The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, ␤-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfurcompound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfurcompound oxidation by ␤-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.Thiobacillus denitrificans, first isolated by Beijerinck over a century ago (4), was one of the first nonfilamentous bacteria ever described to be capable of growth on inorganic sulfur compounds as sole energy sources (47, 49). Characterized by its ability to conserve energy from the oxidation of inorganic sulfur compounds under either aerobic or denitrifying conditions, T. denitrificans is the best studied of the very few obligate chemolithoautotrophic species known to couple denitrification to sulfur-compound oxidation (Thiomicrospira denitrificans and Thioalkalivibrio thiocyanodenitrificans also have this ability [76,85]). Despite many years of work on the biochemistry of inorganic sulfur-compound oxidation by Thiobacillus thioparus and T. denitrificans, the mechanisms of oxidation and how they are coupled to energy conservation are still not well understood in these ␤-proteobacteria, relative to the advances made with facultatively chemolithotrophic ␣-proteobacterial genera, such as Paracoccus and Starkeya (28,39,45,50). The availability of the complete genome sequence should enable elucidation of the sulfur-oxidation pathway(s) and lead to specifically focused biochemical investigations to resolve these knowledge gaps.Rec...

Background: Our aim was to identify and compare modifiable risk factors associated with adverse pregnancy outcomes in women with type 1 and type 2 diabetes and to identify effective maternity clinics. Methods:We included 17,375 pregnancies in 15,290 women with diabetes in a populationbased cohort study across 172 maternity clinics in England, Wales and the Isle of Man.Obstetric complications (preterm delivery, large birthweight) and adverse pregnancy outcomes (congenital anomaly, stillbirth, neonatal death) were obtained for pregnancies completed between 01 January 2014 and 31 December 2018. We assessed associations between modifiable (glycaemia, obesity, clinic) and non-modifiable risk factors (age, deprivation, ethnicity) with pregnancy outcomes.Results: Of 17,375 pregnancies, 8,690 (50.0%) were in women with type 1 and 8,685 (50.0%) in women with type 2 diabetes. The rates of preterm delivery (42.5% type 1, 23.4% type 2), and large birthweight (52.2% type 1, 26.2% type 2) were higher in type 1 diabetes (p<0.001).The prevalence of congenital anomaly (44.8/1000 type 1, 40.5/1000 type 2; p=0.175), and stillbirth (10.4/1000 type 1, 13.5/1000 type 2; p=0.072) did not differ but neonatal death rates (7.4/1000 type 1, 11.2/1000 type 2; p=0.013) were higher in type 2 diabetes. Independent risk factors for perinatal death were third trimester HbA1c > 48mmol/mol (OR 3.06, 95% CI 2.16 to 4.33), living in the highest deprivation quintile (OR 2.29 95% CI 1.16 to 4.52) and having type 2 diabetes (OR 1.65 95% CI 1.18 to 2.31). Variations in glycaemia and large birthweight were associated with maternal characteristics (diabetes duration, deprivation, BMI) without substantial differences between clinics.Interpretation: Our data highlight persistent adverse pregnancy outcomes in type 1 and type 2 diabetes. Maternal glycaemia and obesity are the key modifiable risk factors. No clinics were achieving appreciably better outcomes, suggesting that healthcare system changes are needed

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DNA microarrays encompassing the entire genome of Yersinia pestis were used to characterize global regulatory changes during steady-state vegetative growth occurring after shift from 26 to 37°C in the presence and absence of Ca 2؉ . Transcriptional profiles revealed that 51, 4, and 13 respective genes and open reading frames (ORFs) on pCD, pPCP, and pMT were thermoinduced and that the majority of these genes carried by pCD were downregulated by Ca 2؉ . In contrast, Ca 2؉ had little effect on chromosomal genes and ORFs, of which 235 were thermally upregulated and 274 were thermally downregulated. The primary consequence of these regulatory events is profligate catabolism of numerous metabolites available in the mammalian host.Bubonic plague caused by Yersinia pestis is generally recognized as the most devastating acute infectious disease experienced by mankind. It is therefore of interest that this organism has evolved within the last 10,000 years from Yersinia pseudotuberculosis (1), known to cause chronic enteropathogenic disease. Despite their very close resemblance, plague bacilli have both lost central genes of intermediary metabolism retained in its predecessor and acquired unique genes by lateral transfer (7). For example, even though early studies showed that Y. pestis possesses functional Embden-Meyerhof (28) and Entner-Doudoroff (20) pathways plus a complete tricarboxylic acid (TCA) cycle (13, 27), the species-specific absence of detectable glucose 6-phosphate dehydrogenase (Zwf) prevents use of hexose via the pentose-phosphate pathway (21). Similarly, loss of aspartase (AspA) activity in Y. pestis but not Y. pseudotuberculosis prevents complete catabolism of L-glutamic acid, which undergoes conversion and excretion as L-aspartate (12). In addition, Y. pestis possesses additional species-specific mutations that cause nutritional requirements at 26°C, prevent utilization of potential metabolites, and eliminate host cell invasins and adhesins (7); these events are now characterized by genomic sequencing (11, 23). The nature of nutritional requirements at 37°C is more complex and, as noted below, dependent upon plasmid profile, the presence or absence of Ca 2ϩ , Na ϩ , dicarboxylic amino acids, and regulatory functions addressed in this report.Established functions unique to Y. pestis are encoded by species-specific ϳ10-kb pPCP and ϳ100-kb pMT. The former encodes plasminogen activator (Pla) required for tissue invasion from dermal sites infected by fleabite whereas the structural genes for anti-phagocytic capsular fraction 1 (Caf1) and murine toxin (MT), required for survival in the flea, reside on pMT (7,25). Plague bacilli and the enteropathogenic yersiniae share a ϳ70-kb plasmid (pCD in Y. pestis) encoding a type III protein secretion system (TTSS) that delivers cytotoxins termed Yops to the cytosol of professional and nonprofessional phagocytes (8) and excretes soluble LcrV (V antigen), which inhibits generation of proinflammatory cytokines by upregulating interleukin-10 (6). These functions provide th...

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for α-smooth muscle actin and several other contractile proteins; 2) ‘inflammatory’ CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

BackgroundValue-based health care aims to optimize the balance of patient outcomes and health care costs. To improve value in perinatal care using this strategy, standard outcomes must first be defined. The objective of this work was to define a minimum, internationally appropriate set of outcome measures for evaluating and improving perinatal care with a focus on outcomes that matter to women and their families.MethodsAn interdisciplinary and international Working Group was assembled. Existing literature and current measurement initiatives were reviewed. Serial guided discussions and validation surveys provided consumer input. A series of nine teleconferences, incorporating a modified Delphi process, were held to reach consensus on the proposed Standard Set.ResultsThe Working Group selected 24 outcome measures to evaluate care during pregnancy and up to 6 months postpartum. These include clinical outcomes such as maternal and neonatal mortality and morbidity, stillbirth, preterm birth, birth injury and patient-reported outcome measures (PROMs) that assess health-related quality of life (HRQoL), mental health, mother-infant bonding, confidence and success with breastfeeding, incontinence, and satisfaction with care and birth experience. To support analysis of these outcome measures, pertinent baseline characteristics and risk factor metrics were also defined.ConclusionsWe propose a set of outcome measures for evaluating the care that women and infants receive during pregnancy and the postpartum period. While validation and refinement via pilot implementation projects are needed, we view this as an important initial step towards value-based improvements in care.Electronic supplementary materialThe online version of this article (10.1186/s12913-018-3732-3) contains supplementary material, which is available to authorized users.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

The present work describes a general method for the selective attachment of proteins to solid surfaces through their C-termini that can be used for the efficient creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal alpha-thioester and a modified surface containing N-terminal Cys residues. alpha-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing engineered inteins. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain using a protein microarrayer.