Automated and Accurate Estimation of Gene Family Abundance from Shotgun Metagenomes.

Shotgun metagenomic DNA sequencing is a widely applicable tool for characterizing the functions that are encoded by microbial communities. Several bioinformatic tools can be used to functionally annotate metagenomes, allowing researchers to draw inferences about the functional potential of the community and to identify putative functional biomarkers. However, little is known about how decisions made during annotation affect the reliability of the results. Here, we use statistical simulations to rigorously assess how to optimize annotation accuracy and speed, given parameters of the input data like read length and library size. We identify best practices in metagenome annotation and use them to guide the development of the Shotgun Metagenome Annotation Pipeline (ShotMAP). ShotMAP is an analytically flexible, end-to-end annotation pipeline that can be implemented either on a local computer or a cloud compute cluster. We use ShotMAP to assess how different annotation databases impact the interpretation of how marine metagenome and metatranscriptome functional capacity changes across seasons. We also apply ShotMAP to data obtained from a clinical microbiome investigation of inflammatory bowel disease. This analysis finds that gut microbiota collected from Crohn's disease patients are functionally distinct from gut microbiota collected from either ulcerative colitis patients or healthy controls, with differential abundance of metabolic pathways related to host-microbiome interactions that may serve as putative biomarkers of disease.

Progress in the reprogramming of somatic cells.

Pluripotent stem cells can differentiate into nearly all types of cells in the body. This unique potential provides significant promise for cell-based therapies to restore tissues or organs destroyed by injuries, degenerative diseases, aging, or cancer. The discovery of induced pluripotent stem cell (iPSC) technology offers a possible strategy to generate patient-specific pluripotent stem cells. However, because of concerns about the specificity, efficiency, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use. A chemical approach is regarded as a promising strategy to improve and change the iPSC process. Dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve iPSC reprogramming. In addition to the prospect of deriving patient-specific tissues and organs from iPSCs, another attractive strategy for regenerative medicine is transdifferentiation-the direct conversion of one somatic cell type to another. Recent studies revealed a new paradigm of transdifferentiation: using transcription factors used in iPSC generation to induce transdifferentiation or called iPSC transcription factor-based transdifferentiation. This type of transdifferentiation not only reveals and uses the developmentally plastic intermediates generated during iPSC reprogramming but also produces a wide range of cells, including expandable tissue-specific precursor cells. Here, we review recent progress of small molecule approaches in the generation of iPSCs. In addition, we summarize the new concept of iPSC transcription factor-based transdifferentiation and discuss its application in generating various lineage-specific cells, especially cardiovascular cells.

Small molecules, big roles -- the chemical manipulation of stem cell fate and somatic cell reprogramming.

Despite the great potential of stem cells for basic research and clinical applications, obstacles - such as their scarce availability and difficulty in controlling their fate - need to be addressed to fully realize their potential. Recent achievements of cellular reprogramming have enabled the generation of induced pluripotent stem cells (iPSCs) or other lineage-committed cells from more accessible and abundant somatic cell types by defined genetic factors. However, serious concerns remain about the efficiency and safety of current genetic approaches to cell reprogramming and traditional culture systems that are used for stem cell maintenance. As a complementary approach, small molecules that target specific signaling pathways, epigenetic processes and other cellular processes offer powerful tools for manipulating cell fate to a desired outcome. A growing number of small molecules have been identified to maintain the self-renewal potential of stem cells, to induce lineage differentiation and to facilitate reprogramming by increasing the efficiency of reprogramming or by replacing genetic reprogramming factors. Furthermore, mechanistic investigations of the effects of these chemicals also provide new biological insights. Here, we examine recent achievements in the maintenance of stem cells, including pluripotent and lineage-specific stem cells, and in the control of cell fate conversions, including iPSC reprogramming, conversion of primed to naïve pluripotency, and transdifferentiation, with an emphasis on manipulation with small molecules.

Conversion of human fibroblasts to functional endothelial cells by defined factors.

OBJECTIVE: Transdifferentiation of fibroblasts to endothelial cells (ECs) may provide a novel therapeutic avenue for diseases, including ischemia and fibrosis. Here, we demonstrate that human fibroblasts can be transdifferentiated into functional ECs by using only 2 factors, Oct4 and Klf4, under inductive signaling conditions. APPROACH AND RESULTS: To determine whether human fibroblasts could be converted into ECs by transient expression of pluripotency factors, human neonatal fibroblasts were transduced with lentiviruses encoding Oct4 and Klf4 in the presence of soluble factors that promote the induction of an endothelial program. After 28 days, clusters of induced endothelial (iEnd) cells seemed and were isolated for further propagation and subsequent characterization. The iEnd cells resembled primary human ECs in their transcriptional signature by expressing endothelial phenotypic markers, such as CD31, vascular endothelial-cadherin, and von Willebrand Factor. Furthermore, the iEnd cells could incorporate acetylated low-density lipoprotein and form vascular structures in vitro and in vivo. When injected into the ischemic limb of mice, the iEnd cells engrafted, increased capillary density, and enhanced tissue perfusion. During the transdifferentiation process, the endogenous pluripotency network was not activated, suggesting that this process bypassed a pluripotent intermediate step. CONCLUSIONS: Pluripotent factor-induced transdifferentiation can be successfully applied for generating functional autologous ECs for therapeutic applications.

The Rickettsia Endosymbiont of Ixodes pacificus Contains All the Genes of De Novo Folate Biosynthesis.

Ticks and other arthropods often are hosts to nutrient providing bacterial endosymbionts, which contribute to their host's fitness by supplying nutrients such as vitamins and amino acids. It has been detected, in our lab, that Ixodes pacificus is host to Rickettsia species phylotype G021. This endosymbiont is predominantly present, and 100% maternally transmitted in I. pacificus. To study roles of phylotype G021 in I. pacificus, bioinformatic and molecular approaches were carried out. MUMmer genome alignments of whole genome sequence of I. scapularis, a close relative to I. pacificus, against completely sequenced genomes of R. bellii OSU85-389, R. conorii, and R. felis, identified 8,190 unique sequences that are homologous to Rickettsia sequences in the NCBI Trace Archive. MetaCyc metabolic reconstructions revealed that all folate gene orthologues (folA, folC, folE, folKP, ptpS) required for de novo folate biosynthesis are present in the genome of Rickettsia buchneri in I. scapularis. To examine the metabolic capability of phylotype G021 in I. pacificus, genes of the folate biosynthesis pathway of the bacterium were PCR amplified using degenerate primers. BLAST searches identified that nucleotide sequences of the folA, folC, folE, folKP, and ptpS genes possess 98.6%, 98.8%, 98.9%, 98.5% and 99.0% identity respectively to the corresponding genes of Rickettsia buchneri. Phylogenetic tree constructions show that the folate genes of phylotype G021 and homologous genes from various Rickettsia species are monophyletic. This study has shown that all folate genes exist in the genome of Rickettsia species phylotype G021 and that this bacterium has the genetic capability for de novo folate synthesis.

A taxonomic signature of obesity in the microbiome? Getting to the guts of the matter.

Obesity is an important and intractable public health problem. In addition to the well-known risk factors of behavior, diet, and genetics, gut microbial communities were recently identified as another possible source of risk and a potential therapeutic target. However, human and animal-model studies have yielded conflicting results about the precise nature of associations between microbiome composition and obesity. In this paper, we use publicly available data from the Human Microbiome Project (HMP) and MetaHIT, both surveys of healthy adults that include obese individuals, plus two smaller studies that specifically examined lean versus obese adults. We find that inter-study variability in the taxonomic composition of stool microbiomes far exceeds differences between lean and obese individuals within studies. Our analyses further reveal a high degree of variability in stool microbiome composition and diversity across individuals. While we confirm the previously published small, but statistically significant, differences in phylum-level taxonomic composition between lean and obese individuals in several cohorts, we find no association between BMI and taxonomic composition of stool microbiomes in the larger HMP and MetaHIT datasets. We explore a range of different statistical techniques and show that this result is robust to the choice of methodology. Differences between studies are likely due to a combination of technical and clinical factors. We conclude that there is no simple taxonomic signature of obesity in the microbiota of the human gut.

Dual-color HIV reporters trace a population of latently infected cells and enable their purification.

HIV latency constitutes the main barrier for clearing HIV infection from patients. Our inability to recognize and isolate latently infected cells hinders the study of latent HIV. We engineered two HIV-based viral reporters expressing different fluorescent markers: one HIV promoter-dependent marker for productive HIV infection, and a second marker under a constitutive promoter independent of HIV promoter activity. Infection of cells with these viruses allows the identification and separation of latently infected cells from uninfected and productively infected cells. These reporters are sufficiently sensitive and robust for high-throughput screening to identify drugs that reactivate latent HIV. These reporters can be used in primary CD4 T lymphocytes and reveal a rare population of latently infected cells responsive to physiological stimuli. In summary, our HIV-1 reporters enable visualization and purification of latent-cell populations and open up new perspectives for studies of latent HIV infection.

Concise review: chemical approaches for modulating lineage-specific stem cells and progenitors.

Generation and manipulation of lineage-restricted stem and progenitor cells in vitro and/or in vivo are critical for the development of stem cell-based clinical therapeutics. Lineage-restricted stem and progenitor cells have many advantageous qualities, including being able to efficiently engraft and differentiate into desirable cell types in vivo after transplantation, and they are much less tumorigenic than pluripotent cells. Generation of lineage-restricted stem and progenitor cells can be achieved by directed differentiation from pluripotent stem cells or lineage conversion from easily obtained somatic cells. Small molecules can be very helpful in these processes since they offer several important benefits. For example, the risk of tumorigenesis is greatly reduced when small molecules are used to replace integrated transcription factors, which are widely used in cell fate conversion. Furthermore, small molecules are relatively easy to apply, optimize, and manufacture, and they can more readily be developed into conventional pharmaceuticals. Alternatively, small molecules can be used to expand or selectively control the differentiation of lineage-restricted stem and progenitor cells for desirable therapeutics purposes in vitro or in vivo. Here we summarize recent progress in the use of small molecules for the expansion and generation of desirable lineage-restricted stem and progenitor cells in vitro and for selectively controlling cell fate of lineage-restricted stem and progenitor cells in vivo, thereby facilitating stem cell-based clinical applications.

Cellular reprogramming: a small molecule perspective.

The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the expression of a few transcription factors has attracted enormous interest in biomedical research and the field of regenerative medicine. iPSCs nearly identically resemble embryonic stem cells (ESCs) and can give rise to all cell types in the body, and thus have opened new opportunities for personalized regenerative medicine and new ways of modeling human diseases. Although some studies have raised concerns about genomic stability and epigenetic memory in the resulting cells, better understanding and control of the reprogramming process should enable enhanced efficiency and higher fidelity in reprogramming. Therefore, small molecules regulating reprogramming mechanisms are valuable tools to probe the process of reprogramming and harness cell fate transitions for various applications.

Leadership behaviors of athletic training leaders compared with leaders in other fields.

CONTEXT: Athletic trainers are in positions of leadership. OBJECTIVE: To determine self-reported leadership practices of head athletic trainers (HATCs) and program directors (PDs). DESIGN: Cross-sectional study. SETTING: Respondents' academic institutions. PATIENTS OR OTHER PARTICIPANTS: A total of 238 athletic training leaders completed the Leadership Practices Inventory. Of these, 50.4% (n = 120) were HATCs and 49.6% (n = 118) were PDs; 69.3% (n = 165) were men and 30.7% (n = 73) were women; almost all respondents (97.1%, n = 231) were white. Respondents typically reported having 11 to 15 years of experience as an athletic trainer (n = 57, 23.9%) and being between the ages of 30 and 39 years (n = 109, 45.8%). MAIN OUTCOME MEASURE(S): Categories of leadership behaviors (ie, Model, Inspire, Challenge, Encourage, and Enable) were scored from 1 (almost never) to 10 (almost always). Item scores were summed to compute mean category scores. We analyzed demographic information; used t ratios to compare the data from athletic training leaders (PDs and HATCs) with normative data; compared sex, age, position, ethnicity, and years of experience with leadership practices; and computed mean scores. RESULTS: Athletic training leaders reported using leadership behaviors similar to those of other leaders. The PDs reported using inspiring, challenging, enabling, and encouraging leadership behaviors more often than did the HATCs. No differences were found by ethnicity, age, years of experience, or leadership practices. CONCLUSIONS: Athletic training leaders are transformational leaders. Athletic training education program accreditation requirements likely account for the difference in leadership practices between PDs and HATCs.