Architecture of skin inflammation in psoriasis revealed by spatial transcriptomics.

Psoriasis is a chronic inflammatory skin disease, thought to be predominantly mediated by TH17 cells. Significance of other inflammatory pathways and the innate immune system is not well understood and the spatial heterogeneity of inflammation in the skin has largely been overlooked. Our aim was to create a comprehensive map of skin inflammation in psoriasis, exploring the tissue patterning of inflammation. In situ whole transcriptome sequencing (spatial sequencing) was performed on lesional psoriatic skin in four patients with moderate-to-severe disease to quantify all expressed genes within a tissue section. Transcriptional analysis revealed three major inflammatory niches in psoriasis skin, each with distinct cytokine circuits and chemokines: the hyperplastic epidermis, upper (papillary) dermis, and reticular dermis. Interestingly, key cytokines such as IL-23, IL-17 s, and TNFα were not notably present in the skin's transcriptomic signature. Unexpectedly, IL-32 showed strong expression in the dermis. Our findings underscore the complexity of psoriatic inflammation, highlighting its architectural heterogeneity and the roles of innate cytokines. Both IL-32 and IL-1 family cytokines appear to play critical roles in the dermal and epidermal inflammation, respectively, and may provide pharmacological targets to improve the control of the inflammatory process.

Is Cutaneous T-Cell Lymphoma Caused by Ultraviolet Radiation? A Comparison of UV Mutational Signatures in Malignant Melanoma and Mycosis Fungoides.

Ultraviolet (UV) radiation is a strong environmental carcinogen responsible for the pathogenesis of most skin cancers, including malignant melanoma (MM) and non-melanoma (keratinocyte) skin cancers. The carcinogenic role of UV was firmly established based on epidemiological evidence and molecular findings of the characteristic mutation signatures which occur during the excision repair of cyclobutane pyrimidine dimers and 6,4-photoproducts. The role of UV in the pathogenesis of mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphoma, remains controversial. Here, we performed whole-exome sequencing of 61 samples of MF cells microdissected from cutaneous lesions, and compared their mutational signatures to 340 MMs. The vast majority of MM mutations had a typical UV mutational signature (SBS 7, SBS 38, or DSB 1), underscoring the key role of ultraviolet as a mutagen. In contrast, the SBS 7 signature in MF comprised < 5% of all mutations. SBS 7 was higher in the intraepidermal MF cells (when compared to the dermal cells) and in the cells from tumors as compared to that in early-stage plaques. In conclusion, our data do not support the pathogenic role of UV in the pathogenesis of MF and suggest that the UV mutations are the result of the cumulative environmental ultraviolet exposure of cutaneous lesions rather than an early mutagenic event.

Genomic instability in early systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is associated with secondary malignancies. Previous studies have suggested that mutated cancer proteins, such as RNA polymerase III, are autoantigens promoting an inflammatory response in SSc. However, it has never been previously investigated whether non-neoplastic tissue in SSc harbors mutations which may play a role in SSc pathogenesis. METHODS: Skin biopsies were obtained from 8 sequential patients with a progressive form of early stage SSc (with severe skin and/or lung involvement). Areas of dermal fibrosis were microdissected and analyzed with deep, whole exome sequencing. Gene mutation patterns were compared to autologous buccal mucosal cells as a control. RESULTS: SSc skin biopsies were hypermutated with an average of 58 mutations/106 base pairs. The mutational pattern in all samples exhibited a clock-like signature, which is ubiquitous in cancers and in senescent cells. Of the 1997 genes we identified which were mutated in at least two SSc patients, 39 genes represented cancer drivers (i.e. tumor suppressor genes or oncogenes) which are commonly found in gynecological, squamous and gastrointestinal cancer signatures. Of all the mutations, the most common mutated genes were important in regulating pathways related to epigenetic histone modifications, DNA repair and genome integrity. CONCLUSIONS: Somatic hypermutation occurs in fibrotic skin in patients with early progressive SSc. Cancer driver gene mutations may potentially play a fundamental role in the pathogenesis of SSc.

Metagenomics Versus Metatranscriptomics of the Murine Gut Microbiome for Assessing Microbial Metabolism During Inflammation.

Shotgun metagenomics studies have improved our understanding of microbial population dynamics and have revealed significant contributions of microbes to gut homeostasis. They also allow in silico inference of the metagenome. While they link the microbiome with metabolic abnormalities associated with disease phenotypes, they do not capture microbial gene expression patterns that occur in response to the multitude of stimuli that constantly ambush the gut environment. Metatranscriptomics closes that gap, but its implementation is more expensive and tedious. We assessed the metabolic perturbations associated with gut inflammation using shotgun metagenomics and metatranscriptomics. Shotgun metagenomics detected changes in abundance of bacterial taxa known to be SCFA producers, which favors gut homeostasis. Bacteria in the phylum Firmicutes were found at decreased abundance, while those in phyla Bacteroidetes and Proteobacteria were found at increased abundance. Surprisingly, inferring the coding capacity of the microbiome from shotgun metagenomics data did not result in any statistically significant difference, suggesting functional redundancy in the microbiome or poor resolution of shotgun metagenomics data to profile bacterial pathways, especially when sequencing is not very deep. Obviously, the ability of metatranscriptomics libraries to detect transcripts expressed at basal (or simply low) levels is also dependent on sequencing depth. Nevertheless, metatranscriptomics informed about contrasting roles of bacteria during inflammation. Functions involved in nutrient transport, immune suppression and regulation of tissue damage were dramatically upregulated, perhaps contributed by homeostasis-promoting bacteria. Functions ostensibly increasing bacteria pathogenesis were also found upregulated, perhaps as a consequence of increased abundance of Proteobacteria. Bacterial protein synthesis appeared downregulated. In summary, shotgun metagenomics was useful to profile bacterial population composition and taxa relative abundance, but did not inform about differential gene content associated with inflammation. Metatranscriptomics was more robust for capturing bacterial metabolism in real time. Although both approaches are complementary, it is often not possible to apply them in parallel. We hope our data will help researchers to decide which approach is more appropriate for the study of different aspects of the microbiome.

Prevalence of Angina Among Primary Care Patients With Coronary Artery Disease.

Importance: Angina pectoris is associated with morbidity and mortality. Angina prevalence and frequency among contemporary US populations with coronary artery disease (CAD) remain incompletely defined. Objective: To ascertain the angina prevalence and frequency among stable outpatients with CAD. Design, Setting, and Participants: This cross-sectional survey study involved telephone-based administration of the Seattle Angina Questionnaire-7 (SAQ-7) between February 1, 2017, and July 31, 2017, to a nonconvenience sample of adults with established CAD who receive primary care through a large US integrated primary care network. Data analysis was performed from August 2017 to August 2019. Exposure: SAQ-7 administration. Main Outcomes and Measures: Angina prevalence and frequency were assessed using SAQ-7 question 2. Covariates associated with angina were assessed in univariable and multivariable regression. Results: Of 4139 eligible patients, 1612 responded to the survey (response rate, 38.9%). The mean (SD) age of the respondents was 71.8 (11.0) years, 577 (35.8%) were women, 1447 (89.8%) spoke English, 147 (9.1%) spoke Spanish, 1336 (82.8%) were White, 76 (4.7%) were Black, 92 (5.7%) were Hispanic, 974 (60.4%) had Medicare, and 83 (5.2%) had Medicaid. Among respondents, 342 (21.2%) reported experiencing angina at least once monthly; among those, 201 (12.5%) reported daily or weekly angina, and 141 respondents (8.7%) reported monthly angina. The mean (SD) SAQ-7 score was 93.7 (13.7). After multivariable adjustment, speaking a language other than Spanish or English (odds ratio [OR], 5.07; 95% CI, 1.39-18.50), Black race (OR, 2.01; 95% CI, 1.08-3.75), current smoking (OR, 1.88; 95% CI, 1.27-2.78), former smoking (OR, 1.69; 95% CI, 1.13-2.51), atrial fibrillation (OR, 1.52; 95% CI, 1.02-2.26), and chronic obstructive pulmonary disease (OR, 1.61; 95% CI, 1.18-2.18) were associated with more frequent angina. Male sex (OR, 0.63; 95% CI, 0.47-0.86), peripheral artery disease (OR, 0.63; 95% CI, 0.44-0.90), and novel oral anticoagulant use (OR, 0.19; 95% CI, 0.08-0.48) were associated with less frequent angina. Conclusions and Relevance: Among stable outpatients with CAD receiving primary care through an integrated primary care network, 21.2% of surveyed patients reported experiencing angina at least once monthly. Several objective demographic and clinical characteristics were associated with angina frequency. Proactive assessment of angina symptoms using validated assessment tools and estimation of patients at higher risk of suboptimally controlled angina may be associated with reduced morbidity.

The Neoantigen Landscape of Mycosis Fungoides.

Background: Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma, for which there is no cure. Immune checkpoint inhibitors have been tried in MF but the results have been inconsistent. To gain insight into the immunogenicity of MF we characterized the neoantigen landscape of this lymphoma, focusing on the known predictors of responses to immunotherapy: the quantity, HLA-binding strength and subclonality of neoantigens. Methods: Whole exome and whole transcriptome sequences were obtained from 24 MF samples (16 plaques, 8 tumors) from 13 patients. Bioinformatic pipelines (Mutect2, OptiType, MuPeXi) were used for mutation calling, HLA typing, and neoantigen prediction. PhyloWGS was used to subdivide malignant cells into stem and clades, to which neoantigens were matched to determine their clonality. Results: MF has a high mutational load (median 3,217 non synonymous mutations), resulting in a significant number of total neoantigens (median 1,309 per sample) and high-affinity neoantigens (median 328). In stage I disease most neoantigens were clonal but with stage progression, 75% of lesions had >50% subclonal antigens and 53% lesions had CSiN scores <1. There was very little overlap in neoantigens across patients or between different lesions on the same patient, indicating a high degree of heterogeneity. Conclusions: The neoantigen landscape of MF is characterized by high neoantigen load and significant subclonality which could indicate potential challenges for immunotherapy in patients with advanced-stage disease.

Independent evolution of cutaneous lymphoma subclones in different microenvironments of the skin.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma. Lesions of MF are formed by hematogenous seeding the skin with polyclonal (clonotypically diverse) neoplastic T-cells which accumulate numerous mutations and display a high degree of mutational, intratumoral heterogeneity (ITH). A characteristic but poorly studied feature of MF is epidermotropism, the tendency to infiltrate skin epithelial layer (epidermis) in addition to the vascularized dermis. By sequencing the exomes of the microdissected clusters of lymphoma cells from the epidermis and the dermis, we found that those microenvironments comprised different malignant clonotypes. Subclonal structure witnessed the independent mutational evolution in the epidermis and dermis. Thus, the epidermal involvement in MF could not be explained by gradual infiltration from the dermis but was caused by a separate seeding process followed by a quasi-neutral, branched evolution. In conclusion, tissue microenvironments shape the subclonal architecture in MF leading to "ecological heterogeneity" which contributes to the total ITH. Since ITH adversely affects cancer prognosis, targeting the microenvironment may present therapeutic opportunities in MF and other cancers.

Branched evolution and genomic intratumor heterogeneity in the pathogenesis of cutaneous T-cell lymphoma.

Mycosis fungoides (MF) is a slowly progressive cutaneous T-cell lymphoma (CTCL) for which there is no cure. In the early plaque stage, the disease is indolent, but development of tumors heralds an increased risk of metastasis and death. Previous research into the genomic landscape of CTCL revealed a complex pattern of >50 driver mutations implicated in more than a dozen signaling pathways. However, the genomic mechanisms governing disease progression and treatment resistance remain unknown. Building on our previous discovery of the clonotypic heterogeneity of MF, we hypothesized that this lymphoma does not progress in a linear fashion as currently thought but comprises heterogeneous mutational subclones. We sequenced exomes of 49 cases of MF and identified 28 previously unreported putative driver genes. MF exhibited extensive intratumoral heterogeneity (ITH) of a median of 6 subclones showing a branched phylogenetic relationship pattern. Stage progression was correlated with an increase in ITH and redistribution of mutations from stem to clades. The pattern of clonal driver mutations was highly variable, with no consistent mutations among patients. Similar intratumoral heterogeneity was detected in leukemic CTCL (Sézary syndrome). Based on these findings, we propose a model of MF pathogenesis comprising divergent evolution of cancer subclones and discuss how ITH affects the efficacy of targeted drug therapies and immunotherapies for CTCL.

Skin colonization by circulating neoplastic clones in cutaneous T-cell lymphoma.

Mycosis fungoides (MF) is a mature T-cell lymphoma currently thought to develop primarily in the skin by a clonal expansion of a transformed, resident memory T cell. However, this concept does not explain the key characteristics of MF, such as the debut with multiple, widespread skin lesions or inability of skin-directed therapies to provide cure. The testable inference of the mature T-cell theory is the clonality of MF with respect to all rearranged T-cell receptor (TCR) genes. Here, we used a whole-exome sequencing approach to detect and quantify TCR-α, ß, and γ clonotypes in tumor cell clusters microdissected from MF lesions. This method allowed us to calculate the tumor cell fraction of the sample and therefore an unequivocal identification of the TCR clonotypes as neoplastic. Analysis of TCR sequences from 29 patients with MF stage I to IV proved the existence of multiple T-cell clones within the tumor cell fraction, with a considerable variation between patients and between lesions from the same patient (median, 11 clones; range, 2-80 clones/sample). We have also detected multiple neoplastic clones in the peripheral blood in all examined patients. Based on these findings, we propose that circulating neoplastic T-cell clones continuously replenish the lesions of MF, thus increasing their heterogeneity by a mechanism analogous to the consecutive tumor seeding. We hypothesize that circulating neoplastic clones might be a promising target for therapy and could be exploited as a potential biomarker in MF.

Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing.

Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, is believed to represent a clonal expansion of a transformed skin-resident memory T cell. T-cell receptor (TCR) clonality (ie, identical sequences of rearranged TCRα, TCRß, and TCRγ), the key premise of this hypothesis, has been difficult to document conclusively because malignant cells are not readily distinguishable from the tumor-infiltrating reactive lymphocytes that contribute to the TCR clonotypic repertoire of MF. Here, we have successfully adopted targeted whole-exome sequencing (WES) to identify the repertoire of rearranged TCR genes in tumor-enriched samples from patients with MF. Although some of the investigated MF biopsies had the expected frequency of monoclonal rearrangements of TCRγ corresponding to that of tumor cells, the majority of the samples presented multiple TCRγ, TCRα, and TCRß clonotypes by WES. Our findings are compatible with the model in which the initial malignant transformation in MF does not occur in mature memory T cells but rather at the level of T-lymphocyte progenitors before TCRß or TCRα rearrangements. We have also shown that WES can be combined with whole-transcriptome sequencing in the same sample, which enables comprehensive characterization of the TCR repertoire in relation to tumor content. WES/whole-transcriptome sequencing might be applicable to other types of T-cell lymphomas to determine clonal dominance and clonotypic heterogeneity in these malignancies.

An Audit and Feedback Intervention to Improve Internal Medicine Residents' Performance on Ambulatory Quality Measures: A Randomized Controlled Trial.

Audit and feedback is an effective method to improve attending physician performance. However, there are limited data on how audit and feedback impacts care provided by resident physicians. The authors conducted a 3-arm randomized clinical trial among internal medicine resident physicians to examine the impact of an audit and feedback intervention on ambulatory quality measures (AQMs). Residents in all 3 groups received an email containing the contact information of a population health coordinator and a list of AQMs (control). In addition, the Practice Target group received individual AQM data compared to the target AQM goals for all primary care practices. The Peer Comparison group received information on individual AQM data compared to the average performance of residents in the same postgraduate year. Residents in each intervention group received updated information 6 months later. Ten AQMs related to diabetes care, hypertension management, lipid control, and cancer screening, as well as a composite quality score, were examined at baseline, 6 months, and 13 months. At 13 months follow-up, the Practice Target group had statistically significant improvement in cervical cancer screening rate (77% vs. 65.3%), colorectal cancer screening rate (72.5% vs. 64.6%), and composite quality score (71.7% vs 65.4%) compared to baseline. Providing internal medicine residents with individual AQMs data compared to target goal for the practice led to statistically significant improvement in cancer screening rates and the composite quality score. Audit and feedback may be a relatively simple yet effective tool to improve population health in the resident clinic setting.

A Survey of Molecular Heterogeneity in Hepatocellular Carcinoma.

Understanding the heterogeneity of dysregulated pathways associated with the development of hepatocellular carcinoma (HCC) may provide prognostic and therapeutic avenues for disease management. As HCC involves a complex process of genetic and epigenetic modifications, we evaluated expression of both polyadenylated transcripts and microRNAs from HCC and liver samples derived from two cohorts of patients undergoing either partial hepatic resection or liver transplantation. Copy number variants were inferred from whole genome low-pass sequencing data, and a set of 56 cancer-related genes were screened using an oncology panel assay. HCC was associated with marked transcriptional deregulation of hundreds of protein-coding genes. In the partially resected livers, diminished transcriptional activity was observed in genes associated with drug catabolism and increased expression in genes related to inflammatory responses and cell proliferation. Moreover, several long noncoding RNAs and microRNAs not previously linked with HCC were found to be deregulated. In liver transplant recipients, down-regulation of genes involved in energy production and up-regulation of genes associated with glycolysis were detected. Numerous copy number variants events were observed, with hotspots on chromosomes 1 and 17. Amplifications were more common than deletions and spanned regions containing genes potentially involved in tumorigenesis. Colony stimulating factor 1 receptor (CSF1R), fibroblast growth factor receptor 3 (FGFR3), fms-like tyrosine kinase 3 (FLT3), nucleolar phosphoprotein B23 (NPM1), platelet-derived growth factor receptor alpha polypeptide (PDGFRA), phosphatase and tensin homolog (PTEN), G-protein-coupled receptors-like receptor Smoothened (SMO), and tumor protein P53 (TP53) were mutated in all tumors; another 26 cancer-related genes were mutated with variable penetrance. Conclusion: Our results underscore the marked molecular heterogeneity between HCC tumors and reinforce the notion that precision medicine approaches are needed for management of individual HCC. These data will serve as a resource to generate hypotheses for further research to improve our understanding of HCC biology. (Hepatology Communications 2018; 00:000-000).

Impact of a Population Health Management Intervention on Disparities in Cardiovascular Disease Control.

BACKGROUND: Healthcare systems use population health management programs to improve the quality of cardiovascular disease care. Adding a dedicated population health coordinator (PHC) who identifies and reaches out to patients not meeting cardiovascular care goals to these programs may help reduce disparities in cardiovascular care. OBJECTIVE: To determine whether a program that used PHCs decreased racial/ethnic disparities in LDL cholesterol and blood pressure (BP) control. DESIGN: Retrospective difference-in-difference analysis. PARTICIPANTS: Twelve thousdand five hundred fifty-five primary care patients with cardiovascular disease (cohort for LDL analysis) and 41,183 with hypertension (cohort for BP analysis). INTERVENTION: From July 1, 2014-December 31, 2014, 18 practices used an information technology (IT) system to identify patients not meeting LDL and BP goals; 8 practices also received a PHC. We examined whether having the PHC plus IT system, compared with having the IT system alone, decreased racial/ethnic disparities, using difference-in-difference analysis of data collected before and after program implementation. MAIN MEASURES: Meeting guideline concordant LDL and BP goals. KEY RESULTS: At baseline, there were racial/ethnic disparities in meeting LDL (p = 0.007) and BP (p = 0.0003) goals. Comparing practices with and without a PHC, and accounting for pre-intervention LDL control, non-Hispanic white patients in PHC practices had improved odds of LDL control (OR 1.20 95% CI 1.09-1.32) compared with those in non-PHC practices. Non-Hispanic black (OR 1.15 95% CI 0.80-1.65) and Hispanic (OR 1.29 95% CI 0.66-2.53) patients saw similar, but non-significant, improvements in LDL control. For BP control, non-Hispanic white patients in PHC practices (versus non-PHC) improved (OR 1.13 95% CI 1.05-1.22). Non-Hispanic black patients (OR 1.17 95% CI 0.94-1.45) saw similar, but non-statistically significant, improvements in BP control, but Hispanic (OR 0.90 95% CI 0.59-1.36) patients did not. Interaction testing confirmed that disparities did not decrease (p = 0.73 for LDL and p = 0.69 for BP). CONCLUSIONS: The population health management intervention did not decrease disparities. Further efforts should explicitly target improving both healthcare equity and quality. Clinical Trials #: NCT02812303 ( ClinicalTrials.gov ).

Chronic disease outcomes from primary care population health program implementation.

OBJECTIVES: We implemented a health information technology-enabled population health management program for chronic disease management in academic hospital-affiliated primary care practices, then compared quality-of-care outcome measures among practices assigned a central population health coordinator (PHC) and those not assigned a PHC. STUDY DESIGN: Quasi-experimental. METHODS: Central PHCs were nonrandomly assigned to 8 of 18 practices. They met with physicians, managed lists of patients not at goal in chronic disease registries, and performed administrative tasks. In non-PHC practices, existing staff remained responsible for these tasks. The primary outcome was difference-in-differences over the 6-month follow-up period between PHC and non-PHC practices for outcome measures for diabetes (low-density lipoprotein cholesterol [LDL-C], glycated hemoglobin [A1C], and blood pressure [BP] goal attainment), cardiovascular disease (LDL-C goal attainment), and hypertension (BP goal attainment). Secondary outcomes included process measures only (obtaining LDL-C, A1C, and BP readings) and cancer screening test completion. RESULTS: The difference in the percentage point (PP) increase in outcome measures over follow-up was greater in PHC practices than non-PHC practices for all measures among patients with diabetes (LDL-C, 4.6 PP; A1C, 4.8 PP; BP, 4.7 PP), cardiovascular disease (LDL-C, 3.3 PP), and hypertension (BP, 2.3 PP) (adjusted P all <.001). Changes in cancer screening outcomes, which were not a focus of PHC efforts, were similar between PHC and non-PHC practices. CONCLUSIONS: Use of central PHCs led to greater improvement in short-term chronic disease outcome measures compared with patients in practices not assigned a central PHC.

Development and Implementation of a Collaborative Team Care Model for Effective Insulin Use in an Academic Medical Center Primary Care Network.

Improving glycemic control across a primary care diabetes population is challenging. This article describes the development, implementation, and outcomes of the Diabetes Care Collaborative Model (DCCM), a collaborative team care process focused on promoting effective insulin use targeting patients with hyperglycemia in a patient-centered medical home model. After a pilot, the DCCM was implemented in 18 primary care practices affiliated with an academic medical center. Its implementation was associated with improvements in glycemic control and increase in insulin prescription longitudinally and across the entire population, with a >1% reduction in the proportion of glycated hemoglobin >9% at 2 years after the implementation compared with the 2 years prior ( P < .001). Facilitating factors included diverse stakeholder engagement, institutional alignment of priorities, awarding various types of credits for participation and implementation to providers, and a strong theoretical foundation using the principles of the collaborative care model.

Text to Move: A Randomized Controlled Trial of a Text-Messaging Program to Improve Physical Activity Behaviors in Patients With Type 2 Diabetes Mellitus.

BACKGROUND: Text messages are increasingly being used because of the low cost and the ubiquitous nature of mobile phones to engage patients in self-care behaviors. Self-care is particularly important in achieving treatment outcomes in type 2 diabetes mellitus (T2DM). OBJECTIVE: This study examined the effect of personalized text messages on physical activity, as measured by a pedometer, and clinical outcomes in a diverse population of patients with T2DM. METHODS: Text to Move (TTM) incorporates physical activity monitoring and coaching to provide automated and personalized text messages to help patients with T2DM achieve their physical activity goals. A total of 126 English- or Spanish-speaking patients with glycated hemoglobin A1c (HbA1c) >7 were enrolled in-person to participate in the study for 6 months and were randomized into either the intervention arm that received the full complement of the intervention or a control arm that received only pedometers. The primary outcome was change in physical activity. We also assessed the effect of the intervention on HbA1c, weight, and participant engagement. RESULTS: All participants (intervention: n=64; control: n=62) were included in the analyses. The intervention group had significantly higher monthly step counts in the third (risk ratio [RR] 4.89, 95% CI 1.20 to 19.92, P=.03) and fourth (RR 6.88, 95% CI 1.21 to 39.00, P=.03) months of the study compared to the control group. However, over the 6-month follow-up period, monthly step counts did not differ statistically by group (intervention group: 9092 steps; control group: 3722 steps; RR 2.44, 95% CI 0.68 to 8.74, P=.17). HbA1c decreased by 0.07% (95% CI -0.47 to 0.34, P=.75) in the TTM group compared to the control group. Within groups, HbA1c decreased significantly from baseline in the TTM group by -0.43% (95% CI -0.75 to -0.12, P=.01), but nonsignificantly in the control group by -0.21% (95% CI -0.49 to 0.06, P=.13). Similar changes were observed for other secondary outcomes. CONCLUSION: Personalized text messaging can be used to improve outcomes in patients with T2DM by employing optimal patient engagement measures.

Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics.

The advent of next generation sequencing (NGS) has enabled investigations of the gut microbiome with unprecedented resolution and throughput. This has stimulated the development of sophisticated bioinformatics tools to analyze the massive amounts of data generated. Researchers therefore need a clear understanding of the key concepts required for the design, execution and interpretation of NGS experiments on microbiomes. We conducted a literature review and used our own data to determine which approaches work best. The two main approaches for analyzing the microbiome, 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics, are illustrated with analyses of libraries designed to highlight their strengths and weaknesses. Several methods for taxonomic classification of bacterial sequences are discussed. We present simulations to assess the number of sequences that are required to perform reliable appraisals of bacterial community structure. To the extent that fluctuations in the diversity of gut bacterial populations correlate with health and disease, we emphasize various techniques for the analysis of bacterial communities within samples (α-diversity) and between samples (ß-diversity). Finally, we demonstrate techniques to infer the metabolic capabilities of a bacteria community from these 16S and shotgun data.

Cerebrospinal Fluid in a Small Cohort of Patients with Multiple Sclerosis Was Generally Free of Microbial DNA.

Multiple sclerosis (MS) is a common cause of non-traumatic neurologic disability with high incidence in many developed countries. Although the etiology of the disease remains elusive, it is thought to entail genetic and environmental causes, and microbial pathogens have also been envisioned as contributors to the phenotype. We conducted a metagenomic survey in cerebrospinal fluid (CSF) from 28 MS patients and 15 patients suffering other type of neurological conditions. We detected bacterial reads in eight out of the 15 non-MS patients and in a single MS patient, at an abundance >1% of total classified reads. Two patients were of special interest: one non-MS patient harbored ~73% bacterial reads, while an MS patient had ~83% bacterial reads. In the former case, Veillonella parvula, a bacterium occasionally found associated with meningitis was the predominant species, whilst Kocuria flava, apparently an environmental bacterium, predominated in the latter case. Thirty-four out of 43 samples contained <1% bacterial reads, which we regard as cross- or environmental contamination. A few viral reads corresponding to Epstein-Barr virus, cytomegalovirus, and parvovirus were also identified. Our results suggest that CSF of MS patients is often (but not always) free of microbial DNA.

Identification of hepatotropic viruses from plasma using deep sequencing: a next generation diagnostic tool.

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.