植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...     

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. β-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.     

Receptor for advanced glycation end products aggravates cognitive deficits in type 2 diabetes through binding of C‐terminal AAs 2‐5 to mitogen‐activated protein kinase kinase 3 (MKK3) and facilitation of MEKK3‐MKK3‐p38 module assembly

In this study, we explored the precise mechanisms underlying the receptor for advanced glycation end products (RAGE)‐mediated neuronal loss and behavioral dysfunction induced by hyperglycemia. We used immunoprecipitation (IP) and GST pull‐down assays to assess the interaction between RAGE and mitogen‐activated protein kinase kinase 3 (MKK3). Then, we investigated the effect of specific mutation of RAGE on plasticity at hippocampal synapses and behavioral deficits in db/db mice through electrophysiological recordings, morphological assays, and behavioral tests. We discovered that RAGE binds MKK3 and that this binding is required for assembly of the MEKK3‐MKK3‐p38 signaling module. Mechanistically, we found that activation of p38 mitogen‐activated protein kinase (MAPK)/NF‐κB signaling depends on mediation of the RAGE‐MKK3 interaction by C‐terminal RAGE (ctRAGE) amino acids (AAs) 2‐5. We found that ctRAGE R2A‐K3A‐R4A‐Q5A mutation suppressed neuronal damage, improved synaptic plasticity, and alleviated behavioral deficits in diabetic mice by disrupting the RAGE‐MKK3 conjugation. High glucose induces direct binding of RAGE and MKK3 via ctRAGE AAs 2‐5, which leads to assembly of the MEKK3‐MKK3‐p38 signaling module and subsequent activation of the p38MAPK/NF‐κB pathway, and ultimately results in diabetic encephalopathy (DE).     

CHCHD2 and CHCHD10 regulate mitochondrial dynamics and integrated stress response

Mitochondrial dysfunction is becoming one of the main pathology factors involved in the etiology of neurological disorders. Recently, mutations of the coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) which encode two homologous proteins that belong to the mitochondrial CHCH domain protein family, are linked to Parkinson’s disease and amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), respectively. However, the physiological and pathological roles of these twin proteins have not been well elaborated. Here, we show that, in physiological conditions, CHCHD2 and CHCHD10 interact with OMA1 and suppress its enzyme activity, which not only restrains the initiation of the mitochondrial integrated response stress (mtISR), but also suppresses the processing of OPA1 for mitochondrial fusion. Further, during mitochondria stress-induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, CHCHD2 and CHCHD10 translocate to the cytosol and interacte with eIF2a, which attenuates mtISR overactivation by suppressing eIF2a phosphorylation and its downstream response. As such, knockdown of CHCHD2 and CHCHD10 triggers mitochondrial ISR, and such cellular response is enhanced by CCCP treatment. Therefore, our findings demonstrate the first “mtISR suppressor” localized in mitochondria for regulating stress responses in mammalian cells, which has a profound pathological impact on the CHCH2/CHCH10-linked neurodegenerative disorder.Subject terms: Stress signalling, Mitochondria     

海巴戟ACO基因启动子的克隆及功能初步验证

为了进一步明确海巴戟(Morinda citrifolia Linn.)ACO基因(GenBank登录号:ARB05681.1)功能,本研究通过染色体步移法克隆得到2 066 bp启动子.该区域除了含有大量TATA-box、CAAT-box等启动子核心元件外,还含有乙烯等植物激素响应元件、胁迫和防御相关元件、光相关元件...     

The mRNA–miRNA–lncRNA Regulatory Network and Factors Associated with Prognosis Prediction of Hepatocellular Carcinoma

The aim of this study was to identify novel prognostic mRNA and microRNA (miRNA) biomarkers for hepatocellular carcinoma (HCC) using methods in systems biology. Differentially expressed mRNAs, miRNAs, and long non-coding RNAs (lncRNAs) were compared between HCC tumor tissues and normal liver tissues in The Cancer Genome Atlas (TCGA) database. Subsequently, a prognosis-associated mRNA co-expression network, an mRNA–miRNA regulatory network, and an mRNA–miRNA–lncRNA regulatory network were constructed to identify prognostic biomarkers for HCC through Cox survival analysis. Seven prognosis-associated mRNA co-expression modules were obtained by analyzing these differentially expressed mRNAs. An expression module including 120 mRNAs was significantly correlated with HCC patient survival. Combined with patient survival data, several mRNAs and miRNAs, including CHST4, SLC22A8, STC2, hsa-miR-326, and hsa-miR-21 were identified from the network to predict HCC patient prognosis. Clinical significance was investigated using tissue microarray analysis of samples from 258 patients with HCC. Functional annotation of hsa-miR-326 and hsa-miR-21-5p indicated specific associations with several cancer-related pathways. The present study provides a bioinformatics method for biomarker screening, leading to the identification of an integrated mRNA–miRNA–lncRNA regulatory network and their co-expression patterns in relation to predicting HCC patient survival.     

Determination of nalbuphine by high-performance liquid chromatography with ultraviolet detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with ultraviolet detection was developed for the determination of nalbuphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane-isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of nalbuphine and ethylmorphine (internal standard) were greater than 86%. Calibration graphs were linear over the concentration range 0.75–150 ng/ml with a coefficient of variation, both within-day and between-day, of less than 10% at any level. The limit of quantitation was 0.75 ng/ml of plasma based on a signal-to-noise ratio of 3. Seven other clinically used analgesics were investigated to check for potential interferences and their analytical conditions. The specificity of this assay was checked with a metabolite of nalbuphine (noroxymorphine). Nalbuphine in plasma did not decompose significantly at −20°C for six weeks. Pharmacokinetic application in three surgical patients and four rabbits revealed that nalbuphine followed a linear three-compartment model with two distribution phases. The two distribution and one elimination half-lives and the plasma clearance of nalbuphine were 0.9, 5.8 and 157 min and 370 ml/min in human, and 3.5, 28 and 117 min and 21 166 ml/min in rabbits.     

Characterization of polyclonal antibodies against ovine adipocyte plasma membranes.

1. Antisera against ovine adipocyte plasma membranes were developed in a mare. 2. These antisera showed a high degree of specificity to adipocyte plasma membranes and cross-reacted with other tissues. 3. Antisera cross-reactivity can be removed by adsorption of the antiserum with various tissue plasma membranes without significant reduction in their reactivity to adipocyte plasma membranes. 4. Antisera reacted with different affinity to adipocyte plasma membranes from different sites and from different species of animals.     

Receptome profiling identifies KREMEN1 and ASGR1 as alternative functional receptors of SARS-CoV-2

Host cellular receptors play key roles in the determination of virus tropism and pathogenesis.However,little is known about SARS-CoV-2 host receptors with the e...