagnosis for twin pregnancies.

miR-31-5p from placental and peripheral blood exosomes is a potential biomarker to diagnose preeclampsia.

BACKGROUND: Preeclampsia, a multisystem disorder of unknown etiology, is one of the leading causes of maternal and perinatal morbidity and mortality. Identifying sensitive, noninvasive markers can aid its prevention and improve prognosis. microRNAs (miRs), which function as negative regulators of gene expression, are closely related to preeclampsia occurrence and development. Herein we investigated the relationship between the DLK1-Dio3 imprinted miR cluster derived from placental and peripheral blood exosomes of pregnant women with preeclampsia and routine clinical diagnostic indicators, and also determined its potential as a noninvasive diagnostic marker. METHODS: Exosomes were extracted from the placenta and peripheral blood of pregnant women with preeclampsia. RESULTS: qPCR data indicated that the expression level of miRs, such as miR-134, miR-31-5p, miR-655, miR-412, miR-539, miR-409, and miR-496, in pregnant women with preeclampsia was significantly lower than that in healthy controls; miR-31-5p expression was the most different. Gene ontology analysis predicted that genes negatively regulated by miR-31-5p were mainly enriched in cellular entity, cellular process, and binding; moreover, Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that genes were involved in gonadotropin-releasing hormone receptor pathway and other signaling pathways. Correlation analysis revealed that miR-31-5p was significantly negatively correlated with clinical indicators of preeclampsia, such as systolic and diastolic pressure, lactate dehydrogenase, and proteinuria. CONCLUSION: We believe that exosome-derived miR-31-5p can serve as an effective and sensitive biomarker to determine the course of preeclampsia in pregnant women.

The Value of Exome Sequencing in Thoracoamniotic Shunt for Severe Pleural Effusion with Fetal Hydrops: A Retrospective Clinical Study.

OBJECTIVE: We aimed to study the value of exome sequencing (ES) in severe pleural effusion with nonimmune hydrops fetalis (NIHF) that underwent thoracoamniotic shunt (TAS). METHODS: It was a retrospective study of NIHF that underwent TAS between 2012 and 2020 at Shanghai First Maternity and Infant Hospital. After a detailed assessment, NIHF cases with aneuploidies, infections, and structural anomalies were excluded, and TAS was offered to cases with severe pleural effusion. Quantitative fluorescence polymerase chain reaction (QF-PCR) was conducted to exclude Trisomy 21, 18, and 13 before fetal therapy, and chromosomal microarray analysis (CMA) was offered to all the cases. Before 2019, ES was retrospectively performed using stored fetal DNA extracted from prenatal samples; from 2019 onward, ES was discussed and offered before intrauterine therapies. RESULTS: A total of 18 NIHF cases underwent TAS with negative CMA and continuing pregnancy were included. Fetal hydrops was relieved in 16 cases (88.9%). The median gestational ages at intervention and at delivery were 31.2 (22.0-33.1) weeks and 34.3 (29.7-38.6) weeks, respectively. The neonatal survival rate was 72.2% (13/18), and no causative gene variants were identified from ES in any survivors. Pathogenic or likely pathogenic variants were detected in 3 out of 5 neonatal deaths. If rapid ES could have been available to guide fetal therapy, the neonatal survival rate after TAS would have increased from 72.2% to 86.7%. CONCLUSIONS: Single-gene disorders were one of the major causes of perinatal death in NIHF cases that underwent fetal therapy. Prenatal rapid ES may be of good promise in NIHF to explore precise etiology and guide fetal therapy.

Bright and Near-Unity Polarized Light Emission Enabled by Highly Luminescent Cu2I2-Dimer Cluster-Based Hybrid Materials.

As one fundamental property of light, polarization has a huge impact in quantum optics and optoelectronics through light-matter interactions. However, the bright and near-unity polarized light emissions in the visible range by solid crystalline materials are scantly realized. Here, we report well-defined quasi two-dimensional (2D) hybrid crystals based on the linear alignment of Cu2I2-dimer/bidentate ligand hybrid clusters for achieving bright and near-unity linearly polarized light emissions. Using first-principle calculations, we demonstrate that the superaligned transition dipole moments are the key for the observed excellent polarized light emissions. To further enhance the photoluminescence (PL) polarization degree, we fabricate Cu2I2-dimer-based hybrid nanobelts, which display high PL quantum yield (up to 64%) and ultrahigh PL polarization degree (∼0.99). Our reported copper iodine cluster-based luminescent hybrid materials for bright and highly polarized light emissions will have great potential for future quantum optics applications.

Deletion of PDK1 in oligodendrocyte lineage cells causes white matter abnormality and myelination defect in the central nervous system.

PDK1 (3-Phosphoinositide dependent protein kinase-1) is a member in the PI3K (phosphatidylinositol 3 kinase) pathway and is implicated in neurodevelopmental disease with microcephaly. Although the role of PDK1 in neurogenesis has been broadly studied, it remains unknown how PDK1 may regulate oligogenesis in the central nervous system (CNS). To address this question, we generated oligodendrocyte (OL) lineage cells specific PDK1 conditional knockout (cKO) mice. We find that PDK1 cKOs display abnormal white matter (WM), massive loss of mature OLs and severe defect in myelination in the CNS. In contrast, these mutants exhibit normal neuronal development and unchanged apoptosis in the CNS. We demonstrate that deletion of PDK1 severely impairs OL differentiation. We show that genetic or pharmacological inhibition of PDK1 causes deficit in the mammalian target of rapamycin (mTor) signaling and down-regulation of Sox10. Together, these results highlight a critical role of PDK1 in OL differentiation during postnatal CNS development.

Noncoding mutation in RPGRIP1 contributes to inherited retinal degenerations.

Purpose: Despite the extensive use of next-generation sequencing (NGS) technology to identify disease-causing genomic variations, a major gap in our understanding of Mendelian diseases is the unidentified molecular lesion in a significant portion of patients. For inherited retinal degenerations (IRDs), although currently close to 300 disease-associated genes have been identified, the mutations in approximately one-third of patients remain unknown. With mounting evidence that noncoding mutations might contribute significantly to disease burden, we aimed to systematically investigate the contributions of noncoding regions in the genome to IRDs. Methods: In this study, we focused on RPGRIP1, which has been linked to various IRD phenotypes, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and macular dystrophy (MD). As several noncoding mutant alleles have been reported in RPGRIP1, and we observed that the mutation carrier frequency of RPGRIP1 is higher in patient cohorts with unsolved IRDs, we hypothesized that mutations in the noncoding regions of RPGRIP1 might be a significant contributor to pathogenicity. To test this hypothesis, we performed whole-genome sequencing (WGS) for 25 patients with unassigned IRD who carry a single mutation in RPGRIP1. Results: Three noncoding variants in RPGRIP1, including a 2,890 bp deletion and two deep-intronic variants (c.2710+233G>A and c.1468-263G>C), were identified as putative second hits of RPGRIP1 in three patients with LCA. The mutant alleles were validated with direct sequencing or in vitro assays. Conclusions: The results highlight the significance of the contribution of noncoding pathogenic variants to unsolved IRD cases.

Fetal growth standards for Chinese twin pregnancies.

BACKGROUND: The common use of singleton fetal growth standard to access twin growth might lead to over-monitoring and treatment. We aimed to develop fetal growth standards for Chinese twins based on ultrasound measurements, and compare it with Zhang's and other twin fetal growth charts. METHODS: A cohort of uncomplicated twin pregnancies were prospectively followed in 2014-2017. Smoothed estimates of fetal growth percentiles for both monochorionic (MC) and dichorionic (DC) twins were obtained using a linear mixed model. We also created growth charts for twins using a model-based approach proposed by Zhang et al. Our twin standards were compared with Hadlock's (singleton) in predicting adverse perinatal outcomes. RESULTS: A total of 398 twin pregnancies were included, with 214 MC and 582 DC live-born twins. The MC twins were slightly lighter than the DC twins, with small differences throughout the gestation. Our ultrasound-based fetal weight standards were comparable to that using Zhang's method. Compared with previous references/standards from the US, Brazil, Italy and UK, our twins had very similar 50th percentiles, but narrower ranges between the 5th and 95th or 10th and 90th percentiles. Compared with the Hadlock's standard, the risks of neonatal death and adverse perinatal outcomes for small for gestational age (SGA) versus non-SGA were substantially elevated using our standards. CONCLUSIONS: A normal fetal growth standard for Chinese twins was created. The differences between MC and DC twins were clinically insignificant. The 50th weight percentiles of the Chinese twins were identical to those in other races/ethnicities but the ranges were markedly narrower. Our standard performed much better than the Hadlock's in predicting low birth weight infants associated with adverse perinatal outcomes in twin pregnancies. The present study also indicated that Zhang's method is applicable to Chinese twins, and other areas may use Zhang's method to generate their own curves for twins if deemed necessary.

The abnormal fractional amplitude of low-frequency fluctuation changes in patients with diabetic optic neuropathy: a steady-state fMRI study.

The spontaneous changes in brain activity in patients with diabetic optic neuropathy using steady-state fMRI. The fractional amplitude of the low-frequency fluctuation method was applied to evaluate neural activity changes. The Hospital Anxiety and Depression Scale was used to assess the anxiety and depression status of participants. The independent sample t-test and chi-squared test were applied to analyze the demographics of diabetic optic neuropathy patients and healthy controls. Receiver operating characteristic curves were applied to analyze the variation in mean fractional amplitude of low-frequency fluctuation values between diabetic optic neuropathy patients and healthy controls. Pearson's correlation analysis analyzed the relationships between the fractional amplitude of low-frequency fluctuation values of brain regions and clinical behaviors in the diabetic optic neuropathy group. The fractional amplitude of low-frequency fluctuation value of diabetic optic neuropathy patients was significantly higher than healthy controls in the right precentral gyrus. However, the fractional amplitude of low-frequency fluctuation values in the right anterior cingulate gyrus and left middle cingulate gyrus were markedly decreased in diabetic optic neuropathy patients. The area under the curve of receiver operating characteristics for each brain region showed high accuracy. Pearson's correlation analysis showed that fractional amplitude of low-frequency fluctuation values of the right anterior cingulate gyrus and left middle cingulate gyrus was negatively correlated with Hospital Anxiety and Depression Scale scores. The fractional amplitude of low-frequency fluctuation values of the left middle cingulate gyrus was negatively correlated with diabetic optic neuropathy disease duration. In conclusion, we found abnormal spontaneous brain activities in regions related to cognitive and emotional dysfunction, eye movement disorder, and vision loss in patients with diabetic optic neuropathy. These results may indicate the underlying neuropathological mechanisms of diabetic optic neuropathy and show that fractional amplitude of low-frequency fluctuation may be an effective method to distinguish patients with diabetic optic neuropathy from healthy individuals.

Quadriceps strength and knee joint function in patients with severe knee extension contracture following arthroscopic-assisted mini-incision quadricepsplasty.

PURPOSE: To evaluate reserve quadriceps function and improve knee activity in patients with severe knee extension contracture following arthroscopic-assisted mini-incision quadricepsplasty as well as post-operative complications. METHODS: From 2012 to 2019, 32 patients with severe knee extension contractures (less than 45° range of flexion) were treated with an all-arthroscopic release technique. The clinical results, including range of motion (ROM), quadriceps function (quadriceps index, QI), and knee function, were evaluated, and MRI of the healed tendon after partial quadricepsplasty was performed. The patellar track and length during knee flexion were measured on three normal knees under fluoroscopy. Three formalin-fixed lower limbs were used to mimic severely contracted quadriceps to evaluate the extension of the patellar track. RESULTS: The median follow-up time was 2.1 years (1-5 years). The average QI was 92.0 ± 6.2, and the quadriceps muscle strength was increased from 3.28 to 4.72. At the final follow-up, 90% of the patients had no difficulty going upstairs, going downstairs, or rising from a chair. The ROM improved by 25.69 ± 3.6 preoperatively to 105.88 ± 6.6 at the final follow-up (P < 0.001). The open surgery showed that a 2-cm extension could be achieved by partly cutting the quadriceps tendon, and two cuts achieved a total extension of 5.2 ± 0.52 cm. The patellar tracking distance was 7.7 ± 0.43 cm, and the gap between the patella and femur was also reduced. CONCLUSION: Partial quadricepsplasty of the rectus femoris extended the contracted quadriceps and maintained quadriceps strength, allowing for full knee flexion and satisfactory clinical outcomes of knee function with few complications.

Development of Novel Anti-influenza Thiazolides with Relatively Broad-Spectrum Antiviral Potentials.

Seasonal and pandemic influenza causes 650,000 deaths annually in the world. The emergence of drug resistance to specific anti-influenza virus drugs such as oseltamivir and baloxavir marboxil highlights the urgency of novel anti-influenza chemical entity discovery. In this study, we report a series of novel thiazolides derived from an FDA-approved drug, nitazoxanide, with antiviral activity against influenza and a broad range of viruses. The preferred candidates 4a and 4d showed significantly enhanced anti-influenza virus potentials, with 10-fold improvement compared to results with nitazoxanide, and were effective against a variety of influenza virus subtypes including oseltamivir-resistant strains. Notably, the combination using compounds 4a/4d and oseltamivir carboxylate or zanamivir displayed synergistic antiviral effects against oseltamivir-resistant strains. Mode-of-action analysis demonstrated that compounds 4a/4d acted at the late phase of the viral infection cycle through inhibiting viral RNA transcription and replication. Further experiments showed that treatment with compounds 4a/4d significantly inhibited influenza virus infection in human lung organoids, suggesting the druggability of the novel thiazolides. In-depth transcriptome analysis revealed a series of upregulated cellular genes that may contribute to the antiviral activities of 4a/4d. Together, the results of our study indicated the direction to optimize nitazoxanide as an anti-influenza drug and discovered two candidates with novel structures, compounds 4a/4d, that have relatively broad-spectrum antiviral potentials.

Ebola virus replication is regulated by the phosphorylation of viral protein VP35.

Ebola virus (EBOV) is a zoonotic pathogen, the infection often results in severe, potentially fatal, systematic disease in human and nonhuman primates. VP35, an essential viral RNA-dependent RNA polymerase cofactor, is indispensable for Ebola viral replication and host innate immune escape. In this study, VP35 was demonstrated to be phosphorylated at Serine/Threonine by immunoblotting, and the major phosphorylation sites was S187, S205, T206, S208 and S317 as revealed by LC-MS/MS. By an EBOV minigenomic system, EBOV minigenome replication was shown to be significantly inhibited by the phosphorylation-defective mutant, VP35 S187A, but was potentiated by the phosphorylation mimic mutant VP35 S187D. Together, our findings demonstrate that EBOV VP35 is phosphorylated on multiple residues in host cells, especially on S187, which may contribute to efficient viral genomic replication and viral proliferation.

miR-544 promotes maturity and antioxidation of stem cell-derived endothelial like cells by regulating the YY1/TET2 signalling axis.

BACKGROUND: Inflammation and oxidative stress induced by oxidized low density lipoprotein are the main causes of vascular endothelial injury and atherosclerosis. Endothelial cells are important for the formation and repair of blood vessels. However, the detailed mechanism underlying the regulation of maturity and antioxidation of stem cell-derived endothelial like cells remains unclear. Besides, YY1 and TET2 play a key role on epigenetic modifications of proliferation and differentiation of stem cells. However, the regulatory mechanism of epigenetic modification induced by YY1 and TET2 on stem cells to iECICs is also not clear. AIM: Here, we want to investigate detailed mechanism underlying the regulation of maturity and antioxidation of stem cell-derived iECICs by by YY1 and TET2. METHODS: The qPCR, Western blot, immunohistochemical staining and flow cytometric analysis were used to analyze the expression level of each gene. Luciferase reporter assay was used to detect the binding sites between microRNA and target genes. The hMeDIP-sequence, ChIP-PCR and dot blot were used to detect the 5-hydroxymethylcytosine modification of genomic DNA. ATP, ROS, SOD assay were used to evaluate of oxidative stress in cells. The iECICs transplantation group The ApoE-/- mice were intravenous injected of iECICs to evaluation of therapeutic effect in vivo. RESULTS: Our studies have found that as the differentiation of human amniotic epithelial cells (HuAECs) is directed towards iECICs in vitro, the expression levels of vascular endothelial cell markers and miR-544 increase significantly and the expression level of YinYang 1 (YY1) decreases significantly. The luciferase reporter assay suggests that Yy1 is one of the targets of miR-544. Hydroxymethylated DNA immunoprecipitation sequencing showed that compared with HuAECs, iECICs had 174 protein-coding DNA sequences with extensive hydroxymethylation modifications. Overexpression of miR-544 inhibits the activity of the YY1/PRC2 complex and promotes the transcription and expression of the ten-eleven translocation 2 (TET2) gene, thereby activating the key factors of the serotonergic synapse pathway, CACNA1F, and CYP2D6. In addition, it promotes ability of maturity, antioxidation and vascular formation in vitro. Meanwhile, transplantation for miR-544-iECICs can significantly relieve oxidative stress injury on ApoE-/- atherosclerotic mice in vivo. CONCLUSIONS: miR-544 regulates the maturity and antioxidation of iECICs derived from HuAECs by regulating the YY1/TET2/serotonergic synapse signalling axis. Video abstract.