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Little is known about the response of arbuscular mycorrhizal fungal communities to ecosystem development. We use a long‐term soil chronosequence that includes ecosystem progression and retrogression to quantify the importance of host plant identity as a factor driving fungal community composition during ecosystem development. We identified arbuscular mycorrhizal fungi and plant species from 50 individual roots from each of 10 sites spanning 5–120 000 yr of ecosystem age using terminal restriction fragment length polymorphism (T‐RFLP), Sanger sequencing and pyrosequencing. Arbuscular mycorrhizal fungal communities were highly structured by ecosystem age. There was strong niche differentiation, with different groups of operational taxonomic units (OTUs) being characteristic of early succession, ecosystem progression and ecosystem retrogression. Fungal alpha diversity decreased with ecosystem age, whereas beta diversity was high at early stages and lower in subsequent stages. A total of 39% of the variance in fungal communities was explained by host plant and site age, 29% of which was attributed to host and the interaction between host and site (24% and 5%, respectively). The strong response of arbuscular mycorrhizal fungi to ecosystem development appears to be largely driven by plant host identity, supporting the concept that plant and fungal communities are tightly coupled rather than independently responding to habitat.

Past studies of the human intestinal microbiota are potentially confounded by the common practice of using bowel-cleansing preparations. We examined if colonic lavage changes the natural state of enteric mucosal-adherent microbes in healthy human subjects. Twelve healthy individuals were divided into three groups; experimental group, control group one, and control group two. Subjects in the experimental group underwent an un-prepped flexible sigmoidoscopy with biopsies. Within two weeks, subjects were given a standard polyethylene glycol-based bowel cleansing preparation followed by a second flexible sigmoidoscopy. Subjects in control group one underwent two un-prepped flexible sigmoidoscopies within one week. Subjects in the second control group underwent an un-prepped flexible sigmoidoscopy followed by a second flexible sigmoidoscopy after a 24-hour clear liquid diet within one week. The mucosa-associated microbial communities from the two procedures in each subject were compared using 16S rRNA gene based terminal restriction fragment length polymorphism (T-RFLP), and library cloning and sequencing. Clone library sequencing analysis showed that there were changes in the composition of the mucosa-associated microbiota in subjects after colonic lavage. These changes were not observed in our control groups. Standard bowel preparation altered the diversity of mucosa-associated microbiota. Taxonomic classification did not reveal significant changes at the phylum level, but there were differences observed at the genus level. Standard bowel cleansing preparation altered the mucosal-adherent microbiota in all of our subjects, although the degree of change was variable. These findings underscore the importance of considering the confounding effects of bowel preparation when designing experiments exploring the gut microbiota.

Antibiotic administration is the standard treatment for the bacterium Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer. However, the long-term consequences of this treatment on the human indigenous microbiota are relatively unexplored. Here we studied short- and long-term effects of clarithromycin and metronidazole treatment, a commonly used therapy regimen against H. pylori, on the indigenous microbiota in the throat and in the lower intestine. The bacterial compositions in samples collected over a four-year period were monitored by analyzing the 16S rRNA gene using 454-based pyrosequencing and terminal-restriction fragment length polymorphism (T-RFLP). While the microbial communities of untreated control subjects were relatively stable over time, dramatic shifts were observed one week after antibiotic treatment with reduced bacterial diversity in all treated subjects in both locations. While the microbiota of the different subjects responded uniquely to the antibiotic treatment some general trends could be observed; such as a dramatic decline in Actinobacteria in both throat and feces immediately after treatment. Although the diversity of the microbiota subsequently recovered to resemble the pre treatment states, the microbiota remained perturbed in some cases for up to four years post treatment. In addition, four years after treatment high levels of the macrolide resistance gene erm(B) were found, indicating that antibiotic resistance, once selected for, can persist for longer periods of time than previously recognized. This highlights the importance of a restrictive antibiotic usage in order to prevent subsequent treatment failure and potential spread of antibiotic resistance.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in Japan. The etiology of CRC has been linked to numerous factors including genetic mutation, diet, life style, inflammation, and recently, the gut microbiota. However, CRC-associated gut microbiota is still largely unexamined. This study used terminal restriction fragment length polymorphism (T-RFLP) and next-generation sequencing (NGS) to analyze and compare gut microbiota of Japanese control subjects and Japanese patients with carcinoma in adenoma. Stool samples were collected from 49 control subjects, 50 patients with colon adenoma, and 9 patients with colorectal cancer (3/9 with invasive cancer and 6/9 with carcinoma in adenoma) immediately before colonoscopy; DNA was extracted from each stool sample. Based on T-RFLP analysis, 12 subjects (six control and six carcinoma in adenoma subjects) were selected; their samples were used for NGS and species-level analysis. T-RFLP analysis showed no significant differences in bacterial population between control, adenoma and cancer groups. However, NGS revealed that i), control and carcinoma in adenoma subjects had different gut microbiota compositions, ii), one bacterial genus (Slackia) was significantly associated with the control group and four bacterial genera (Actinomyces, Atopobium, Fusobacterium, and Haemophilus) were significantly associated with the carcinoma-in-adenoma group, and iii), several bacterial species were significantly associated with each type (control: Eubacterium coprostanoligens; carcinoma in adenoma: Actinomyces odontolyticus, Bacteroides fragiles, Clostridium nexile, Fusobacterium varium, Haemophilus parainfluenzae, Prevotella stercorea, Streptococcus gordonii, and Veillonella dispar). Gut microbial properties differ between control subjects and carcinoma-in-adenoma patients in this Japanese population, suggesting that gut microbiota is related to CRC prevention and development.

Detection and analysis of genetic variation can help us to understand the molecular basis of various biological phenomena in plants. Since the entire plant kingdom cannot be covered under sequencing projects, molecular markers and their correlation to phenotypes provide us with requisite landmarks for elucidation of genetic variation. Genetic or DNA based marker techniques such as RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) are routinely being used in ecological, evolutionary, taxonomical, phylogenic and genetic studies of plant sciences. These techniques are well established and their advantages as well as limitations have been realized. In recent years, a new class of advanced techniques has emerged, primarily derived from combination of earlier basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques. The newer methods also incorporate modifications in the methodology of basic techniques to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. The advanced marker techniques also utilize newer class of DNA elements such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The review details account of techniques used in identification of markers and their applicability in plant sciences.

The impact of various agricultural practices on soil biodiversity and, in particular, on arbuscular mycorrhizal fungi (AMF), is still poorly understood, although AMF can provide benefit to plants and ecosystems. Here, we tested whether organic farming enhances AMF diversity and whether AMF communities from organically managed fields are more similar to those of species-rich grasslands or conventionally managed fields. To address this issue, the AMF community composition was assessed in 26 arable fields (13 pairs of organically and conventionally managed fields) and five semi-natural grasslands, all on sandy soil. Terminal restriction fragment length polymorphism community fingerprinting was used to characterize AMF community composition. The average number of AMF taxa was highest in grasslands (8.8), intermediate in organically managed fields (6.4) and significantly lower in conventionally managed fields (3.9). Moreover, AMF richness increased significantly with the time since conversion to organic agriculture. AMF communities of organically managed fields were also more similar to those of natural grasslands when compared with those under conventional management, and were less uniform than their conventional counterparts, as expressed by higher β-diversity (between-site diversity). We suggest that organic management in agro-ecosystems contributes to the restoration and maintenance of these important below-ground mutualists.

The association between atherosclerosis and gut microbiota has been attracting increased attention. We previously demonstrated a possible link between gut microbiota and coronary artery disease. Our aim of this study was to clarify the gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism (T-RFLP). This study included 39 coronary artery disease (CAD) patients and 30 age- and sex- matched no-CAD controls (Ctrls) with coronary risk factors. Bacterial DNA was extracted from their fecal samples and analyzed by T-RFLP and data mining analysis using the classification and regression algorithm. Five additional CAD patients were newly recruited to confirm the reliability of this analysis. Data mining analysis could divide the composition of gut microbiota into 2 characteristic nodes. The CAD group was classified into 4 CAD pattern nodes (35/39 = 90 %), while the Ctrl group was classified into 3 Ctrl pattern nodes (28/30 = 93 %). Five additional CAD samples were applied to the same dividing model, which could validate the accuracy to predict the risk of CAD by data mining analysis. We could demonstrate that operational taxonomic unit 853 (OTU853), OTU657, and OTU990 were determined important both by the data mining method and by the usual statistical comparison. We classified the gut microbiota profiles in coronary artery disease patients using data mining analysis of T-RFLP data and demonstrated the possibility that gut microbiota is a diagnostic marker of suffering from CAD.

Since the application of molecular methods, culture-independent methods (CIM) have been developed to study microbial communities from various environments. In the past 20 years, several methods based on the direct amplification and analyses of the small subunit ribosomal RNA gene have been developed to directly study environmental microorganisms. These methods include denaturing/temperature gradient gel electrophoresis, single-strand-conformation polymorphism, restriction fragment length polymorphism, terminal restriction fragment length polymorphism, and quantitative polymerase chain reaction (PCR). Similarly, non-PCR-based molecular techniques, such as microarray and fluorescence in situ hybridization have also been adopted. In recent years, several novel fields of investigation such as metagenomics, metatranscriptomics, metaproteomics, and single-cell genomics were developed, largely propelled by the innovation and application of next-generation sequencing methods. Several single-cell-based technologies such as Raman microspectroscopy and nano-scale secondary ion mass spectrometry are also increasingly used in the fields of microbial ecology and environmental microbiology. The application of these methods has revolutionized microbiology by allowing scientists to directly analyze natural microbial communities in situ, including their genes, transcripts, proteins, and metabolites and how their interactions impact their distribution patterns. In this review, we present an up-to-date review on different CIMs and their applications, our focuses are on the comparison of different CIMs and their application in the analyses of microbial diversities and communities. [PUBLICATION ABSTRACT]

Background Aedes albopictus is a primary vector of multiple arboviruses, including dengue, chikungunya, yellow fever, and Zika virus. Its control relies heavily on pyrethroid insecticides. The V1016G mutation in the voltage-gated sodium channel (VGSC) is a well-documented mechanism conferring pyrethroid resistance in Ae. albopictus , which directly challenges the efficacy of pyrethroid-based control. Understanding of the status of insecticide resistance will offer insights to inform evidence-based vector management. However, current phenotypic monitoring is laborious and time-consuming, highlighting the need for rapid and reliable genotyping tools. Methods To detect the V1016G mutation, we developed a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay. This assay was then applied to genotype 208 field-collected Ae. albopictus mosquitoes. These samples were collected in 2024 from seven counties/districts within Guangyuan City, a prefecture in northern Sichuan, China. Results The PCR–RFLP assay demonstrated 100% concordance with Sanger sequencing results. Genotyping confirmed the widespread presence of the 1016G allele, with frequencies ranging from 3.13% to 14.06%. The resistance allele (1016G) was exclusively detected in heterozygotes, and all populations conformed to Hardy–Weinberg equilibrium ( P  > 0.05). Furthermore, no significant temporal changes in allele frequencies were detected between 2020 and 2024 across the populations ( P  > 0.05). Conclusions This study established a cost-effective and reliable PCR–RFLP assay for detecting the V1016G mutation in Ae. albopictus , and demonstrated the widespread distribution of this mutation across Guangyuan City, Sichuan Province of China. Graphical abstract

Plasma omentin levels have been shown to be associated with circulating adiponectin concentrations and cardiometabolic disease-related outcomes. In this study, we aim to examine the association of omentin gene polymorphism with serum adiponectin levels and cardiometabolic health status using a genetic approach, and investigate whether these associations are modified by lifestyle factors. The study included 945 normal glucose tolerant and 941 unrelated individuals with type 2 diabetes randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Study participants were classified into cardiometabolically healthy and unhealthy, where cardiometabolically healthy were those without hypertension, diabetes, and dyslipidemia. Fasting serum adiponectin levels were measured by radioimmunoassay. The omentin A326T (rs2274907) single nucleotide polymorphism (SNP) was screened by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. The 'A' allele of the omentin SNP was significantly associated with lower adiponectin concentrations after adjusting for age, sex, body mass index (BMI), waist circumference (WC) and cardiometabolic health status (p = 1.90 x 10-47). There was also a significant association between circulating adiponectin concentrations and cardiometabolic health status after adjusting for age, sex, BMI, WC and Omentin SNP (p = 7.47x10-10). However, after adjusting for age, sex, BMI, WC and adiponectin levels, the association of 'A' allele with cardiometabolic health status disappeared (p = 0.79) suggesting that adiponectin serves as a mediator of the association between omentin SNP and cardiometabolic health status. There were no significant interactions between the SNP and dietary factors on adiponectin levels and cardiometabolic health status (p>0.25, for all comparisons). Our findings show that adiponectin might function as a mechanistic link between omentin SNP and increased risk of cardiometabolic diseases independent of common and central obesity in Asian Indians. Before strategies to promote adiponectin modulation could be implemented, further studies are required to confirm the molecular mechanisms involved in this triangular relationship between omentin gene, adiponectin and cardiometabolic diseases.