Community structure and biogeochemical impacts of microbial life on floating pumice.
Volcanic eruptions are a widespread force of geological and ecological disturbance and present recurrent opportunities for the study of biological responses to novel habitat formation. However, scientific study of such events is difficult given their short duration and often distant location. Here we report results from opportunistic sampling of unique volcano-generated habitats formed during the 2011 explosive eruption in the Puyehue-Cordón Caulle complex (Chile), when massive amounts of pumice were ejected, creating novel floating substrata that have never before been characterized from a microbiological perspective. DNA sequencing revealed a dynamic community of microbes that came to inhabit the pumice, with a unique composition distinct from that of the lakes' surface waters and with suggestions of ecological convergence across lakes and sampling times. Furthermore, biogeochemical studies of net nutrient fluxes showed that, while the fresh pumice arriving to the lakes was an initial source of phosphorus (P), colonized pumice had high rates of nitrogen (N) and P uptake and was sufficiently abundant to represent a significant lake-wide nutrient sink. These findings highlight the remarkable versatility of microbes in exploiting novel environments and are consistent with a recent proposal of floating pumice as a favorable environment for the initial origins of life on early Earth.


The genus Boreviulisoma Brolemann, 1928--an Iberian-N African outlier of a mainly tropical tribe of millipedes (Diplopoda: Polydesmida: Paradoxosomatidae.
The genus Boreviulisoma Brolemann, 1928, is revised. The synonymy of Liliputia Attems, 1952, under Boreviulisoma is confirmed, but L. badia Attems, 1951, from Spain, is resurrected, as Boreviulisoma badium, from synonymy under B. liouvillei Brolemann, 1928 (the type species of Boreviulisoma) from Morocco. Boreviulisoma barrocalense n. sp. is described from the subterranean environment of the Algarve, the southernmost province of Portugal. The distribution of the three known species of Boreviulisoma is mapped, main characters of the genus and its species are reviewed and a key to species of the genus is included. The isolated occurrence of Boreviulisoma badium and B. barrocalense n. sp. in the southern Iberian Peninsula, together with B. liouvillei and Eviulisoma abadi Mauries, 1985, in Morocco, as northern outposts of the chiefly Afrotropical-Neotropical tribe Eviulisomatini, is discussed.


Associations between antimicrobial use and the faecal resistome on broiler farms from nine European countries.
To determine associations between farm- and flock-level antimicrobial usage (AMU), farm biosecurity status and the abundance of faecal antimicrobial resistance genes (ARGs) on broiler farms. In the cross-sectional pan-European EFFORT study, conventional broiler farms were visited and faeces, AMU information and biosecurity records were collected. The resistomes of pooled faecal samples were determined by metagenomic analysis for 176 farms. A meta-analysis approach was used to relate total and class-specific ARGs (expressed as fragments per kb reference per million bacterial fragments, FPKM) to AMU (treatment incidence per DDD, TIDDDvet) per country and subsequently across all countries. In a similar way, the association between biosecurity status (Biocheck.UGent) and the resistome was explored. Sixty-six (38%) flocks did not report group treatments but showed a similar resistome composition and roughly similar ARG levels to antimicrobial-treated flocks. Nevertheless, we found significant positive associations between β-lactam, tetracycline, macrolide and lincosamide, trimethoprim and aminoglycoside antimicrobial flock treatments and ARG clusters conferring resistance to the same class. Similar associations were found with purchased products. In gene-level analysis for β-lactams and macrolides, lincosamides and streptogramins, a significant positive association was found with the most abundant gene clusters blaTEM and erm(B). Little evidence was found for associations with biosecurity. The faecal microbiome in European broilers contains a high diversity of ARGs, even in the absence of current antimicrobial selection pressure. Despite this, the relative abundance of genes and the composition of the resistome is positively related to AMU in European broiler farms for several antimicrobial classes.


Relative effects of fat-, carbohydrate- and protein-containing liquid diets on cardiac output in healthy adult subjects.
1. Nine healthy adult subjects consumed four types of proprietary liquid diet of similar volume and calorific value but of different nutritional composition. The effects on resting cardiac output, mean blood pressure and pulse rate were measured. 2. A significant rise in cardiac output occurred with the balanced, protein and carbohydrate diets but not with the fat diet. The greatest rise was seen with the balanced diet. Water alone had no effect on cardiac output. 3. The average time taken to reach peak cardiac output was shortest with the carbohydrate diet and longest with the fat diet. 4. The increases in cardiac output resulted from a rise in both pulse rate and stroke volume. The carbohydrate diet produced the most sustained rise in pulse rate but the least sustained elevation in stroke volume. 5. No significant changes were seen in mean blood pressure when each liquid meal was compared with water. 6. Our data show that the increase in cardiac output with liquid ingestion is related to the dietary components. These effects are additive.


Genetic Screens Identify Additional Genes Implicated in Envelope Remodeling during the Engulfment Stage of Bacillus subtilis Sporulation.
During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.


Structure-based analysis of the metal-dependent mechanism of H-N-H endonucleases.
Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn(2+), Zn(2+).DNA, and Mg(2+).DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg(2+)-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn(2+) but active with other transition metal ions such as Ni(2+). This is because of coordination of the Zn(2+) ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.


Impact of hydraulic loading rate and media type on removal of bulk organic matter and nitrogen from primary effluent in a laboratory-scale soil aquifer treatment system.
The effect of hydraulic loading rate (HLR) and media type on the removal of bulk organic matter and nitrogen from primary effluent during soil aquifer treatment was investigated by conducting laboratory-scale soil column studies. Two soil columns packed with silica sand were operated at HLRs of 0.625 and 1.25 m/d, while a third column was packed with dune filtering material and operated at HLR of 1.25 m/d. Bulk organic matter was effectively removed by 47.5 ± 1.2% and 45.1 ± 1.2% in silica sand columns operated at 0.625 and 1.25 m/d, respectively and 57.3 ± 7.6% in dune filtering material column operated at 1.25 m/d. Ammonium-nitrogen reduction of 74.5 ± 18.0% was achieved at 0.625 m/d compared to 39.1 ± 4.3% at 1.25 m/d in silica sand columns, whereas 49.2 ± 5.2% ammonium-nitrogen reduction was attained at 1.25 m/d in the dune filtering material column. Ammonium-nitrogen reduction in the first 3 m was assumed to be dominated by nitrification process evidenced by corresponding increase in nitrate. Part of the ammonium-nitrogen was adsorbed onto the media, which was observed at higher rates between 3 and 5 m in silica sand column operated at HLR of 0.625 m/d and dune filtering material column operated at 1.25 m/d compared to 1.25 m/d silica.


Ecology and biogeography of bacterial communities associated with chloroethene-contaminated aquifers.
Massive usage, along with careless handling, storage, spills, and leakages made chloroethenes (CEs) one of the most abundant classes of groundwater contaminants. Anaerobic organohalide respiring bacteria (OHRB) can couple reductive dechlorination of CEs with energy conservation, a central microbial process in (enhanced) natural attenuation of CE-contaminated aquifers. Spatial variability of OHRB guild members present in contaminated sites has not yet been investigated in detail and it is not known whether the spatial localization of contaminated sites could impact differentially remediation capacities. The goal of this study was to investigate how spatially distant microbial communities responded to the presence of CEs. Bacterial communities associated with five geographically distant European CE-contaminated aquifers were analyzed with terminal restriction fragment length polymorphism. Numerical ecology tools were used to assess the separate and combined effects on the communities of their spatial localization, their local environmental conditions and their contaminant concentrations. Three spatial scales were used for the assessment of the structuration of the communities as a function of geographical distances, namely at the aquifer scale, at medium (50 km) and long (ca. 1000 km) distances between aquifers. As a result, bacterial communities were structured with an almost identical contribution by both the geographical position of the aquifer and local environmental variables, especially electron donors and acceptors. The impact of environmental factors decreased with distance between aquifers, with the concomitant increase in importance of a geographical factor. Contrastingly, CEs contributed at a low extent at the medium scale and became important only when all aquifers were considered together, at a large geographical scale, suggesting that distant communities were structured partially by a common niche specialization in organohalide respiration.


Quality of life and sexual functıon in obese women with pelvic floor dysfunction.
This prospective study was conducted in the urogynecology and gynecology outpatient clinics of Istanbul Faculty of Medicine from December 2014 to March 2015. The objective was to identify the association between obesity and sexual function and quality of life in women with pelvic floor dysfunction (PFD). A total of 387 sexually active women diagnosed with urinary incontinence and/or pelvic organ prolapse were included and categorized as obese (n = 200) or nonobese (n = 187). Mean body mass indexes were 25.7 ± 2.41 kg/m2 for nonobese women and 34.9 ± 3.92 kg/m2 for obese women. The Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12 total score was significantly lower in obese (27.66 ± 7.12) than in nonobese women (30.18 ± 6.54) (p < .05). Quality of life mean scores were higher in obese women for both the Incontinence Impact Questionnaire total score (67.24 ± 26.8 versus 49.12 ± 27.5) and Urogenital Distress Inventory total score (65.02 ± 21.4 versus 55.07 ± 24.7) (p < .001). Obese women with PFD had symptoms for longer durations, had more frequent urinary incontinence, and worse sexual function and quality of life than nonobese women. Health-care professionals caring for obese women should be aware of the coexistence of obesity and PFD. Future studies should evaluate whether obesity-associated PFD can be reduced through successful weight reduction interventions.


Bovine mammary stem cells: cell biology meets production agriculture.
Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and alter the function of bovine MaSC. In this review, we provide an overview of current knowledge of MaSC gained from studies using mouse and human model systems and present research on bovine MaSC within that context. Recent data indicate that MaSC retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. Relying on this long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine MaSC. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%). They are predominantly estrogen receptor (ER)-negative and localized in a basal or suprabasal layer of the epithelium throughout the gland. Thus, the response of MaSC to estrogen, the major mitogen in mammary gland, is likely mediated by paracrine factors released by cells that are ER-positive. This is consistent with considerable evidence for cross-talk within and between epithelial cells and surrounding stromal cells. Excision of classes of cells by laser microdissection and subsequent microarray analysis will hopefully provide markers for MaSC and insights into their regulation. Preliminary analyses of gene expression in laser-microdissected LREC and non-LREC are consistent with the concept that LREC represent populations of stem cells and progenitor cells that differ with regard to their properties and location within the epithelial layer. We have attempted to modulate the MaSC number by infusing a solution of xanthosine through the teat canal and into the ductal network of the mammary glands of prepubertal heifers. This treatment increased the number of putative stem cells, as evidenced by an increase in the percentage of LREC and increased telomerase activity within the tissue. The exciting possibility that stem cell expansion can influence milk production is currently under investigation.


Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4.
Pulmonary hypertension (PH) is a progressive disorder whose cellular pathogenesis involves enhanced smooth muscle cell (SMC) proliferation and resistance to apoptosis signals. Existing evidence demonstrates that the tumor suppressor programmed cell death 4 (PDCD4) affects patterns of cell growth and repair responses in the systemic vasculature following experimental injury. In the current study, the regulation PDCD4 and its functional effects on growth and apoptosis susceptibility in pulmonary artery smooth muscle cells were explored. We previously demonstrated that pharmacological activation of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) attenuated hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (HPASMCs) by inhibiting the expression and mitogenic functions of microRNA-21 (miR-21). In the current study, we hypothesize that PPARγ stimulates PDCD4 expression and HPASMC apoptosis by inhibiting miR-21. Our findings demonstrate that PDCD4 is reduced in the mouse lung upon exposure to chronic hypoxia (10% O2 for 3 wk) and in hypoxia-exposed HPASMCs (1% O2). HPASMC apoptosis was reduced by hypoxia, by miR-21 overexpression, or by siRNA-mediated PPARγ and PDCD4 depletion. Activation of PPARγ inhibited miR-21 expression and resultant proliferation, while restoring PDCD4 levels and apoptosis to baseline. Additionally, pharmacological activation of PPARγ with rosiglitazone enhanced PDCD4 protein expression and apoptosis in a dose-dependent manner as demonstrated by increased annexin V detection by flow cytometry. Collectively, these findings demonstrate that PPARγ confers growth-inhibitory signals in hypoxia-exposed HPASMCs through suppression of miR-21 and the accompanying derepression of PDCD4 that augments HPASMC susceptibility to undergo apoptosis.


Relations of self-regulation and self-efficacy for exercise and eating and BMI change: A field investigation.
This study aimed to assess relations of self-regulatory skill use with self-efficacy for exercise and appropriate eating, and the resulting change in weight associated with participation in a nutrition and exercise treatment supported by cognitive-behavioral methods. Adults with severe obesity (N = 95; mean BMI = 40.5 ± 3.9 kg/m2) participated in a 6-month exercise and nutrition treatment emphasizing self-regulatory skills. Changes in self-regulatory skills usage, self-efficacy, overall mood, and BMI were measured. Relations of changes in self-regulatory skill use and self-efficacy, for both physical activity and appropriate eating, were assessed, as was the possibility of mood change being a mediator of these relationships. Indirect effects of the variables associated with the present treatment on BMI change were then estimated. For both exercise and appropriate eating, changes in self-regulation were associated with self-efficacy change. Mood change partially mediated the relationship between changes in self-regulation for appropriate eating and self-efficacy for appropriate eating. Self-efficacy changes for physical activity and controlled eating, together, explained a significant portion of the variance in BMI change (R2 = 0.26, p < 0.001). The total indirect effect of the treatment on BMI change was 0.20. Findings suggest that training in self-regulation for exercise and eating may benefit self-efficacy and weight-loss outcomes. Thus, these variables should be considered in both the theory and behavioral treatment of obesity.


Fizeau Fourier transform imaging spectroscopy: missing data reconstruction.
Fizeau Fourier transform imaging spectroscopy yields both spatial and spectral information about an object. Spectral information, however, is not obtained for a finite area of low spatial frequencies. A nonlinear reconstruction algorithm based on a gray-world approximation is presented. Reconstruction results from simulated data agree well with ideal Michelson interferometer-based spectral imagery. This result implies that segmented-aperture telescopes and multiple telescope arrays designed for conventional imaging can be used to gather useful spectral data through Fizeau FTIS without the need for additional hardware.


Evaluation of zinc-doped magnetite nanoparticle toxicity in the liver and kidney of mice after sub-chronic intragastric administration.
Super-paramagnetic iron oxide nanoparticles (SPIONs) have been approved for clinical use due to their salient super-paramagnetic properties and low toxicity. Zn2+ doped SPIONs possess significantly higher magnetic susceptibility than that of conventional SPIONs. Here we evaluated the potential toxicity of Zn2+ doped Fe3O4 nanoparticles (Zn0.4Fe2.6O4 NPs) in the liver and kidney of mice after repeated intragastric administration for 30 days. Zn0.4Fe2.6O4 NPs did not cause significant changes in their body weights and the coefficients of the liver and kidney, but increased the levels of Fe and Zn in the two organs. Zn0.4Fe2.6O4 NP induced slight oxidative stress in the liver and kidney, which could be successfully counteracted by their intrinsic antioxidant systems and had no observable hazardous effects on the histopathology, ultrastructure and functions of the two organs. These results demonstrated that high-performance magnetic Zn0.4Fe2.6O4 NPs did not produce apparent toxicity in the liver and kidney of mice even after sub-chronic intragastric administration. In addition, Zn2+ doping not only markedly enhanced magnetic susceptibility of Zn0.4Fe2.6O4 NPs but also significantly increased the stability of Zn0.4Fe2.6O4 NPs in biological conditions, making them appropriate for use in magnetic resonance imaging and drug delivery by the oral route.


Chromatin dynamics underlying latent responses to xenobiotics.
Pleiotropic xenobiotics can trigger dynamic alterations in mammalian chromatin structure and function but many of these are likely non-adverse and simply reflect short-term changes in DNA transactions underlying normal homeostatic, adaptive and protective cellular responses. However, it is plausible that a subset of xenobiotic-induced perturbations of somatic tissue or germline epigenomes result in delayed-onset and long-lasting adverse effects, in particular if they occur during critical stages of growth and development. These could include reprogramming, dedifferentiation, uncontrolled growth, and cumulative toxicity effects through molecular memory of prior xenobiotic exposures or altered susceptibility to subsequent xenobiotic exposures. Here we discuss the current evidence for epigenetic mechanisms underlying latent responses to xenobiotics, and the potential for identifying molecular epigenetic changes that are prodromal to overt morphologic or functional toxicity phenotypes.


Y and W Chromosome Assemblies: Approaches and Discoveries.
Hundreds of vertebrate genomes have been sequenced and assembled to date. However, most sequencing projects have ignored the sex chromosomes unique to the heterogametic sex - Y and W - that are known as sex-limited chromosomes (SLCs). Indeed, haploid and repetitive Y chromosomes in species with male heterogamety (XY), and W chromosomes in species with female heterogamety (ZW), are difficult to sequence and assemble. Nevertheless, obtaining their sequences is important for understanding the intricacies of vertebrate genome function and evolution. Recent progress has been made towards the adaptation of next-generation sequencing (NGS) techniques to deciphering SLC sequences. We review here currently available methodology and results with regard to SLC sequencing and assembly. We focus on vertebrates, but bring in some examples from other taxa.


Geographical distribution of selected and putatively neutral SNPs in Southeast Asian malaria parasites.
Loci targeted by directional selection are expected to show elevated geographical population structure relative to neutral loci, and a flurry of recent papers have used this rationale to search for genome regions involved in adaptation. Studies of functional mutations that are known to be under selection are particularly useful for assessing the utility of this approach. Antimalarial drug treatment regimes vary considerably between countries in Southeast Asia selecting for local adaptation at parasite loci underlying resistance. We compared the population structure revealed by 10 nonsynonymous mutations (nonsynonymous single-nucleotide polymorphisms [nsSNPs]) in four loci that are known to be involved in antimalarial drug resistance, with patterns revealed by 10 synonymous mutations (synonymous single-nucleotide polymorphisms [sSNPs]) in housekeeping genes or genes of unknown function in 755 Plasmodium falciparum infections collected from 13 populations in six Southeast Asian countries. Allele frequencies at known nsSNPs underlying resistance varied markedly between locations (F(ST) = 0.18-0.66), with the highest frequencies on the Thailand-Burma border and the lowest frequencies in neighboring Lao PDR. In contrast, we found weak but significant geographic structure (F(ST) = 0-0.14) for 8 of 10 sSNPs. Importantly, all 10 nsSNPs showed significantly higher F(ST) (P < 8 x 10(-5)) than simulated neutral expectations based on observed F(ST) values in the putatively neutral sSNPs. This result was unaffected by the methods used to estimate allele frequencies or the number of populations used in the simulations. Given that dense single-nucleotide polymorphism (SNP) maps and rapid SNP assay methods are now available for P. falciparum, comparing genetic differentiation across the genome may provide a valuable aid to identifying parasite loci underlying local adaptation to drug treatment regimes or other selective forces. However, the high proportion of polymorphic sites that appear to be under balancing selection (or linked to selected sites) in the P. falciparum genome violates the central assumption that selected sites are rare, which complicates identification of outlier loci, and suggests that caution is needed when using this approach.


Determinants of different deep and superficial CA1 pyramidal cell dynamics during sharp-wave ripples.
Sharp-wave ripples represent a prominent synchronous activity pattern in the mammalian hippocampus during sleep and immobility. GABAergic interneuronal types are silenced or fire during these events, but the mechanism of pyramidal cell (PC) participation remains elusive. We found opposite membrane polarization of deep (closer to stratum oriens) and superficial (closer to stratum radiatum) rat CA1 PCs during sharp-wave ripples. Using sharp and multi-site recordings in combination with neurochemical profiling, we observed a predominant inhibitory drive of deep calbindin (CB)-immunonegative PCs that contrasts with a prominent depolarization of superficial CB-immunopositive PCs. Biased contribution of perisomatic GABAergic inputs, together with suppression of CA2 PCs, may explain the selection of CA1 PCs during sharp-wave ripples. A deep-superficial gradient interacted with behavioral and spatial effects to determine cell participation during sleep and awake sharp-wave ripples in freely moving rats. Thus, the firing dynamics of hippocampal PCs are exquisitely controlled at subcellular and microcircuit levels in a cell type-selective manner.


Crystal structure of the alpha subunit of human translation initiation factor 2B.
Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the alpha subunit of human eIF2B at 2.65 A resolution. The eIF2Balpha structure consists of the N-terminal alpha-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 A in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 A in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Balpha. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Balpha. Interestingly, the yeast eIF2Balpha missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Balpha missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The beta and delta subunits of eIF2B are homologous to eIF2Balpha and may have tertiary structures similar to the present eIF2Balpha structure. The sulfate-ion-binding residues of eIF2Balpha are well conserved in eIF2Bbeta/delta. The abovementioned yeast and human missense mutations of eIF2Bbeta/delta were also mapped on the eIF2Balpha structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.


The role of primary care non-physician clinic staff in e-mail communication with patients.
Few studies have explicitly addressed how e-mail technology may affect non-physician clinic staff, even though these staff typically manage tasks well suited to e-mail communication such as requests for prescription renewals, laboratory and test results, and referral authorizations. We conducted a survey of staff members at 10 primary care clinics in Boston to further evaluate non-physician staff attitudes towards e-mail use with patients. We subsequently re-surveyed staff at three of these clinics after the implementation of Patient Gateway, an application designed to facilitate secure electronic communication between patients and the clinics. Before Patient Gateway implementation, 88% of surveyed staff were already using e-mail at least once a day for work-related communication. Many of these staff members (24%) were already using e-mail with patients. Forty-eight percent of staff members thought that increasing e-mail use with patients could improve the quality of care their practices delivered. However, staff reported having some hesitations about increasing e-mail use with patients, mostly relating to security, confidentiality, and workload. After Patient Gateway implementation, users reported high satisfaction with the application and staff in general (users and non-users of Patient Gateway) felt more enthusiastic about increasing e-mail use with patients. In order to maximize the potential of staff-patient e-mail, it is important that concerns relating to security, confidentiality, and workflow are addressed, and patients must be given guidelines for the appropriate use of e-mail. Secure applications designed with these issues in mind are likely to be well received by staff members, and in turn physicians.


Gene expression in developing fibres of Upland cotton (Gossypium hirsutum L.) was massively altered by domestication.
Understanding the evolutionary genetics of modern crop phenotypes has a dual relevance to evolutionary biology and crop improvement. Modern upland cotton (Gossypium hirsutum L.) was developed following thousands of years of artificial selection from a wild form, G. hirsutum var. yucatanense, which bears a shorter, sparser, layer of single-celled, ovular trichomes ('fibre'). In order to gain an insight into the nature of the developmental genetic transformations that accompanied domestication and crop improvement, we studied the transcriptomes of cotton fibres from wild and domesticated accessions over a developmental time course. Fibre cells were harvested between 2 and 25 days post-anthesis and encompassed the primary and secondary wall synthesis stages. Using amplified messenger RNA and a custom microarray platform designed to interrogate expression for 40,430 genes, we determined global patterns of expression during fibre development. The fibre transcriptome of domesticated cotton is far more dynamic than that of wild cotton, with over twice as many genes being differentially expressed during development (12,626 versus 5273). Remarkably, a total of 9465 genes were diagnosed as differentially expressed between wild and domesticated fibres when summed across five key developmental time points. Human selection during the initial domestication and subsequent crop improvement has resulted in a biased upregulation of components of the transcriptional network that are important for agronomically advanced fibre, especially in the early stages of development. About 15% of the differentially expressed genes in wild versus domesticated cotton fibre have no homology to the genes in databases. We show that artificial selection during crop domestication can radically alter the transcriptional developmental network of even a single-celled structure, affecting nearly a quarter of the genes in the genome. Gene expression during fibre development within accessions and expression alteration arising from evolutionary change appears to be 'modular' - complex genic networks have been simultaneously and similarly transformed, in a coordinated fashion, as a consequence of human-mediated selection. These results highlight the complex alteration of the global gene expression machinery that resulted from human selection for a longer, stronger and finer fibre, as well as other aspects of fibre physiology that were not consciously selected. We illustrate how the data can be mined for genes that were unwittingly targeted by aboriginal and/or modern domesticators during crop improvement and/or which potentially control the improved qualities of domesticated cotton fibre.See Commentary: http://www.biomedcentral.com/1741-7007/8/137.


Intelligence and executive functions in frontotemporal dementia.
Recently (Roca et al. (2010), we used the relationship with general intelligence (Spearman's g) to define two sets of frontal lobe or "executive" tests. For one group, including Wisconsin card sorting and verbal fluency, reduction in g entirely explained the deficits found in frontal patients. For another group, including tests of social cognition and multitasking, frontal deficits remained even after correction for g. Preliminary evidence suggested a link of the latter tasks to more anterior frontal regions. Here we develop this distinction in the context of behavioural-variant frontotemporal dementia (bvFTD), a disorder which progressively affects frontal lobe cortices. In bvFTD, some executive tests, including tests of social cognition and multitasking, decline from the early stage of the disease, while others, including classical executive tests such as Wisconsin card sorting, verbal fluency or Trail Making Test part B, show deficits only later on. Here we show that, while deficits in the classical executive tests are entirely explained by g, deficits in the social cognition and multitasking tests are not. The results suggest a relatively selective cognitive deficit at mild stages of the disease, followed by more widespread cognitive decline well predicted by g.


Expert modeling, expert/self-modeling versus lecture: a comparison of learning, retention, and transfer of rescue skills in health professions students.
It is unclear whether traditional lecture followed by simulation leads to the best learning and knowledge and skill retention over time. A 3×4 mixed design study used three modes of education-traditional lecture with self-guided learning, expert modeling/dual viewing with brief questioning, and expert plus self-modeling-at four time points to compare knowledge, time to treat, and correct steps over time. No significant differences were found in knowledge or time to treat between training methods. An expert modeling/ dual viewing group with brief questioning performed more steps correctly (p = 0.05) than did the other two groups. Expert modeling may help students remember and perform a complex series of tasks in a scenario. Further research is needed to explore expert modeling for novice learners.


Weight consistency specifies regularities of macaque cortical networks.
To what extent cortical pathways show significant weight differences and whether these differences are consistent across animals (thereby comprising robust connectivity profiles) is an important and unresolved neuroanatomical issue. Here we report a quantitative retrograde tracer analysis in the cynomolgus macaque monkey of the weight consistency of the afferents of cortical areas across brains via calculation of a weight index (fraction of labeled neurons, FLN). Injection in 8 cortical areas (3 occipital plus 5 in the other lobes) revealed a consistent pattern: small subcortical input (1.3% cumulative FLN), high local intrinsic connectivity (80% FLN), high-input form neighboring areas (15% cumulative FLN), and weak long-range corticocortical connectivity (3% cumulative FLN). Corticocortical FLN values of projections to areas V1, V2, and V4 showed heavy-tailed, lognormal distributions spanning 5 orders of magnitude that were consistent, demonstrating significant connectivity profiles. These results indicate that 1) connection weight heterogeneity plays an important role in determining cortical network specificity, 2) high investment in local projections highlights the importance of local processing, and 3) transmission of information across multiple hierarchy levels mainly involves pathways having low FLN values.


Is digital cognitive behavioural therapy for insomnia effective in treating sub-threshold insomnia: a pilot RCT.
Many patients find cognitive behavioral therapy for insomnia (CBT-I) useful. However, it is currently unknown if those with sub-threshold insomnia also benefit. Here we assessed whether CBT-I is both feasible and acceptable in participants with sub-threshold insomnia. The primary aims were to evaluate participation rates and treatment acceptability, and to establish an effect size for symptom improvement. A total of 199 female participants (Mage 20 ± 5 years) took part. Following baseline assessments, participants were randomly allocated to either a six-week digital CBT-I intervention or a six-week control group receiving puzzles. Additional assessments were performed three-weeks, six-weeks, and six-months later. Participation rates at each survey assessment wave did not differ between the groups (ps > 0.140), though adherence to completing each weekly task was lower in the CBT-I group, p = 0.02. Treatment acceptability was high (M (SD) = 33.61 (4.82), theoretical range 6-42). The CBT-I group showed greater improvement in insomnia symptoms at the end of the intervention compared to the control group (p = 0.013, d = 0.42), with significant variation in outcome (M = 4.69, SD = 5.41). Sub-threshold participants showed a similar pattern of results, whilst those meeting insomnia criteria showed a smaller between-group difference. CBT-I led to improvements in anxiety, paranoia and perceived stress between baseline and end of intervention. Changes in insomnia symptoms were mediated by cognitions about sleep and somatic pre-sleep arousal. CBT-I provides a benefit even in sub-threshold insomnia. CBT-I may be useful to tackle insomnia symptoms even when they are sub-threshold.


Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.
We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.


Retinal vascular network architecture in low-birth-weight men.
Low birth weight is associated with hypertension and increased cardiovascular mortality, but the mechanism of this association is not known. Hypertension is accompanied by abnormalities of the microvasculature including rarefaction. To test the hypothesis that low birth weight is associated with an alteration in microvascular architecture. A stratified random sample of 100 men aged 64-74 years was selected from a cohort of men whose birth weights were known. They were of relatively high or low birth weight ('high' > or = 3700 g, 'low' < or = 3200 g) and high or low systolic blood pressure (high > or = 160 mmHg, low < or = 140 mmHg). Retinal arteriolar geometry was defined in terms of arteriolar bifurcation angles and junction exponents (a measure of the relative diameters of parent and daughter vessels), measured from photographic diapositives using operator-directed image analysis. Members of low-birth-weight groups had significantly narrower bifurcation angles than did members of high-birth-weight groups (74 +/- 1 degree versus 78 +/- 1 degree, P= 0.017 by analysis of variance). There was no significant difference between angles in members of groups with high and low blood pressures. Neither birth weight nor blood pressure grouping affected junction exponents. Narrower bifurcation angles are associated with increased circulatory energy costs and may be related to a lower than normal microvascular density. Our finding of differences in retinal microvascular architecture might reflect a persistent alteration in vascular architecture as a result of an impairment of foetal development and could provide a mechanistic link between low birth weight and subsequently increased cardiovascular risk.


Prostate Cancer Mortality-To-Incidence Ratios Are Associated with Cancer Care Disparities in 35 Countries.
The variation in mortality-to-incidence ratios (MIRs) among countries reflects the clinical outcomes and the available interventions for colorectal cancer treatments. The association between MIR of prostate cancer and cancer care disparities among countries is an interesting issue that is rarely investigated. For the present study, cancer incidence and mortality rates were obtained from the GLOBOCAN 2012 database. The rankings and total expenditures on health of various countries were obtained from the World Health Organization (WHO). The association between variables was analyzed by linear regression analyses. In this study, we estimated the role of MIRs from 35 countries that had a prostate cancer incidence greater than 5,000 cases per year. As expected, high prostate cancer incidence and mortality rates were observed in more developed regions, such as Europe and the Americas. However, the MIRs were 2.5 times higher in the less developed regions. Regarding the association between MIR and cancer care disparities, countries with good WHO ranking and high total expenditures on health/gross domestic product (GDP) were significant correlated with low MIR. The MIR variation for prostate cancer correlates with cancer care disparities among countries further support the role of cancer care disparities in clinical outcome.


Pitx2c orchestrates embryonic axis extension via mesendodermal cell migration.
Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration during late stages of gastrulation. Transplant data indicate that Pitx2c functions cell non-autonomously to regulate this cell behavior by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2c that are required during early embryogenesis and reveal novel functions for Pitx2c as a regulator of morphogenesis.


Synaptonemal complexes from DNase-treated rat pachytene chromosomes contain (GT)n and LINE/SINE sequences.
Purified chromosome cores (synaptonemal complexes) of rat pachytene chromosomes, from which the chromatin is removed by extensive DNase II digestion, retain a residual class of DNA, presumably the bases of chromatin loops. This synaptonemal complex-associated DNA, isolated by proteinase digestion and phenol extraction of purified DNase-treated synaptonemal complexes, and cloned in plasmid vector pEMBL18, has a length distribution of 50-500 bp. From a library of these fragments, 21 fragments were sequenced. Present in this sample are short 40-200-bp segments with greater than 80% identity to "long" and "short" interspersed repeated elements (LINE/SINEs), an excess of GT/CA tandem repeats and a number of unidentified sequences. The LINE/SINE segments may play a role in homology vs. nonhomology recognition during meiosis and the alternating purine-pyrimidine sequences have been implicated in genetic recombination. Their enrichment in synaptonemal complexes may be related to the synapsis and recombination functions of meiosis.


Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population.
The forensic application of mitochondrial DNA (mtDNA) typing requires large and regionally well-defined databases. To expand the database for forensic identification purposes in Slovenia, the mtDNA control region sequences of the hypervariable regions HVI and HVII were determined in a population of 129 maternally unrelated Slovenians, using a fluorescent-based capillary electrophoresis sequencing method. A total of 111 different haplotypes resulting from 124 polymorphic positions (80 polymorphic positions in HVI and 44 in HVII) were found. Of these, 101 mtDNA types were unique, 6 haplotypes were shared by 2 individuals, 1 haplotype by 3 individuals, 2 haplotypes by 4 individuals, and the most common haplotype was found in 5 individuals. The most frequent haplotypes in the Slovenian population,263(G), 315.1(C) and 263(G), 309.1(C), 315.1(C) are also the most common in other European populations. The data support the concept that these haplotypes may represent a common European mtDNA sequence types. The sequence poymorphisms were compared to the databases of west Austria and central Italy and the HVI and HVII sequence matching probabilities within and between populations were calculated. It is 1.1-4.5 times more likely to find a sequence match in a random pair of Slovenians than in a random Slovenian-Italian pair and in a random Slovenian-Austrian pair. The length heteroplasmy in the homopolymeric C-stretch regions located at nucleotide positions 16184-16193 in HVI and at positions 303-315 in HVII was observed in 17% and 8% of individuals, respectively. A statistical estimate of the results for this population showed the random match probability and the genetic diversity of 1.16% and 0.996, respectively.


Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage.
The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.


Positive association between plasma homocysteine level and chronic kidney disease.
Increasing experimental evidence, including recently developed animal models, supports a role for homocysteine in the development of chronic kidney disease (CKD). However, relatively few clinical/epidemiological studies have examined this hypothesis in humans. We examined the relationship between plasma homocysteine level and CKD in a population-based study of older Australians. Community-based study (1992-1994) among 2,609 individuals (58.6% women), aged 49-98 years, free of clinical cardiovascular disease in the Blue Mountains region, west of Sydney, Australia. The main outcome-of-interest was CKD (n = 461), defined as estimated glomerular filtration rate of <60 ml/min/1.73 m(2). Higher plasma homocysteine levels were positively associated with CKD, independent of smoking, body mass index, diabetes mellitus, hypertension, cholesterol levels, and other confounders. The multivariable odds ratio (OR; 95% confidence intervals, CI) comparing quartile 4 of plasma homocysteine (>14 micromol/l) to quartile 1 (< or =9 micromol/l) was 10.44 (6.99-15.60), p-trend <0.0001. This association persisted in both men and women separately. The results were also consistent in subgroup analyses by categories of diabetes mellitus and hypertension. Higher plasma homocysteine levels are associated with CKD in a community-based sample of older Australians. This association appeared to be independent of diabetes mellitus and hypertension.


Calibration and standardization of extracellular vesicle measurements by flow cytometry for translational prostate cancer research.
Extracellular vesicles (EVs) are microscopic particles released naturally in biofluids by all cell types. Since EVs inherits genomic and proteomic patterns from the cell of origin, they are emerging as promising liquid biomarkers for human diseases. Flow cytometry is a popular method that is able to detect, characterize and determine the concentration of EVs with minimal sample preparation. However, the limited awareness of the scientific community to utilize standardization and calibration methods of flow cytometers is an important roadblock for data reproducibility and inter-laboratory comparison. A significant collaborative effort by the Extracellular Vesicle Flow Cytometry Working Group has led to the development of guidelines and best practices for using flow cytometry and reporting data in a way to improve rigor and reproducibility in EV research. At first look, standardization and calibration of flow cytometry for EV detection may seem burdensome and technically challenging for non-academic laboratories with limited technical training and knowledge in EV flow cytometry. In this study, we build on prior research efforts and provide a systematic approach to evaluate the performance of a high sensitivity flow cytometer (herein Apogee A60-Micro Plus) and fine-tune settings to improve detection sensitivity for EVs. We performed calibration of our flow cytometer to generate data with comparable units (nanometers, MESF). Finally, we applied our optimized protocol to measure the concentrations of prostate-derived EVs in healthy individuals and prostate cancer patients. In conclusion, our proof-of-feasibility study can serve as a scientific and technical framework for other groups motivated in using flow cytometry for EV research.


Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial.
Stem cell therapy offers potential in the regeneration of craniofacial bone defects; however, it has not been studied clinically. Tissue repair cells (TRCs) isolated from bone marrow represent a mixed stem and progenitor population enriched in CD90- and CD14-positive cells. In this phase I/II, randomized, controlled feasibility trial, we investigated TRC cell therapy to reconstruct localized craniofacial bone defects. Twenty-four patients requiring localized reconstruction of jawbone defects participated in this longitudinal trial. For regenerative therapy, patients were randomized to receive either guided bone regeneration (GBR) or TRC transplantation. At 6 or 12 weeks following treatment, clinical and radiographic assessments of bone repair were performed. Bone biopsies were harvested and underwent quantitative micro-computed tomographic (μCT) and bone histomorphometric analyses. Oral implants were installed, subsequently restored, and functionally loaded with tooth restorations. Reconstructed sites were assessed for 1 year following therapy. No study-related, serious adverse events were reported. Following therapy, clinical, radiographic, tomographic, and histological measures demonstrated that TRC therapy accelerated alveolar bone regeneration compared to GBR therapy. Additionally, TRC treatment significantly reduced the need for secondary bone grafting at the time of oral implant placement with a five fold decrease in implant bony dehiscence exposure (residual bone defects) as compared to GBR-treated sites(p < 0.01). Transplantation of TRCs for treatment of alveolar bone defects appears safe and accelerates bone regeneration, enabling jawbone reconstruction with oral implants. The results from this trial support expanded studies of TRC therapy in the treatment of craniofacial deformities (ClinicalTrials.gov number CT00755911).


Social participation among older adults not engaged in full- or part-time work is associated with more physical activity and less sedentary time.
Social participation provides health benefits for older adults. However, there is the need to identify whether higher social participation is associated with older adults being more physically active and less sedentary (sitting time). We examined the associations of social participation with physical activity, and sedentary time, in a population-based sample of older Japanese adults. A population-based, cross-sectional mail survey carried out in 2010 was used to collect data on social participation, physical activity, sedentary time and sociodemographic characteristics. Data were examined from 1146 community-dwelling, unemployed older adults (mean age 70.1 years, 43% men). Multivariate logistic regression analyses were used to calculate odds ratios (OR) for associations of social participation with physical activity and total sedentary time; and, for associations with passive and mentally-active sedentary (sitting) time. For both men and women, those with higher social participation were more physically active (OR 2.10, 95% CI 1.44-3.06 among men; OR 1.93, 95% CI 1.39-2.68 among women). Total sedentary time had significant associations among men (OR 0.62, 95% CI 0.42-0.90), but not among women (OR 0.80, 95% CI 0.58-1.11). Social participation was associated with less passive sedentary time (OR 0.55, 95% CI 0.38-0.81 for men; OR 0.72, 95% CI 0.51-0.99 for women). Promoting social participation among older adults could contribute to increasing physical activity and reducing sedentary time, with potential benefits for chronic disease. Further research is required to elucidate the deleterious and beneficial roles of passive and mentally-active sedentary time for older adults. Geriatr Gerontol Int 2017; 17: 1921-1927.


Oral or intravenous proton pump inhibitor in patients with peptic ulcer bleeding after successful endoscopic epinephrine injection.
We aimed to assess the clinical effectiveness of oral vs. intravenous (i.v.) regular-dose proton pump inhibitor (PPI) after endoscopic injection of epinephrine in patients with peptic ulcer bleeding. Peptic ulcer patients with active bleeding, nonbleeding visible vessels, or adherent clots were enrolled after successful endoscopic haemostasis achieved by epinephrine injection. They were randomized to receive either oral rabeprazole (RAB group, 20 mg twice daily for 3 days) or i.v. omeprazole (OME group, 40 mg i.v. infusion every 12 h for 3 days). Subsequently, the enrolled patients receive oral PPI for 2 months (rabeprazole 20 mg or esomeprazole 40 mg once daily). The primary end-point was recurrent bleeding up to 14 days. The hospital stay, blood transfusion, surgery and mortality within 14 days were compared as well. A total of 156 patients were enrolled, with 78 patients randomly allocated in each group. The two groups were well matched for factors affecting the clinical outcomes. Primary end-points (recurrent bleeding up to 14 days) were reached in 12 patients (15.4%) in the OME group and 13 patients (16.7%) in the RAB group [95% confidence interval (CI) of difference -12.82, 10.22]. All the rebleeding events occurred within 3 days of enrolment. The two groups were not different in hospital stay, volume of blood transfusion, surgery or mortality rate (1.3% of the OME group and 2.6% of the RAB group died, 95% CI of difference -5.6, 3.0). Oral rabeprazole and i.v. regular-dose omeprazole are equally effective in preventing rebleeding in patients with high-risk bleeding peptic ulcers after successful endoscopic injection with epinephrine.


The labour market integration of migrants and refugees: career guidance and the newly arrived.
This scoping paper sets out to consider various aspects of the phenomenon of people on the move, and to examine some of the ways in which career guidance has responded to the challenge of integrating the newly arrived. The paper is informed by a recognition of the dignity of individuals above any classificatory system that places persons in a hierarchy, with some having access to rights that others are denied. This stance requires career practitioners to confront the prevalent vocabulary, discourses and attitudes circulating in the mainstream in order to become aware of-and to overcome-personal prejudice, thus opening up possibilities for more socially just forms of service. A synthesis of relevant literature concerning the integration of migrants and refugees in the labour market is provided in order to signpost emancipatory forms of career guidance, ones that move beyond an emphasis on individual resilience to take seriously the impact of exclusionary social practices and structures. The paper concludes by highlighting a number of initiatives that suggest that career guidance is rising to the challenge of catering for a diverse group of newly arrived by mobilising theoretical frameworks that are fit for purpose, by developing bespoke training programmes for practitioners, and by sharing the lessons learnt from the field.


Ambivalent helpers and unhealthy choices: public health practitioners' narratives of Indigenous ill-health.
Public health practitioners in Australian indigenous health work in a complex political environment. Public health training is limited in providing them with conceptual tools needed to unpack the postcolonial nexus of 'fourth-world' health. A workshop was designed by the authors to facilitate critical reflection on how the concepts of race and culture are used in constructions of indigenous ill-health. It was attended by researchers, students, clinicians and bureaucrats working in public health in northern Australia. A thematic analysis of the workshop minutes provided insight into public health practitioners' narratives of Indigenous ill-health. The major themes that emerged included tension between structure and agency and between sameness and difference, and ambivalence surrounding the 'helper' identity of public health practitioners. We suggest that these narratives can be understood as attempts to maintain the moral integrity of both Indigenous people and practitioners. This task is necessitated by the specter of cultural relativism intrinsic to contemporary liberal discourses of multiculturalism that attempt to reconcile the universal rights of the citizen with the special rights of minority groups. We argue that the concepts of self-determination and neocolonialism mark the spaces where universal and particular discourses overlap and clash. Practitioners who seek to escape neocolonialism must inhabit only the discursive space of public health congruent with self-determination, leaving them in a bind common to many postcolonial situations. They must relieve the ill-health of indigenous people without acting upon them; change them without declaring that change is required.


Diversification of bacterial genome content through distinct mechanisms over different timescales.
Bacterial populations often consist of multiple co-circulating lineages. Determining how such population structures arise requires understanding what drives bacterial diversification. Using 616 systematically sampled genomes, we show that Streptococcus pneumoniae lineages are typically characterized by combinations of infrequently transferred stable genomic islands: those moving primarily through transformation, along with integrative and conjugative elements and phage-related chromosomal islands. The only lineage containing extensive unique sequence corresponds to a set of atypical unencapsulated isolates that may represent a distinct species. However, prophage content is highly variable even within lineages, suggesting frequent horizontal transmission that would necessitate rapidly diversifying anti-phage mechanisms to prevent these viruses sweeping through populations. Correspondingly, two loci encoding Type I restriction-modification systems able to change their specificity over short timescales through intragenomic recombination are ubiquitous across the collection. Hence short-term pneumococcal variation is characterized by movement of phage and intragenomic rearrangements, with the slower transfer of stable loci distinguishing lineages.


Algae biostimulants: A critical look at microalgal biostimulants for sustainable agricultural practices.
For the growing human population to be sustained during present climatic changes, enhanced quality and quantity of crops are essential to enable food security worldwide. The current consensus is that we need to make a transition from a petroleum-based to a bio-based economy via the development of a sustainable circular economy and biorefinery approaches. Both macroalgae (seaweeds) and microalgae have been long considered a rich source of plant biostimulants with an attractive business opportunity in agronomy and agro-industries. To date, macroalgae biostimulants have been well explored. In contrast, microalgal biostimulants whilst known to have positive effects on development, growth and yields of crops, their commercial implementation is constrained by lack of research and cost of production. The present review highlights the current knowledge on potential biostimulatory compounds, key sources and their quantitative information from algae. Specifically, we provide an overview on the prospects of microalgal biostimulants to advance crop production and quality. Key aspects such as specific biostimulant effects caused by extracts of microalgae, feasibility and potential of co-cultures and later co-application with other biostimulants/biofertilizers are highlighted. An overview of the current knowledge, recent advances and achievements on extraction techniques, application type, application timing, current market and regulatory aspects are also discussed. Moreover, aspects involved in circular economy and biorefinery approaches are also covered, such as: integration of waste resources and implementation of high-throughput phenotyping and -omics tools in isolating novel strains, exploring synergistic interactions and illustrating the underlying mode of microalgal biostimulant action. Overall, this review highlights the current and future potential of microalgal biostimulants, algal biochemical components behind these traits and finally bottlenecks and prospects involved in the successful commercialisation of microalgal biostimulants for sustainable agricultural practices.


B cells turn off virgin but not memory T cells.
There are three possible outcomes when a T cell recognizes a cell bearing a self or foreign antigen. (i) The T cell is not sufficiently signaled and is unaffected. (ii) The T cell is activated. (iii) The T cell is turned off. The differentiation state of the T cell is critical to the outcome. Although both virgin and memory T cells can be activated by antigens presented by "professional" antigen-presenting cells such as dendritic cells, they differ in their responses to B cells. Experienced T cells respond to antigen presented by B cells, whereas virgin T cells are rendered tolerant. These findings may relate to the phenomena of low- and high-zone tolerance, neonatal tolerance, and the beneficial effect of blood transfusions on allograft survival.


REVEILLE8 and PSEUDO-REPONSE REGULATOR5 form a negative feedback loop within the Arabidopsis circadian clock.
Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE-binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE-containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.


Factors related to occurrence and distribution of selected bacterial and protozoan pathogens in Pennsylvania streams.
The occurrence and distribution of fecal indicator bacteria (FIB) and bacterial and protozoan pathogens are controlled by diverse factors. To investigate these factors in Pennsylvania streams, 217 samples were collected quarterly from a 27-station water-quality monitoring network from July 2007 through August 2009. Samples were analyzed for concentrations of Escherichia coli (EC) and enterococci (ENT) indicator bacteria, concentrations of Cryptosporidium oocysts and Giardia cysts, and the presence of four genes related to pathogenic types of EC (eaeA, stx2, stx1, rfb(O157)) plus three microbial source tracking (MST) gene markers that are also associated with pathogenic ENT and EC (esp, LTIIa, STII). Water samples were concurrently analyzed for basic water chemistry, physical measures of water quality, nutrients, metals, and a suite of 79 organic compounds that included hormones, pharmaceuticals, and antibiotics. For each sample location, stream discharge was measured by using standardized methods at the time of sample collection, and ancillary sample site information, such as land use and geological characteristics, was compiled. Samples exceeding recreational water quality criteria were more likely to contain all measured pathogen genes but not Cryptosporidium or Giardia (oo)cysts. FIB and Giardia density and frequency of eaeA gene occurrence were significantly related to season. When discharge at a sampling location was high (>75th percentile of daily mean discharge), there were greater densities of FIB and Giardia, and the stx2, rfb(O157), STII, and esp genes were found more frequently than at other discharge conditions. Giardia occurrence was likely related to nonpoint sources, which are highly influential during seasonal overland transport resulting from snowmelt and elevated precipitation in late winter and spring in Pennsylvania. When MST markers of human, swine, or bovine origin were present, samples more frequently carried the eaeA, stx2, stx1, and rfb(O157) genes, but no genes were related exclusively to an individual MST marker. The human source pharmaceuticals (HSPs) acetaminophen and caffeine were correlated with Giardia, and the presence of HSPs proved to be more useful than MST markers in distinguishing the occurrence of Giardia. The HSPs caffeine and carbamazepine were correlated with the sum total of pathogen genes detected in a sample, demonstrating the value of using HSPs as an indicator of fecally derived pathogens. Sites influenced by urban land use with less forest were more likely to have greater FIB and Giardia densities and sum of the array of pathogen genes. Sites dominated by shallow carbonate bedrock in the upstream catchment were likely to have greater FIB densities and higher sum totals of pathogen genes but no correlation with Giardia detection. Our study provides a range of specific environmental, chemical, geologic, and land-use variables related to occurrence and distribution of FIB and selected bacterial and protozoan pathogens in Pennsylvania streams. The information presented could be useful for resource managers in understanding bacterial and protozoan pathogen occurrence and their relation to fecal indicator bacteria in similar settings.


Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.
To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.


SU-E-T-310: Micro-Dosimetry Study of the Radiation Dose Enhancement at the Gold-Tissue Interface for Nanoparticle-Aided Radiation Therapy.
Gold nanoparticles (AuNP) have been proposed to be utilized for local dose enhancement in radiation therapy. Due to a very sharp spatial fall-off of the effect, the dosimetry associated with such an approach is difficult to implement in a direct measurement. This study is aimed at establishing a micro-dosimetry technique for experimental verification of dose enhancement in the vicinity of gold-tissue interface. The spatial distribution of the dose enhancement near the gold-tissue interface is modeled with Monte Carlo (MC) package MCNP5 in a 1-dimentional approach of a thin gold slab placed in an ICRU-4 component tissue phantom. The model is replicating the experiment, where the dose enhancement due to gold foils having thicknesses of 1, 10, and 100μm and areas of 12.5×25mm2 are placed at a short distance from clinical HDR brachytherapy (Ir-192) source. The measurements are carried out with a thin-film CdTe-based photodetector, having thickness <10μm, allowing for high spatial resolution at progressively increasing distances from the foil. Our MC simulation results indicate that for Ir-192 energy spectrum the dose enhancement region extends over ∼1 mm distance from the foil, changing from several hundred at the interface to just a few percent. The trend in the measured dose enhancement closely follows the results obtained from MC simulations. AuNP's have been established as promising candidates for dose enhancement in nanoparticle-aided radiation therapy, particularly, in the energy range relevant to brachytherapy applications. Most researchers study the dose enhancement with MC simulations, or experimental approaches involving biological systems, where achievable dose enhancements are difficult to quantify. Successful development of micro-dosimetry approaches will pave a way for direct assessment of the dose in experiments on biological models, shedding some light on apparent discrepancy between physical dose enhancement and biological effect established in studies of AuNP-aided radiation therapy. No conflict of interest.


Study of the synergistic anti-inflammatory activity of Solanum xanthocarpum Schrad and Wendl and Cassia fistula Linn.
Nonsteroidal and steroidal drugs are generally used as a part of drug therapy in inflammation. However, these drugs have severe side-effects like nausea and vomiting. Therefore, there is a need to identify anti-inflammatory compounds that will be effective with a better safety profile. Solanum xanthocarpum Schrad and Wendl and Cassia fistula Linn has many therapeutic uses mentioned in Ayurveda and therefore we aimed to study its anti-inflammatory activity both alone and in combination. The water extract of dried fruits of Solanum xanthocarpum Schrad and Wendl and dried pulp of Cassia fistula Linn was prepared. The anti-inflammatory activity of these extracts was investigated using the carragenan-induced paw edema model in rats individually and in two different combinations. ED50 of both the extracts singly and in combination were calculated by dose-response curves, and this information was then plotted on the isobologram. The interaction index of the extracts was also investigated to determine whether both the extracts in combination show synergistic or antagonistic or additive effects. It was observed that extracts of dried fruits of Solanum xanthocarpum showed more anti-inflammatory activity than dried fruits of Cassia fistula Linn. Both the extracts showed maximum anti-inflammatory activity at 500 mg/kg dose. Among the different dose combinations of both the extracts, the 1:1 combination at the 500 mg/kg dose showed maximum percentage inhibition of 75%, which was comparable with the positive control, diclofenac sodium, which showed 81% inhibition. As revealed by the isobolograms, both the combinations fell below the additivity line, which indicates synergistic interactions between Solanum xanthocarpum and Cassia fistula extracts. Interaction indices of both combinations were observed to be <1, which re-demonstrated the synergistic effects of the combination.


Genomic Analysis of Carbapenem-Resistant Acinetobacter baumannii Isolates Belonging to Major Endemic Clones in South America.
Carbapenem-resistant Acinetobacter baumannii (CRAB) are emerging worldwide. In South America, clinical isolates presenting such a phenotype usually do not belong to the globally distributed international clone 2 (IC2). The majority of these isolates are also resistant to multiple other antimicrobials and are often designated extremely drug-resistant (XDR). The aim of this study was to characterize the resistance mechanisms presented by 18 carbapenem-resistant A. baumannii isolates from five different Brazilian hospitals. Species identification was determined by rpoB sequencing, and antimicrobial susceptibility was determined by broth microdilution. Isolates were submitted to whole genome sequencing using Illumina platform and genetic similarity was determined by PFGE, MLST, and cgMLST. Genome analysis was used to identify intrinsic and acquired resistance determinants, including mutations in the AdeRSABC efflux system and in outer membrane proteins (OMPs). All isolates were identified as A. baumannii and grouped into 4 pulsotypes by PFGE, which belonged to clonal complexes (CC) 15 Pas /103 Ox (n = 4) and 79 Pas /113 Ox (n = 14), corresponding to IC4 and IC5, respectively. High MIC values to carbapenems, broad-spectrum cephalosporins, amikacin, and ciprofloxacin were observed in all isolates, while MICs of ampicillin/sulbactam, gentamicin, and tigecycline varied among the isolates. Minocycline was the most active antimicrobial agent tested. Moreover, 12 isolates (66.7%) were considered resistant to polymyxins. Besides intrinsic OXA-51 and ADC variants, all isolates harbored an acquired carbapenem-hydrolyzing class D β-lactamase (CHDL) encoding gene, either bla OXA- 23 or bla OXA- 72. A diversity of aminoglycoside modifying enzymes and resistance determinants to other antimicrobial classes were found, as well as mutations in gyrA and parC. Non-synonymous mutations have also been identified in the AdeRSABC efflux system and in most OMPs, but they were considered natural polymorphisms. Moreover, resistance to polymyxins among isolates belonging to IC5 were associated to non-synonymous mutations in pmrB, but no known polymyxin resistance mechanism was identified in isolates belonging to IC4. In conclusion, A. baumannii clinical isolates belonging to South America's major clones present a myriad of antimicrobial resistance determinants. Special attention should be paid to natural polymorphisms observed in each clonal lineage, especially regarding non-synonymous mutations in constitutive genes associated with distinct resistance phenotypes.


Triplet state homoaromaticity: concept, computational validation and experimental relevance.
Cyclic conjugation that occurs through-space and leads to aromatic properties is called homoaromaticity. Here we formulate the homoaromaticity concept for the triplet excited state (T1) based on Baird's 4n rule and validate it through extensive quantum-chemical calculations on a range of different species (neutral, cationic and anionic). By comparison to well-known ground state homoaromatic molecules we reveal that five of the investigated compounds show strong T1 homoaromaticity, four show weak homoaromaticity and two are non-aromatic. Two of the compounds have previously been identified as excited state intermediates in photochemical reactions and our calculations indicate that they are also homoaromatic in the first singlet excited state. Homoaromaticity should therefore have broad implications in photochemistry. We further demonstrate this by computational design of a photomechanical "lever" that is powered by relief of homoantiaromatic destabilization in the first singlet excited state.


Innate and adaptive processes in the spleen.
The spleen is a lymphatic organ interposed in the blood stream. It remains a largely neglected aggregate of lymphoid tissue. The spleen immune system is responsible for protecting the body from invading pathogens and detecting senescent, mechanically damaged, displaced and aberrant cells that could lead to tumor formation. Recent studies prove its dominant role in a simultaneous dual reaction to bacterial and allogeneic antigens. Spleen is the site of innate and adaptive immune processes. Microbial penetration of tissues evokes an immediate reaction of the innate system, whereas the adaptive immune response involves the interaction of cells that recognize a particular antigen in context with MHC molecules presented by antigen-presenting cells. This review gives some insight into the immune events in the spleen and its neglected systemic role in the allogeneic reaction.


A new neodiplostomid (Digenea) from the intestine of chicks infected with metacercariae from the grass snake, Rhabdophis tigrina.
Three species of neodiplostomula are known to inhabit the European grass snake, Rhabdophis tigrina, in the Republic of Korea: Pharyngostomum cordatum (large-sized neodiplostomula), an intestinal trematode of cats; Neodiplostomum seoulense (small-sized neodiplostomula), an intestinal trematode of humans and rodents; and Neodiplostomum leei (small-sized neodiplostomula), which migrates to the livers of rodents and is an intestinal trematode of birds. The present study describes a fourth species, Neodiplostomum (Conodiplostomum) boryongense n. sp. (Digenea: Neodiplostomidae), based on adult flukes recovered from the small intestines of chicks experimentally infected with small-sized neodiplostomula from the grass snake. The new species differs from 13 previously known species. It also differs from N. seoulense in its larger body size, severely bilobed testes, and smaller genital atrium, and from N. leei in its larger body size, smaller ventral sucker, presence of a genital cone, and vitelline follicles distributed chiefly in the forebody. The new species does not migrate to the livers of rodents nor does it develop to adulthood in the rodent intestines. However, the neodiplostomula of the new species are indistinguishable from those of the other 2 species. Results show that at least 4 species of neodiplostomula inhabit the grass snake in the Republic of Korea.


Caffeine Stabilises Fission Yeast Wee1 in a Rad24-Dependent Manner but Attenuates Its Expression in Response to DNA Damage.
The widely consumed neuroactive compound caffeine has generated much interest due to its ability to override the DNA damage and replication checkpoints. Previously Rad3 and its homologues was thought to be the target of caffeine's inhibitory activity. Later findings indicate that the Target of Rapamycin Complex 1 (TORC1) is the preferred target of caffeine. Effective Cdc2 inhibition requires both the activation of the Wee1 kinase and inhibition of the Cdc25 phosphatase. The TORC1, DNA damage, and environmental stress response pathways all converge on Cdc25 and Wee1. We previously demonstrated that caffeine overrides DNA damage checkpoints by modulating Cdc25 stability. The effect of caffeine on cell cycle progression resembles that of TORC1 inhibition. Furthermore, caffeine activates the Sty1 regulated environmental stress response. Caffeine may thus modulate multiple signalling pathways that regulate Cdc25 and Wee1 levels, localisation and activity. Here we show that the activity of caffeine stabilises both Cdc25 and Wee1. The stabilising effect of caffeine and genotoxic agents on Wee1 was dependent on the Rad24 chaperone. Interestingly, caffeine inhibited the accumulation of Wee1 in response to DNA damage. Caffeine may modulate cell cycle progression through increased Cdc25 activity and Wee1 repression following DNA damage via TORC1 inhibition, as TORC1 inhibition increased DNA damage sensitivity.


Collagen architecture in pregnancy-induced protection from breast cancer.
The reduction in breast cancer risk attributed to early-age pregnancy is mediated in part by changes in the mammary epithelium. Here, we address the role of the mammary stroma in this protection. Utilizing tumor cells capable of transitioning from indolent to proliferative or invasive states, we demonstrate that mammary extracellular matrix (ECM) from parous rats (parous matrix) decreases tumor growth and impedes cellular phenotypes associated with tumor cell invasion compared with that observed using nulliparous matrix. Proteomic analysis identifies an increased abundance of collagen I in parous matrix, an observation extended to breast tissue of parous women. Given the pro-tumorigenic attributes of fibrillar collagen, these results were unexpected. Second-harmonic generation imaging and atomic force microscopy revealed that the abundant collagen observed in the mammary glands of parous rats is less linearized and associated with a decrease in stromal stiffness, implicating collagen organization and stiffness in parity-induced protection. Using 3D cell culture models, we demonstrate that linearized (fibrillar) collagen I induces cellular phenotypes consistent with an invasive behavior in mammary tumor cells and alters the subcellular distribution of β1 integrin. Conversely, high-density non-fibrillar collagen I induces tumor-suppressive attributes, including increases in junctional E-cadherin in tumor cells, upregulation of genes encoding components of cell-cell junctions, and downregulation of mesenchymal-specific and metalloproteinase-encoding genes. These data show that collagen organization, rather than density alone, is a key contributor to the invasive phenotype. Furthermore, our data show that parity alters the composition and organization of mammary ECM, particularly fibrillar collagen, in a manner consistent with tumor suppression.


Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction.
To identify human proteins that bind to the Smac and caspase-9 binding pocket on the baculoviral inhibitor of apoptosis protein (IAP) repeat 3 (BIR3) domain of human XIAP, we used BIR3 as an affinity reagent, followed by elution with the BIR3 binding peptide AVPIA, microsequencing, and mass spectrometry. The mature serine protease Omi (also known as HtrA2) was identified as a mitochondrial direct BIR3-binding protein and a caspase activator. Like mature Smac (also known as Diablo), mature Omi contains a conserved IAP-binding motif (AVPS) at its N terminus, which is exposed after processing of its N-terminal mitochondrial targeting sequence upon import into the mitochondria. Mature Omi is released together with mature Smac from the mitochondria into the cytosol upon disruption of the outer mitochondrial membrane during apoptosis. Finally, mature Omi can induce apoptosis in human cells in a caspase-independent manner through its protease activity and in a caspase-dependent manner via its ability to disrupt caspase-IAP interaction. Our results provide clear evidence for the involvement of a mitochondrial serine protease in the apoptotic pathway, emphasizing the critical role of the mitochondria in cell death.


Application of chronic intravascular blood glucose sensor in dogs.
An intravenous glucose sensor was implanted in six dogs for 1-15 wk. The glucose sensor is a flexible cylinder, approximately 0.2 cm diam and 30 cm long, with a tip containing immobilized glucose oxidase and catalase coupled to a potentiostatic O2 sensor. The sensor and a similar O2 reference sensor were implanted in the superior vena cava near the entrance of the right atrium. The sensor response was conveyed externally either by a telemetry system implanted nearby, surgically accessed leads, or chronically maintained percutaneous leads. Summing over the six implants, there was a total implantation period of 333 days during which glucose sensors were functional on demand. The sensor response showed agreement with conventionally assayed blood samples after accounting for a response lag. Sensor response to glucose showed little change over the implant period. Biocompatibility, enzyme lifetime, O2 availability, O2 sensor stability, and biochemical interference were not limitations. Results demonstrated that this sensor can function effectively as an implant in dogs for a period of months and has the potential for long-term operation.


Aggregation induced emission in one easy step: pyridinium AIEgens and counter ion effect.
Protonation of pyridines with a strong acid is a general and straightforward approach to achieve efficient aggregation induced emission (AIE) in structurally remarkably simple organic molecules that lack any of the conventional luminophores. The relationship between the nature of counter ion and the AIE efficiency is demonstrated. The superiority of the perchlorate counter ion is attributed to efficient stabilization of the key intermolecular π+-π interactions between neighboring luminophore molecules in the crystal lattice.


Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins.
Ras proteins participate as a molecular switch in the early steps of the signal transduction pathway that is associated with cell growth and differentiation. When the protein is in its GTP complexed form it is active in signal transduction, whereas it is inactive in its GDP complexed form. A comparison of eight three-dimensional structures of ras proteins in four different crystal lattices, five with a nonhydrolyzable GTP analog and three with GDP, reveals that the "on" and "off" states of the switch are distinguished by conformational differences that span a length of more than 40 A, and are induced by the gamma-phosphate. The most significant differences are localized in two regions: residues 30 to 38 (the switch I region) in the second loop and residues 60 to 76 (the switch II region) consisting of the fourth loop and the short alpha-helix that follows the loop. Both regions are highly exposed and form a continuous strip on the molecular surface most likely to be the recognition sites for the effector and receptor molecule(or molecules). The conformational differences also provide a structural basis for understanding the biological and biochemical changes of the proteins due to oncogenic mutations, autophosphorylation, and GTP hydrolysis, and for understanding the interactions with other proteins.


Ocular motor abnormalities in neurodegenerative disorders.
Eye movements are a source of valuable information to both clinicians and scientists as abnormalities of them frequently act as clues to the localization of a disease process. Classically, they are divided into two main types: those that hold the gaze, keeping images steady on the retina (vestibulo-ocular and optokinetic reflexes) and those that shift gaze and redirect the line of sight to a new object of interest (saccades, vergence, and smooth pursuit). Here we will review some of the major ocular motor abnormalities present in neurodegenerative disorders.


Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity.
HIF (hypoxia-inducible factor) is an alphabeta transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-alpha are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-alpha (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340-->Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344-->Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.


Prediction of on-treatment disability worsening in RRMS with the MAGNIMS score.
The magnetic resonance imaging in multiple sclerosis (MAGNIMS) score combines relapses and magnetic resonance imaging (MRI) lesions to predict disability outcomes in relapsing-remitting multiple sclerosis (RRMS) treated with interferon-β. To validate the MAGNIMS score and extend to other disease-modifying therapies (DMTs). To examine the prognostic value of gadolinium contrast-enhancing (Gd+) lesions. This RRMS MSBase cohort study (n = 2293) used a Cox model to examine the prognostic value of relapses, MRI activity and the MAGNIMS score for disability worsening during treatment with interferon-β and three other DMTs. Three new T2 lesions (hazard ratio (HR) = 1.60, p = 0.028) or two relapses (HR = 2.24, p = 0.002) on interferon-β (for 12 months) were predictive of disability worsening over 4 years. MAGNIMS score = 2 (1 relapse and ⩾3 T2 lesions or ⩾2 relapses) was associated with a greater risk of disability worsening on interferon-β (HR = 2.0, p = 0.001). In pooled cohort of four DMTs, similar associations were seen (MAGNIMS score = 2: HR = 1.72, p = 0.001). Secondary analyses demonstrated that the addition of Gd+ to the MAGNIMS did not materially improve its prediction of disability worsening. We have validated the MAGNIMS score in RRMS and extended its application to three other DMTs: 1 relapse and ⩾3 T2 lesions or ⩾2 relapses predicted worsening of disability. Contrast-enhancing lesions did not substantially improve the prognostic score.


Longevity and lifespan control in mammals: lessons from the mouse.
Aging, which affects all organ systems, is one of the most complex phenotypes. Recent discoveries in long-lived mutant mice have revealed molecular mechanisms of longevity in mammals which may contribute to our understanding of why humans age. These mutations include naturally occurring spontaneous mutations, and those of mice genetically modified by modern genomic technologies. It is generally believed that the most fundamental mechanisms of aging are evolutionarily conserved across species. The following types of longevity mechanisms have been intensively studied: suppression of the somatotropic (growth hormone/insulin-like growth factor 1) axis, decreased metabolism and increased resistance of oxidative stress, reduced insulin secretion and increased insulin sensitivity, and delayed reproductive maturation and reduced fertility. In addition, many of the mutations have a sex-dependent effect on lifespan, and when present in different genetic backgrounds, the effects of the same gene mutation can vary considerably. The present review discusses these phenotypic variations as well as describing the known longevity genes in long-lived mutant mice and the molecular mechanisms specifying longevity. We anticipate that these mouse studies will ultimately provide clues about how to delay the aging and prolong lifespan, and help to develop therapies for healthier human aging.