CRISPR-iPAS: a novel dCAS13-based method for alternative polyadenylation interference.

Alternative polyadenylation (APA) plays an important role in gene regulation. With the recent application of novel sequencing technology in APA profiling, an ever-increasing number of APA genes/sites have been identified. However, the phenotypic relevance of most of these APA isoforms remains elusive, which is largely due to the lack of a convenient genetics tool for APA interference. To address this issue, herein, an efficient method is developed based on the CRISPR-dCas13 system, termed as CRISPR-iPAS. Out of eight different dCas13 proteins, Porphyromonas gulae (Pgu) dCas13b, is identified as the most effective one in blocking the usage of the polyadenylation site (PAS). With guide RNAs targeting at core regulatory elements, dPguCas13b enabled APA regulation of endogenous genes with different APA types, including tandem 3'UTR, alternative terminal exon, as well as intronic PAS. Finally, we demonstrated that the proposed APA perturbation tool could be used to investigate the functional relevance of APA isoforms.

Green space exposure on depression and anxiety outcomes: A meta-analysis.

BACKGROUND: The development of urbanization has led to emerging mental health issues. Green space was becoming increasingly important for mental health. Previous studies have demonstrated the value of green space for a variety of outcomes connected to mental health. However, uncertainty remains regarding the association between green spaces and the risk of depression and anxiety outcomes. This study aimed to integrate present evidence from observational studies to define the association of exposure to green space with depression and anxiety. METHODS: A thorough electronic search of PubMed, Web of Science and Embase database was performed. We transformed the odds ratio (OR) of different green increments into per 0.1 unit increase in normalized difference vegetation index (NDVI) and per 10% increase in percentage of green space. Cochrane's Q and I2 statistics were used to assess study heterogeneity, and random-effects models were employed to calculate combined effect estimation OR with 95% confidence intervals (CIs). Pooled analysis was completed using Stata 15.0. RESULTS: According to this meta-analysis, a 10% increase in the proportion of green space was linked to a lower risk of depression (merged OR (95% CI) = 0.963 (0.948, 0.979)) and anxiety (merged OR (95% CI) = 0.938 (0.858, 1.025)) and a 0.1 unit increase in NDVI was linked to a lower risk of depression (merged OR (95% CI) = 0.931 (0.887, 0.977)). CONCLUSIONS: Results of this meta-analysis supported improving green space exposure in preventing depression and anxiety. Higher green space exposure might be helpful for depression and anxiety disorders. Therefore, improving or preserving green space should be regarded as a promising intervention for public health.

Circulating Pulmonary-Originated Epithelial Biomarkers for Acute Respiratory Distress Syndrome: A Systematic Review and Meta-Analysis.

Previous studies have found several biomarkers for acute respiratory distress syndrome (ARDS), but the accuracy of most biomarkers is still in doubt due to the occurrence of other comorbidities. In this systematic review and meta-analysis, we aimed to explore ideal ARDS biomarkers which can reflect pathophysiology features precisely and better identify at-risk patients and predict mortality. Web of Science, PubMed, Embase, OVID, and the Cochrane Library were systematically searched for studies assessing the reliability of pulmonary-originated epithelial proteins in ARDS. A total of 32 studies appeared eligible for meta-analysis, including 2654 ARDS/ALI patients in this study. In the at-risk patients' identification group, the highest pooled effect size was observed in Krebs von den Lungren-6 (KL-6) (SMD: 1.17 [95% CI: 0.55, 1.79]), followed by club cell proteins 16 (CC16) (SMD: 0.74 [95% CI: 0.01, 1.46]), and surfactant proteins-D (SP-D) (SMD: 0.71 [95% CI: 0.57, 0.84]). For the mortality prediction group, CC16 exhibited the largest effect size with SMD of 0.92 (95% CI: 0.42, 1.43). Meanwhile, the summary receiver operating characteristic (SROC) of CC16 for ARDS diagnosis reached an AUC of 0.80 (95% CI: 0.76, 0.83). In conclusion, this study provides a ranking system for pulmonary-originated epithelial biomarkers according to their association with distinguishing at-risk patients and predicting mortality. In addition, the study provides evidence for the advantage of biomarkers over traditional diagnostic criteria. The performance of biomarkers may help to clinically improve the ARDS diagnosis and mortality prediction.

Prenatal phthalate exposure and birth size: a systematic review and meta-analysis.

Phthalates are common endocrine disruptors. The placental barrier can be crossed by phthalates and may have a negative impact on the health of the fetus. However, the association between prenatal exposure to phthalates and birth size is still debatable. Here, we performed this meta-analysis to assess the relationship between prenatal phthalates exposure and birth size. Eighteen studies were finally included by searching PubMed, Embase, Scopus, Ovid, and Web of Science databases and standardized regression coefficients and standard errors were used to pool effect size. Our results showed that prenatal exposure to MMP (=-0.04, 95%CI: -0.08, -0.01) and MEP (=-0.01, 95%CI: -0.01, -0.002) was significantly associated with birth weight. However, no significant associations were identified for phthalate exposure with birth length, head circumference and chest circumference. Because the limiting of studies, more high-quality case-control studies or cohort studies are urgently needed to draw the best conclusions.

CASB: a concanavalin A-based sample barcoding strategy for single-cell sequencing.

Sample multiplexing facilitates single-cell sequencing by reducing costs, revealing subtle difference between similar samples, and identifying artifacts such as cell doublets. However, universal and cost-effective strategies are rather limited. Here, we reported a concanavalin A-based sample barcoding strategy (CASB), which could be followed by both single-cell mRNA and ATAC (assay for transposase-accessible chromatin) sequencing techniques. The method involves minimal sample processing, thereby preserving intact transcriptomic or epigenomic patterns. We demonstrated its high labeling efficiency, high accuracy in assigning cells/nuclei to samples regardless of cell type and genetic background, and high sensitivity in detecting doublets by three applications: 1) CASB followed by scRNA-seq to track the transcriptomic dynamics of a cancer cell line perturbed by multiple drugs, which revealed compound-specific heterogeneous response; 2) CASB together with both snATAC-seq and scRNA-seq to illustrate the IFN-γ-mediated dynamic changes on epigenome and transcriptome profile, which identified the transcription factor underlying heterogeneous IFN-γ response; and 3) combinatorial indexing by CASB, which demonstrated its high scalability.

CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6 A and m5 C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m5 C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m5 C sites and together contributed to almost all the m5 C modification in mRNA. Finally, using m1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.

Aerobic Oxidative Cascade Thiolation and Cyclization to Construct Indole-Fused Isoquinolin-6(5H)-one Derivatives in EtOH.

A practical method to construct sulfenylated indole-fused isoquinolin-6(5H)-one derivatives has been developed. Using eco-friendly ethanol as the solvent and air as the oxidant, this reaction could be compatible with sensitive molecular framework. The utility of the product was well illustrated by further transformations. Moreover, the reaction mechanism was investigated by control experiments.

Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region.

BACKGROUND: Recombinant proteins are often engineered with an N-terminal signal peptide, which facilitates their secretion to the oxidising environment of the periplasm (gram-negative bacteria) or the culture supernatant (gram-positive bacteria). A commonly encountered problem is that the signal peptide influences the synthesis and secretion of the recombinant protein in an unpredictable manner. A molecular understanding of this phenomenon is highly sought after, as it could lead to improved methods for producing recombinant proteins in bacterial cell factories. RESULTS: Herein we demonstrate that signal peptides contribute to an unpredictable translation initiation region. A directed evolution approach that selects a new translation initiation region, whilst leaving the amino acid sequence of the signal peptide unchanged, can increase production levels of secreted recombinant proteins. The approach can increase production of single chain antibody fragments, hormones and other recombinant proteins in the periplasm of E. coli. CONCLUSIONS: The study demonstrates that signal peptide performance is coupled to the efficiency of the translation initiation region.

Overexpression of endothelin B receptor in glioblastoma: a prognostic marker and therapeutic target?

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with median survival of 12-15 months. Owing to uncertainty in clinical outcome, additional prognostic marker(s) apart from existing markers are needed. Since overexpression of endothelin B receptor (ETBR) has been demonstrated in gliomas, we aimed to test whether ETBR is a useful prognostic marker in GBM and examine if the clinically available endothelin receptor antagonists (ERA) could be useful in the disease treatment. METHODS: Data from The Cancer Genome Atlas and the Gene Expression Omnibus database were analyzed to assess ETBR expression. For survival analysis, glioblastoma samples from 25 Swedish patients were immunostained for ETBR, and the findings were correlated with clinical history. The druggability of ETBR was assessed by protein-protein interaction network analysis. ERAs were analyzed for toxicity in in vitro assays with GBM and breast cancer cells. RESULTS: By bioinformatics analysis, ETBR was found to be upregulated in glioblastoma patients, and its expression levels were correlated with reduced survival. ETBR interacts with key proteins involved in cancer pathogenesis, suggesting it as a druggable target. In vitro viability assays showed that ERAs may hold promise to treat glioblastoma and breast cancer. CONCLUSIONS: ETBR is overexpressed in glioblastoma and other cancers and may be a prognostic marker in glioblastoma. ERAs may be useful for treating cancer patients.

Ficellomycin: an aziridine alkaloid antibiotic with potential therapeutic capacity.

Ficellomycin is an aziridine antibiotic produced by Streptomyces ficellus, which displays high in vitro activity against Gram-positive bacteria including multidrug resistant strains of Staphylococcus aureus. Compared to currently available antibiotics, ficellomycin exhibits a unique mechanism of action-it impairs the semiconservative DNA replication by inducing the formation of deficient 34S DNA fragments, which lack the ability to integrate into larger DNA pieces and eventually the complete bacterial chromosome. Until recently, some important progress has been made in research on ficellomycin synthesis and biosynthesis, opening the perspective to develop a new generation of antibiotics with better clinical performance than the currently used ones. In this review, we will cover the discovery and biological activity of ficellomycin, its biosynthesis, mode of action, and related synthetic analogs. The role of ficellomycin and its analogs as an important source of drug prototypes will be discussed together with future research prospects.

Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA.

DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure.

[Discussion on the standard of clinical genetic testing report and the consensus of gene testing industry].

The widespread application of next generation sequencing (NGS) in clinical settings has enabled testing, diagnosis, treatment and prevention of genetic diseases. However, many issues have arisen in the meanwhile. One of the most pressing issues is the lack of standards for reporting genetic test results across different service providers. The First Forum on Standards and Specifications for Clinical Genetic Testing was held to address the issue in Shenzhen, China, on October 28, 2017. Participants, including geneticists, clinicians, and representatives of genetic testing service providers, discussed problems of clinical genetic testing services across in China and shared opinions on principles, challenges, and standards for reporting clinical genetic test results. Here we summarize expert opinions presented at the seminar and report the consensus, which will serve as a basis for the development of standards and guidelines for reporting of clinical genetic testing results, in order to promote the standardization and regulation of genetic testing services in China.

Efficacy and safety of mycophenolate mofetil in patients with active moderate-to-severe Graves' orbitopathy.

CONTEXT: Graves' orbitopathy (GO) is a potentially sight-threatening disease for which currently available medical therapy is not reliably successful. Mycophenolate mofetil (MMF) is a selective immunosuppressant used widely in many autoimmune diseases. Preliminary studies have shown that MMF is effective in the treatment of active GO. OBJECTIVE: To evaluate the efficacy and safety of MMF in patients with active moderate-to-severe GO. PATIENTS: One hundred and 74 patients with active moderate-to-severe GO were randomized to receive either MMF or glucocorticoids (GC). MAIN OUTCOME MEASURES: The primary outcome was overall response at the 12th and 24th weeks; the outcome assessments included clinical activity score (CAS), soft tissue involvement, pain, visual acuity, proptosis, diplopia and reduction in eye movements. The secondary outcome was changes in those individual parameters. Adverse effects were recorded at each visit. RESULTS: A greater overall response rate was found in the MMF group compared with the GC group at the 24th week (91·3% vs 67·9%, P = 0·000). MMF therapy showed a better CAS response than GC (92·5% vs 70·5% improved, P < 0·05). Patients treated with MMF showed a significantly improved rate of diplopia and proptosis than patients treated with GC at the 24th week (90·4% and 68·8% improved, respectively). Disease reactivation was not observed in the patients treated with MMF but was observed in five patients after GC therapy. Adverse events occurred in 4 of 80 patients treated with MMF (5%), all of which were mild to moderate. A severe adverse event was only observed in one patient treated with GC but not at all in patients treated with MMF. CONCLUSION: Compared with GC treatment, MMF therapy is more effective and safer for patients with active moderate-to-severe GO.

Metal-free Oxidative Coupling of Aromatic Alkenes with Thiols Leading to (E)-Vinyl Sulfones.

A facile I2O5-mediated direct oxidative coupling of aromatic alkenes with thiols toward vinyl sulfones has been developed under metal-free conditions. This methodology provides a convenient and efficient approach to various (E)-vinyl sulfones from readily available starting materials with excellent regioselectivity. The present oxidative coupling reaction, not only expands the scope of functionalization of alkenes with thiols, but also makes it a practical and powerful complement to traditional methods for the synthesis of (E)-vinyl sulfones.

Small GTPase ARF6 Regulates Endocytic Pathway Leading to Degradation of ATP-Binding Cassette Transporter A1.

OBJECTIVE: ABCA1 (ATP-binding cassette transporter A1) is the principal protein responsible for cellular cholesterol efflux. Abundance and functionality of ABCA1 is regulated both transcriptionally and post-translationally, with endocytosis of ABCA1 being an important element of post-translational regulation. Functional ABCA1 resides on the plasma membrane but can be internalized and either degraded or recycled back to the plasma membrane. The interaction between the degradative and recycling pathways determines the abundance of ABCA1 and may contribute to the efflux of intracellular cholesterol. APPROACH AND RESULTS: Here, we show that the principal pathway responsible for the internalization of ABCA1 leading to its degradation in macrophages is ARF6-dependent endocytic pathway. This pathway was predominant in the regulation of ABCA1 abundance and efflux of plasma membrane cholesterol. Conversely, the efflux of intracellular cholesterol was predominantly controlled by ARF6-independent pathways, and inhibition of ARF6 shifted ABCA1 into recycling endosomes enhancing efflux of intracellular cholesterol. CONCLUSIONS: We conclude that ARF6-dependent pathway is the predominant route responsible for the ABCA1 internalization and degradation, whereas ARF6-independent endocytic pathways may contribute to ABCA1 recycling and efflux of intracellular cholesterol.

Metal-free I2O5-mediated direct construction of sulfonamides from thiols and amines.

A simple and convenient method has been developed for the construction of sulfonamides via I2O5-mediated sulfonylation of amines with arylthiols. The present protocol provides an attractive approach to sulfonamides in moderate to good yields from readily accessible and easy to handle starting materials under mild and metal-free conditions.

MicroRNA-223 coordinates cholesterol homeostasis.

MicroRNAs (miRNAs) regulate a wide variety of biological processes and contribute to metabolic homeostasis. Here, we demonstrate that microRNA-223 (miR-223), an miRNA previously associated with inflammation, also controls multiple mechanisms associated with cholesterol metabolism. miR-223 promoter activity and mature levels were found to be linked to cellular cholesterol states in hepatoma cells. Moreover, hypercholesterolemia was associated with increased hepatic miR-223 levels in athero-prone mice. miR-223 was found to regulate high-density lipoprotein-cholesterol (HDL-C) uptake, through direct targeting and repression of scavenger receptor BI, and to inhibit cholesterol biosynthesis through the direct repression of sterol enzymes 3-hydroxy-3-methylglutaryl-CoA synthase 1 and methylsterol monooxygenase 1 in humans. Additionally, miR-223 was found to indirectly promote ATP-binding cassette transporter A1 expression (mRNA and protein) through Sp3, thereby enhancing cellular cholesterol efflux. Finally, genetic ablation of miR-223 in mice resulted in increased HDL-C levels and particle size, as well as increased hepatic and plasma total cholesterol levels. In summary, we identified a critical role for miR-223 in systemic cholesterol regulation by coordinated posttranscriptional control of multiple genes in lipoprotein and cholesterol metabolism.

Molecular Iodine-Mediated Difunctionalization of Alkenes with Nitriles and Thiols Leading to ß-Acetamido Sulfides.

A direct difunctionalization protocol of alkenes with nitriles and thiols toward ß-acetamido sulfide derivatives has been proposed under metal-free synthesis conditions. The present protocol provides the facile and highly efficient synthesis of various ß-acetamido sulfides in a scaled-up manner with good to excellent yields simply using inexpensive molecular iodine as a catalyst, DMSO as a mild oxidant, and readily available thiols as thiolating reagents.

NBS/DBU mediated one-pot synthesis of α-acyloxyketones from benzylic secondary alcohols and carboxylic acids.

A simple and efficient one-pot NBS/DBU-mediated method has been developed for the synthesis of α-acyloxyketones from various benzylic secondary alcohols and carboxylic acids. Through this methodology, a series of α-acyloxyketones could be obtained in good to excellent yields under mild conditions. Importantly, this new reaction avoids the direct usage of toxic metal catalysts or potentially dangerous peroxide oxidants.

Prion infection impairs cholesterol metabolism in neuronal cells.

Conversion of prion protein (PrP(C)) into a pathological isoform (PrP(Sc)) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrP(C) to the pathological isoform led to PrP(Sc) accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.