In most industrialized countries, screening programs for cervical cancer have shifted from cytology (Pap smear or ThinPrep) alone on clinician-obtained samples to the addition of screening for human papillomavirus (HPV), its main causative agent. For HPV testing, self-sampling instead of clinician-sampling has proven to be equally accurate, in particular for assays that use nucleic acid amplification techniques. In addition, HPV testing of self-collected samples in combination with a follow-up Pap smear in case of a positive result is more effective in detecting precancerous lesions than a Pap smear alone. Self-sampling for HPV testing has already been adopted by some countries, while others have started trials to evaluate its incorporation into national cervical cancer screening programs. Self-sampling may result in more individuals willing to participate in cervical cancer screening, because it removes many of the barriers that prevent women, especially those in low socioeconomic and minority populations, from participating in regular screening programs. Several studies have shown that the majority of women who have been underscreened but who tested HPV-positive in a self-obtained sample will visit a clinic for follow-up diagnosis and management. In addition, a self-collected sample can also be used for vaginal microbiome analysis, which can provide additional information about HPV infection persistence as well as vaginal health in general.

Changes in the relative abundances of many intestinal microorganisms, both those that naturally occur in the human gut microbiome and those that are considered pathogens, have been associated with a range of diseases. To more accurately diagnose health conditions, medical practitioners could benefit from a molecular, culture-independent assay for the quantification of these microorganisms in the context of a healthy reference range. Here we present the targeted sequencing of the microbial 16S rRNA gene of clinically relevant gut microorganisms as a method to provide a gut screening test that could assist in the clinical diagnosis of certain health conditions. We evaluated the possibility of detecting 46 clinical prokaryotic targets in the human gut, 28 of which could be identified with high precision and sensitivity by a bioinformatics pipeline that includes sequence analysis and taxonomic annotation. These targets included 20 commensal, 3 beneficial (probiotic), and 5 pathogenic intestinal microbial taxa. Using stool microbiome samples from a cohort of 897 healthy individuals, we established a reference range defining clinically relevant relative levels for each of the 28 targets. Our assay quantifies 28 targets in the context of a healthy reference range and correctly reflected 38/38 verification samples of real and synthetic stool material containing known gut pathogens. Thus, we have established a method to determine microbiome composition with a focus on clinically relevant taxa, which has the potential to contribute to patient diagnosis, treatment, and monitoring. More broadly, our method can facilitate epidemiological studies of the microbiome as it relates to overall human health and disease.

The human distal gut is home to a rich and dense microbial community with representatives of all three domains of life which are intricately connected with our physiology and health. The combined genomes of these microbes, collectively called the human microbiome, vastly expand the metabolic capacities of our own genome, allowing us to break down and extract energy from dietary compounds that human enzymes cannot digest. In addition, the variable composition of these communities and their biotransformations might explain inter‐individual differences in toxicities, tolerances and efficacies for certain drugs. Recent advances in sequencing technologies and bioinformatics have provided exciting new insights into the genomes of our microbial symbionts, their functional capacities and the interactions between these microbes and their human host. This review summarizes the metabolic conversions of dietary components and pharmaceuticals that take place in the human distal gut, as well as their implications for human health.Linked ArticlesThis article is part of a themed section on When Pharmacology Meets the Microbiome: New Targets for Therapeutics? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.24/issuetoc

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman’s reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive vaginal health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 31 bacterial taxa of clinical importance, including Lactobacillus , Sneathia , Gardnerella , and 3 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, reference ranges were determined in a group of 50 self-reported healthy women. The HPV sequencing portion of the test was evaluated against the digene High-Risk HPV HC2 DNA test. For hrHPV genotyping, agreement was 95.3% with a kappa of 0.804 (601 samples); after removal of samples in which the digene hrHPV probe showed cross-reactivity with lrHPV types, the sensitivity and specificity of the hrHPV genotyping assay were 94.5% and 96.6%, respectively, with a kappa of 0.841. For lrHPV genotyping, agreement was 93.9% with a kappa of 0.788 (148 samples), while sensitivity and specificity were 100% and 92.9%, respectively. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

Systems level approaches to analyzing complex emergent behavior require quantitative characterization of alterations of behavior on both the microscale and macroscale. Here we consider the problem of cellular organization and describe a statistical methodology for quantitative comparison of the internal organization between different populations of similar physical objects, such as cells. This comparison is achieved with several steps of analysis. Starting with three-dimensional or two-dimensional images of cells, images are segmented to identify individual cells. Locations of internal points of interest, such as organelles or proteins, are recorded. To define the configuration of internal points in each cell, the individual cells are subjected to bounded Voronoi tessellation: subdividing the bounded volume or area of the cell into subvolumes determined by the locations of the internal points of interest. A statistical methodology is applied to yield a metric for similarity in degree of organization between populations. We applied this methodology to test whether centrioles play a role in global cellular organization, using mutants of the green alga Chlamydomonas reinhardtii with known alterations in centriole number, structure, and position as a model system. Comparing mutant populations and wild-type populations revealed a dramatic difference in the degree of organization in the mutant strains. These computational and experimental results provide statistical support for prior observational studies and support the idea that centrioles play a role in generating or maintaining global cellular organization. Our results confirm that this method can be used to sensitively compare the extent and type of organization within cells.cell shape | image analysis | microscopy | cytoskeleton A major outstanding problem in basic biology is how cells generate and regulate their 3D geometry on the molecular level (1). In addition to being an interesting fundamental science question, there are clinical implications involved. In development, differentiation of stem cells into distinct functional cell types is accompanied by characteristic changes in cellular organization (2). The disruption of cellular organization (dysplasia) is a major hallmark of cancer and the basis of cytopathology (3). The biochemistry canon presupposes cell organization is mechanistically generated from molecular networks and molecular self-assembly (4). Although entire genomes have been sequenced and genome-wide molecular-interaction maps exist for model organisms (5), it remains unclear which molecules regulate the intracellular organization or how they do it (1, 6, 7).Traditionally, cell organization has been investigated visually by identifying mutants or perturbations that cause gross changes in cell appearance. However, such an approach will only identify the most dramatic phenotypes, and it is likely that many mutations may exist that play more subtle roles in cell organization and that are only distinguishable statistically by considering large num...

26In most industrialized countries, screening programs for cervical cancer have 27 shifted from cytology (Pap smear or ThinPrep) alone on clinician-obtained samples to 28 the addition of screening for human papillomavirus (HPV), its main causative agent. For 29 HPV testing, self-sampling instead of clinician-sampling has proven to be equally 30 accurate, in particular for assays that use nucleic acid amplification techniques. In 31 addition, HPV testing of self-collected samples in combination with a follow-up Pap 32 smear in case of a positive result is more effective in detecting precancerous lesions 33 than a Pap smear alone. Self-sampling for HPV testing has already been adopted by 34 some countries, while others have started trials to evaluate its incorporation into 35 national cervical cancer screening programs. Self-sampling may result in more 36 individuals willing to participate in cervical cancer screening, because it removes many 37 of the barriers that prevent women, especially those in low socioeconomic and minority

11Microbial community analysis can be biased by multiple technical factors, such as 12 storage conditions, DNA extraction, or amplification conditions. In a high-throughput 13 laboratory that relies on samples obtained from thousands of different subjects, 14 knowledge of the extent of subject-introduced sampling and storage variation on the 15 outcome of the inferred microbiome, as well as the effect of laboratory-introduced 16 variation caused by reagent batches, equipment, or operator on the consistency of 17 these processes within the laboratory is paramount. Here, we analyzed the effect of 18 sampling from different parts of the same stool specimen or on different consecutive 19 days, as well as short-term storage of samples at different temperatures on microbiome 20 profiles obtained by 16S rRNA gene amplification. Each of these factors had relatively 21 little effect on the microbial composition. In addition, replicate amplification of 44 stool 22 samples showed reproducible results. Finally, 363 independent replicate extractions 23 and amplifications of a single human homogenized stool (HS) specimen showed 24 reproducible results (average Lin's correlation = 0.95), with little variation introduced by 25 HS batch, operator, extraction equipment, or DNA sequencer. In all cases, variations 26 between replicates were significantly smaller than those between individual samples; 27 subject identity always was the largest determinant. We propose that homogenized 28 stool specimens could be used as quality control to routinely monitor the laboratory 29 process and to validate new methods. 30

We describe the characterization of the gastrointestinal tract (gut) and oral microbiota (bacteria) in 32 urban Pakistani adults representing seven major geographies and six ethnicities in the country. Study participants were between ages 18 and 40, had body mass index between 18 and 25 Kg/m 2 , and were early-career students or professionals belonging to 25 major cities of the country. These individuals donated a total of 61 samples (32 gut and 29 oral) that were subjected to 16S ribosomal RNA (rRNA) gene sequencing. Microbiome composition of Pakistani individuals was compared against the uBiome database of selected individuals who self-reported to be in excellent health. Using the crude measure of percentage overlap or similarity between the gut microbiota profile of Pakistani and uBiome dataset as proxy for health, our sequencing indicated that the Pakistani gut microbiota was moderately healthy relative to the uBiome dataset and Pakistani women appeared healthier relative to men. The Pakistani gut microbiome seemed susceptible to obesity and weight gain, levels of probiotics was very high likely due to the popularity of milk-based and fermented foods in the Pakistani diet, and bacteria that metabolize starch and carbohydrates (typically seen in the gut microbiota of honey bee) were abnormally enriched in the gut of Pakistani men. Our investigations reveal serious issues with the dietary habits and lifestyle of Pakistani individuals of consuming food enriched in high carbohydrates and fats, overcooked in oil and spices, following a sedentary lifestyle, little or no daily intake of fresh fruits, over-consumption of antibiotics from a very early age, and health and hygiene standards that do not meet international standards. Our sequencing is the first step towards generating a countrywide understanding of the impact of the local diet and lifestyle on Pakistani gut microbiota and can help understand its overall association with health and wellness.