High-throughput Molecular Approaches Research Articles

Current and emerging biomarkers in ovarian cancer diagnosis; CA125 and beyond.

Ovarian cancer (OC) is one of the most common causes of cancer-related death in women worldwide. Its five-year survival rates are worse than the two most common gynecological cancers, cervical and endometrial. This is because it is asymptomatic in the early stages and usually detected in the advanced metastasized stage. Thus, survival is increasingly dependent on timely diagnosis. The delay in detection is contributed partly by the occurrence of non-specific clinical symptoms in the early stages and the lack of effective biomarkers and detection approaches. This underlines the need for biomarker identification and clinical validation, enabling earlier diagnosis, effective prognosis, and response to therapy. Apart from the traditional diagnostic biomarkers for OC, several new biomarkers have been delineated using advanced high-throughput molecular approaches in recent years. They are currently being clinically evaluated for their true diagnostic potential. In this chapter, we document the commonly utilized traditional screening markers and recently identified emerging biomarkers in OC diagnosis, focusing on secretory and protein biomarkers. We also briefly reviewed the recent advances and prospects in OC diagnosis.

Assessing the diagnostic performance of a novel RT-PCR fluorescence method for the detection of human plasmodium species.

BackgroundMalaria elimination effort is hampered not only by the lack of effective medication but also due to the lack of sensitive diagnostic tools to detect infections with low levels of parasitemia. Therefore, more sensitive and specific high-throughput molecular diagnostic approaches are needed for accurate malaria diagnosis.MethodsIn the present study, the performance of a novel single-tube MC004 real-time polymerase chain reaction (PCR) assay (MRC-Holland, Amsterdam, the Netherlands) was assessed for the detection of infection and discrimination of Plasmodium species. Blood samples (n = 150) were collected from malaria suspected patients at Adama malaria diagnosis and treatment centre, Adama, central Ethiopia. The positive predictive value (PPV), negative predictive value (NPV), analytical sensitivity and specificity of the assay were assessed against the conventional microscopic method.ResultsPlasmodium species were detected in 59 (39.3%) of the samples by microscopy and in 62 (41.3%) by the novel MC004 RT-PCR. Plasmodium vivax, Plasmodium falciparum and mixed infections with Plasmodium falciparum & Plasmodium vivax accounted for 47.5%, 40.6% and 11.9% respectively as detected by microscopy. The MC004 RT-PCR assay identified 59.7% and 40.3% of the samples positive for Plasmodium vivax and Plasmodium falciparum respectively. The sensitivity, specificity, PPV, and NPV of the MC004 RT-PCR assay were 95.8%, 97.8%, 92%, and 98.9%, respectively. No mixed infections were detected using the MC004 assay.ConclusionThe MC004 RT-PCR assay is a useful tool for the early detection of malaria and identification of Plasmodium species with a high degree of sensitivity and specificity. Due to its high sensitivity, and simplicity (being a single-tube assay), the MC004 is suitable for use in clinical settings and epidemiological studies.

Hematopoietic Stem Cells Undergo Immune Training and Constitute a Long-Term Reservoir for Hyper-Inflammatory Macrophages in a Mouse Model of Chronic Autoimmunity

Auto-immune diseases (AD) are characterized by repeated flares of disease activity separated by periods of remission. Cycles of AD remission and relapse can occur even with therapeutic intervention and contribute to AD morbidity. Paradoxically, during remission myeloid cells retain increased expression of genes related to interferon signaling and antigen presentation. Given the relatively short lifespan of myeloid cells, these observations imply the existence of a clonal reservoir fueling AD relapse. Recent literature describes hematopoietic stem cells (HSC) as a cellular source for trained myeloid cells in response to immune stimuli. Thus, we hypothesize that HSC may also retain a long-term memory of chronic autoimmune inflammation, thereby providing a continuous supply of myeloid cells that promote AD pathogenesis.To test this hypothesis, we have combined high-throughput molecular and cytokine profiling approaches with functional assays to address heritable changes in immune function using the pristane-induced mouse model of systemic lupus erythematosus (SLE). Eight weeks after pristane injection, we observe significant myeloid lineage expansion in the bone marrow (BM), including mature myeloid cells, granulocyte/monocyte progenitors (GMP) and multipotent progenitor (MPP) populations. To understand the impact of SLE-like disease on the molecular programming of the hematopoietic system, we performed RNA-seq analyses of BM Mon and HSC. As anticipated, BM Mon activated inflammatory programs and antigen presentation genes, which overlapped with gene signatures of human monocytes from SLE patients. HSC also activated innate defense gene programs resembling signatures of trained immunity, thus establishing the potential for autoimmune inflammation to induce immune training in HSC.To establish whether these molecular programs potentiate myeloid cell function, we generated BM-derived macrophages (BMDM) from control and pristane-induced mice. BMDMs from pristane-induced mice exhibited significantly increased capacity to kill Mycobacterium avium. Further, co-culture of T cells with BMDMs from pristane-induced mice significantly boosted T cell proliferation, indicative of enhanced antigen presentation. To establish whether HSC from pristane-induced mice propagate molecular memory of SLE-like disease to myeloid progeny, we transplanted stringently enriched (LSK/SLAM/CD34 -/EPCR +) long-term (LT)-HSC into lethally irradiated recipient mice. 18 weeks post transplant, mice transplanted with LT-HSC from pristane-induced donors had a small but significant reduction in donor BM HSC chimerism but did not exhibit overt changes in lineage output. Strikingly, BMDMs from pristane-induced donors showed increased bacterial killing and inflammatory cytokine generation following M. avium challenge, as well as increased capacity to induce antigen-specific T cell proliferation. Thus, LT-HSC retain and pass on altered functional properties to myeloid cells, even in the absence of AD activity.To characterize the molecular mechanisms underlying HSC-propagated alterations in myeloid cell function, we ran RNA-seq on donor-derived GMP and BMDMs. Notably, GMP derived from pristane-induced donor LT-HSC had increased expression of Fos and Jun/b/d, key molecular drivers of immune training in stem cells. Likewise, pristane-induced donor BMDMs maintained increased expression of IFN-regulated genes including MHC-I, a gene also overexpressed in PBMCs from human SLE patients. Consistent with these findings, re-stimulation of recipient mice with pristane led to a significant increase in cytokine-producing lymph node T cells versus recipient mice transplanted with control HSC, further supporting a model of immune training.Overall, these data show that chronic autoimmune inflammation can induce in HSC a heritable trained immunity phenotype that is transmitted to myeloid progeny, enhancing their functional activity. Ongoing studies are establishing the capacity for this phenotype to exacerbate AD. We are also testing the importance of molecular players identified above in establishing AD-related immune training and assessing the potential for therapeutic interventions to disrupt HSC memory in this setting. Our data thus stand to establish a new paradigm for trained immunity in HSC as a key contributor to AD pathology and relapse. DisclosuresNo relevant conflicts of interest to declare.

Paramyxoviruses from neotropical bats suggest a novel genus and nephrotropism.

Paramyxoviruses have a broad host range and geographic distribution, including human pathogens transmitted by bats, such as Nipah and Hendra viruses. In this study, we combined high-throughput sequencing and molecular approaches to investigate the presence of paramyxoviruses in neotropical bats (Microchiroptera suborder) in Brazil. We discovered and characterized three novel paramyxoviruses in the kidney tissues of apparently healthy common vampire bats (D. rotundus) and Seba's short-tailed bats (C. perspicillata), which we tentatively named Kanhgág virus (KANV), Boe virus (BOEV), and Guató virus (GUATV). In this study, we classified these viruses as putative species into the Macrojêvirus genus, a newly proposed genus of the Orthoparamyxovirinae subfamily. Using RT-PCR, we detected these viruses in 20.9% (9 out of 43) of bats tested, and viral RNA was detected exclusively in kidney tissues. Attempts to isolate infectious virus were successful for KANV and GUATV. Our results expand the viral diversity, host range, and geographical distribution of the paramyxoviruses.

Plant-microbiome interactions for sustainable agriculture: a review.

The online version contains supplementary material available at. 10.1007/s12298-021-00927-1.

Inversion Genotyping in the Anopheles gambiae Complex Using High-Throughput Array and Sequencing Platforms.

Chromosomal inversion polymorphisms have special importance in the Anopheles gambiae complex of malaria vector mosquitoes, due to their role in local adaptation and range expansion. The study of inversions in natural populations is reliant on polytene chromosome analysis by expert cytogeneticists, a process that is limited by the rarity of trained specialists, low throughput, and restrictive sampling requirements. To overcome this barrier, we ascertained tag single nucleotide polymorphisms (SNPs) that are highly correlated with inversion status (inverted or standard orientation). We compared the performance of the tag SNPs using two alternative high throughput molecular genotyping approaches vs. traditional cytogenetic karyotyping of the same 960 individual An. gambiae and An. coluzzii mosquitoes sampled from Burkina Faso, West Africa. We show that both molecular approaches yield comparable results, and that either one performs as well or better than cytogenetics in terms of genotyping accuracy. Given the ability of molecular genotyping approaches to be conducted at scale and at relatively low cost without restriction on mosquito sex or developmental stage, molecular genotyping via tag SNPs has the potential to revitalize research into the role of chromosomal inversions in the behavior and ongoing adaptation of An. gambiae and An. coluzzii to environmental heterogeneities.

The Role of Next Generation Sequencing in Diagnosis of Brain Tumors: A Review Study

: Since the number of prognostic and predictive neuro-oncologic genetic markers is steadily increasing, a comprehensive analysis of the molecular techniques used to examine neuro-oncology samples is vastly required. Molecular analysis and profiling of brain malignancies result in improved diagnostic accuracy, target identification, and predictive prognosis. Application of high-throughput molecular approaches, such as next generation sequencing (NGS), to analyze brain tumors has provided a large amount of molecular information. In the clinical practice, the application of NGS has been increased, which has consequently improved the treatment option for brain cancer as well as other types of malignancies. Target therapy has recently become one of the most promising treatment options for various tumors, especially brain tumors. In this review, we provided and summarized high-throughput genomic studies, such as NGS technique, that could independently identify the integrated management and diagnosis of primary human brain tumors across a variety of entities with pathognomonic genetic alterations.

A Metabolomics Approach to Investigate Kukoamine B-A Potent Natural Product With Anti-diabetic Properties.

Due to the surge in type 2 diabetes mellitus (T2DM), treatments for chronic metabolic dysregulations with fewer side-effects are sought. Lycii Cortex (LyC), a traditional Chinese Medicine (TCM) herb has a long history of being widely prescribed to treat T2DM as alternative medicine; however, the bioactive molecules and working mechanism remained unknown. Previous studies revealed kukoamine B (KB) as a major and featured compound for LyC with bioactivities for anti-oxidation and acute inflammation, which may be related to anti-diabetes properties. This study aims to understand the efficacy and the mode of action of KB in the diabetic (db/db) mouse model using a metabolomics approach. Parallel comparison was conducted using the first-line anti-diabetic drugs, metformin and rosligtazone, as positive controls. The db/db mice were treated with KB (50 mg kg−1 day−1) for 9 weeks. Bodyweight and fasting blood glucose were monitored every 5 and 7 days, respectively. Metabolomics and high-throughput molecular approaches, including lipidomics, targeted metabolomics (Biocrates p180), and cytokine profiling were applied to measure the alteration of serum metabolites and inflammatory biomarkers between different treatments vs. control (db/db mice treated with vehicle). After 9 weeks of treatment, KB lowered blood glucose, without the adverse effects of bodyweight gain and hepatomegaly shown after rosiglitazone treatment. Lipidomics analysis revealed that KB reduced levels of circulating triglycerides, cholesterol, phosphatidylethanolamine, and increased levels of phosphatidylcholines. KB also increased acylcarnitines, and reduced systemic inflammation (cytokine array). Pathway analysis suggested that KB may regulate nuclear transcription factors (e.g., NF-κB and/or PPAR) to reduce inflammation and facilitate a shift toward metabolic and inflammatory homeostasis. Comparison of KB with first-line drugs suggests that rosiglitazone may over-regulate lipid metabolism and anti-inflammatory responses, which may be associated with adverse side effects, while metformin had less impact on lipid and anti-inflammation profiles. Our research from holistic and systemic views supports the conclusion that KB is the bioactive compound of LyC for managing T2DM, and suggests KB as a nutraceutical or a pharmaceutical candidate for T2D treatment. In addition, our research provides insights related to metformin and rosiglitazone action, beyond lowering blood glucose.

WITHDRAWN: Environmental toxicology and omics: A question of sex

Clinical toxicology recognizes that sex is an important variable, as pharmacokinetics (ADME; absorption, distribution, metabolism, and excretion) can differ between females and males. However, few studies in toxicology have explored the implication of sex in relation to the transcriptome and proteome of whole organisms. High-throughput molecular approaches are becoming more frequently applied in toxicity screens (e.g. pre-clinical experiments, fish embryos, cell lines, synthetic tissues) and such data are expected to build upon reporter-based cell assays (e.g. receptor activation, enzyme inhibition) used in toxicant screening programs (i.e. Tox21, ToxCast, REACH). Thus, computational models can more accurately predict the diversity of adverse effects that can occur from chemical exposure within the biological system. Our studies and those synthesized from the literature suggest that the transcriptome and proteome of females and males respond quite differentially to chemicals. This has significant implications for predicting adverse effects in one sex when using molecular data generated in the other sex. While molecular initiating events are not expected to differ dramatically between females and males (i.e. an estrogen binds estrogen receptors in both sexes), it is important to acknowledge that the downstream transcriptomic and proteomic responses can differ based upon the presence/absence of co-regulators and inherent sex-specific variability in regulation of transcriptional and translational machinery. Transcriptomic and proteomic studies also reveal that cell processes affected by chemicals can differ due to sex, and this can undoubtedly lead to sex-specific physiological responses.

Reviewing the effects of food provisioning on wildlife immunity.

While urban expansion increasingly encroaches on natural habitats, many wildlife species capitalize on anthropogenic food resources, which have the potential to both positively and negatively influence their responses to infection. Here we examine how food availability and key nutrients have been reported to shape innate and adaptive immunity in wildlife by drawing from field-based studies, as well as captive and food restriction studies with wildlife species. Examples of food provisioning and key nutrients enhancing immune function were seen across the three study type distinctions, as were cases of trace metals and pharmaceuticals impairing the immunity of wildlife species. More generally, food provisioning in field studies tended to increase innate and adaptive responses to certain immune challenges, whereas patterns were less clear in captive studies. Mild food restriction often enhanced, whereas severe food restriction frequently impaired immunity. However, to enable stronger conclusions we stress a need for further research, especially field studies, and highlight the importance of integrating nutritional manipulation, immune challenge, and functional outcomes. Despite current gaps in research on this topic, modern high throughput molecular approaches are increasingly feasible for wildlife studies and offer great opportunities to better understand human influences on wildlife health.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'.

Identification and expression profiling of microRNAs involved in the stigma exsertion under high-temperature stress in tomato

BackgroundAutogamy in cultivated tomato varieties is a derived trait from wild type tomato plants, which are mostly allogamous. However, environmental stresses can cause morphological defects in tomato flowers and hinder autogamy. Under elevated temperatures, tomato plants usually exhibit the phenotype of stigma exsertion, with severely hindered self-pollination and fruit setting, whereas the inherent mechanism of stigma exsertion have been hitherto unknown. Numerous small RNAs (sRNAs) have been shown to play significant roles in plant development and stress responses, however, none of them have been studied with respect to stamen and pistil development under high-temperature conditions. We investigated the associations between stigma exsertion and small RNAs using high-throughput sequencing technology and molecular biology approaches.ResultsSixteen sRNA libraries of Micro-Tom were constructed from plants stamen and pistil samples and sequenced after 2 d and 12 d of exposure to heat stress, respectively, from which a total of 110 known and 84 novel miRNAs were identified. Under heat stress conditions, 34 known and 35 novel miRNAs were differentially expressed in stamens, and 20 known and 10 novel miRNAs were differentially expressed in pistils. GO and KEGG pathway analysis showed that the predicted target genes of differentially expressed miRNAs were significantly enriched in metabolic pathways in both stamen and pistil libraries. Potential miRNA-target cleavage cascades that correlated with the regulation of stigma exsertion under heat stress conditions were found and validated through qRT-PCR and RLM-5′ RACE.ConclusionOverall, a global spectrum of known and novel miRNAs involved in tomato stigma exsertion and induced by high temperatures were identified using high-throughput sequencing and molecular biology approaches, laying a foundation for revealing the miRNA-mediated regulatory network involved in the development of tomato stamens and pistils under high-temperature conditions.

Environmental toxicology and omics: A question of sex

Molecular initiating events and downstream transcriptional/proteomic responses provide valuable information for adverse outcome pathways, which can be used predict the effects of chemicals on physiological systems. There has been a paucity of research that addresses sex-specific expression profiling in toxicology and due to cost, time, and logistical considerations, sex as a variable has not been widely considered. In response to this deficiency, federal agencies in the United States, Canada, and Europe have highlighted the importance of including sex as a variable in scientific investigations. Using case studies from both aquatic and mammalian toxicology, we report that there can be less than ~20–25% consensus in how the transcriptome and proteome of each sex responds to chemicals. Chemicals that have been shown to elicit sex-specific responses in the transcriptome or proteome include pharmaceuticals, anti-fouling agents, anticorrosive agents, and fungicides, among others. Sex-specific responses in the transcriptome and proteome are not isolated to whole animals, as investigations demonstrate that primary cell cultures isolated from each sex responds differently to toxicants. This signifies that sex is important, even in cell lines. Sex has significant implications for predictive toxicology, and both male and female data are required to improve robustness of adverse outcome pathways. Biological significanceClinical toxicology recognizes that sex is an important variable, as pharmacokinetics (ADME; absorption, distribution, metabolism, and excretion) can differ between females and males. However, few studies in toxicology have explored the implication of sex in relation to the transcriptome and proteome of whole organisms. High-throughput molecular approaches are becoming more frequently applied in toxicity screens (e.g. pre-clinical experiments, fish embryos, cell lines, synthetic tissues) and such data are expected to build upon reporter-based cell assays (e.g. receptor activation, enzyme inhibition) used in toxicant screening programs (i.e. Tox21, ToxCast, REACH). Thus, computational models can more accurately predict the diversity of adverse effects that can occur from chemical exposure within the biological system. Our studies and those synthesized from the literature suggest that the transcriptome and proteome of females and males respond quite differently to chemicals. This has significant implications for predicting adverse effects in one sex when using molecular data generated in the other sex. While molecular initiating events are not expected to differ dramatically between females and males (i.e. an estrogen binds estrogen receptors in both sexes), it is important to acknowledge that the downstream transcriptomic and proteomic responses can differ based upon the presence/absence of co-regulators and inherent sex-specific variability in regulation of transcriptional and translational machinery. Transcriptomic and proteomic studies also reveal that cell processes affected by chemicals can differ due to sex, and this can undoubtedly lead to sex-specific physiological responses.

Barcoding the largest animals on Earth: ongoing challenges and molecular solutions in the taxonomic identification of ancient cetaceans.

Over the last few centuries, many cetacean species have witnessed dramatic global declines due to industrial overharvesting and other anthropogenic influences, and thus are key targets for conservation. Whale bones recovered from archaeological and palaeontological contexts can provide essential baseline information on the past geographical distribution and abundance of species required for developing informed conservation policies. Here we review the challenges with identifying whale bones through traditional anatomical methods, as well as the opportunities provided by new molecular analyses. Through a case study focused on the North Sea, we demonstrate how the utility of this (pre)historic data is currently limited by a lack of accurate taxonomic information for the majority of ancient cetacean remains. We then discuss current opportunities presented by molecular identification methods such as DNA barcoding and collagen peptide mass fingerprinting (zooarchaeology by mass spectrometry), and highlight the importance of molecular identifications in assessing ancient species’ distributions through a case study focused on the Mediterranean. We conclude by considering high-throughput molecular approaches such as hybridization capture followed by next-generation sequencing as cost-effective approaches for enhancing the ecological informativeness of these ancient sample sets.This article is part of the themed issue ‘From DNA barcodes to biomes’.

Alterations in fecal microbiota composition by probiotic supplementation in healthy adults: a systematic review of randomized controlled trials

BackgroundThe effects of probiotic supplementation on fecal microbiota composition in healthy adults have not been well established. We aimed to provide a systematic review of the potential evidence for an effect of probiotic supplementation on the composition of human fecal microbiota as assessed by high-throughput molecular approaches in randomized controlled trials (RCTs) of healthy adults.MethodsThe survey of peer-reviewed papers was performed on 17 August 2015 by a literature search through PubMed, SCOPUS, and ISI Web of Science. Additional papers were identified by checking references of relevant papers. Search terms included healthy adult, probiotic, bifidobacterium, lactobacillus, gut microbiota, fecal microbiota, intestinal microbiota, intervention, and (clinical) trial. RCTs of solely probiotic supplementation and placebo in healthy adults that examined alteration in composition of overall fecal microbiota structure assessed by shotgun metagenomic sequencing, 16S ribosomal RNA sequencing, or phylogenetic microarray methods were included. Independent collection and quality assessment of studies were performed by two authors using predefined criteria including methodological quality assessment of reports of the clinical trials based on revised tools from PRISMA/Cochrane and by the Jadad score.ResultsSeven RCTs investigating the effect of probiotic supplementation on fecal microbiota in healthy adults were identified and included in the present systematic review. The quality of the studies was assessed as medium to high. Still, no effects were observed on the fecal microbiota composition in terms of α-diversity, richness, or evenness in any of the included studies when compared to placebo. Only one study found that probiotic supplementation significantly modified the overall structure of the fecal bacterial community in terms of β-diversity when compared to placebo.ConclusionsThis systematic review of the pertinent literature demonstrates a lack of evidence for an impact of probiotics on fecal microbiota composition in healthy adults. Future studies would benefit from pre-specifying the primary outcome and transparently reporting the results including effect sizes, confidence intervals, and P values as well as providing a clear distinction of between-group and within-group comparisons.

Seasonal Changes in Bacterial Communities Cause Foaming in a Wastewater Treatment Plant.

Bio-foaming is a major problem in solid separation in activated sludge (AS) wastewater treatment systems. Understanding the changes in bacterial communities during sludge foaming is vital for explaining foam formation. Changes in bacterial communities in the foam, corresponding foaming AS, and non-foaming AS in a seasonal foaming wastewater treatment plant (WWTP) in Northern China were investigated by high-throughput pyrosequencing and molecular quantification-based approaches. We found that bacterial communities of the foam and the corresponding foaming AS were similar but markedly different from those of the non-foaming AS. Actinobacteria was the predominant phylum in the foam and the corresponding foaming AS, whereas Proteobacteria was predominant in the non-foaming AS. Similar to the results of most previous studies, our results showed that Candidatus "Microthrix parvicella" was the predominant filamentous bacteria in the foam and the corresponding foaming AS and was significantly enriched in the foam compared to the corresponding foaming AS. Its abundance decreased gradually with a slow disappearance of sludge foaming, indicating that its overgrowth had a direct relationship with sludge foaming. In addition to Candidatus M. parvicella, Tetrasphaera and Trichococcus might play a role in sludge foaming, because they supported the changes in AS microbial ecology for foam formation. The effluent water quality of the surveyed plant remained stable during the period of sludge foaming, but the microbial consortia responsible for nitrogen and phosphorus transformation and removal markedly changed compared to that in the non-foaming AS. This study adds to the previous understanding of bacterial communities causing foaming in WWTPs.

Systems biology and gene networks in neurodevelopmental and neurodegenerative disorders.

Genetic and genomic approaches have implicated hundreds of genetic loci in neurodevelopmental disorders and neurodegeneration, but mechanistic understanding continues to lag behind the pace of gene discovery. Understanding the role of specific genetic variants in the brain involves dissecting a functional hierarchy that encompasses molecular pathways, diverse cell types, neural circuits and, ultimately, cognition and behaviour. With a focus on transcriptomics, this Review discusses how high-throughput molecular, integrative and network approaches inform disease biology by placing human genetics in a molecular systems and neurobiological context. We provide a framework for interpreting network biology studies and leveraging big genomics data sets in neurobiology.

Meiofaunal community analysis by high-throughput sequencing: Comparison of extraction, quality filtering, and clustering methods

Using molecular tools to examine community composition of meiofauna, animals 45μm to 1mm in size living between sediment grains in aquatic environments, is relatively new in comparison to bacterial and archaeal microbial studies. Although high-throughput molecular approaches are starting to be applied to these ccommunities, effectiveness of different approaches for nucleic acid extraction from meiofauna is poorly known and bioinformatic pipelines vary between studies. Given this situation, there is a need for protocols to be developed that promote consistency in sample collection and processing, sequence quality filtering, and Operational Taxonomic Unit (OTU) clustering methods. Herein, we assess different approaches used for DNA extraction (DNA extracted directly from sediment versus elutriated material retained on a 45μm sieve) as well as how different quality filtering methods of sequences and OTU clustering algorithms impact genetic assessment of meiofauna community composition. DNA extracted directly from sediment resulted in higher presence of non-metazoan eukaryotic taxa; in contrast, an elutriation (resuspension with decanting) approach increased meiofauna abundance and enriched metazoan OTUs. In regards to bioinformatics analyses, the number of overall OTUs varied by clustering algorithm, primarily due to the applied method of sequence quality filtering. However, alpha and beta diversity analyses showed similar trends regardless of bioinformatics pipeline utilized. Based on our results, we recommend studies of meiofauna communities first elutriate samples prior to DNA extraction and include multiple biological replicates to account for variation in community-level composition. The quality filtering method should be carefully considered as this step accounted for large discrepancy in the number of OTUs inferred.

Profiling invasiveness in head and neck cancer: recent contributions of genomic and transcriptomic approaches.

High-throughput molecular profiling approaches have emerged as precious research tools in the field of head and neck translational oncology. Such approaches have identified and/or confirmed the role of several genes or pathways in the acquisition/maintenance of an invasive phenotype and the execution of cellular programs related to cell invasion. Recently published new-generation sequencing studies in head and neck squamous cell carcinoma (HNSCC) have unveiled prominent roles in carcinogenesis and cell invasion of mutations involving NOTCH1 and PI3K-patwhay components. Gene-expression profiling studies combined with systems biology approaches have allowed identifying and gaining further mechanistic understanding into pathways commonly enriched in invasive HNSCC. These pathways include antigen-presenting and leucocyte adhesion molecules, as well as genes involved in cell-extracellular matrix interactions. Here we review the major insights into invasiveness in head and neck cancer provided by high-throughput molecular profiling approaches.

Conservation and utilization of African Oryza genetic resources.

Africa contains a huge diversity of both cultivated and wild rice species. The region has eight species representing six of the ten known genome types. Genetic resources of these species are conserved in various global germplasm repositories but they remain under collected and hence underrepresented in germplasm collections. Moreover, they are under characterized and therefore grossly underutilized. The lack of in situ conservation programs further exposes them to possible genetic erosion or extinction. In order to obtain maximum benefits from these resources, it is imperative that they are collected, efficiently conserved and optimally utilized. High throughput molecular approaches such as genome sequencing could be employed to more precisely study their genetic diversity and value and thereby enhance their use in rice improvement. Oryza sativa was the first crop plant to have its reference genome sequence released marking a major milestone that opened numerous opportunities for functional characterization of the entire rice genome. Studies have however demonstrated that one reference genome sequence is not enough to fully explore the genetic variation in the Oryza genus, hence the need to have reference sequences for other species in the genus. An overview of the state of conservation and utilization of African Oryza is hereby presented. Progress in the release of reference genome sequences for these species is also highlighted.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-29) contains supplementary material, which is available to authorized users.

New insights into the structure and function of phyllosphere microbiota through high-throughput molecular approaches

The phyllosphere is an ecologically and economically important ecosystem that hosts a large and diverse microbial community. Phyllosphere microbiota play a critical role in protecting plants from diseases as well as promoting their growth by various mechanisms. There are serious gaps in our understanding of how and why microbiota composition varies across spatial and temporal scales, the ecology of leaf surface colonizers and their interactions with their host, and the genetic adaptations that enable phyllosphere survival of microorganisms. These gaps are due in large part to past technical limitations, as earlier studies were restricted to the study of culturable bacteria only and used low-throughput molecular techniques to describe community structure and function. The availability of high-throughput and cost-effective molecular technologies is changing the field of phyllosphere microbiology, enabling researchers to begin to address the dynamics and composition of the phyllosphere microbiota across a large number of samples with high, in-depth coverage. Here, we discuss and connect the most recent studies that have used next-generation molecular techniques such as metagenomics, proteogenomics, genome sequencing, and transcriptomics to gain new insights into the structure and function of phyllosphere microbiota and highlight important challenges for future research.