mRNA-based parallel detection of active methanotroph populations by use of a diagnostic microarray

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.     

Isolation of pure human mucosal epithelium for RNA analysis

Techniques for isolating the desired cell populations from complex tissues are essential for characterizing cells through mRNA analysis. We established a procedure for isolating pure mucosal epithelium from the human alimentary tract. To do this, we made rotating hooks that hold mucosal strips and detach the epithelial sheets from the irregular mucosae surface in medium containing EDTA. An additional step using a cell strainer was required to reduce contamination by lymphoid cells. Sheets of epithelial cells were detached successfully from mucosal samples derived from five different parts of the human alimentary tract. Contamination by lymphoid cells or fibroblasts was monitored by competitive RT-PCR and was no more than 0.5% of the total cells. Total RNA yields were 12.5-17 micrograms for each separation, and the integrity of the RNA was as good as that of RNAs extracted from mucosa immediately after resection. In conclusion, our method permits isolation of RNAs from a pure population of epithelial cells that can be used for mRNA-based gene expression analyses.     

Covaxin: An overview of its immunogenicity and safety trials in India

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global coronavirus disease-19 (COVID-19) pandemic. Several vaccine types, such as inactivated, viral vector-, or mRNA-based, have received approval against SARS-CoV-2. The ability to induceT-helper-1 cell (Th1) responses is desirable from an effective vaccine against this virus. Covaxin (BBV152) is a wholevirion inactivated SARS-CoV-2 vaccine adjuvanted with Algel-Imidazoquinoline (IMDG) molecule, a toll-like receptor (TLR) 7/8 agonist. The mRNA-based vaccine use is hindered because of cold storage requirement, whereas covaxin is stored between 2°C and 8°C, making it suitable for countries with limited resources. The Drug Controller General of India (DCGI) has approved the BBV152 vaccine. Therefore, it is of interest to document known data on BBV152 vaccine phase I, phase II and phase III human clinical trials to evaluate the safety, reactogenicity, tolerance, and immunogenicity of the whole-virion inactivated SARS-CoV-2 vaccine (BBV152).     

Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances

Microbial ecology examines the diversity and activity of micro-organisms in Earth's biosphere. In the last 20 years, the application of genomics tools have revolutionized microbial ecological studies and drastically expanded our view on the previously underappreciated microbial world. This review first introduces the basic concepts in microbial ecology and the main genomics methods that have been used to examine natural microbial populations and communities. In the ensuing three specific sections, the applications of the genomics in microbial ecological research are highlighted. The first describes the widespread application of multilocus sequence typing and representational difference analysis in studying genetic variation within microbial species. Such investigations have identified that migration, horizontal gene transfer and recombination are common in natural microbial populations and that microbial strains can be highly variable in genome size and gene content. The second section highlights and summarizes the use of four specific genomics methods (phylogenetic analysis of ribosomal RNA, DNA-DNA re-association kinetics, metagenomics, and micro-arrays) in analysing the diversity and potential activity of microbial populations and communities from a variety of terrestrial and aquatic environments. Such analyses have identified many unexpected phylogenetic lineages in viruses, bacteria, archaea, and microbial eukaryotes. Functional analyses of environmental DNA also revealed highly prevalent, but previously unknown, metabolic processes in natural microbial communities. In the third section, the ecological implications of sequenced microbial genomes are briefly discussed. Comparative analyses of prokaryotic genomic sequences suggest the importance of ecology in determining microbial genome size and gene content. The significant variability in genome size and gene content among strains and species of prokaryotes indicate the highly fluid nature of prokaryotic genomes, a result consistent with those from multilocus sequence typing and representational difference analyses. The integration of various levels of ecological analyses coupled to the application and further development of high throughput technologies are accelerating the pace of discovery in microbial ecology.     

RT-PCR检测金黄色葡萄球菌

目的探讨检测金黄色葡萄球菌及其活菌的RT-PCR方法。方法用RT-PCR方法对金黄色葡萄球菌的spa基因进行检测,并做灵敏度和特异性测定,用RT-PCR检测细菌灭活前后的spa基因。结果用spa基因检测金黄色葡萄球菌灵敏度为1.5×104CFU/mL;Spa引物能特异性扩增出金黄色葡萄球菌的标准株和14株临床株的目的片段,对大肠埃希菌、铜绿假单胞菌、表皮葡萄球菌和化脓性链球菌则无特异性扩增条带,而对白色念珠菌有较弱条带扩出;细菌灭活前可以检测出目的基因,灭活后4℃放置24、48和72 h均无目的基因片段扩出。结论可以用spa基因对金黄色葡萄球菌进行活菌检测。     

Microorganisms relevant to bioremediation

Naturally occurring microbial consortia have been utilized in a variety of bioremediation processes. Recent developments in molecular microbial ecology offer new tools that facilitate molecular analyses of microbial populations at contaminated and bioremediated sites. Information provided by such analyses aids in the evaluation of the effectiveness of bioremediation and the formulation of strategies that might accelerate bioremediation.     

Antiphospholipid antibodies and risk of post-COVID-19 vaccination thrombophilia: The straw that breaks the camel's back?

Antiphospholipid antibodies (aPLs), present in 1–5 % of healthy individuals, are associated with the risk of antiphospholipid syndrome (APS), which is the most common form of acquired thrombophilia. APLs may appear following infections or vaccinations and have been reported in patients with COronaVIrus Disease-2019 (COVID-19). However, their association with COVID-19 vaccination is unclear. Notably, a few cases of thrombocytopenia and thrombotic events resembling APS have been reported to develop in recipients of either adenoviral vector- or mRNA-based COVID-19 vaccines.The aim of this review is therefore to speculate on the plausible role of aPLs in the pathogenesis of these rare adverse events.Adenoviral vector-based vaccines can bind platelets and induce their destruction in the reticuloendothelial organs. Liposomal mRNA-based vaccines may instead favour activation of coagulation factors and confer a pro-thrombotic phenotype to endothelial cells and platelets. Furthermore, both formulations may trigger a type I interferon response associated with the generation of aPLs. In turn, aPLs may lead to aberrant activation of the immune response with participation of innate immune cells, cytokines and the complement cascade. NETosis, monocyte recruitment and cytokine release may further support endothelial dysfunction and promote platelet aggregation. These considerations suggest that aPLs may represent a risk factor for thrombotic events following COVID-19 vaccination, and deserve further investigations.     

Molecular phylogenetic and chemical analyses of the microbial mats in deep-sea cold seep sediments at the northeastern Japan Sea

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier (¹³C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.     

Influences of space, soil, nematodes and plants on microbial community composition of chalk grassland soils

Microbial communities respond to a variety of environmental factors related to resources (e.g. plant and soil organic matter), habitat (e.g. soil characteristics) and predation (e.g. nematodes, protozoa and viruses). However, the relative contribution of these factors on microbial community composition is poorly understood. Here, we sampled soils from 30 chalk grassland fields located in three different chalk hill ridges of Southern England, using a spatially explicit sampling scheme. We assessed microbial communities via phospholipid fatty acid (PLFA) analyses and PCR-denaturing gradient gel electrophoresis (DGGE) and measured soil characteristics, as well as nematode and plant community composition. The relative influences of space, soil, vegetation and nematodes on soil microorganisms were contrasted using variation partitioning and path analysis. Results indicate that soil characteristics and plant community composition, representing habitat and resources, shape soil microbial community composition, whereas the influence of nematodes, a potential predation factor, appears to be relatively small. Spatial variation in microbial community structure was detected at broad (between fields) and fine (within fields) scales, suggesting that microbial communities exhibit biogeographic patterns at different scales. Although our analysis included several relevant explanatory data sets, a large part of the variation in microbial communities remained unexplained (up to 92% in some analyses). However, in several analyses, significant parts of the variation in microbial community structure could be explained. The results of this study contribute to our understanding of the relative importance of different environmental and spatial factors in driving the composition of soil-borne microbial communities.     

Highly efficient mRNA-based gene transfer in feeder-free cultured H9 human embryonic stem cells

Several protocols have been described for virus-based gene transfer in human embryonic stem (hES) cells, while efficient non-viral methods are currently non-existing. In this study, we investigated the efficiency of mRNA-based gene transfer in feeder-free cultured H9 hES cells, based on electroporation of in vitro transcribed mRNA encoding the enhanced green fluorescent protein (EGFP). Optimisation of culture and electroporation conditions for feeder-free cultured H9 hES cells resulted a highly pure, transgene-expressing (90% positive cells) H9 hES cell population.     

Population genomics in natural microbial communities

Little is known about the evolutionary processes that structure and maintain microbial diversity because, until recently, it was difficult to explore individual-level patterns of variation at the microbial scale. Now, community-genomic sequence data enable such variation to be assessed across large segments of microbial genomes. Here, we discuss how population-genomic analysis of these data can be used to determine how selection and genetic exchange shape the evolution of new microbial lineages. We show that once independent lineages have been identified, such analyses enable the identification of genome changes that drive niche differentiation and promote the coexistence of closely related lineages within the same environment. We suggest that understanding the evolutionary ecology of natural microbial populations through population-genomic analyses will enhance our understanding of genome evolution across all domains of life.     

Application of Meta-Mesh on the analysis of microbial communities from human associated-habitats

With the current fast accumulation of microbial community samples and related metagenomic sequencing data, data integration and analysis system is urgently needed for in-depth analysis of large number of metagenomic samples (also referred to as “microbial communities”) of interest. Although several existing databases have collected a large number of metagenomic samples, they mostly serve as data repositories with crude annotations, and offer limited functionality for analysis. Moreover, the few available tools for comparative analysis in the literature could only support the comparison of a few pre-defined set of metagenomic samples. To facilitate comprehensive comparative analysis on large amount of diverse microbial community samples, we have designed a Meta-Mesh system for a variety of analyses including quantitative analysis of similarities among microbial communities and computation of the correlation between the meta-information of these samples. We have used Meta-Mesh for systematically and efficiently analyses on diverse sets of human associate-habitat microbial community samples. Results have shown that Meta-Mesh could serve well as an efficient data analysis platform for discovery of clusters, biomarker and other valuable biological information from a large pool of human microbial samples.     

土壤微生物生态学研究中的非培养方法

土壤微生物种类和数量是评价土壤健康质量的重要指标之一。然而环境中90%以上的微生物不能够通过传统的培养基培养方法获得。最近发展起来的分析方法如磷脂脂肪酸(PLFA)、BIOLOG微孔板和分子生物学的方法可从不同方面对土壤微生物群落进行更为详尽的分析。论文就土壤微生物生态学研究中使用的主要方法进行了综述。     

An invitation to the marriage of metagenomics and metabolomics

Metagenomics seeks to characterize the composition of microbial communities, their operations, and their dynamically coevolving relationships with the habitats they occupy without having to culture community members. Uniting metagenomics with analyses of the products of microbial community metabolism (metabolomics) will shed light on how microbial communities function in a variety of environments, including the human body.     

应用DGGE研究微生物群落时的常见问题分析

变性梯度凝胶电泳(DGGE)是通过核酸片段对微生物群落进行研究,可以监测未培养细菌及其功能基因,被广泛地应用于微生物群落多样性和动态分析,并成为微生物分子生态学研究中的重要手段之一。文中论述了DGGE操作过程中遇到的常见问题,并提出了相应的解决方法。全面分析了样品预处理过程和PCR扩增效果对DGGE分析的影响,探讨了DGGE图谱的优化过程和图谱分析方法,并对DGGE的应用前景进行了综述。     

MAR-FISH技术及其在环境微生物群落与功能研究中的应用

对复杂环境中微生物群落结构和功能的研究是微生物生态学的重要任务。尽管现代分子生物学技术已经成功地用于解析环境中微生物的群落结构, 但是这些方法并不能提供微生物的原位生理学信息。而一种新的方法, 微观放射自显影和荧光原位杂交集成技术(MAR-FISH)则能够同时在单细胞水平上, 检测复杂环境中微生物的系统发育信息及其生理特性。本文总结了MAR-FISH方法的原理, 实验步骤及其在环境微生物群落与功能研究中的应用。     

Development of compost maturity and Actinobacteria populations during full-scale composting of organic household waste

AIMS: This study investigates changes in microbiological and physicochemical parameters during large-scale, thermophilic composting of a single batch of municipal organic waste. The inter-relationships between the microbial biomass and community structure as well as several physicochemical parameters and estimates of maturation were evaluated. METHODS AND RESULTS: Analyses of signature fatty acids with the phospholipid fatty acid and ester-linked methods showed that the total microbial biomass was highest during the early thermophilic phase. The contribution of signature 10Me fatty acids from Actinobacteria indicated a relatively constant proportion around 10% of the microbial community. However, analyses of the Actinobacteria species composition with a PCR-denaturing gradient gel electrophoresis approach targeting 16S rRNA genes demonstrated clear shifts in the community structure. CONCLUSIONS: This study demonstrates that compost quality, particularly maturity, is linked to the composition of the microbial community structure, but further studies in other full-scale systems are needed to validate the generality of these findings. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of signature lipid and nucleic acid-based analyses greatly expands the specificity and the scope for assessing the microbial community composition in composts. The results presented in this study give new information on how the development of the compost microbial community is connected to curing and maturation in the later stages of composting, and emphasizes the role of Actinobacteria in this respect.