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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.

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.

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF¹⁰³ of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Background Polycystic ovary syndrome (PCOS) is the main cause of female infertility. Interactions among genetic, biochemical, and immunological factors can affect the pathogenesis of PCOS. As a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) plays an important role in this regard. The present study aimed to evaluate the association of the rs361525 gene single-nucleotide polymorphism (SNP) and TNF-α serum levels with the hormonal and biochemical characteristics of PCOS in Iranian individuals. Methods The SNP rs361525 in the TNF-α gene was analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) in a total of 111 PCOS patients and 105 healthy females. Serum levels of TNF-α, lipid and hormone profiles, and biochemical factors were measured using enzyme-linked immunosorbent assay (ELISA) and calorimetric methods, as appropriate. Results The TNF-α serum level was higher in women with PCOS compared with the control group ( p  <  0.0001), and it was significantly correlated with the homeostasis model assessment (HOMA) factor ( r  = 0.138, p  <  0.05). No significant differences were found in the genotype and allelic frequencies between the two groups ( p  >  0.05). Higher levels and significant differences were found for the HOMA factor, luteinizing hormone/follicle-stimulating hormone (LH/FSH), testosterone, and body mass index (BMI) in the PCOS group compared with the control group ( p  <  0.0001). High LH/FSH ratios (odds ratio [OR] = 1.98, 95% confidence interval [CI] = 1.20–3.28, p  <  0.01), and high HOMA factor (OR = 5.04, 95% CI = 2.82–9.01, p  <  0.001) were significantly associated with an increased risk of PCOS. Conclusions Despite the lack of significant difference between rs361525 polymorphism of the TNF-α gene and PCOS, the serum level of TNF-α was increased in PCOS patients and positively correlated with the HOMA factor. Elevation of the LH/FSH ratio and HOMA for insulin resistance (HOMA-IR) increased the risk of PCOS. Therefore, TNF-α could indirectly contribute to PCOS progression.

The Fusarium species are a widely spread phytopathogen identified in an extensive variety of hosts. The Fusarium genus is one of the most heterogeneous fungi and is difficult to classify. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is a useful method in detection of DNA polymorphism in objective sequences. The aim of the present study was to identify the phylogenetic associations and usefulness of the internal transcribed spacer (ITS) region as a genetic marker within the most clinically important strain of the Fusarium species. A total of 50 strains of Fusarium spp. were used in the study, including environmental, clinical and reference isolates. The primers ITS1 and ITS4 were used in the study. Two restriction enzymes, HaeIII and SmaI, were assessed for the digestion of PCR products. A PCR product of ~550-base pairs was generated for each Fusarium species. The digested products with HaeIII and SmaI demonstrated that the bands generated for the medically significant Fusarium species, including F. solani, F. oxysporum, F. verticillidea, F. proliferatum and F. fujikuri, have different restriction enzyme patterns. In conclusion, it appears that the PCR-RFLP method used in the present study produces a sufficient restriction profile for differentiation of the most medically significant Fusarium species.

How nitrogen (N) deposition impacts ectomycorrhizal (EM) fungal communities has been little studied in deciduous forests or across spatial scales. Here, it was tested whether N addition decreases species richness and shifts species composition across spatial scales in temperate deciduous oak forests. Combined molecular (terminal restriction fragment length polymorphism (T-RFLP), sequencing) and morphological approaches were used to measure EM fungal operational taxon unit (OTU) richness, community structure and composition at the spatial scale of the root, soil core and forest during a 3-yr N fertilization experiment in Quercus-dominated forests near Chicago, IL, USA. In N treatments, significantly lower OTU richness at the largest but not smaller spatial scales and a different community structure were detected. The effects of N appeared to be immediate, not cumulative. Ordination indicated the composition of EM fungal communities was determined by forest site and N fertilization. The EM fungi responded to a N increase that was low compared with other fertilization studies, suggesting that moderate increases in N deposition can affect EM fungal communities at larger spatial scales in temperate deciduous ecosystems. While responses at large spatial scales indicate that environmental factors can drive changes in these communities, untangling the impacts of abiotic from biotic factors remain limited by detection issues.

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.

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.

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]

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.