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Abstract Background Retrospective and descriptive molecular epidemiology studies have shown that Mycobacterium tuberculosis whole genome sequencing can identify outbreaks and disease transmission events with higher resolution than conventional epidemiological investigations. Those studies have strengthened our understanding of genomic polymorphisms correlating with person-to-person transmission and helped resolve putative transmission clusters. To date, systematic genomic surveillance programs implemented for M. tuberculosis were only implemented in low-incidence settings. The purpose of this study is to determine whether there is an impact of routine M. tuberculosis whole genome sequencing on tuberculosis case detection in a high-incidence setting. Methods A cluster randomized controlled trial will be performed. Forty-eight rural village groups (or Fokontany) in the Vohibato district of Madagascar will be randomized to one of three interventions arms. Arm 1 (standard of care) involves healthcare facility-based passive case detection with smear microscopy testing. Arm 2 (best practice) consists of active case finding and Xpert MTB/RIF Ultra PCR testing followed by household contact investigations. Arm 3 (novel intervention) includes the same interventions as arm 2, with addition of sputum culture and M. tuberculosis whole genome sequencing for all newly diagnosed cases. In arm 3, molecular suggested putative outbreaks are investigated, and additional TB suspects are appropriately tested. The intervention observational period will be 2 years. The primary outcome will be the number of detected cases/100,000/year in each arm after 1 year of intervention. Discussion This study is designed to determine whether there is an impact of prospective whole genome sequencing-based molecular typing on tuberculosis case detection in high-incidence settings. Investigating potential outbreaks and focusing active case finding in spatiotemporal settings where disease transmission is suggested by genomic typing is hypothesized to improve case detection in rural communities. Trial registration ClinicalTrials.gov NCT05406453 . Retrospectively registered on June 6, 2022.

Abstract Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821–1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs.

Background Innovative restructuring of cardiac rehabilitation (CR) delivery remains critical to reduce barriers and improve access to diverse populations. Destination Cardiac Rehab is a novel virtual world technology‐based CR program delivered through the virtual world platform, Second Life, which previously demonstrated high acceptability as an extension of traditional center‐based CR. This study aims to evaluate efficacy and adherence of the virtual world–based CR program compared with center‐based CR within a community‐informed, implementation science framework. Methods Using a noninferiority, hybrid type 1 effectiveness‐implementation, randomized controlled trial, 150 patients with an eligible cardiovascular event will be recruited from 6 geographically diverse CR centers across the United States. Participants will be randomized 1:1 to either the 12‐week Destination Cardiac Rehab or the center‐based CR control groups. The primary efficacy outcome is a composite cardiovascular health score based on the American Heart Association Life's Essential 8 at 3 and 6 months. Adherence outcomes include CR session attendance and participation in exercise sessions. A diverse patient/caregiver/stakeholder advisory board was assembled to guide recruitment, implementation, and dissemination plans and to contextualize study findings. The institutional review board–approved randomized controlled trial will enroll and randomize patients to the intervention (or control group) in 3 consecutive waves/year over 3 years. The results will be published at data collection and analyses completion. Conclusions The Destination Cardiac Rehab randomized controlled trial tests an innovative and potentially scalable model to enhance CR participation and advance health equity. Our findings will inform the use of effective virtual CR programs to expand equitable access to diverse patient populations. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT05897710.

Abstract Background Preterm birth is a global problem with about 12% of births in sub-Saharan Africa occurring before 37 weeks of gestation. Several studies have explored a potential association between vaginal microbiota and preterm birth, and some have found an association while others have not. We performed a study designed to determine whether there is an association with vaginal microbiota and/or placental microbiota and preterm birth in an African setting. Methods Women presenting to the study hospital in labor with a gestational age of 26 to 36 weeks plus six days were prospectively enrolled in a study of the microbiota in preterm labor along with controls matched for age and parity. A vaginal sample was collected at the time of presentation to the hospital in active labor. In addition, a placental sample was collected when available. Libraries were constructed using PCR primers to amplify the V6/V7/V8 variable regions of the 16S rRNA gene, followed by sequencing with an Illumina MiSeq machine and analysis using QIIME2 2022.2. Results Forty-nine women presenting with preterm labor and their controls were enrolled in the study of which 23 matched case–control pairs had sufficient sequence data for comparison. Lactobacillus was identified in all subjects, ranging in abundance from 99%, with Lactobacillus iners and Lactobacillus crispatus the most common species. Over half of the vaginal samples contained Gardnerella and/or Prevotella; both species were associated with preterm birth in previous studies. However, we found no significant difference in composition between mothers with preterm and those with full-term deliveries, with both groups showing roughly equal representation of different Lactobacillus species and dysbiosis-associated genera. Placental samples generally had poor DNA recovery, with a mix of probable sequencing artifacts, contamination, and bacteria acquired during passage through the birth canal. However, several placental samples showed strong evidence for the presence of Streptococcus species, which are known to infect the placenta. Conclusions The current study showed no association of preterm birth with composition of the vaginal community. It does provide important information on the range of sequence types in African women and supports other data suggesting that women of African ancestry have an increased frequency of non-Lactobacillus types, but without evidence of associated adverse outcomes.

Summary: Background: Identifying novel epigenetic signatures associated with serum immunoglobulin E (IgE) may improve our understanding of molecular mechanisms underlying asthma and IgE-mediated diseases. Methods: We performed an epigenome-wide association study using whole blood from Framingham Heart Study (FHS; n = 3,471, 46% females) participants and validated results using the Childhood Asthma Management Program (CAMP; n = 674, 39% females) and the Genetic Epidemiology of Asthma in Costa Rica Study (CRA; n = 787, 41% females). Using the closest gene to each IgE-associated CpG, we highlighted biologically plausible pathways underlying IgE regulation and analyzed the transcription patterns linked to IgE-associated CpGs (expression quantitative trait methylation loci; eQTMs). Using prior UK Biobank summary data from genome-wide association studies of asthma and allergy, we performed Mendelian randomization (MR) for causal inference testing using the IgE-associated CpGs from FHS with methylation quantitative trait loci (mQTLs) as instrumental variables. Findings: We identified 490 statistically significant differentially methylated CpGs associated with IgE in FHS, of which 193 (39.3%) replicated in CAMP and CRA (FDR < 0.05). Gene ontology analysis revealed enrichment in pathways related to transcription factor binding, asthma, and other immunological processes. eQTM analysis identified 124 cis-eQTMs for 106 expressed genes (FDR < 0.05). MR in combination with drug-target analysis revealed CTSB and USP20 as putatively causal regulators of IgE levels (Bonferroni adjusted P

Accurate and precise regulation of gene expression programmes in eukaryotes involves the coordinated control of transcription, mRNA stability and translation. In recent years, significant progress has been made about the role of sequence elements in the 3′ untranslated region for the regulation of mRNA degradation, and a model has emerged in which recruitment of the Ccr4-Not complex is the critical step in the regulation of mRNA decay. Recruitment of the Ccr4-Not complex to a target mRNA results in deadenylation mediated by the Caf1 and Ccr4 catalytic subunits of the complex. Following deadenylation, the 5′ cap structure is removed, and the mRNA subjected to 5′-3′ degradation. Here, the role of the human Ccr4-Not complex in cytoplasmic deadenylation of mRNA is reviewed, with a particular focus on mechanisms of its recruitment to mRNA by sequence motifs in the 3′ untranslated region, codon usage, as well as general mechanisms involving the poly(A) tail.

Introduction: NADH pyrophosphatase, a hydrolase catalyzing the phosphate bond of NADH to reduced nicotinamide mononucleotide, has potential applications in the food, cosmetic and pharmaceutical industry.Methods: Here, we investigated the effects of vector screening, promoter and RBS strategies on NADH pyrophosphatase expression and protein engineering on its enzymatic activity and thermal stability.Results: In this study, we describe a NADH pyrophosphatase derived from Escherichia coli (EcNudc). Strategies focusing on expression regulation including screening vectors, optimizing promoters and ribosome binding sites were utilized to enhance the productivity of EcNudc (1.8 U/mL). Moreover, protein engineering was adopted to further improve the catalytic properties of EcNudc, achieving 3.3-fold higher activity and 3.6-fold greater thermostability at 50°C. Furthermore, fermentation for the combined mutant R148A-H149E (EcNudc-M) production in a 7 L fermenter was implemented and the enzyme activity of EcNudc-M reached 33.0 U/mL. Finally, the EcNudc-M was applied in the catalysis of NADH with the highest NMNH yield of 16.65 g/L.Discussion: In conclusion, we constructed a commercially available genetically engineered strain with high activity and thermal stability of NADH pyrophosphatase, laying a broad foundation for the biocatalytic industrial production of NMNH and expand its application range.

Renal artery aneurysm (RAA) and renal arteriovenous fistula are rare vascular pathologies with reported incidences of 0.3% to 1.0% and 0.04% in the general population, respectively. We describe a 61-year-old Caucasian man who presented to the hospital with symptoms of right flank pain. Imaging demonstrated a right RAA with concurrent hilar RAA and renal arteriovenous fistula. He ultimately underwent an open right nephrectomy, ligation of the fistula, and bovine patch repair of the aortic defect.

The relationship between a university and its city is considered strategical to achieve university targeted ambitions and visions. The university-city relationship is also encouraged for the benefit of the city, as universities unleash their respective cities’ potentials to act as driving forces not only for their local communities, but also for the whole nation. Therefore, maintaining a mutual relationship between the university and the city is considered essential to accomplish strategic goals for both. However, the nature of this relationship is quite complex, overlapped, interconnected, and diverse. Therefore, this paper conducted a systematic review of the literature on university-city and campus-city relationships to evaluate recent research trends to uncover the aspects that connect universities with their respective cities. The search included articles published in 4 different databases from January 1990 to January 2021. A total number of 50 articles were selected in this review. The findings uncovered different aspects that could help or hinder university-city relationship based on the physical and functional linkage between the campus and the city. Moreover, findings have shown that it is necessary to understand universities according to their contextual differences, as universities have shown different impacts on their respective cities in terms of their sizes and locations. Results have also shown that the impact of the physical connection between the university and the city goes far beyond campus’s accessibility as it deeply affects students’ social life as well. Therefore, decision makers, stakeholders, and university administrators need to co-design campus development process especially in the early stages to maximize the mutual benefits of campus-city relationship. The main conclusions of this paper address several perspectives and lessons for a more sustainable campus-city relationship.

Aortic aneurysms (AA) are a common complication in patients with large-vessel vasculitis, such as chronic phase Takayasu arteritis, that often require surgical management to prevent a lethal rupture. Historically, mainstay of treatment for AA in the setting of arteritis was traditional open repair. However, in this case study an alternative surgical approach was devised to successfully treat an extent III thoracoabdominal AA in a patient with a diagnosis of Takayasu arteritis and a complex surgical history that made her high risk for an open surgical intervention. This case study summarizes a hybrid surgical approach that successfully excluded a thoracoabdominal AA and revascularized the superior mesenteric artery and left renal artery, by directly accessing the infrarenal aorta and using a bifurcated abdominal aortic endograft as a two-vessel branched device.

The fossil record for Cretaceous birds in Australia has been limited to rare skeletal material, feathers, and two tracks, a paucity shared with other Gondwanan landmasses. Hence the recent discovery of 27 avian footprints and other traces in the Early Cretaceous (Barremian-Aptian, 128-120 Ma) Wonthaggi Formation of Victoria, Australia amends their previous rarity there, while also confirming the earliest known presence of birds in Australia and the rest of Gondwana. The avian identity of these tracks is verified by their tridactyl forms, thin digits relative to track lengths, wide divarication angles, and sharp claws; three tracks also have hallux imprints. Track forms and sizes indicate a variety of birds as tracemakers, with some among the largest reported from the Early Cretaceous. Although continuous trackways are absent, close spacing and similar alignments of tracks on some bedding planes suggest gregariousness. The occurrence of this avian trace-fossil assemblage in circumpolar fluvial-floodplain facies further implies seasonal behavior, with trackmakers likely leaving their traces on floodplain surfaces during post-thaw summers.

Systemic ATTR amyloidosis causes the abnormal accumulation of ATTR fibrils formed from the human plasma protein transthyretin (TTR) in multiple organs including the eye. Here, the authors present a 3.2 Å cryo-EM structure of an ATTR fibril isolated from the vitreous body of an ATTR patient’s eye and discuss the mechanism for the structural conversion of TTR into a fibrillar form.

Rationale & Objective: Recent literature suggests improvement in kidney function after percutaneous valvular replacement therapies, implying a pathophysiological contribution of valvular heart disease to chronic kidney disease (CKD). However, this association has not been investigated epidemiologically. We aimed to assess the association of valvular abnormality with prevalent and incident CKD. Study Design: Cross-sectional and prospective analyses. Setting & Participants: Community-dwelling participants (mean age 75.5 [standard deviation 5.1] years) from the Atherosclerosis Risk in Communities study (2011-2013). Exposure: Valvular abnormality defined as echocardiography-based aortic stenosis, aortic regurgitation, and mitral regurgitation. Outcomes: Prevalent CKD was defined as estimated glomerular filtration rate (eGFR])

Abstract In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated Mn4CaO5-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.

This case study uses three different frameworks of inquiry to examine Turtles All the Way Down by John Green (2017) with a disability lens. The analysis extends beyond the traditional medical/social dichotomy and considers how disability is tied to both agency and identity. Narratives and counter-narratives of disability are also investigated, as well as disability markers used in previous scholarship. The discussion concludes with an argument to include the novel in secondary English classes to create mental health allies. A consideration for medical humanities scholars is also included to use Green’s text with patients with OCD, as a way for readers to find an identifiable protagonist.

Robotic positioning systems are used in a variety of chemical instruments, primarily for liquid handling purposes, such as autosamplers from vials or well plates. Here, two approaches to the design of open-source autosampler positioning systems for use with 96-well plates are described and compared. The first system, a 3-axis design similar to many low-cost 3D printers that are available on the market, is constructed using an aluminum frame and stepper motors. The other system relies upon a series of 3D printed parts to achieve movement with a series of linker arms based on Selective Compliance Assembly Robot Arm (SCARA) design principles. Full printer design files, assembly instructions, software, and user directions are included for both samplers. The positioning precision of the 3-axis system is better than the SCARA mechanism due to finer motor control, albeit with a slightly higher cost of materials. Based on the improved precision of this approach, the 3-axis autosampler system was used to demonstrate the generation of a segmented flow droplet stream from adjacent wells within a 96-well plate.

The world is currently in a pandemic of COVID-19 (Coronavirus disease-2019) caused by a novel positive-sense, single-stranded RNA β-coronavirus referred to as SARS-CoV-2. Here we investigated rates of SARS-CoV-2 infection in the greater Cincinnati, Ohio, USA metropolitan area from August 13 to December 8, 2020, just prior to initiation of the national vaccination program. Examination of 9,550 adult blood donor volunteers for serum IgG antibody positivity against the SARS-CoV-2 Spike protein showed an overall prevalence of 8.40%, measured as 7.56% in the first 58 days and 9.24% in the last 58 days, and 12.86% in December 2020, which we extrapolated to ~20% as of March, 2021. Males and females showed similar rates of past infection, and rates among Hispanic or Latinos, African Americans and Whites were also investigated. Donors under 30 years of age had the highest rates of past infection, while those over 60 had the lowest. Geographic analysis showed higher rates of infectivity on the West side of Cincinnati compared with the East side (split by I-75) and the lowest rates in the adjoining region of Kentucky (across the Ohio river). These results in regional seroprevalence will help inform efforts to best achieve herd immunity in conjunction with the national vaccination campaign.

Introduction Prognosis of biliary tract cancers (BTC) remains dismal and novel treatment strategies are needed to improve survival. BRCA mutations are known to occur in BTC but their frequency and the molecular landscape in which they are observed in distinct sites of BTC remain unknown.Material and methods Tumour samples from 1292 patients with BTC, comprising intrahepatic cholangiocarcinoma (IHC, n=746), extrahepatic cholangiocarcinoma (EHC, n=189) and gallbladder cancer (GBC, n=353), were analysed using next-generation sequencing (NGS). Tumour mutational burden (TMB) was calculated based on somatic non-synonymous missense mutations. Determination of tumour mismatch repair (MMR) or microsatellite instability (MSI) status was done by fragment analysis, immunohistochemistry and the evaluation of known microsatellite loci by NGS. Programmed death ligand 1 expression was analysed using immunohistochemistry.Results Overall, BRCA mutations were detected in 3.6% (n=46) of samples (BRCA1: 0.6%, BRCA2: 3%) with no significant difference in frequency observed based on tumour site. In GBC and IHC, BRCA2 mutations (4.0% and 2.7%) were more frequent than BRCA1 (0.3% and 0.4%, p

Abstract Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (CP) and, if present, the resolving Cys (CR). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 Å resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.

Abstract Dimethylfumarate (DMF) has long been used as part of a fixed combination of fumaric acid esters (FAE) in some European countries and is now available as an oral monotherapy for psoriasis. The present investigation determined whether DMF and its main metabolite monomethylfumarate (MMF) interact with hepatic cytochrome P450 (CYP) enzymes and the P‐glycoprotein (P‐gp) transporter, and was performed as part of DMF's regulatory commitments. Although referred to in the available product labels/summary of product characteristics, the actual data have not yet been made publicly available. In vitro inhibition experiments using CYP‐selective substrates with human liver microsomes showed 50% inhibitory concentrations (IC50) of >666 µmol/L for DMF and >750 µmol/L for MMF. MMF (≤250 μmol/L; 72 hours) was not cytotoxic in cultured human hepatocyte experiments and mRNA expression data indicated no CYP induction by MMF (1‐250 µmol/L). DMF (≤6.66 mmol/L) showed moderate‐to‐high absorption (apparent permeability [Papp] ≥2.3‐29.7 x 10−6 cm/s) across a Caucasian colon adenocarcinoma (Caco‐2) cell monolayer, while MMF (≤7.38 mmol/L) demonstrated low‐to‐moderate permeability (Papp 1.2‐8.9 × 10−6 cm/s). DMF was not a substrate for P‐gp (net efflux ratios ≤1.22) but was a weak inhibitor of P‐gp at supratherapeutic concentrations (estimated IC50 relative to solvent control of 1.5 mmol/L; [3H]digoxin efflux in Caco‐2 cells). This inhibition is unlikely to be clinically relevant. MMF was not a substrate or inhibitor of P‐gp. Thus, DMF and MMF should not affect the absorption, distribution, metabolism or excretion of coadministered drugs that are CYP and P‐gp substrates.

The BH3-only apoptosis agonists BAD and NOXA target BCL-2 and MCL-1 respectively and co-operate to induce apoptosis. On this basis, therapeutic drugs targeting BCL-2 and MCL-1 might have enhanced activity if used in combination. We identified anti-leukaemic drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism using the technique of dynamic BH3 profiling, whereby cells were primed with drugs to discover whether this would elicit mitochondrial outer membrane permeabilisation in response to BCL-2-targeting BAD-BH3 peptide or MCL-1-targeting MS1-BH3 peptide. We found that a broad range of anti-leukaemic agents-notably MCL-1 inhibitors, DNA damaging agents and FLT3 inhibitors-sensitise leukaemia cells to BAD-BH3. We further analysed the BCL-2 inhibitors ABT-199 and JQ1, the MCL-1 inhibitors pladienolide B and torin1, the FLT3 inhibitor AC220 and the DNA double-strand break inducer etoposide to correlate priming responses with co-operative induction of apoptosis. ABT-199 in combination with pladienolide B, torin1, etoposide or AC220 strongly induced apoptosis within 4 hours, but the MCL-1 inhibitors did not co-operate with etoposide or AC220. In keeping with the long half-life of BCL-2, the BET domain inhibitor JQ1 was found to downregulate BCL-2 and to prime cells to respond to MS1-BH3 at 48, but not at 4 hours: prolonged priming with JQ1 was then shown to induce rapid cytochrome C release when pladienolide B, torin1, etoposide or AC220 were added. In conclusion, dynamic BH3 profiling is a useful mechanism-based tool for understanding and predicting co-operative lethality between drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism. A plethora of agents sensitised cells to BAD-BH3-mediated mitochondrial outer membrane permeabilisation in the dynamic BH3 profiling assay and this was associated with effective co-operation with the BCL-2 inhibitory compounds ABT-199 or JQ1.

Blasts from different patients with acute myeloid leukemia (AML) vary in the agent(s) to which they are most responsive. With a myriad of novel agents to evaluate, there is a lack of predictive biomarkers to precisely assign targeted therapies to individual patients. Primary AML cells often survive poorly in vitro, thus confounding conventional cytotoxicity assays. The purpose of this work was to assess the potential of two same-day functional predictive assays in AML cell lines to predict long-term response to chemotherapy. (i) Ribosomal protein S6 (rpS6) is a downstream substrate of PI3K/akt/mTOR/ kinase and MAPK kinase pathways and its dephosphorylation is also triggered by DNA double strand breaks. Phospho-rpS6 is reliably measurable by flow cytometry and thus has the potential to function as a biomarker of responsiveness to several therapeutic agents. (ii) A cell's propensity for apoptosis can be interrogated via a functional assay termed "Dynamic BH3 Profiling" in which mitochondrial outer membrane permeabilization in drug-treated cells can be driven by pro-apoptotic BH3 domain peptides such as PUMA-BH3. The extent to which a particular cell is primed for apoptosis by the drug can be determined by measuring the amount of cytochrome C released on addition of BH3 peptide. We demonstrate that phospho-rpS6 expression and PUMA-BH3 peptide-induced cytochrome C release after 4 hours both predict long term chemoresponsiveness to tyrosine kinase inhibitors and DNA double strand break inducers in AML cell lines. We also describe changes in expression levels of the prosurvival BCL-2 family member Mcl-1 and the pro-apoptotic protein BIM after short term drug culture.

A new small-bodied ornithopod dinosaur, Diluvicursor pickeringi, gen. et sp. nov., is named from the lower Albian of the Eumeralla Formation in southeastern Australia and helps shed new light on the anatomy and diversity of Gondwanan ornithopods. Comprising an almost complete tail and partial lower right hindlimb, the holotype (NMV P221080) was deposited as a carcass or body-part in a log-filled scour near the base of a deep, high-energy river that incised a faunally rich, substantially forested riverine floodplain within the Australian–Antarctic rift graben. The deposit is termed the ‘Eric the Red West Sandstone.’ The holotype, interpreted as an older juvenile ∼1.2 m in total length, appears to have endured antemortem trauma to the pes. A referred, isolated posterior caudal vertebra (NMV P229456) from the holotype locality, suggests D. pickeringi grew to at least 2.3 m in length. D. pickeringi is characterised by 10 potential autapomorphies, among which dorsoventrally low neural arches and transversely broad caudal ribs on the anterior-most caudal vertebrae are a visually defining combination of features. These features suggest D. pickeringi had robust anterior caudal musculature and strong locomotor abilities. Another isolated anterior caudal vertebra (NMV P228342) from the same deposit, suggests that the fossil assemblage hosts at least two ornithopod taxa. D. pickeringi and two stratigraphically younger, indeterminate Eumeralla Formation ornithopods from Dinosaur Cove, NMV P185992/P185993 and NMV P186047, are closely related. However, the tail of D. pickeringi is far shorter than that of NMV P185992/P185993 and its pes more robust than that of NMV P186047. Preliminary cladistic analysis, utilising three existing datasets, failed to resolve D. pickeringi beyond a large polytomy of Ornithopoda. However, qualitative assessment of shared anatomical features suggest that the Eumeralla Formation ornithopods, South American Anabisetia saldiviai and Gasparinisaura cincosaltensis, Afro-Laurasian dryosaurids and possibly Antarctic Morrosaurus antarcticus share a close phylogenetic progenitor. Future phylogenetic analysis with improved data on Australian ornithopods will help to test these suggested affinities.

Increases in the spatial scale and intensity of activities that produce marine anthropogenic sound highlight the importance of understanding the impacts and effects of sound on threatened species such as marine turtles. Marine turtles detect and behaviorally respond to low-frequency sounds, however few studies have directly examined their behavioral responses to specific types or intensities of anthropogenic or natural sounds. Recent advances in the development of bio-logging tools, which combine acoustic and fine-scale movement measurements, have allowed for evaluations of animal responses to sound. Here, we describe these tools and present a case study demonstrating the potential application of a newly developed technology (ROTAG, Loggerhead Instruments, Inc.) to examine behavioral responses of freely swimming marine turtles to sound. The ROTAG incorporates a three-axis accelerometer, gyroscope, and magnetometer to record the turtle's pitch, roll, and heading; a pressure sensor to record turtle depth; a hydrophone to record the turtle's received underwater acoustic sound field; a temperature gauge; and two VHF radio telemetry transmitters and antennas for tag and turtle tracking. Tags can be programmed to automatically release via a timed corrodible link several hours or days after deployment. We describe an example of the data collected with these tags and present a case study of a successful ROTAG deployment on a juvenile green turtle (Chelonia mydas) in the Paranaguá Estuary Complex, Brazil. The tag was deployed for 221 min, during which several vessels passed closely (

Porphyromonas gingivalis expresses two forms of fimbriae, FimA and Mfa1. Each fimbria consists of five proteins; FimA-E and Mfa1-5. While the assembly of the type-1 fimbriae from Escherichia coli is well studied; the chaperone-usher pathway, very little is known about the polymerization of P. gingivalis fimbriae. The P. gingivalis fimbrial proteins form lipidated precursors that have an N-terminal extension, which is not found in the mature protein. In order to obtain an understanding of the structure, function and assembly mechanism of the P. gingivalis fimbriae we are studying the Mfa proteins using X-ray crystallography. The Mfa proteins have been crystallized in their precursor forms and their crystal structures reveal that the proteins are structurally related despite low sequence similarity. The proteins consist of two β-sandwich domains where the N-terminal extension forms a β-strand that is tightly integrated in the first β-sheet. An arginine, recognized by a protease, is exposed on a long flexible loop. The structures raise questions if the N-terminal extension functions as an internal chaperone that stabilizes the protein before polymerization is initiated. We further speculate which structural part of the protein that can function as a donor strand upon fimbrial polymerization.

The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat) as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2–3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2–3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2–3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes.

Hand-held near-infrared (NIR) optical imagers are developed by various researchers towards non-invasive clinical breast imaging. Unlike these existing imagers that can perform only reflectance imaging, a generation-2 (Gen-2) hand-held optical imager has been recently developed to perform both reflectance and transillumination imaging. The unique forked design of the hand-held probe head(s) allows for reflectance imaging (as in ultrasound) and transillumination or compressed imaging (as in X-ray mammography). Phantom studies were performed to demonstrate two-dimensional (2D) target detection via reflectance and transillumination imaging at various target depths (1–5 cm deep) and using simultaneous multiple point illumination approach. It was observed that 0.45 cc targets were detected up to 5 cm deep during transillumination, but limited to 2.5 cm deep during reflectance imaging. Additionally, implementing appropriate data post-processing techniques along with a polynomial fitting approach, to plot 2D surface contours of the detected signal, yields distinct target detectability and localization. The ability of the gen-2 imager to perform both reflectance and transillumination imaging allows its direct comparison to ultrasound and X-ray mammography results, respectively, in future clinical breast imaging studies.

In this conceptual article we suggest that understanding clients’ self-reflexive processes enables coaches to become even more effective in helping clients make changes in how they think, feel and act. Our aim is also to throw light on the relationship between metacognition, change and coaching. We begin with an overview of theories of metacognition and then set out 10 principles of meta-level processing which, together with an understanding of NLP (neuro-linguistic programming), lead Hall to propose the Meta-States model of self-reflexivity (Hall 1995/2000). We then describe the Meta-States model and how it in turn led to the development of the Axes of Change model (Hall and Duval 2004). Following that we outline how the NLP, Meta-States and Axes of Change models underpin the Meta-coaching methodology and we illustrate with case studies. Finally we reflect on how the ideas presented here address issues raised in the coaching literature.

Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His(6)-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, k(cat), compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.

Nesta entrevista a autora discorre sobre as relações entre classes sociais e gêneros, sobre as influências de Foucault em sua obra, sobre a atualidade das greves como forma de luta e sobre a participação da mulher no sindicalismo da virada do século.

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.

In plants oxygenic photosynthesis is performed by large protein complexes found in the thylakoid membranes of chloroplasts. The soluble thylakoid lumen space is a narrow and compressed region within the thylakoid membrane which contains 80-200 proteins. Because the thylakoid lumen proteins are in close proximity to the protein complexes of photosynthesis, it is reasonable to assume that the lumen proteins are highly influenced by the presence of light. To identify light regulated proteins in the thylakoid lumen of Arabidopsis thaliana we developed a faster thylakoid preparation and combined this with difference gel electrophoresis (DIGE) of dark-adapted and light-adapted lumen proteomes. The DIGE experiments revealed that 19 lumen proteins exhibit increased relative protein levels after eight hour light exposure. Among the proteins showing increased abundance were the PsbP and PsbQ subunits of Photosystem II, major plastocyanin and several other proteins of known or unknown function. In addition, co-expression analysis of publicly available transcriptomic data showed that the co-regulation of lumen protein expression is not limited to light but rather that lumen protein genes exhibit a high uniformity of expression. The large proportion of thylakoid lumen proteins displaying increased abundance in light-adapted plants, taken together with the observed uniform regulation of transcription, implies that the majority of thylakoid lumen proteins have functions that are related to photosynthetic activity. This is the first time that an analysis of the differences in protein level during a normal day/night cycle has been performed and it shows that even a normal cycle of light significantly influences the thylakoid lumen proteome. In this study we also show for the first time, using co-expression analysis, that the prevalent lumenal chloroplast proteins are very similarly regulated at the level of transcription.

Unintended teenage pregnancy in the United States is a public health concern with ramifications that include a variety of social, economic and health costs. It has been estimated that adolescents giving birth before the age of 18 cost the United States at least $9.1 billion dollars annually (NCPTUP, 2008). Latest available national data indicate a slight increase in rates of unintended teen pregnancy after a 15 year period of steady decline. The unintended teen pregnancy rate in Troup County, Georgia in 2006 was 51.9/1,000 which was higher than the national average of 41.9/1,000(Kids Count, 2008). The purpose of this study was to review the Circle of Care intervention program, a collaborative multi-agency teen pregnancy prevention program. The Circle of Care program was developed in 1997 through the efforts of multiple community partner organizations. These organizations included the local school system, the Division of Family and Children Services, Public Health, Troup County Family Connection, the local teen clinic, the local hospital and other organizations. Participants in the Circle of Care program receive multiple services, including case management, a family assessment, parenting classes, home visits from the case manager, family planning assistance, services from the teen health clinic and the Division of Family and Children Services. Preliminary data indicate that Circle of Care participants gained social, economic and health benefits from participation in the program including: higher rates of high school enrollment, no repeat pregnancies, and no reported incidences of child abuse or child neglect. Projected cost savings from these outcomes are also reported. Preliminary examination of the Circle of Care program supports the efficacy of multi-level, collaborative efforts to reduce unintended teen pregnancy and subsequent social, economic and health risks. Future research should examine longer term outcomes of this program.

Interest in the use of neural tissue transplantation for the study of CNS development and maturation and the potential use of this technique for the treatment of certain degenerative CNS disorders has led to our use of transplantation of neural tissue across species lines. Prior to extensive transplantation studies using athymic rats as recipients, we wished to evaluate the currently available strains of athymic rat for their suitability as host animals for xenografts of neural tissue. Fetal cerebellar and cerebral cortex tissue from rabbit brain of gestational age 20-25 days was dissected and transplanted to the anterior chamber of the eye of Harlan Wisconsin, Fisher 344 Jnu, or NCI-Harlan athymic nude rat strains. The brain tissue grafts were allowed to mature for 3 mo during which time the size and vascularity of each graft was monitored through the cornea of anesthetized hosts. In each group all of the transplants survived and grew to varying extents in the anterior chamber of the eye. Following the growth study in vivo extracellular recording of single neuronal activity was performed. Spontaneous neural activity was found in most transplants in all three groups with no difference in the viability or discharge rates of neurons between the groups. Illumination of the ipsilateral eye increased the firing rate of neurons in all three groups, suggesting excitatory cholinergic innervation of the grafted neurons from the host parasympathetic iris ground plexus. Antibodies directed against neurofilament protein, glial fibrillary acidic protein, synapsin, and tyrosine hydroxylase were used to characterize the transplants immunocytochemically and revealed no differences between the grafts in the three groups of recipients. All transplants contained significant numbers of glial and neuronal elements with the distribution resembling that in adult brain tissue. Some of the transplants contained a sparse innervation of tyrosine hydroxylase–positive fibers from the sympathetic plexus of the host iris. Furthermore, synapsin-immunoreactivity suggested that synaptogenesis had taken place within the grafts. Histological examination of the grafts revealed that 67% of the grafts had been infiltrated, to varying extents, by lymphocytes which led to areas of cell lysis and necrosis. All host animals had populations of T-cell receptor positive cells, most of which also expressed the T-cell surface antigens CD4 and CD8. However, no transplants were overtly rejected over the 15 wk period of study. Our investigation demonstrates that all of the athymic strains used in this study are able to mount an immune response against grafted fetal tissue, despite the absence of rejection, and that none of these strains is superior to the others with respect to suitability as a host for the long-term study of fetal CNS xenografts in oculo.

The integration of second home owners into rural communities poses a complex challenge that remains little addressed in conservative developing countries' context. Guided by integration theory, this qualitative study explores the integration of second home owners in Iran. The findings demonstrate that second home owners' integration processes are manifested in three distinct forms: environmental-physical, socio-cultural, and mental-psychological. These types of integration significantly influence the dynamics of tourism development and community cohesion in the studied rural communities. Overall, this research contributes to integration theory by exploring second home owner adaption to rural environmental, sociocultural norms and deepens understanding of the social implications of second home ownership within conservative rural communities. The study also expands knowledge of place attachment and identity formation by investigating how the presence of second home owners influences the identity and sense of place for both tourists and local residents. By recognizing the complex nature of integration, the findings offer practical implications for improving the integrating second home owners into rural destinations. [ABSTRACT FROM AUTHOR]