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Introduction: Biochemical alterations in the esophagus of patients with eosinophilic esophagitis (EoE) are poorly understood. We used Raman spectroscopy through a pediatric endoscope to identify key Raman features reflective of the esophageal biochemical composition to differentiate between children with EoE from non-EoE controls and between children with active (aEoE) and inactive EoE (iEoE)., Methods: Spectral measurements were obtained using a customized pediatric endoscope-compatible fiber-optic Raman probe in real time during an esophagogastroduodenoscopy. Chemometric analysis was performed to identify key Raman features associated with EoE. Pearson correlation analysis was used to assess relationship between the key Raman features and EoE activity indices. Their diagnostic utility was ascertained using the receiver operator characteristic curve analysis., Results: Forty-three children were included in the study (EoE = 32 [74%] and non-EoE control = 11 [26%]; aEoE = 20 [63%] and iEoE = 12 [37%]). Raman intensities assigned to lipids, proteins, and glycogen:protein ratio accurately distinguished children with EoE from those without EoE and aEoE from iEoE. They significantly correlated with EoE activity indices. The Raman peak ratio for lipids had 90.6% sensitivity, 100% specificity, and an area under the curve of 0.95 to differentiate children with EoE from non-EoE controls. The glycogen:protein ratio had 70% sensitivity, 91.7% specificity, and an area under the curve of 0.75 to distinguish children with aEoE from iEoE., Discussion: Real-time intraendoscopy Raman spectroscopy is an effective method for identifying spectral markers reflective of the esophageal biochemical composition in children with EoE. This technique may aid in the diagnosis and monitoring of EoE and help to elucidate EoE pathogenesis., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

The majority of disease-associated variants identified through genome-wide association studies are located outside of protein-coding regions. Prioritizing candidate regulatory variants and gene targets to identify potential biological mechanisms for further functional experiments can be challenging. To address this challenge, we developed FORGEdb ( https://forgedb.cancer.gov/ ; https://forge2.altiusinstitute.org/files/forgedb.html ; and https://doi.org/10.5281/zenodo.10067458 ), a standalone and web-based tool that integrates multiple datasets, delivering information on associated regulatory elements, transcription factor binding sites, and target genes for over 37 million variants. FORGEdb scores provide researchers with a quantitative assessment of the relative importance of each variant for targeted functional experiments., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Since spatially offset Raman spectroscopy (SORS) can acquire biochemical measurements of tissue quality through light scattering materials, we investigated the feasibility of this technique to acquire Raman bands related to the fracture resistance of bone. Designed to maximize signals at different offsets, a SORS probe was used to acquire spectra from cadaveric bone with and without skin-like tissue phantoms attenuating the light. Autoclaving the lateral side of femur mid-shafts from 5 female and 5 male donors at 100 °C and again at 120 °C reduced the yield stress of cortical beams subjected to three-point bending. It did not affect the volumetric bone mineral density or porosity. Without tissue phantoms, autoclaving affected more Raman characteristics of the organic matrix when determined by peak intensity ratios, but fewer matrix properties depended on the three offsets (5 mm, 6 mm, and 7 mm) when determined by band area ratios. The cut-off in the thickness of the tissue phantom layers was ∼4 mm for most properties, irrespective of offset. Matching trends when spectra were acquired without phantom layers between bone and the probe, ν 1 PO 4 3- /Amide III and ν 1 PO 4 3- /(proline + OH-proline) were higher and lower in the non-treated bone than in the autoclaved bone, respectively, when the thickness of tissue phantom layers was 4 mm. The layers, however, caused a loss of sensitivity to autoclaving-related changes in ν 3 CO 3 /ν 1 PO 4 3- and crystallinity. Without advanced post-processing of Raman spectra, SORS acquisition through turbid layers can detect changes in Raman properties of bone that accompany a loss in bone strength., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jeffry S Nyman reports financial support and equipment, drugs, or supplies were provided by National Institute of Arthritis and Musculoskeletal and Skin Diseases. Jeffry S Nyman reports financial support and administrative support were provided by US Department of Veterans Affairs., (Published by Elsevier B.V.)

Objective: Nutrition therapy for gestational diabetes mellitus (GDM) has conventionally focused on carbohydrate restriction. In a randomized controlled trial (RCT), we tested the hypothesis that a diet (all meals provided) with liberalized complex carbohydrate (60%) and lower fat (25%) (CHOICE diet) could improve maternal insulin resistance and 24-h glycemia, resulting in reduced newborn adiposity (NB%fat; powered outcome) versus a conventional lower-carbohydrate (40%) and higher-fat (45%) (LC/CONV) diet., Research Design and Methods: After diagnosis (at ∼28-30 weeks' gestation), 59 women with diet-controlled GDM (mean ± SEM; BMI 32 ± 1 kg/m2) were randomized to a provided LC/CONV or CHOICE diet (BMI-matched calories) through delivery. At 30-31 and 36-37 weeks of gestation, a 2-h, 75-g oral glucose tolerance test (OGTT) was performed and a continuous glucose monitor (CGM) was worn for 72 h. Cord blood samples were collected at delivery. NB%fat was measured by air displacement plethysmography (13.4 ± 0.4 days)., Results: There were 23 women per group (LC/CONV [214 g/day carbohydrate] and CHOICE [316 g/day carbohydrate]). For LC/CONV and CHOICE, respectively (mean ± SEM), NB%fat (10.1 ± 1 vs. 10.5 ± 1), birth weight (3,303 ± 98 vs. 3,293 ± 81 g), and cord C-peptide levels were not different. Weight gain, physical activity, and gestational age at delivery were similar. At 36-37 weeks of gestation, CGM fasting (86 ± 3 vs. 90 ± 3 mg/dL), 1-h postprandial (119 ± 3 vs. 117 ± 3 mg/dL), 2-h postprandial (106 ± 3 vs. 108 ± 3 mg/dL), percent time in range (%TIR; 92 ± 1 vs. 91 ± 1), and 24-h glucose area under the curve values were similar between diets. The %time >120 mg/dL was statistically higher (8%) in CHOICE, as was the nocturnal glucose AUC; however, nocturnal %TIR (63-100 mg/dL) was not different. There were no between-group differences in OGTT glucose and insulin levels at 36-37 weeks of gestation., Conclusions: A ∼100 g/day difference in carbohydrate intake did not result in between-group differences in NB%fat, cord C-peptide level, maternal 24-h glycemia, %TIR, or insulin resistance indices in diet-controlled GDM., (© 2023 by the American Diabetes Association.)

Significance: Successful differentiation of carcinoma in situ (CIS) from inflammation in the bladder is key to preventing unnecessary biopsies and enabling accurate therapeutic decisions. Current standard-of-care diagnostic imaging techniques lack the specificity needed to differentiate these states, leading to false positives., Aim: We introduce multiparameter interferometric polarization-enhanced (MultiPIPE) imaging as a promising technology to improve the specificity of detection for better biopsy guidance and clinical outcomes., Approach: In this ex vivo study, we extract tissue attenuation-coefficient-based and birefringence-based parameters from MultiPIPE imaging data, collected with a bench-top system, to develop a classifier for the differentiation of benign and CIS tissues. We also analyze morphological features from second harmonic generation imaging and histology slides and perform imaging-to-morphology correlation analysis., Results: MultiPIPE enhances specificity to differentiate CIS from benign tissues by nearly 20% and reduces the false-positive rate by more than four-fold over clinical standards. We also show that the MultiPIPE measurements correlate well with changes in morphological features in histological assessments., Conclusions: The results of our study show the promise of MultiPIPE imaging to be used for better differentiation of bladder inflammation from flat tumors, leading to a fewer number of unnecessary procedures and shorter operating room (OR) time., (© 2023 The Authors.)

Iatrogenic nerve injuries contribute significantly to postoperative morbidity across various surgical disciplines and occur in approximately 500,000 cases annually in the US alone. Currently, there are no clinically adopted means to intraoperatively visualize nerves beyond the surgeon's visual assessment. Here, we report a label-free method for nerve detection using diffuse reflectance spectroscopy (DRS). Starting with an in vivo rat model, fiber- and imaging-based DRS independently identified similar wavelengths that provided optimal contrast for nerve identification with an accuracy of 92%. Optical property measurements of rat and human cadaver tissues verify that the source of contrast between nerve and surrounding tissues is largely due to higher scattering in nerve and differences in oxygenated hemoglobin content. Clinical feasibility was demonstrated in patients undergoing thyroidectomies using both probe-based and imaging-based approaches where the nerve were identified with 91% accuracy. Based on our preliminary results, DRS has the potential to both provide surgeons with a label-free, intraoperative means of nerve visualization and reduce the incidence of iatrogenic nerve injuries along with its detrimental complications., (© 2023. The Author(s).)

Biochemical insights into varying breast cancer (BC) phenotypes can provide a fundamental understanding of BC pathogenesis, while identifying novel therapeutic targets. Raman spectroscopy (RS) can gauge these biochemical differences with high specificity. For routine RS, cells are traditionally seeded onto calcium fluoride (CaF 2 ) substrates that are costly and fragile, limiting its widespread adoption. Stainless steel has been interrogated previously as a less expensive alternative to CaF 2 substrates, while reporting increased Raman signal intensity than the latter. We sought to further investigate and compare the Raman signal quality measured from stainless steel versus CaF 2 substrates by characterizing different BC phenotypes with altered human epidermal growth factor receptor 2 (HER2) expression. Raman spectra were obtained on stainless steel and CaF 2 substrates for HER2 negative cells - MDA-MB-231, MDA-MB-468 and HER2 overexpressing cells - AU565, SKBr3. Upon analyzing signal-to-noise ratios (SNR), stainless steel provided a stronger Raman signal, improving SNR by 119% at 1450 cm -1 and 122% at 2925 cm -1 on average compared to the CaF 2 substrate. Utilizing only 22% of laser power on sample relative to the CaF 2 substrate, stainless steel still yielded improved spectral characterization over CaF 2 , achieving 96.0% versus 89.8% accuracy in BC phenotype discrimination and equivalent 100.0% accuracy in HER2 status classification. Spectral analysis further highlighted increased lipogenesis and altered metabolism in HER2 overexpressing cells, which was subsequently visualized with coherent anti-Stokes Raman scattering microscopy. Our findings demonstrate that stainless steel substrates deliver improved Raman signal and enhanced spectral characterization, underscoring its potential as a cost-effective alternative to CaF 2 for non-invasively monitoring cellular biochemical dynamics in translational cancer research.

Background: During adrenalectomy, surgeons have traditionally relied on their subjective visual skills to distinguish adrenal glands (AGs) from retroperitoneal fat and surrounding structures, while ultrasound and exogenous contrast agents have been employed for intraoperative AG visualization, all of which have their limitations. We present a novel label-free approach that uses near-infrared autofluorescence (NIRAF) detection, which demonstrates potential for enhanced intraoperative AG visualization and efficient tumor resection during adrenalectomies., Methods: Patients undergoing adrenalectomy or nephrectomy were enrolled for this feasibility study. NIRAF emitted beyond 800 nm was detected in vivo from AGs and surrounding tissues during open adrenalectomies or nephrectomies. NIRAF was also measured ex vivo in excised AGs following robotic adrenalectomies. NIRAF images of tissues were captured using near-infrared (NIR) camera systems, whereas NIRAF intensities were recorded concurrently using fiber-optic probe-based NIR devices. Normalized NIRAF intensities (expressed as mean ± standard error) were analyzed and compared., Results: Among the 55 enrolled patients, NIRAF intensity was elevated significantly for AGs versus retroperitoneal fat and other structures. NIR images of AGs also revealed a distinct demarcation of NIRAF between adrenal cortex and other periadrenal structures. NIRAF intensity in AGs was decreased markedly in malignant adrenal tumors, while benign adrenal cortical tumors and healthy adrenal cortex exhibited the strongest NIRAF levels., Conclusions: Our preliminary findings indicate that NIRAF detection could be a promising label-free technology to enhance intraoperative AG visualization and holds immense potential for effective tumor demarcation during cortical-sparing adrenalectomies or adrenal-conserving surgeries., (© 2022. The Author(s) under exclusive licence to Société Internationale de Chirurgie.)

Background: The coronavirus disease 2019 (COVID-19) pandemic is dominated by variant viruses; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the hospitalization risk following infection with 7 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants., Methods: Our study includes individuals with positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) in the Washington Disease Reporting System with available viral genome data, from 1 December 2020 to 14 January 2022. The analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for hospitalization risk following infection with a variant, adjusting for age, sex, calendar week, and vaccination., Results: In total, 58 848 cases were sequenced through sentinel surveillance, of which 1705 (2.9%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.20, 95% confidence interval [CI] 2.40-4.26), Beta (HR 2.85, 95% CI 1.56-5.23), Delta (HR 2.28 95% CI 1.56-3.34), or Alpha (HR 1.64, 95% CI 1.29-2.07) compared to infections with ancestral lineages; Omicron (HR 0.92, 95% CI .56-1.52) showed no significant difference in risk. Following Alpha, Gamma, or Delta infection, unvaccinated patients show higher hospitalization risk, while vaccinated patients show no significant difference in risk, both compared to unvaccinated, ancestral lineage cases. Hospitalization risk following Omicron infection is lower with vaccination., Conclusions: Infection with Alpha, Gamma, or Delta results in a higher hospitalization risk, with vaccination attenuating that risk. Our findings support hospital preparedness, vaccination, and genomic surveillance., Competing Interests: Potential conflicts of interests. A. L. G. reports central testing lab contract from Abbott and research funding from Merck and Gilead. K. A. became an employee of Biobot Analytics after the initial manuscript submission. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. Analyzing genome-wide data from 733 human cell and tissue samples, we identified 2 prototypical regions that exhibit high or absent hypersensitivity to deoxyribonuclease I, respectively. These regulatory active or inactive regions were examined in the lymphoblast cell line K562 by using high-throughput super-resolution microscopy. In both regions, we systematically measured the physical distance of 2 fluorescence in situ hybridization spots spaced by only 5 kb of DNA. Unexpectedly, the resulting distance distributions range from very compact to almost elongated configurations of more than 200-nm length for both the active and inactive regions. Monte Carlo simulations of a coarse-grained model of these chromatin regions based on published data of nucleosome occupancy in K562 cells were performed to understand the underlying mechanisms. There was no parameter set for the simulation model that can explain the microscopically measured distance distributions. Obviously, the chromatin state given by the strength of internucleosomal interaction, nucleosome occupancy, or amount of histone H1 differs from cell to cell, which results in the observed broad distance distributions. This large variability was not expected, especially in inactive regions. The results for the mechanisms for different distance distributions on this scale are important for understanding the contacts that mediate gene regulation. Microscopic measurements show that the inactive region investigated here is expected to be embedded in a more compact chromatin environment. The simulation results of this region require an increase in the strength of internucleosomal interactions. It may be speculated that the higher density of chromatin is caused by the increased internucleosomal interaction strength., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) are known to preferentially co-locate to active regulatory elements in tissues and cell types relevant to disease aetiology. Further characterisation of associated cell type-specific regulation can broaden our understanding of how GWAS signals may contribute to disease risk., Results: To gain insight into potential functional mechanisms underlying GWAS associations, we developed FORGE2 ( https://forge2.altiusinstitute.org/ ), which is an updated version of the FORGE web tool. FORGE2 uses an expanded atlas of cell type-specific regulatory element annotations, including DNase I hotspots, five histone mark categories and 15 hidden Markov model (HMM) chromatin states, to identify tissue- and cell type-specific signals. An analysis of 3,604 GWAS from the NHGRI-EBI GWAS catalogue yielded at least one significant disease/trait-tissue association for 2,057 GWAS, including > 400 associations specific to epigenomic marks in immune tissues and cell types, > 30 associations specific to heart tissue, and > 60 associations specific to brain tissue, highlighting the key potential of tissue- and cell type-specific regulatory elements. Importantly, we demonstrate that FORGE2 analysis can separate previously observed accessible chromatin enrichments into different chromatin states, such as enhancers or active transcription start sites, providing a greater understanding of underlying regulatory mechanisms. Interestingly, tissue-specific enrichments for repressive chromatin states and histone marks were also detected, suggesting a role for tissue-specific repressed regions in GWAS-mediated disease aetiology., Conclusion: In summary, we demonstrate that FORGE2 has the potential to uncover previously unreported disease-tissue associations and identify new candidate mechanisms. FORGE2 is a transparent, user-friendly web tool for the integrative analysis of loci discovered from GWAS., (© 2021. The Author(s).)

Electronic relaxation in organic chromophores often proceeds via states not directly accessible by photoexcitation. We report on the photoinduced dynamics of pyrazine that involves such states, excited by a 267 nm laser and probed with X-ray transient absorption spectroscopy in a table-top setup. In addition to the previously characterized 1 B 2u (ππ*) (S 2 ) and 1 B 3u (nπ*) (S 1 ) states, the participation of the optically dark 1 A u (nπ*) state is assigned by a combination of experimental X-ray core-to-valence spectroscopy, electronic structure calculations, nonadiabatic dynamics simulations, and X-ray spectral computations. Despite 1 A u (nπ*) and 1 B 3u (nπ*) states having similar energies at relaxed geometry, their X-ray absorption spectra differ largely in transition energy and oscillator strength. The 1 A u (nπ*) state is populated in 200 ± 50 femtoseconds after electronic excitation and plays a key role in the relaxation of pyrazine to the ground state., (© 2021. The Author(s).)

The purpose of this study was to investigate the tissue regenerating and biomechanical properties of processed eggshell membrane powder (PEP) for use in 3D-scaffolds. PEP is a low-cost, natural biomaterial with beneficial bioactive properties. Most importantly, this material is available as a by-product of the chicken egg processing (breaking) industry on a large scale, and it could have potential as a low-cost ingredient for therapeutic scaffolds. Scaffolds consisting of collagen alone and collagen combined with PEP were produced and analyzed for their mechanical properties and the growth of primary fibroblasts and skeletal muscle cells. Mechanical testing revealed that a PEP/collagen-based scaffold increased the mechanical hardness of the scaffold compared with a pure collagen scaffold. Scanning electron microscopy (SEM) demonstrated an interconnected porous structure for both scaffolds, and that the PEP was evenly distributed in dense clusters within the scaffold. Fibroblast and skeletal muscle cells attached, were viable and able to proliferate for 1 and 2 weeks in both scaffolds. The cell types retained their phenotypic properties expressing phenotype markers of fibroblasts (TE7, alpha-smooth muscle actin) and skeletal muscle (CD56) visualized by immunostaining. mRNA expression of the skeletal muscle markers myoD, myogenin, and fibroblasts marker (SMA) together with extracellular matrix components supported viable phenotypes and matrix-producing cells in both types of scaffolds. In conclusion, PEP is a promising low-cost, natural biomaterial for use in combination with collagen as a scaffold for 3D-tissue engineering to improve the mechanical properties and promote cellular adhesion and growth of regenerating cells.

DNase I hypersensitive sites (DHSs) are generic markers of regulatory DNA 1-5 and contain genetic variations associated with diseases and phenotypic traits 6-8 . We created high-resolution maps of DHSs from 733 human biosamples encompassing 438 cell and tissue types and states, and integrated these to delineate and numerically index approximately 3.6 million DHSs within the human genome sequence, providing a common coordinate system for regulatory DNA. Here we show that these maps highly resolve the cis-regulatory compartment of the human genome, which encodes unexpectedly diverse cell- and tissue-selective regulatory programs at very high density. These programs can be captured comprehensively by a simple vocabulary that enables the assignment to each DHS of a regulatory barcode that encapsulates its tissue manifestations, and global annotation of protein-coding and non-coding RNA genes in a manner orthogonal to gene expression. Finally, we show that sharply resolved DHSs markedly enhance the genetic association and heritability signals of diseases and traits. Rather than being confined to a small number of distal elements or promoters, we find that genetic signals converge on congruently regulated sets of DHSs that decorate entire gene bodies. Together, our results create a universal, extensible coordinate system and vocabulary for human regulatory DNA marked by DHSs, and provide a new global perspective on the architecture of human gene regulation.

Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits 1 , but it remains challenging to distinguish variants that affect regulatory function 2 . Genomic DNase I footprinting enables the quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin 3-6 . However, only a small fraction of such sites have been precisely resolved on the human genome sequence 6 . Here, to enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate about 4.5 million compact genomic elements that encode transcription factor occupancy at nucleotide resolution. We map the fine-scale structure within about 1.6 million DNase I-hypersensitive sites and show that the overwhelming majority are populated by well-spaced sites of single transcription factor-DNA interaction. Cell-context-dependent cis-regulation is chiefly executed by wholesale modulation of accessibility at regulatory DNA rather than by differential transcription factor occupancy within accessible elements. We also show that the enrichment of genetic variants associated with diseases or phenotypic traits in regulatory regions 1,7 is almost entirely attributable to variants within footprints, and that functional variants that affect transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find increased density of human genetic variation within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N 6 -adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: The purpose of this study was to investigate the effect of advising sleep health optimization on anthropomorphic variables following 10 weeks resistance exercise training., Methods: Thirty untrained healthy men were recruited for the study and 23 were included in the final analysis. Participants were randomly assigned to exercise and sleep optimization: ExS (N.=10) or exercise only: Ex (N.=12). Both groups performed a whole-body resistance exercise program twice a week for 10 weeks. The ExS group received sleep health (SH) education on how to improve both sleep quantity and quality., Results: After 10 weeks of training both groups had increased lean body mass by a similar amount. The ExS group experienced an increase of 1.7±1.1 kg while the Ex group experienced an increase of 1.3±0.8 kg (P=0.29 for difference between groups). The men in the ExS group reduced fat mass significantly (-1.8±0.8 kg) while the Ex group did not (0.8±1.0 kg). P value was 0.02 for difference between groups., Conclusions: This randomized controlled trial suggests that combining regular resistance exercise training with optimization of sleep health provide significant added benefits to body composition. This optimization provides a simple and cheap tool, applicable to the general healthy population.

Background: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior., Methods: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA)., Findings: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC., Interpretation: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The Izu-Bonin-Mariana (IBM) fore arc preserves igneous rock assemblages that formed during subduction initiation circa 52 Ma. International Ocean Discovery Program (IODP) Expedition 352 cored four sites in the fore arc near the Ogasawara Plateau in order to document the magmatic response to subduction initiation and the physical, petrologic, and chemical stratigraphy of a nascent subduction zone. Two of these sites (U1440 and U1441) are underlain by fore-arc basalt (FAB). FABs have mid-ocean ridge basalt (MORB)-like compositions, however, FAB are consistently lower in the high-field strength elements (TiO 2 , P 2 O 5 , Zr) and Ni compared to MORB, with Na 2 O at the low end of the MORB field and FeO* at the high end. Almost all FABs are light rare earth element depleted, with low total REE, and have low ratios of highly incompatible to less incompatible elements (Ti/V, Zr/Y, Ce/Yb, and Zr/Sm) relative to MORB. Chemostratigraphic trends in Hole U1440B are consistent with the uppermost lavas forming off axis, whereas the lower lavas formed beneath a spreading center axis. Axial magma of U1440B becomes more fractionated upsection; overlying off-axis magmas return to more primitive compositions. Melt models require a two-stage process, with early garnet field melts extracted prior to later spinel field melts, with up to 23% melting to form the most depleted compositions. Mantle equilibration temperatures are higher than normal MORB (1,400 °C-1,480 °C) at relatively low pressures (1-2 GPa), which may reflect an influence of the Manus plume during subduction initiation. Our data support previous models of FAB origin by decompression melting but imply a source more depleted than normal MORB source mantle.

Objectives: In this substudy of the DETO2X-AMI (An Efficacy and Outcome Study of Supplemental Oxygen Treatment in Patients With Suspected Myocardial Infarction) trial, the authors aimed to assess the analgesic effect of moderate-flow oxygen supplementation in patients with suspected acute myocardial infarction (AMI) treated with percutaneous coronary intervention (PCI) and to study the effect of oxygen supplementation on the use of opiates and sedatives during PCI., Background: Routine oxygen in normoxemic patients with AMI does not provide clinical benefit. However, oxygen may relieve ischemic pain., Methods: Patients were randomly allocated to oxygen or ambient air according to the main study protocol. After PCI, peak level of pain during PCI was measured by the Visual Analogue Scale. The total amount of opiates and sedatives was reported., Results: A total of 622 patients were enrolled: 330 in the oxygen group and 292 in the ambient air group. There was no significant difference in peak level of pain (oxygen 4.0 [1.0 to 6.0] vs. air 3.0 [0.6 to 6.0]; p = 0.37), use of opiates (mg) (oxygen 0.0 [0.0 to 3.0] vs. air 0.0 [0.0 to 3.0]; p = 0.31), or use of sedatives between the groups (median [interquartile range]) (oxygen 2.5 [0.0 to 2.5] vs. air 2.5 [0.0 to 2.5]; p = 0.74)., Conclusions: In the present study, the authors did not find any analgesic effect of routine oxygen as compared with ambient air, and no differences in the use of sedatives and opiates during PCI. Our results indicate that moderate-flow oxygen supplementation does not relieve pain in normoxemic patients with suspected AMI undergoing treatment with PCI and should thus not be used for this purpose., (Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Marriage and family therapy educators increasingly emphasize training competencies. What we know less about is what makes family therapy education meaningful to marriage and family therapy (MFT) graduate students and what does not. In this study, through an Internet survey, we explored the most and least meaningful learning experiences of 68 MFT graduate students and recent graduates of Commission on Accreditation for Marriage and Family Therapy Education-accredited programs. We used thematic analysis to identify and illustrate resulting themes, which included the importance of experiential and personal components to learning, the professor-student alliance, tying theory to practice, and the experiences of students with their clients, among others. We discuss the implications of these findings to support family therapy education and offer tentative suggestions for formative discussions both within and across programs. Video Abstract is found in the online version of the article., (© 2016 American Association for Marriage and Family Therapy.)

Background: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death., Objectives: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass., Methods: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment., Results: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo., Conclusions: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets., (Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Alterations in serotonin signaling are suspected in the pathophysiology of irritable bowel syndrome (IBS). By modulating the extracellular reuptake of serotonin, the serotonin transporter (SERT) acts as a key regulator of the bioavailability of serotonin. This study is the first to investigate the impact of rare SERT variants (i.e., those with a minor allele frequency of < 1%) on the risk for IBS, gastrointestinal (GI) symptom level, response to cognitive-behavioral treatment, and psychiatric comorbidity. We sequenced a 0.19 megabase chromosomal stretch containing the SERT gene and surrounding regions in a community sample of 304 IBS patients and 83 controls. We found no significant associations between rare variants in and around the SERT gene and IBS risk, GI symptom profile, or response to treatment. We found preliminary evidence, however, that IBS subjects with a history of either depression or anxiety were significantly more likely to carry multiple rare likely functional variant alleles than IBS patients without psychiatric comorbidity., (© The Author(s) 2016.)

The function of human regulatory regions depends exquisitely on their local genomic environment and on cellular context, complicating experimental analysis of common disease- and trait-associated variants that localize within regulatory DNA. We use allelically resolved genomic DNase I footprinting data encompassing 166 individuals and 114 cell types to identify >60,000 common variants that directly influence transcription factor occupancy and regulatory DNA accessibility in vivo. The unprecedented scale of these data enables systematic analysis of the impact of sequence variation on transcription factor occupancy in vivo. We leverage this analysis to develop accurate models of variation affecting the recognition sites for diverse transcription factors and apply these models to discriminate nearly 500,000 common regulatory variants likely to affect transcription factor occupancy across the human genome. The approach and results provide a new foundation for the analysis and interpretation of noncoding variation in complete human genomes and for systems-level investigation of disease-associated variants.

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes., (Copyright © 2014, American Association for the Advancement of Science.)

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone 3 lysine 4 trimethylation (H3K4me3) is known to be associated with transcriptionally active or poised genes and required for postnatal neurogenesis within the subventricular zone (SVZ) in the rodent model. Previous comparisons have shown significant correlation between baboon (Papio anubis) and human brain. In this study, we demonstrate that chromatin activation mark H3K4me3 is present in undifferentiated progenitor cells within the SVZ of adult baboon brain. To identify the targets and regulatory role of H3K4me3 within the baboon SVZ, we developed a technique to purify undifferentiated SVZ cells while preserving the endogenous nature without introducing culture artifact to maintain the in vivo chromatin state for genome-wide studies (ChIP-Seq and RNA-Seq). Overall, H3K4me3 is significantly enriched for genes involved in cell cycle, metabolism, protein synthesis, signaling pathways, and cancer mechanisms. Additionally, we found elevated levels of H3K4me3 in the MRI-classified SVZ-associated Glioblastoma Multiforme (GBM), which has a transcriptional profile that reflects the H3K4me3 modifications in the undifferentiated progenitor cells of the baboon SVZ. Our findings highlight the importance of H3K4me3 in coordinating distinct networks and pathways for life-long neurogenesis, and suggest that subtypes of GBM could occur, at least in part, due to aberrant H3K4me3 epigenetic regulation.

This study is, to the best of our knowledge, the first application of whole transcriptome sequencing (RNA-seq) to cells isolated from postmortem human brain by laser capture microdissection. We investigated the transcriptome of dentate gyrus (DG) granule cells in postmortem human hippocampus in 79 subjects with mental illness (schizophrenia, bipolar disorder, major depression) and nonpsychiatric controls. We show that the choice of normalization approach for analysis of RNA-seq data had a strong effect on results; under our experimental conditions a nonstandard normalization method gave superior results. We found evidence of disrupted signaling by miR-182 in mental illness. This was confirmed using a novel method of leveraging microRNA genetic variant information to indicate active targeting. In healthy subjects and those with bipolar disorder, carriers of a high- vs those with a low-expressing genotype of miR-182 had different levels of miR-182 target gene expression, indicating an active role of miR-182 in shaping the DG transcriptome for those subject groups. By contrast, comparing the transcriptome between carriers of different genotypes among subjects with major depression and schizophrenia suggested a loss of DG miR-182 signaling in these conditions.

Carcinogenesis and neoplastic progression are mediated by the accumulation of somatic mutations. Here we report that the local density of somatic mutations in cancer genomes is highly reduced specifically in accessible regulatory DNA defined by DNase I hypersensitive sites. This reduction is independent of any known factors influencing somatic mutation density and is observed in diverse cancer types, suggesting a general mechanism. By analyzing individual cancer genomes, we show that the reduced local mutation density within regulatory DNA is linked to intact global genome repair machinery, with nearly complete abrogation of the hypomutation phenomenon in individual cancers that possess mutations in components of the nucleotide excision repair system. Together, our results connect chromatin structure, gene regulation and cancer-associated somatic mutation.

Genomes contain both a genetic code specifying amino acids and a regulatory code specifying transcription factor (TF) recognition sequences. We used genomic deoxyribonuclease I footprinting to map nucleotide resolution TF occupancy across the human exome in 81 diverse cell types. We found that ~15% of human codons are dual-use codons ("duons") that simultaneously specify both amino acids and TF recognition sites. Duons are highly conserved and have shaped protein evolution, and TF-imposed constraint appears to be a major driver of codon usage bias. Conversely, the regulatory code has been selectively depleted of TFs that recognize stop codons. More than 17% of single-nucleotide variants within duons directly alter TF binding. Pervasive dual encoding of amino acid and regulatory information appears to be a fundamental feature of genome evolution.

Cellular-state information between generations of developing cells may be propagated via regulatory regions. We report consistent patterns of gain and loss of DNase I-hypersensitive sites (DHSs) as cells progress from embryonic stem cells (ESCs) to terminal fates. DHS patterns alone convey rich information about cell fate and lineage relationships distinct from information conveyed by gene expression. Developing cells share a proportion of their DHS landscapes with ESCs; that proportion decreases continuously in each cell type as differentiation progresses, providing a quantitative benchmark of developmental maturity. Developmentally stable DHSs densely encode binding sites for transcription factors involved in autoregulatory feedback circuits. In contrast to normal cells, cancer cells extensively reactivate silenced ESC DHSs and those from developmental programs external to the cell lineage from which the malignancy derives. Our results point to changes in regulatory DNA landscapes as quantitative indicators of cell-fate transitions, lineage relationships, and dysfunction., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa can develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistant phoQ mutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of the colRS genes, individually or in tandem, abrogated the polymyxin resistance of a ΔphoQ mutant, as did individual or tandem deletion of cprRS. Individual deletion of colR or colS in a ΔphoQ mutant also suppressed 4-amino-L-arabinose addition to lipid A, consistent with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion of colRS or cprRS in the ΔphoQ mutant or individual deletion of cprR or cprS failed to suppress 4-amino-L-arabinose addition to lipid A, indicating that this modification alone is not sufficient for PhoPQ-mediated polymyxin resistance in P. aeruginosa. Episomal expression of colRS or cprRS in tandem or of cprR individually complemented the Pm resistance phenotype in the ΔphoQ mutant, while episomal expression of colR, colS, or cprS individually did not. Highly polymyxin-resistant phoQ mutants of P. aeruginosa isolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of colRS and cprS; when expressed in a ΔphoQ background, these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance in P. aeruginosa, indicate that addition of 4-amino-L-arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate that colRS and cprS mutations can contribute to high-level clinical resistance.

Here we report the complete, accurate 1.89-Mb genome sequence of Francisella tularensis subsp. holarctica strain FSC200, isolated in 1998 in the Swedish municipality Ljusdal, which is in an area where tularemia is highly endemic. This genome is important because strain FSC200 has been extensively used for functional and genetic studies of Francisella and is well-characterized.

Genome-wide association studies have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by deoxyribonuclease I (DNase I) hypersensitive sites (DHSs). Eighty-eight percent of such DHSs are active during fetal development and are enriched in variants associated with gestational exposure-related phenotypes. We identified distant gene targets for hundreds of variant-containing DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrated tissue-selective enrichment of more weakly disease-associated variants within DHSs and the de novo identification of pathogenic cell types for Crohn's disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease and provide pathogenic insights into diverse disorders.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

Unlabelled: The large and growing number of genome-wide datasets highlights the need for high-performance feature analysis and data comparison methods, in addition to efficient data storage and retrieval techniques. We introduce BEDOPS, a software suite for common genomic analysis tasks which offers improved flexibility, scalability and execution time characteristics over previously published packages. The suite includes a utility to compress large inputs into a lossless format that can provide greater space savings and faster data extractions than alternatives., Availability: http://code.google.com/p/bedops/ includes binaries, source and documentation.

Cholera is a disease which shows a clear blood group profile, with blood group O individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group O individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: S100A8/A9 complex is a new inflammation-related protein and has a positive correlation with C-reaction protein level. However its role in chronic heart failure (CHF) remains unclear., Methods and Results: Circulating levels of S100A8/A9 complex and other biomarkers (IL-6, IL-8, TNF-α, and BNP) were measured in CHF (n = 54) and hypertensive without CHF (n = 31) as well as healthy subjects (n = 23), with follow up to 1480 days. During follow-up, cumulative mortality rate for CHF patients was 63%. Plasma levels of S100A8/A9 complex, IL-6, IL-8 and TNF-α were significantly higher in CHF than the hypertensive patients and healthy subjects. A significant positive correlation was found between S100A8/A9 complex and IL-6 and IL-8 respectively. Cox regression analysis showed that IL-6 and IL-8 were predictors for mortality for 6 months, and S100A8/A9 complex, IL-6, IL-8 and age were predictors for mortality for one year whereas BNP, TNF-α, IL-6 and IL-8 remained predictors for mortality for two years. A combination of S100A8/A9 complex and IL-6 provided powerful predictive value in mortality for both 6 and 12 months., Conclusions: S100A8/A9 complex is a useful biomarker as a predictor for one year mortality and its combination with IL-6 is able to provide additive prognostic information in this vulnerable heart failure population in the elderly., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx(1). The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx(1), indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx(1) and a propensity for HUS.

Altered myocardial Ca(2+) and Na(+) handling in congestive heart failure (CHF) may be expected to decrease the tolerance to ischemia by augmenting reperfusion Ca(2+) overload. The aim of the present study was to investigate tolerance to hypoxia-reoxygenation by measuring enzyme release, cell death, ATP level, and cell Ca(2+) and Na(+) in cardiomyocytes from failing rat hearts. CHF was induced in Wistar rats by ligation of the left coronary artery during isoflurane anesthesia, after which cardiac failure developed within 6 wk. Isolated cardiomyocytes were cultured for 24 h and subsequently exposed to 4 h of hypoxia and 2 h of reoxygenation. Cell damage was measured as lactate dehydrogenase (LD) release, cell death as propidium iodide uptake, and ATP by firefly luciferase assay. Cell Ca(2+) and Na(+) were determined with radioactive isotopes, and free intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo-3 AM. CHF cells showed less increase in LD release and cell death after hypoxia-reoxygenation and had less relative reduction in ATP level after hypoxia than sham cells. CHF cells accumulated less Na(+) than sham cells during hypoxia (117 vs. 267 nmol/mg protein). CHF cells maintained much lower [Ca(2+)](i) than sham cells during hypoxia (423 vs. 1,766 arbitrary units at 4 h of hypoxia), and exchangeable Ca(2+) increased much less in CHF than in sham cells (1.4 vs. 6.7 nmol/mg protein) after 120 min of reoxygenation. Ranolazine, an inhibitor of late Na(+) current, significantly attenuated both the increase in exchangeable Ca(2+) and the increase in LD release in sham cells after reoxygenation. This supports the suggestion that differences in Na(+) accumulation during hypoxia cause the observed differences in Ca(2+) accumulation during reoxygenation. Tolerance to hypoxia and reoxygenation was surprisingly higher in CHF than in sham cardiomyocytes, probably explained by lower hypoxia-mediated Na(+) accumulation and subsequent lower Ca(2+) accumulation in CHF after reoxygenation.

Background: Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared., Methodology/principal Findings: The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name "island integration determinant" (iid)., Conclusion/significance: These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.

Background: Tumor necrosis factor alpha (TNFalpha) has been shown to be a prognostic marker in heart failure, but recent clinical trials using TNFalpha antagonists in patients with severe heart failure have been disappointing. Hypertension is one of most common causes to chronic heart failure in humans., Hypothesis: Suppression of a single cytokine in CHF is not an effective treatment strategy because it leads to the upregulation of other proinflammatory cytokines., Objectives: The aim of the present study was to investigate the effect of chronic treatment with a TNFalpha antagonist in a rat model of the early stage of heart failure due to hypertension., Methods: Spontaneously hypertensive rats (SHR, n=30) and healthy Wistar Kyoto rats (WKY, n=30) were treated with either the TNFalpha antagonist etanercept or placebo for 12 weeks. At the end of the study, the rats were 26 weeks old and indices of cardiac structure, function and cytokines were analyzed., Results: SHR displayed early stage of heart failure as shown by increased heart weight/body weight ratio and relative wall thickness by echocardiography, downregulated myocardial beta(1)-adrenoceptor, and upregulated myocardial brain natriuretic peptide and interleukin-6 (IL6). Chronic treatment with etanercept in SHR resulted in decreased relative wall thickness but also increased cardiac reserve and higher blood pressure. In addition, IL6 was further upregulated compared with placebo treatment., Conclusion: Chronic treatment with etanercept in SHR resulted in favorable cardiac remodeling, but also had a positive inotropic effect and was associated with an upregulation of IL6. These findings indicate that chronic treatment with TNFalpha antagonists is not an effective treatment strategy and may aggravate heart failure in the long term.

Objectives: Despite tumor necrosis factor alpha (TNFalpha) has been shown to be a prognostic marker in patients with heart failure and previous preclinical study with TNFalpha-antagonist has been demonstrated to improve cardiac function in acute heart failure, recent clinical trials using TNFalpha-antagonist in patients with chronic severe heart failure have been disappointing. The aim was to study why TNFalpha-antagonist may not work during long-term treatment in chronic heart failure (CHF) in experimental model., Methods: 49 rats were used at the age of 26 weeks: healthy Whistar Kyoto rats (WKY, n = 26) and diabetic (WKY+D, n = 23). Rats in each group received either a 12-week treatment with TNFalpha-antagonist (Etanercept) or NaCl injections., Results: In diabetic rats, there were increased plasma glucose level and blood pressure. By use of echocardiography diabetic rats displayed not only enlarged and thinned left ventricles but also decreased both systolic and diastolic functions. Moreover, there are increased interleukin-6 (IL6) mRNA levels. However, TNFalpha-antagonist, etanercept, does not improve either cardiac remodelling or cardiac function. IL6 mRNA level remained unchanged after treatment of etanercept., Conclusion: Chronic treatment of TNFalpha-antagonist has no favourable effect on either cardiac remodelling or cardiac function. It is therefore inappropriate to use TNFalpha-antagonist in CHF in diabetes as underlying cause.