Clonal expansion within pneumococcal serotype 6C after use of seven-valent vaccine

Streptococcus pneumoniae causes invasive infections, primarily at the extremes of life. A seven-valent conjugate vaccine (PCV7) is used to protect against invasive pneumococcal disease in children. Within three years of PCV7 introduction, we observed a fourfold increase in serotype 6C carriage, predominantly due to a single clone. We determined the whole-genome sequences of nineteen S. pneumoniae serotype 6C isolates, from both carriage (n = 15) and disease (n = 4) states, to investigate the emergence of serotype 6C in our population, focusing on a single multi-locus sequence type (MLST) clonal complex 395 (CC395). A phylogenetic network was constructed to identify different lineages, followed by analysis of variability in gene sets and sequences. Serotype 6C isolates from this single geographical site fell into four broad phylogenetically distinct lineages. Variation was seen in the 6C capsular locus and in sequences of genes encoding surface proteins. The largest clonal complex was characterised by the presence of lantibiotic synthesis locus. In our population, the 6C capsular locus has been introduced into multiple lineages by independent capsular switching events. However, rapid clonal expansion has occurred within a single MLST clonal complex. Worryingly, plasticity exists within current and potential vaccine-associated loci, a consideration for future vaccine use, target selection and design

RNA sequencing-based single sample predictors of molecular subtype and risk of recurrence for clinical assessment of early-stage breast cancer

BackgroundMultigene expression assays for molecular subtypes and biomarkers can aid clinical management of early invasive breast cancer. Based on RNA-sequencing we aimed to develop single-sample predictor (SSP) models for conventional clinical markers, molecular intrinsic subtype and risk of recurrence (ROR).MethodsA uniformly accrued breast cancer cohort of 7743 patients with RNA-sequencing data from fresh tissue was divided into a training set and a reserved test set. We trained SSPs for PAM50 molecular subtypes and ROR assigned by nearest-centroid (NC) and SSPs for conventional clinical markers from histopathology data. Additionally, SSP classifications were compared with Prosigna® in two external cohorts. Prognostic value was assessed using distant recurrence-free interval.ResultsIn the test set, agreement between SSP and NC classifications for PAM50 (five subtypes) and Subtype (four subtypes) was high (85%, Kappa=0.78) and very high (90%, Kappa=0.84) respectively. Accuracy for ROR risk category was high (84%, Kappa=0.75, weighted Kappa=0.90). The prognostic value for SSP and NC was assessed as equivalent. Agreement for SSP and histopathology was very high or high for receptor status, while moderate and poor for Ki67 status and Nottingham histological grade, respectively. SSP concordance with Prosigna® was high for subtype and moderate and high for ROR risk category. In pooled analysis, concordance between SSP and Prosigna® for emulated treatment recommendation for chemotherapy (yes vs. no) was high (85%, Kappa=0.66). In postmenopausal ER+/HER2-/N0 patients SSP application suggested changed treatment recommendations for up to 17% of patients, with nearly balanced escalation and de-escalation of chemotherapy.ConclusionsSSP models for histopathological variables, PAM50, and ROR classifications can be derived from RNA-sequencing that closely matches clinical tests. Agreement and outcome analyses suggest that NC and SSP models are interchangeable on a group-level and nearly so on a patient level. Retrospective evaluation in postmenopausal ER+/HER2-/N0 patients suggested that molecular testing could lead to a changed therapy recommendation for almost one-fifth of patients

Identification of novel imidazo[1,2-a]pyridine inhibitors targeting M. tuberculosis QcrB.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism 937ACC>937GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization

Tunable phenotypic variability through an autoregulatory alternative sigma factor circuit.

Genetically identical individuals in bacterial populations can display significant phenotypic variability. This variability can be functional, for example by allowing a fraction of stress prepared cells to survive an otherwise lethal stress. The optimal fraction of stress prepared cells depends on environmental conditions. However, how bacterial populations modulate their level of phenotypic variability remains unclear. Here we show that the alternative sigma factor σV circuit in Bacillus subtilis generates functional phenotypic variability that can be tuned by stress level, environmental history and genetic perturbations. Using single-cell time-lapse microscopy and microfluidics, we find the fraction of cells that immediately activate σV under lysozyme stress depends on stress level and on a transcriptional memory of previous stress. Iteration between model and experiment reveals that this tunability can be explained by the autoregulatory feedback structure of the sigV operon. As predicted by the model, genetic perturbations to the operon also modulate the response variability. The conserved sigma-anti-sigma autoregulation motif is thus a simple mechanism for bacterial populations to modulate their heterogeneity based on their environment

Specialized High�Protein Oral Nutrition Supplement Improves Home Nutrient Intake of Malnourished Older Adults Without Decreasing Usual Food Intake

Background: Reduced nutrient intake is common in patients after hospitalization, contributing to increased risk for readmissionand mortality. Oral nutrition supplements can improve nutrition status and clinical outcomes, but intake of food is prioritized byclinicians. This study examines the impact of a high-protein oral nutrition supplement (S-ONS) on nutrient intake post discharge. Methods: In a subset of patients (14 S-ONS and 16 placebo) from the NOURISH (Nutrition effect On Unplanned ReadmIssionsand Survival in Hospitalized patients) trial, 24-hour dietary recalls were conducted on 3 randomly selected days during the weeksof 30, 60, and 90 days post discharge. Nutrient intake was estimated using Nutrition Data System for Research software. Adequateenergy and protein intake were dened as 30 kcal/kg/d and 1.2 g/kg/d, respectively. Dietary Reference Intakes (DRIs) were usedfor other nutrients. Results: Less than half of patients met the requirements for energy, protein, and 12 micronutrients from foodintake alone during the study. Energy and protein intakes from food were not diminished relative to placebo. Considering nutrientintake from both food and S-ONS, 50% and 71% of patients receiving S-ONSs met energy and protein goals respectively at 90 days(compared with 29% and 36%, in the placebo group), and 100% met the DRI for total carbohydrate, iron, phosphorus, copper,selenium, thiamin, and riboavin at all time points, all of which were consumed at higher amounts vs placebo. Conclusion: Threemonths of S-ONS consumption increases intake of numerous nutrients without decreasing nutrient intake from food in oldermalnourished adults post discharge

Hyperkeratotic hand eczema:Eczema or not?

BACKGROUND: Hyperkeratotic hand eczema (HHE) is a typical clinical hand eczema subtype with a largely unknown pathophysiology. OBJECTIVE: To investigate histopathology, expression of keratins (K), epidermal barrier proteins, and adhesion molecules in HHE. METHODS: Palmar skin biopsies (lesional and perilesional) were obtained from seven HHE patients and two healthy controls. Moreover, 135 candidate genes associated with palmoplantar keratoderma were screened for mutations. RESULTS: Immunofluorescence staining showed a significant reduction of K9 and K14 in lesional skin. Upregulation was found for K5, K6, K16, and K17 in lesional skin compared with perilesional and healthy palmar skin. Further, upregulation of involucrin and alternating loricrin staining, both in an extracellular staining pattern, was found. Filaggrin expression was similar in lesional, perilesional, and control skin. No monogenetic mutations were found. CONCLUSION: Currently, the phenotype of HHE is included in the hand eczema classification system; however, it can be argued whether this is justified. The evident expression of filaggrin and involucrin in lesional skin does not support a pathogenesis of atopic eczema. The upregulation of K6, K16, and K17 and reduction of K9 and K14 might contribute to the underlying pathogenesis. Unfortunately, comparison with hand eczema studies is not possible yet, because similar protein expression studies are lacking

Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.</p

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Intrauterine exposures, pregnancy estrogens and breast cancer risk: where do we currently stand?

Since 1990, when a hypothesis on intrauterine influences on breast cancer risk was published, several studies have provided supportive, indirect evidence by documenting associations of birth weight and other correlates of the prenatal environment with breast cancer risk in offspring. Recent results from a unique cohort of women with documented exposure to diethylstilbestrol in utero have provided direct evidence in support of a potential role of pregnancy oestrogens on breast cancer risk in offspring