The interstitial response to renal injury: fibroblast-like cells show phenotypic changes and have reduced potential for erythropoietin gene expression.
To define the potential for erythropoietin gene expression in injured kidneys, marker gene expression was examined in transgenic mice bearing a homologously recombined erythropoietin--simian virus 40 T antigen (Epo-TAg) transgene. Three types of renal injury were studied: ureteric obstruction, global ischemia following clamping of the renal pedicle, and focal needlestick injury. All modes of injury were associated with an expansion of the interstitial space, which contained an increased number of cells. Alterations observed in the interstitial fibroblast-like cells included an increased number and complexity of cellular processes, enhanced expression of contractile elements, particularly of the intermediate filament desmin, and reduced expression of ecto-5'-nucleotidase. Following each type of injury there was a focal or general reduction in the proportion of such cells that could be stimulated to express Epo-TAg. However, some positively staining cells were present even in severely injured regions and more could be recruited to express Epo-TAg by severe anemic or hypoxic stimulation, indicating that cells with the potential for erythropoietin gene expression were neither absent nor completely refractory to stimulation in these regions. In all injured kidneys, Epo-TAg expression was limited to the fibroblast-like population. Double labeling experiments showed that cells expressing Epo-TAg also expressed increased amounts of desmin, demonstrating that the myofibroblast features which develop in response to injury and the capacity for erythropoietin gene expression are not mutually exclusive.


Modelling the joint torques and loadings during squatting at the Smith machine.
An analytical biomechanical model was developed to establish the relevant properties of the Smith squat exercise, and the main differences from the free barbell squat. The Smith squat may be largely patterned to modulate the distributions of muscle activities and joint loadings. For a given value of the included knee angle (θ(knee)), bending the trunk forward, moving the feet forward in front of the knees, and displacing the weight distribution towards the forefoot emphasizes hip and lumbosacral torques, while also reducing knee torque and compressive tibiofemoral and patellofemoral forces (and vice versa). The tibiofemoral shear force φ(t) displays more complex trends that strongly depend on θ(knee). Notably, for 180° ≥ θ(knee) ≥ 130°, φ(t) and cruciate ligament strain forces can be suppressed by selecting proper pairs of ankle and hip angles. Loading of the posterior cruciate ligament increases (decreases) in the range 180° ≥ θ(knee) ≥ 150° (θ(knee) ≤ 130°) with knee extension, bending the trunk forward, and moving the feet forward in front of the knees. In the range 150° > θ(knee) > 130°, the behaviour changes depending on the foot weight distribution. The conditions for the development of anterior cruciate ligament strain forces are explained. This work enables careful use of the Smith squat in strengthening and rehabilitation programmes.


Segmented filamentous bacteria in a defined bacterial cocktail induce intestinal inflammation in SCID mice reconstituted with CD45RBhigh CD4+ T cells.
The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RB(high) subset of CD4+T cells isolated from the spleen of normal BALB/c mice. A CD4+CD45RB(high) subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ-free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen-free (SPF) bacteria. Mice were evaluated 8-12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD). After the transfer of the CD4+CD45RB(high) T-cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO-1 (zona occludens). Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB.


Effects of temperament and acclimation to handling on reproductive performance of Bos taurus beef females.
Two experiments evaluated the effects of temperament and acclimation to handling on reproductive performance of Bos taurus beef females. In Exp. 1, 433 multiparous, lactating Angus × Hereford cows were sampled for blood and evaluated for temperament before the breeding season. Cow temperament was assessed by chute score and exit velocity. Chute score was assessed on a 5-point scale according to behavioral responses during chute restraining. Exit score was calculated by dividing exit velocity into quintiles and assigning cows with a score from 1 to 5 (1 = slowest, 5 = fastest cows). Temperament score was calculated by averaging chute and exit scores. Cows were classified for temperament type according to temperament score (≤ 3 = adequate, > 3 = aggressive). Plasma cortisol concentrations were greater (P < 0.01) in cows with aggressive vs. adequate temperament. Cows with aggressive temperament had reduced (P ≤ 0.05) pregnancy and calving rate and tended to have reduced (P = 0.09) weaning rate compared with cows with adequate temperament. Hence, kilogram of calf born per cow was reduced (P = 0.05) and kilogram of calf weaned per cow tended to be reduced (P = 0.08) in aggressive cows. In Exp. 2, 88 Angus × Hereford heifers (initial age = 206 ± 2 d) were weighed (d 0 and 10) and evaluated for temperament score (d 10). On d 11, heifers were ranked by these variables and assigned to receive or not (control) an acclimation treatment. Acclimated heifers were processed through a handling facility 3 times weekly for 4 wk (d 11 to 39; Mondays, Wednesdays, and Fridays), whereas control heifers remained undisturbed on pasture. Heifer puberty status, evaluated via plasma progesterone concentrations, was assessed on d 0 and 10, d 40 and 50, 70 and 80, 100 and 110, 130 and 140, 160 and 170, and 190 and 200. Blood samples collected on d 10 and 40 were also analyzed for plasma concentrations of cortisol and haptoglobin. Temperament score was assessed again on d 40 and d 200. Acclimated heifers had reduced (P = 0.01) concentrations of cortisol and haptoglobin on d 40 and reduced (P = 0.02) exit velocity on d 200 compared with control heifers. Puberty was hastened in acclimated heifers compared with control (P = 0.01). Results from this study indicate that B. taurus beef cows with aggressive temperament have impaired reproductive performance compared with cohorts with adequate temperament, whereas acclimation to human handling after weaning hastens reproductive development of replacement heifers.


Morphological abnormalities in prefrontal surface area and thalamic volume in attention deficit/hyperactivity disorder.
Although previous morphological studies have demonstrated abnormalities in prefrontal cortical thickness in children with attention deficit/hyperactivity disorder (ADHD), studies investigating cortical surface area are lacking. As the development of cortical surface is closely linked to the establishment of thalam-ocortical connections, any abnormalities in the structure of the thalamus are likely to relate to altered cortical surface area. Using a clinically well-defined sample of children with ADHD (n = 25, 1 female) and typically developing controls (n = 24, 1 female), we studied surface area across the cortex to determine whether children with ADHD had reduced thalamic volume that related to prefrontal cortical surface area. Relative to controls, children with ADHD had a significant reduction in thalamic volume and dorsolateral prefrontal cortical area in both hemispheres. Furthermore, children with ADHD with smaller thalamic volumes were found to have greater reductions in surface area, a pattern not evident in the control children. Our results are further evidence of reduced lateral prefrontal cortical area in ADHD. Moreover, for the first time, we have also shown a direct association between thalamic anatomy and frontal anatomy in ADHD, suggesting the pathophysiological process that alters surface area maturation is likely to be linked to the development of the thalamus.


Alzheimer disease research in the 21st century: past and current failures, new perspectives and funding priorities.
Much of Alzheimer disease (AD) research has been traditionally based on the use of animals, which have been extensively applied in an effort to both improve our understanding of the pathophysiological mechanisms of the disease and to test novel therapeutic approaches. However, decades of such research have not effectively translated into substantial therapeutic success for human patients. Here we critically discuss these issues in order to determine how existing human-based methods can be applied to study AD pathology and develop novel therapeutics. These methods, which include patient-derived cells, computational analysis and models, together with large-scale epidemiological studies represent novel and exciting tools to enhance and forward AD research. In particular, these methods are helping advance AD research by contributing multifactorial and multidimensional perspectives, especially considering the crucial role played by lifestyle risk factors in the determination of AD risk. In addition to research techniques, we also consider related pitfalls and flaws in the current research funding system. Conversely, we identify encouraging new trends in research and government policy. In light of these new research directions, we provide recommendations regarding prioritization of research funding. The goal of this document is to stimulate scientific and public discussion on the need to explore new avenues in AD research, considering outcome and ethics as core principles to reliably judge traditional research efforts and eventually undertake new research strategies.


Human appendix B cells naturally express receptors for and respond to interleukin 6 with selective IgA1 and IgA2 synthesis.
Past studies have shown that freshly isolated human B cells from peripheral blood and tonsils do not express IL-6 receptors (IL-6R); however, mitogen or antigen activation of these B cells induces IL-6R and responsiveness to IL-6. In this study, we have shown that a high proportion of B cells enzymatically dissociated from human appendix, a gut-associated lymphoreticular tissue (GALT), expresses the IL-6R, and that recombinant human IL-6 induces significant increases in the number of Ig-producing cells. The recombinant human IL-6-induced increase in Ig-producing cells is restricted to the IgA isotype. Further, IgA2 is the major subclass; however, significant numbers of IgA1 producing cells are also seen. In contrast, human tonsillar and peripheral blood B cells express low levels of IL-6R, and exogenous IL-6 does not increase numbers of Ig-producing cells. When PBMC or tonsillar cells are stimulated with PWM, the former display an equal distribution of IgA1 and IgA2 secreting cells, while tonsillar B cells are mainly of the IgA1 subclass. The distribution of surface Ig-positive (sIg+) B cells in the appendix B cell population is sIgA+ greater than sIgG+ greater than sIgM+, and the sIgA+ B cells express higher levels of IL-6R when compared with sIgG+ or sIgM+ B cells. These studies show that human appendix contains B cell subsets that constitutively express IL-6R, and that a high proportion of these cells are committed to the IgA isotype. Furthermore, higher numbers of IL-6 responsive IgA2 B cells are present in the human appendix as compared to tonsils or PBMC.


Localization of MEG human brain responses to retinotopic visual stimuli with contrasting source reconstruction approaches.
Magnetoencephalography (MEG) allows the physiological recording of human brain activity at high temporal resolution. However, spatial localization of the source of the MEG signal is an ill-posed problem as the signal alone cannot constrain a unique solution and additional prior assumptions must be enforced. An adequate source reconstruction method for investigating the human visual system should place the sources of early visual activity in known locations in the occipital cortex. We localized sources of retinotopic MEG signals from the human brain with contrasting reconstruction approaches (minimum norm, multiple sparse priors, and beamformer) and compared these to the visual retinotopic map obtained with fMRI in the same individuals. When reconstructing brain responses to visual stimuli that differed by angular position, we found reliable localization to the appropriate retinotopic visual field quadrant by a minimum norm approach and by beamforming. Retinotopic map eccentricity in accordance with the fMRI map could not consistently be localized using an annular stimulus with any reconstruction method, but confining eccentricity stimuli to one visual field quadrant resulted in significant improvement with the minimum norm. These results inform the application of source analysis approaches for future MEG studies of the visual system, and indicate some current limits on localization accuracy of MEG signals.


The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases.
The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article.


Actinomycetes from Red Sea sponges: sources for chemical and phylogenetic diversity.
The diversity of actinomycetes associated with marine sponges collected off Fsar Reef (Saudi Arabia) was investigated in the present study. Forty-seven actinomycetes were cultivated and phylogenetically identified based on 16S rRNA gene sequencing and were assigned to 10 different actinomycete genera. Eight putatively novel species belonging to genera Kocuria, Mycobacterium, Nocardia, and Rhodococcus were identified based on sequence similarity values below 98.2% to other 16S rRNA gene sequences available in the NCBI database. PCR-based screening for biosynthetic genes including type I and type II polyketide synthases (PKS-I, PKS-II) as well as nonribosomal peptide synthetases (NRPS) showed that 20 actinomycete isolates encoded each at least one type of biosynthetic gene. The organic extracts of nine isolates displayed bioactivity against at least one of the test pathogens, which were Gram-positive and Gram-negative bacteria, fungi, human parasites, as well as in a West Nile Virus protease enzymatic assay. These results emphasize that marine sponges are a prolific resource for novel bioactive actinomycetes with potential for drug discovery.


A natural history of FUT2 polymorphism in humans.
Because pathogens are powerful selective agents, host-cell surface molecules used by pathogens as identification signals can reveal the signature of selection. Most of them are oligosaccharides, synthesized by glycosyltransferases. One known example is balancing selection shaping ABO evolution as a consequence of both, A and B antigens being recognized as receptors by some pathogens, and anti-A and/or anti-B natural antibodies produced by hosts conferring protection against the numerous infectious agents expressing A and B motifs. These antigens can also be found in tissues other than blood if there is activity of another enzyme, FUT2, a fucosyltransferase responsible for ABO biosynthesis in body fluids. Homozygotes for null variants at this locus present the nonsecretor phenotype (se), because they cannot express ABO antigens in secretions. Multiple independent mutations have been shown to be responsible for the nonsecretor phenotype, which is coexisting with the secretor phenotype in most populations. In this study, we have resequenced the coding region of FUT2 in 732 individuals from 39 worldwide human populations. We report a complex pattern of natural selection acting on the gene. Although frequencies of secretor and nonsecretor phenotypes are similar in different populations, the point mutations at the base of the phenotypes are different, with some variants showing a long history of balancing selection among Eurasian and African populations, and one recent variant showing a fast spread in East Asia, likely due to positive selection. Thus, a convergent phenotype composition has been achieved through different mutations with different evolutionary histories.


Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.
We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.


Continuity of care in the cross-border context: insights from a survey of German patients treated abroad.
Continuity of care is important for outcomes and patient satisfaction and includes additional considerations in the context of cross-border health care. Although this has been discussed in research and was picked up in the recently transposed Directive on patients' rights (2011/24/EU), there is limited evidence about related issues actually encountered by patients crossing borders. An anonymous postal survey was carried out by the Techniker Krankenkasse, one of the largest sickness funds in Germany. The questionnaire was sent to 45 189 individuals who had received treatment in EU/EEA countries and included items on relational, management and informational continuity. The survey had a response rate of 41% (n = 17 543). Of those respondents who had travelled for care (n = 3307), ∼19% (n = 570) did so due to a relationship of trust with a given provider. Only 8% of all respondents required emergency follow-up services due to complications, the majority of which was obtained back in Germany. Twelve percentage of those who were prescribed medication abroad (n = 4208) reported problems, spanning unknown products, dispensation and reimbursement. Information exchange between providers across borders was rare and largely carried out by the patients themselves. Although relational continuity may be important to specific groups of patients travelling for care, it is primarily informational continuity and its interrelation with management continuity that need to be addressed in the cross-border context. Information exchange should be endorsed at European level. Additional focus is required on informing patients about documentation rights and requirements and providing health records that are comprehensive and comprehensible.


Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.
Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors.


Hospital admissions for viral meningitis in children in England over five decades: a population-based observational study.
A substantial reduction in bacterial meningitis has occurred in the UK following successful implementation of immunisation programmes. Most childhood meningitis in developed countries is now caused by viruses. Long-term trends in paediatric viral meningitis in England have not previously been reported. The objective of this study is to report on epidemiological trends over time in childhood viral meningitis in England. In this population-based observational study, we used routinely collected hospital discharge records from English National Health Service hospitals from 1968-2011 to analyse annual age-specific admission rates for viral meningitis, including specific viral aetiologies, in children younger than 15 years. We analysed hospital discharge records from Jan 1, 1968, to Dec 31, 2011. Hospital admission rates for viral meningitis from Jan 1, 1968, to Dec 31, 1985, varied annually, with a mean of 13·5 admissions per 100 000 children aged less than 15 years, per year (95% CI 13·0-14·0). Admission rates declined during the late 1980s, and the mean number of admissions from 1989-2011 was 5·2 per 100 000 per year (5·1-5·3). This decrease was entirely in children aged 1-14 years. Admission rates for infants aged less than 1 year increased since 2005, to 70·0 per 100 000 (63·7-76·2) in 2011, which was driven by an increase in admission of infants aged 90 days or less. In 1968-85, the majority of cases in children were in those aged 1-14 years (22 150 [89%] of 24 920 admissions). In 2007-11, 1716 (72%) of 2382 cases were in infants. Admissions for mumps-related meningitis almost disappeared following introduction of the measles-mumps-rubella (MMR) vaccine in 1988. Admissions with a specified viral aetiology have increased since 2000. Trends in viral meningitis admissions have changed substantially over the past 50 years, and probably reflect the impact of the MMR vaccine programme and the use of more sensitive diagnostic techniques. None.


Is Physical Activity Associated With Physical Performance in Adults With Intellectual Disability?
Physical activity (PA) and sedentary behavior may contribute to physical function in adults with intellectual disability (ID). This study examined whether objectively measured PA and sedentary behavior levels are associated with physical performance in adults with ID. Fifty-eight adults with ID (29 women and 29 men, age 44 ± 14 years) underwent a measurement of physical performance with the Short Physical Performance Battery (SPPB) and PA and sedentary time using a hip-worn accelerometer (wGT3X-BT; ActiGraph, Pensacola, FL). Moderate PA and age were significantly associated with the SPPB score (r = .39 and .34, respectively; p < .01). A hierarchical-regression model with moderate PA and age as independent variables indicated that moderate PA was a significant predictor of SPPB (p < .001; R2 = .153), but age was not (p = .123; R2 change = .036). Overall, moderate PA was significantly associated with the SPPB score, even after accounting for age, in adults with ID.


Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family.
The synucleins (α, β, and γ) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signaling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4- to 14-month-old) animals using a combination of behavioral, biochemical, histological, and electrochemical techniques. Adult triple-synuclein-null (TKO) mice displayed no overt phenotype and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover, and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms that differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson's disease may ultimately inform on the selectivity of the disease process.


Dynamics of QoI Sensitivity in Mycosphaerella fijiensis in Costa Rica During 2000 to 2003.
ABSTRACT From 1997 onward, the strobilurin fungicide azoxystrobin was widely used in the main banana-production zone in Costa Rica against Mycosphaerella fijiensis var. difformis causing black Sigatoka of banana. By 2000, isolates of M. fijiensis with resistance to the quinolene oxidase inhibitor fungicides were common on some farms in the area. The cause was a single point mutation from glycine to alanine in the fungal target protein, cytochrome b gene. An amplification refractory mutation system Scorpion quantitative polymerase chain reaction assay was developed and used to determine the frequency of G143A allele in samples of M. fijiensis. Two hierarchical surveys of spatial variability, in 2001 and 2002, found no significant variation in frequency on spatial scales <10 m. This allowed the frequency of G143A alleles on a farm to be estimated efficiently by averaging single samples taken at two fixed locations. The frequency of G143A allele in bulk samples from 11 farms throughout Costa Rica was determined at 2-month intervals. There was no direct relationship between the number of spray applications and the frequency of G143A on individual farms. Instead, the frequency converged toward regional averages, presumably due to the large-scale mixing of ascospores dispersed by wind. Using trap plants in an area remote from the main producing area, immigration of resistant ascospores was detected as far as 6 km away both with and against the prevailing wind.


An evidence-based systematic review of stevia by the Natural Standard Research Collaboration.
The objective of this study was to evaluate the scientific evidence on stevia, including expert opinion, folkloric precedent, history, pharmacology, kinetics/dynamics, interactions, adverse effects, toxicology, and dosing. This review serves as a clinical support tool. Electronic searches were conducted in 10 databases, 20 additional journals (not indexed in common databases), and bibliographies from 50 selected secondary references. No restrictions were placed on the language or quality of the publications. All literature collected pertained to efficacy in humans, dosing, precautions, adverse effects, use in pregnancy and lactation, interactions, alteration of laboratory assays, and mechanisms of action. Standardized inclusion and exclusion criteria were used for selection. Grades were assigned using an evidence-based grading rationale. Based on the availability of scientific data, two indications are discussed in this review: hypertension and hyperglycemia. Evaluation of two long-term studies (1 and 2 years in length, respectively) indicates that stevia may be effective in lowering blood pressure in hypertensive patients, although data from shorter studies (1-3 months) did not support these findings. A pair of small studies also report positive results with respect to glucose tolerance and response, although the relatively low methodological rigor of these experiments limits the strength of these findings. Further investigation is warranted in both indications.


Investigating resting-state functional connectivity in the cervical spinal cord at 3T.
The study of spontaneous fluctuations in the blood-oxygen-level-dependent (BOLD) signal has recently been extended from the brain to the spinal cord. Two ultra-high field functional magnetic resonance imaging (fMRI) studies in humans have provided evidence for reproducible resting-state connectivity between the dorsal horns as well as between the ventral horns, and a study in non-human primates has shown that these resting-state signals are impacted by spinal cord injury. As these studies were carried out at ultra-high field strengths using region-of-interest (ROI) based analyses, we investigated whether such resting-state signals could also be observed at the clinically more prevalent field strength of 3T. In a reanalysis of a sample of 20 healthy human participants who underwent a resting-state fMRI acquisition of the cervical spinal cord, we were able to observe significant dorsal horn connectivity as well as ventral horn connectivity, but no consistent effects for connectivity between dorsal and ventral horns, thus replicating the human 7T results. These effects were not only observable when averaging along the acquired length of the spinal cord, but also when we examined each of the acquired spinal segments separately, which showed similar patterns of connectivity. Finally, we investigated the robustness of these resting-state signals against variations in the analysis pipeline by varying the type of ROI creation, temporal filtering, nuisance regression and connectivity metric. We observed that - apart from the effects of band-pass filtering - ventral horn connectivity showed excellent robustness, whereas dorsal horn connectivity showed moderate robustness. Together, our results provide evidence that spinal cord resting-state connectivity is a robust and spatially consistent phenomenon that could be a valuable tool for investigating the effects of pathology, disease progression, and treatment response in neurological conditions with a spinal component, such as spinal cord injury.


Metabolomic networks connect host-microbiome processes to human Clostridioides difficile infections.
Clostridioides difficile infection (CDI) accounts for a substantial proportion of deaths attributable to antibiotic-resistant bacteria in the United States. Although C. difficile can be an asymptomatic colonizer, its pathogenic potential is most commonly manifested in patients with antibiotic-modified intestinal microbiomes. In a cohort of 186 hospitalized patients, we showed that host and microbe-associated shifts in fecal metabolomes had the potential to distinguish patients with CDI from those with non-C. difficile diarrhea and C. difficile colonization. Patients with CDI exhibited a chemical signature of Stickland amino acid fermentation that was distinct from those of uncolonized controls. This signature suggested that C. difficile preferentially catabolizes branched chain amino acids during CDI. Unexpectedly, we also identified a series of noncanonical, unsaturated bile acids that were depleted in patients with CDI. These bile acids may derive from an extended host-microbiome dehydroxylation network in uninfected patients. Bile acid composition and leucine fermentation defined a prototype metabolomic model with potential to distinguish clinical CDI from asymptomatic C. difficile colonization.


A comprehensive microarray-based DNA methylation study of 367 hematological neoplasms.
Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes--DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1--that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.


Non-alcoholic fatty liver disease (NAFLD) in the Western Cape: a descriptive analysis.
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in Western countries, but the disease profile has not yet been described in South Africa. NAFLD affects all spheres of society, especially the poorest and least educated. Aim. To investigate the demographics and clinical and biochemical features of South African patients diagnosed with non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH) in the Western Cape, South Africa. Overweight/obese subjects were screened by ultrasound and those with fatty liver/hepatomegaly were included. Liver biochemistry, insulin resistance (using the insulin resistance homeostasis model assessment method for insulin resistance, HOMA-IR) and body mass index were assessed and liver biopsies were performed on patients older than 45 years with persistently abnormal liver function and/or hepatomegaly. We screened 233 patients: 69% coloured, 25% Caucasian, 5% black and 1% Asian. The majority (73%) were female. NAFLD was confirmed histologically in 111 patients, of whom 36% had NASH and 17% advanced liver fibrosis. No black patient had advanced fibrosis. Subjects with NASH had higher mean triglyceride (p=0.03) and cholesterol (p=0.01) levels than subjects with NAFL. All patients were insulin resistant/diabetic. HOMA-IR and not the degree of obesity was strongly associated with advanced fibrosis (p=0.09). This study is the first to describe the clinical characteristics of NAFLD in South Africa, albeit only in the Western Cape population. Insulin resistance was the universal factor present. The degree of obesity was not associated with severity of disease. The role of genetic risk factors in disease development and severity remains to be defined.


Disambiguating brain functional connectivity.
Functional connectivity (FC) analyses of correlations of neural activity are used extensively in neuroimaging and electrophysiology to gain insights into neural interactions. However, analyses assessing changes in correlation fail to distinguish effects produced by sources as different as changes in neural signal amplitudes or noise levels. This ambiguity substantially diminishes the value of FC for inferring system properties and clinical states. Network modelling approaches may avoid ambiguities, but require specific assumptions. We present an enhancement to FC analysis with improved specificity of inferences, minimal assumptions and no reduction in flexibility. The Additive Signal Change (ASC) approach characterizes FC changes into certain prevalent classes of signal change that involve the input of additional signal to existing activity. With FMRI data, the approach reveals a rich diversity of signal changes underlying measured changes in FC, suggesting that it could clarify our current understanding of FC changes in many contexts. The ASC method can also be used to disambiguate other measures of dependency, such as regression and coherence, providing a flexible tool for the analysis of neural data.


Quantification of hepatic FOXP3+ T-lymphocytes in HIV/hepatitis C coinfection.
Coinfection with HIV adversely impacts every stage of hepatitis C (HCV) infection. Liver damage in HCV infection results from host antiviral responses rather than direct viral pathogenesis. Despite depressed cellular immunity, coinfected patients show accelerated hepatic fibrosis compared with HCV monoinfected patients. This paradox is poorly understood. T-regulatory (Treg) cells (CD4+ and FOXP3+) are hypothesized to limit hepatic damage in HCV. Our hypothesis was that reduced frequency of hepatic Treg in HIV/HCV coinfection compared with HCV monoinfection may explain poorer outcomes. We quantified FOXP3+, CD4+, CD8+ and CD20+ cells in liver biopsies of 35 male subjects matched by age and ISHAK fibrosis score, 12 HIV monoinfected, 11 HCV monoinfected and 12 HIV/HCV coinfected. Cell counts were performed using indirect immunohistochemical staining and light microscopy. HIV/HCV coinfected subjects had fewer hepatic FOXP3+ (P = 0.031) and CD4+ cells (P = 0.001) than HCV monoinfected subjects. Coinfected subjects had more hepatic CD8+ cells compared with HCV monoinfected (P = 0.023), and a lower ratio of FOXP3+ to CD8+ cells (0.08 vs 0.27, P < 0.001). Multivariate analysis showed number of CD4+ cells controlled for differences in number of FOXP3+ cells. Fewer hepatic FOXP3+ and CD4+ cells in HIV/HCV coinfection compared with HCV monoinfection suggests lower Treg activity, driven by an overall loss of CD4+ cells. Higher number of CD8+ cells in HIV/HCV coinfection suggests higher cytotoxic activity. This may explain poorer outcomes in HIV/HCV coinfected patients and suggests a potential mechanism by which highly active antiretroviral therapy may benefit these patients.


Reduced limbic connections may contraindicate subgenual cingulate deep brain stimulation for intractable depression.
In this study, the authors performed deep brain stimulation (DBS) of the subgenual anterior cingulate cortex (SACC) in a patient with a history of bipolar disorder. After a right thalamic stroke, intractable depression without mood elevation or a mixed state developed in this patient. He underwent bilateral SACC DBS and died 16 months afterwards. Anatomical connections were studied in this patient preoperatively and postmortem using diffusion tractography (DT). A comparison of in vivo and high resolution ex vivo connectivity patterns was performed as a measure of the utility of in vivo DT in presurgical planning for DBS. Diagnostic measures included neuropsychological testing, preoperative and ex vivo DT, and macroscopic neuropathological assessment. Post-DBS depression rating scores did not improve. In vivo and ex vivo DT revealed markedly reduced limbic projections from the thalamus and SACC to the amygdala in the right (stroke-affected) hemisphere. A highly selective right mediothalamic lesion was associated with the onset of refractory depression. Reduced amygdalar-thalamic and amygdalar-SACC connections could be a contraindication to DBS for depression. Correspondence between preoperative and higher resolution ex vivo DT supports the validity of DT as a presurgical planning tool for DBS.


H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis.
Epigenetic changes (i.e., chromatin modifications) occur during chondrogenesis and in osteoarthritis (OA). We investigated the effect of H3K27me3 demethylase inhibition on chondrogenesis and assessed its utility in cartilage tissue engineering and in understanding cartilage destruction in OA. We used a high-content screen to assess the effect of epigenetic modifying compounds on collagen output during chondrogenesis of monolayer human mesenchymal stem cells (MSCs). The impact of GSK-J4 on gene expression, glycosaminoglycan output and collagen formation during differentiation of MSCs into cartilage discs was investigated. Expression of lysine (K)-specific demethylase 6A (UTX) and Jumonji domain-containing 3 (JMJD3), the HEK27Me3 demethylases targeted by GSK-J4, was measured in damaged and undamaged cartilage from patients with OA. The impact of GSK-J4 on ex vivo cartilage destruction and expression of OA-related genes in human articular chondrocytes (HACs) was assessed. H3K27Me3 demethylase regulation of transforming growth factor (TGF)-β-induced gene expression was measured in MSCs and HACs. Treatment of chondrogenic MSCs with the H3K27me3 demethylase inhibitor GSK-J4, which targets JMJD3 and UTX, inhibited collagen output; expression of chondrogenic genes, including SOX9 and COL2A1; and disrupted glycosaminoglycan and collagen synthesis. JMJD3 but not UTX expression was increased during chondrogenesis and in damaged OA cartilage, suggesting a predominant role of JMJD3 in chondrogenesis and OA. GSK-J4 prevented ex vivo cartilage destruction and expression of the OA-related genes MMP13 and PTGS2. TGF-β is a key regulator of chondrogenesis and articular cartilage homeostasis, and TGF-β-induced gene expression was inhibited by GSK-J4 treatment of both chondrogenic MSCs and HACs. Overall, we show that H3K27me3 demethylases modulate chondrogenesis and that enhancing this activity may improve production of tissue-engineered cartilage. In contrast, targeted inhibition of H3K27me3 demethylases could provide a novel approach in OA therapeutics.


Combined pulmonary and left ventricular support with veno-pulmonary ECMO and impella 5.0 for cardiogenic shock after coronary surgery.
Mechanical circulatory support is a common practice nowadays in the management of patients after cardiogenic shock due to myocardial infarction. The single or combined use of one or more devices for mechanical support depends not only on the advantage or disadvantage of these devices but also on the timing of use of these devices before the development of multi organ failure. In our case we used more than one tool for mechanical circulatory support during the prolonged and complicated course of our patient with postcardiotomy cardiogenic shock after coronary artery bypass surgery. We describe the combined use of Impella 5.0 and veno- pulmonary extra corporeal membrane oxygenation (VP-ECMO) for biventricular failure in a 52 years-old man. He presented with cardiogenic shock after inferior wall ST-elevation myocardial infarction. After emergency coronary artery bypass surgery and failure to wean from extracorporeal circulation we employed V-P ECMO and consecutively Impella 5.0 to manage the primarily failing right and secondarily failing left ventricles. He remained hemodynamically stable on both Impella 5.0 and VP-ECMO until Heart Mate II left ventricular assist device implantation on the 14th postoperative day. Right sided support was weaned on 66th postoperative day. The patient remained in the intensive care unit for 77 days. During his prolonged stay, he underwent renal replacement therapy and tracheostomy with complete recovery. Six months later, he was successfully heart transplanted and has completed three and half years of unremarkable follow up. The combined use of VP ECMO and Impella 5.0 is effective in the management of postcardiotomy biventricular failure as a bridge for further mechanical support or heart transplantation.


Paraneoplastic hypoglycemia in a patient with a malignant solitary fibrous tumor.
Hypoglycemia is a common medical emergency. It is the most frequent complication induced by anti-diabetic treatment. However, it can be observed in other conditions unrelated to diabetes such as insulinoma, autoimmune disorders, and neoplasia. Herein, we report the case of a rare cause of severe and recurrent hypoglycemia in a 77-year-old woman with a malignant solitary fibrous tumor (MSFT). A 77-year-old woman was admitted to the emergency department for loss of consciousness induced by severe hypoglycemia. Her standard laboratory findings were unremarkable. HbA1c, albumin, renal, liver, thyroid, and adrenal function tests were normal. Cerebral CT scan was also normal. At the time of confirmed hypoglycemia, the serum level of insulin and C-peptide was low. On the basis of the past medical history and the absence of other comment etiologies, a paraneoplastic cause was suspected. Thus, the diagnosis of a non-islet cell tumor-induced hypoglycemia (NICTH) was established by the presence of incompletely processed precursors of IGF2 (big IGF2) in plasma electrophoresis. However, the IGF1 level was low. Therapy with corticosteroids improved hypoglycemia and clinical symptoms. NICTH is a rare cause of hypoglycemia. It should be considered in patients with mesenchymal or malignant epithelial tumors suffering from recurrent episodes of hypoglycemia. The diagnosis will be established in the case of low serum insulin concentrations and elevated levels of big IGF2. Treatment with corticosteroids, GH, or both can improve hypoglycemic symptoms and restore plasma glucose to normal levels. NICTH is a very rare condition that should be considered in patients known to have mesenchymal or malignant epithelial tumors and suffering from recurrent episodes of hypoglycemia.The diagnosis of an NICTH is established on the basis of the hypoinsulinemic hypoglycemia, the MSFT history, and the presence of paraneoplastic secretion of IGF1 or an immature form of IGF2.Treatment with corticosteroids, GH, or both can improve hypoglycemic symptoms and restore plasma glucose to normal levels in NICTH.


Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure.
Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.


Validation of the psychometric hepatic encephalopathy score (PHES) for identifying patients with minimal hepatic encephalopathy.
The psychometric hepatic encephalopathy score (PHES) is a battery of neuropsychological tests used in the diagnosis of minimal hepatic encephalopathy (MHE). The aim of this study was to construct and validate a dataset of normal values for the PHES. Volunteers and patients with cirrhosis with and without low-grade overt hepatic encephalopathy (OHE) were enrolled. All subjects completed the PHES battery, and possible modifying factors were assessed. Formulas to predict expected scores in cirrhotics were constructed, and MHE was diagnosed whenever a deviation of <-4 SDs occurred across the five tests. Among the 743 volunteers, age and years of education influenced the scores of all tests. Eighty-four patients with cirrhosis lacked evidence of OHE, whereas 20 had OHE: median PHES were -1 [0 to -3] and -9 [-6.5 to -11.8] (P < 0.001), respectively. Thirteen of the 84 patients (15%) with cirrhosis but without OHE had MHE. Patients with MHE were older and less educated than those without MHE (61 ± 8 and 52 ± 10 years old, P = 0.003; 7 ± 4 and 12 ± 5 years education, P = 0.002), whereas liver function was not different (MELD, 8 ± 5 and 8 ± 5). A very strong correlation between these norms and those derived from Spain was observed (r = 0.964, P < 0.001). PHES performance was mostly influenced by age and education, and expected results in cirrhotics need to be adjusted for these factors. Our validation of Mexican norms for PHES (PHES-Mex) establishes a practical method for assessing MHE and contributes to international attempts to standardize diagnostic protocols for MHE.


Uncoupling Malt1 threshold function from paracaspase activity results in destructive autoimmune inflammation.
The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.


Microbial manipulations to improve fish health and production--a Mediterranean perspective.
The interactions between the endogenous gut microbiota and the fish host are integral in mediating the development, maintenance and effective functionality of the intestinal mucosa and gut associated lymphoid tissues (GALTs). These microbial populations also provide a level of protection against pathogenic visitors to the gastrointestinal (GI) tract and aid host digestive function via the production of exogenous digestive enzymes and vitamins. Manipulation of these endogenous populations may provide an alternative method to antibiotics to control disease and promote health management. Applications of probiotics for Mediterranean teleosts can stimulate immune responses, enhance growth performance, feed utilisation, digestive enzyme activities, antioxidant enzyme activities, gene expression, disease resistance, larval survival, gut morphology, modulate GI microbiota and mediate stress responses. Although considerably less information is available regarding prebiotic applications for Mediterranean teleosts, prebiotics also offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature, obtaining consistent and reliable results is often difficult due to our incomplete understanding of indigenous fish GI microbiota and their subsequent host interactions which mediate and drive both localised and systemic host immunological responses. Additionally, the probiotic and prebiotic (biotics) mechanisms which mediate host benefits at the mucosal interface are poorly understood. Future studies focused on these interactions utilising gnotobiotic techniques should provide a better understanding of how to extract the full potential of biotic applications to promote immune function of Mediterranean teleosts.


"Maybe I made up the whole thing": placebos and patients' experiences in a randomized controlled trial.
Patients in the placebo arms of randomized controlled trials (RCT) often experience positive changes from baseline. While multiple theories concerning such "placebo effects" exist, peculiarly, none has been informed by actual interviews of patients undergoing placebo treatment. Here, we report on a qualitative study (n = 27) embedded within a RCT (n = 262) in patients with irritable bowel syndrome. Besides identical placebo acupuncture treatment in the RCT, the qualitative study patients also received an additional set of interviews at the beginning, midpoint, and end of the trial. Interviews of the 12 qualitative subjects who underwent and completed placebo treatment were transcribed. We found that patients (1) were persistently concerned with whether they were receiving placebo or genuine treatment; (2) almost never endorsed "expectation" of improvement but spoke of "hope" instead and frequently reported despair; (3) almost all reported improvement ranging from dramatic psychosocial changes to unambiguous, progressive symptom improvement to tentative impressions of benefit; and (4) often worried whether their improvement was due to normal fluctuations or placebo effects. The placebo treatment was a problematic perturbation that provided an opportunity to reconstruct the experiences of the fluctuations of their illness and how it disrupted their everyday life. Immersion in this RCT was a co-mingling of enactment, embodiment and interpretation involving ritual performance and evocative symbols, shifts in bodily sensations, symptoms, mood, daily life behaviors, and social interactions, all accompanied by self-scrutiny and re-appraisal. The placebo effect involved a spectrum of factors and any single theory of placebo--e.g. expectancy, hope, conditioning, anxiety reduction, report bias, symbolic work, narrative and embodiment--provides an inadequate model to explain its salubrious benefits.


Incomplete Stevens-Johnson syndrome secondary to atypical pneumonia.
Steven-Johnson syndrome is a common condition characterised by erythematous target lesions on the skin and involvement of the oral mucosa, genitals and conjunctivae. It has been documented as one of the extra-pulmonary manifestations of Mycoplasma pneumoniae infection. Recently, there has been several documentation of an incomplete presentation of this syndrome - without the typical rash but with mucosal, conjunctival and genital involvement. Our case illustrates that the incomplete Steven-Johnson syndrome may present with oral mucosal and conjunctival involvement alone without skin or genital involvement. This important clinical diagnosis should not be missed due to its atypical presentation. Treatment of Steven-Johnson syndrome remains supportive along with treating the underlying infection if recognised.


Think twice: understanding the high potency of bis(phenyl)methane inhibitors of thrombin.
Successful design of potent and selective protein inhibitors, in terms of structure-based drug design, strongly relies on the correct understanding of the molecular features determining the ligand binding to the target protein. We present a case study of serine protease inhibitors with a bis(phenyl)methane moiety binding into the S3 pocket. These inhibitors bind with remarkable potency to the active site of thrombin, the blood coagulation factor IIa. A combination of X-ray crystallography and isothermal titration calorimetry provides conclusive insights into the driving forces responsible for the surprisingly high potency of these inhibitors. Analysis of six well-resolved crystal structures (resolution 1.58-2.25 A) along with the thermodynamic data allows an explanation of the tight binding of the bis(phenyl)methane inhibitors. Interestingly, the two phenyl rings contribute to binding affinity for very different reasons - a fact that can only be elucidated by a structure-based approach. The first phenyl moiety occupies the hydrophobic S3 pocket, resulting in a mainly entropic advantage of binding. This observation is based on the displacement of structural water molecules from the S3 pocket that are observed in complexes with inhibitors that do not bind in the S3 pocket. The same classic hydrophobic effect cannot explain the enhanced binding affinity resulting from the attachment of the second, more solvent-exposed phenyl ring. For the bis(phenyl)methane inhibitors, an observed adaptive rotation of a glutamate residue adjacent to the S3 binding pocket attracted our attention. The rotation of this glutamate into salt-bridging distance with a lysine moiety correlates with an enhanced enthalpic contribution to binding for these highly potent thrombin binders. This explanation for the magnitude of the attractive force is confirmed by data retrieved by a Relibase search of several thrombin-inhibitor complexes deposited in the Protein Data Bank exhibiting similar molecular features. Special attention was attributed to putative changes in the protonation states of the interaction partners. For this purpose, two analogous inhibitors differing mainly in their potential to change the protonation state of a hydrogen-bond donor functionality were compared. Buffer dependencies of the binding enthalpy associated with complex formation could be traced by isothermal titration calorimetry, which revealed, along with analysis of the crystal structures (resolution 1.60 and 1.75 A), that a virtually compensating proton interchange between enzyme, inhibitor and buffer is responsible for the observed buffer-independent thermodynamic signatures.


Covert visual spatial orienting and saccades: overlapping neural systems.
We used functional magnetic resonance imaging (fMRI) to investigate the functional anatomical relationship between covert orienting of visual spatial attention and execution of saccadic eye movements. Brain areas engaged by shifting spatial attention covertly and by moving the eyes repetitively toward visual targets were compared and contrasted directly within the same subjects. The two tasks activated highly overlapping neural systems and showed that common parietal and frontal regions are more activated during the covert task than the overt oculomotor condition. The possible nature of the relationship between these two operations is discussed.


Whole-brain magnetic resonance spectroscopic imaging measures are related to disability in ALS.
To demonstrate the sensitivity of a recently developed whole-brain magnetic resonance spectroscopic imaging (MRSI) sequence to cerebral pathology and disability in amyotrophic lateral sclerosis (ALS), and compare with measures derived from diffusion tensor imaging. Whole-brain MRSI and diffusion tensor imaging were undertaken in 13 patients and 14 age-similar healthy controls. Mean N-acetylaspartate (NAA), fractional anisotropy, and mean diffusivity were extracted from the corticospinal tract, compared between groups, and then in relation to disability in the patient group. Significant reductions in NAA were found along the course of the corticospinal tracts on whole-brain MRSI. There were also significant changes in fractional anisotropy (decreased) and mean diffusivity (increased) in the patient group, but only NAA showed a significant relationship with disability (r = 0.65, p = 0.01). Whole-brain MRSI has potential as a quantifiable neuroimaging marker of disability in ALS. It offers renewed hope for a neuroimaging outcome measure with the potential for harmonization across multiple sites in the context of a therapeutic trial.


Factors associated with satisfaction in patients undergoing elbow surgery: a prospective study.
Evidence on factors associated with patient satisfaction with elbow surgery is sparse; outcomes of surgery are not necessarily related to patient satisfaction. This study explored the hypothesis that condition-specific outcome measures would more closely reflect patient satisfaction than generic measures. The prospective cohort comprised 104 consecutive patients/elbows undergoing elbow surgery. Preoperative and 6 month postoperative outcome questionnaires included the Oxford Elbow Score (OES), Disabilities of the Arm, Shoulder and Hand (DASH), and Short Form (SF)-36 general health survey. Clinical assessments used the standard Mayo Elbow Performance Score (MEPS). Patients who were "very pleased" with surgery were compared with others regarding which factors were associated with being "very pleased" at 6 months after surgery. By 6 months, 54% of patients were very pleased with their surgery. Preoperative scores were generally not associated with subsequent patient satisfaction. Postoperative scores and change scores for the OES Pain and Social-Psychological scales and the DASH were moderately to highly correlated with patient satisfaction (r(s), -0.43 to -0.72; all P < .001), whereas SF-36 and MEPS change scores were only correlated to a small extent (r < or = 0.34; all P < .05). A multivariable analysis revealed that patients' age, postoperative elbow pain, and change in elbow function each independently influenced the odds of patients being very pleased with surgery. Associations between outcome measures and patient satisfaction revealed likely differences between patients' and clinicians' perceptions of which aspects of outcome rated as important. Patient-reported results are more likely than clinically assessed outcome measures, and condition-specific are more likely than generic measures, to reflect patient-rated satisfaction with elbow surgery.


The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. The European Polycystic Kidney Disease Consortium.
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently results in renal failure due to progressive cyst development. The major locus, PKD1, maps to 16p13.3. We identified a chromosome translocation associated with ADPKD that disrupts a gene (PBP) encoding a 14 kb transcript in the PKD1 candidate region. Further mutations of the PBP gene were found in PKD1 patients, two deletions (one a de novo event) and a splicing defect, confirming that PBP is the PKD1 gene. This gene is located adjacent to the TSC2 locus in a genomic region that is reiterated more proximally on 16p. The duplicate area encodes three transcripts substantially homologous to the PKD1 transcript. Partial sequence analysis of the PKD1 transcript shows that it encodes a novel protein whose function is at present unknown.


Comparison of the effects of genetic and environmental risk factors on in situ and invasive ductal breast cancer.
Little is known about the etiology of in situ ductal breast cancer (DCIS) or what influences its possible progression to invasive ductal disease. Comparison of risk factors for DCIS and invasive ductal cancer may throw some light on these issues. We estimated relative risks for DCIS and invasive ductal breast cancer according to 12 genetic and eight environmental risk factors among 1.1 million postmenopausal women in a large prospective UK study. There was no strong evidence of a different association with DCIS versus invasive ductal cancer for any of the 12 susceptibility loci examined. We also found similar associations of age at menarche, age at first birth, parity, age at menopause, family history of breast cancer and use of hormone replacement therapy with DCIS and invasive ductal cancer. Only body mass index (BMI) showed a clear difference in association in that it was positively associated with the risk of invasive ductal cancer but not DCIS (RRs per 5 kg/m(2) = 1.20 and 1.01, respectively; p-value for heterogeneity = 0.002). The very similar risk factor profiles observed here for DCIS and invasive ductal cancer suggest that DCIS is a precursor of invasive ductal cancer and most risk factors affect the risk of invasive ductal cancer primarily through their effects on the risk of DCIS. The lack of association between BMI and DCIS suggests a greater influence of BMI on disease progression.


Molecular mechanism for a gateway drug: epigenetic changes initiated by nicotine prime gene expression by cocaine.
In human populations, cigarettes and alcohol generally serve as gateway drugs, which people use first before progressing to marijuana, cocaine, or other illicit substances. To understand the biological basis of the gateway sequence of drug use, we developed an animal model in mice and used it to study the effects of nicotine on subsequent responses to cocaine. We found that pretreatment of mice with nicotine increased the response to cocaine, as assessed by addiction-related behaviors and synaptic plasticity in the striatum, a brain region critical for addiction-related reward. Locomotor sensitization was increased by 98%, conditioned place preference was increased by 78%, and cocaine-induced reduction in long-term potentiation (LTP) was enhanced by 24%. The responses to cocaine were altered only when nicotine was administered first, and nicotine and cocaine were then administered concurrently. Reversing the order of drug administration was ineffective; cocaine had no effect on nicotine-induced behaviors and synaptic plasticity. Nicotine primed the response to cocaine by enhancing its ability to induce transcriptional activation of the FosB gene through inhibition of histone deacetylase, which caused global histone acetylation in the striatum. We tested this conclusion further and found that a histone deacetylase inhibitor simulated the actions of nicotine by priming the response to cocaine and enhancing FosB gene expression and LTP depression in the nucleus accumbens. Conversely, in a genetic mouse model characterized by reduced histone acetylation, the effects of cocaine on LTP were diminished. We achieved a similar effect by infusing a low dose of theophylline, an activator of histone deacetylase, into the nucleus accumbens. These results from mice prompted an analysis of epidemiological data, which indicated that most cocaine users initiate cocaine use after the onset of smoking and while actively still smoking, and that initiating cocaine use after smoking increases the risk of becoming dependent on cocaine, consistent with our data from mice. If our findings in mice apply to humans, a decrease in smoking rates in young people would be expected to lead to a decrease in cocaine addiction.


Enhanced Therapeutic and Long-Term Dynamic Vascularization Effects of Human Pluripotent Stem Cell-Derived Endothelial Cells Encapsulated in a Nanomatrix Gel.
Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3β inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5+ cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis. The resultant hPSC-derived CDH5+ cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.


Tumor inhibition by genomically integrated inducible RNAi-cassettes.
RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes by post-transcriptional silencing of gene expression. In this study we wondered whether inducible RNAi-cassettes integrated into cellular DNA possess the power to trigger neoplastic growth. For this purpose inducible RNAi vectors containing tetracycline (Tet)-responsive derivatives of the H1 promoter for the conditional expression of short hairpin RNA (shRNA) were used to target human polo-like kinase 1 (Plk1), which is overexpressed in a broad spectrum of human tumors. In the absence of doxycycline (Dox) HeLa clones expressing TetR, that carry the RNAi-cassette stably integrated, exhibited no significant alteration in Plk1 expression levels. In contrast, exposure to Dox led to marked downregulation of Plk1 mRNA to 3% and Plk1 protein to 14% in cell culture compared to mismatch shRNA/Plk1-expressing cells. As a result of Plk1 depletion cell proliferation decreased to 17%. Furthermore, for harnessing RNAi for silencing disease-related genes in vivo we transplanted inducible RNAi-HeLa cells onto nude mice. After administration of Dox knockdown of Plk1 expression was observed correlating to a significant inhibition of tumor growth. Taken together, our data revealed that genomically integrated RNAi-elements are suitable to hamper tumor growth by conditional expression of shRNA.


Enabling consistency in pluripotent stem cell-derived products for research and development and clinical applications through material standards.
There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify "signatures" for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes.


Yogic meditation reverses NF-κB and IRF-related transcriptome dynamics in leukocytes of family dementia caregivers in a randomized controlled trial.
Although yoga and meditation have been used for stress reduction with reported improvement in inflammation, little is known about the biological mechanisms mediating such effects. The present study examined if a yogic meditation might alter the activity of inflammatory and antiviral transcription control pathways that shape immune cell gene expression. Forty-five family dementia caregivers were randomized to either Kirtan Kriya Meditation (KKM) or Relaxing Music (RM) listening for 12 min daily for 8 weeks and 39 caregivers completed the study. Genome-wide transcriptional profiles were collected from peripheral blood leukocytes sampled at baseline and 8-week follow-up. Promoter-based bioinformatics analyses tested the hypothesis that observed transcriptional alterations were structured by reduced activity of the pro-inflammatory nuclear factor (NF)-κB family of transcription factors and increased activity of Interferon Response Factors (IRFs; i.e., reversal of patterns previously linked to stress). In response to KKM treatment, 68 genes were found to be differentially expressed (19 up-regulated, 49 down-regulated) after adjusting for potentially confounded differences in sex, illness burden, and BMI. Up-regulated genes included immunoglobulin-related transcripts. Down-regulated transcripts included pro-inflammatory cytokines and activation-related immediate-early genes. Transcript origin analyses identified plasmacytoid dendritic cells and B lymphocytes as the primary cellular context of these transcriptional alterations (both p<.001). Promoter-based bioinformatic analysis implicated reduced NF-κB signaling and increased activity of IRF1 in structuring those effects (both p<.05). A brief daily yogic meditation intervention may reverse the pattern of increased NF-κB-related transcription of pro-inflammatory cytokines and decreased IRF1-related transcription of innate antiviral response genes previously observed in healthy individuals confronting a significant life stressor.


Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015.
Non-fatal outcomes of disease and injury increasingly detract from the ability of the world's population to live in full health, a trend largely attributable to an epidemiological transition in many countries from causes affecting children, to non-communicable diseases (NCDs) more common in adults. For the Global Burden of Diseases, Injuries, and Risk Factors Study 2015 (GBD 2015), we estimated the incidence, prevalence, and years lived with disability for diseases and injuries at the global, regional, and national scale over the period of 1990 to 2015. We estimated incidence and prevalence by age, sex, cause, year, and geography with a wide range of updated and standardised analytical procedures. Improvements from GBD 2013 included the addition of new data sources, updates to literature reviews for 85 causes, and the identification and inclusion of additional studies published up to November, 2015, to expand the database used for estimation of non-fatal outcomes to 60 900 unique data sources. Prevalence and incidence by cause and sequelae were determined with DisMod-MR 2.1, an improved version of the DisMod-MR Bayesian meta-regression tool first developed for GBD 2010 and GBD 2013. For some causes, we used alternative modelling strategies where the complexity of the disease was not suited to DisMod-MR 2.1 or where incidence and prevalence needed to be determined from other data. For GBD 2015 we created a summary indicator that combines measures of income per capita, educational attainment, and fertility (the Socio-demographic Index [SDI]) and used it to compare observed patterns of health loss to the expected pattern for countries or locations with similar SDI scores. We generated 9·3 billion estimates from the various combinations of prevalence, incidence, and YLDs for causes, sequelae, and impairments by age, sex, geography, and year. In 2015, two causes had acute incidences in excess of 1 billion: upper respiratory infections (17·2 billion, 95% uncertainty interval [UI] 15·4-19·2 billion) and diarrhoeal diseases (2·39 billion, 2·30-2·50 billion). Eight causes of chronic disease and injury each affected more than 10% of the world's population in 2015: permanent caries, tension-type headache, iron-deficiency anaemia, age-related and other hearing loss, migraine, genital herpes, refraction and accommodation disorders, and ascariasis. The impairment that affected the greatest number of people in 2015 was anaemia, with 2·36 billion (2·35-2·37 billion) individuals affected. The second and third leading impairments by number of individuals affected were hearing loss and vision loss, respectively. Between 2005 and 2015, there was little change in the leading causes of years lived with disability (YLDs) on a global basis. NCDs accounted for 18 of the leading 20 causes of age-standardised YLDs on a global scale. Where rates were decreasing, the rate of decrease for YLDs was slower than that of years of life lost (YLLs) for nearly every cause included in our analysis. For low SDI geographies, Group 1 causes typically accounted for 20-30% of total disability, largely attributable to nutritional deficiencies, malaria, neglected tropical diseases, HIV/AIDS, and tuberculosis. Lower back and neck pain was the leading global cause of disability in 2015 in most countries. The leading cause was sense organ disorders in 22 countries in Asia and Africa and one in central Latin America; diabetes in four countries in Oceania; HIV/AIDS in three southern sub-Saharan African countries; collective violence and legal intervention in two north African and Middle Eastern countries; iron-deficiency anaemia in Somalia and Venezuela; depression in Uganda; onchoceriasis in Liberia; and other neglected tropical diseases in the Democratic Republic of the Congo. Ageing of the world's population is increasing the number of people living with sequelae of diseases and injuries. Shifts in the epidemiological profile driven by socioeconomic change also contribute to the continued increase in years lived with disability (YLDs) as well as the rate of increase in YLDs. Despite limitations imposed by gaps in data availability and the variable quality of the data available, the standardised and comprehensive approach of the GBD study provides opportunities to examine broad trends, compare those trends between countries or subnational geographies, benchmark against locations at similar stages of development, and gauge the strength or weakness of the estimates available. Bill & Melinda Gates Foundation.


Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.
Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity.


Lymphoid microenvironments and innate lymphoid cells in the gut.
Gut-associated lymphoid tissue (GALT) is a sensor region for luminal content and plays an important role in lymphoid maturation, activation and differentiation. It comprises isolated and aggregated lymphoid follicles, cryptopatches (CPs) and tertiary lymphoid tissue. Innate lymphoid cells (ILCs) play a central role within GALT. Prenatal GALT development is dependent on ILC lymphoid-inducer function. Postnatally, these cells rapidly respond to commensal and pathogenic intestinal bacteria, parasites and food components by polarized cytokine production [such as interleukin (IL)-22, IL-17 or IL-13] and further contribute to GALT formation and function. Here, we discuss how ILCs shape lymphoid intestinal microenvironments and act as amplifier cells for innate and adaptive immune responses.


Peroxisome-derived lipids regulate adipose thermogenesis by mediating cold-induced mitochondrial fission.
Peroxisomes perform essential functions in lipid metabolism, including fatty acid oxidation and plasmalogen synthesis. Here, we describe a role for peroxisomal lipid metabolism in mitochondrial dynamics in brown and beige adipocytes. Adipose tissue peroxisomal biogenesis was induced in response to cold exposure through activation of the thermogenic coregulator PRDM16. Adipose-specific knockout of the peroxisomal biogenesis factor Pex16 (Pex16-AKO) in mice impaired cold tolerance, decreased energy expenditure, and increased diet-induced obesity. Pex16 deficiency blocked cold-induced mitochondrial fission, decreased mitochondrial copy number, and caused mitochondrial dysfunction. Adipose-specific knockout of the peroxisomal β-oxidation enzyme acyl-CoA oxidase 1 (Acox1-AKO) was not sufficient to affect adiposity, thermogenesis, or mitochondrial copy number, but knockdown of the plasmalogen synthetic enzyme glyceronephosphate O-acyltransferase (GNPAT) recapitulated the effects of Pex16 inactivation on mitochondrial morphology and function. Plasmalogens are present in mitochondria and decreased with Pex16 inactivation. Dietary supplementation with plasmalogens increased mitochondrial copy number, improved mitochondrial function, and rescued thermogenesis in Pex16-AKO mice. These findings support a surprising interaction between peroxisomes and mitochondria regulating mitochondrial dynamics and thermogenesis.


16S rRNA gene survey of microbial communities in Winogradsky columns.
A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities.


Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.
To provide an update to the original Surviving Sepsis Campaign clinical management guidelines, "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," published in 2004. Modified Delphi method with a consensus conference of 55 international experts, several subsequent meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. This process was conducted independently of any industry funding. We used the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations. A strong recommendation (1) indicates that an intervention's desirable effects clearly outweigh its undesirable effects (risk, burden, cost) or clearly do not. Weak recommendations (2) indicate that the tradeoff between desirable and undesirable effects is less clear. The grade of strong or weak is considered of greater clinical importance than a difference in letter level of quality of evidence. In areas without complete agreement, a formal process of resolution was developed and applied. Recommendations are grouped into those directly targeting severe sepsis, recommendations targeting general care of the critically ill patient that are considered high priority in severe sepsis, and pediatric considerations. Key recommendations, listed by category, include early goal-directed resuscitation of the septic patient during the first 6 hrs after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm potential source of infection (1C); administration of broad-spectrum antibiotic therapy within 1 hr of diagnosis of septic shock (1B) and severe sepsis without septic shock (1D); reassessment of antibiotic therapy with microbiology and clinical data to narrow coverage, when appropriate (1C); a usual 7-10 days of antibiotic therapy guided by clinical response (1D); source control with attention to the balance of risks and benefits of the chosen method (1C); administration of either crystalloid or colloid fluid resuscitation (1B); fluid challenge to restore mean circulating filling pressure (1C); reduction in rate of fluid administration with rising filing pressures and no improvement in tissue perfusion (1D); vasopressor preference for norepinephrine or dopamine to maintain an initial target of mean arterial pressure > or = 65 mm Hg (1C); dobutamine inotropic therapy when cardiac output remains low despite fluid resuscitation and combined inotropic/vasopressor therapy (1C); stress-dose steroid therapy given only in septic shock after blood pressure is identified to be poorly responsive to fluid and vasopressor therapy (2C); recombinant activated protein C in patients with severe sepsis and clinical assessment of high risk for death (2B except 2C for postoperative patients). In the absence of tissue hypoperfusion, coronary artery disease, or acute hemorrhage, target a hemoglobin of 7-9 g/dL (1B); a low tidal volume (1B) and limitation of inspiratory plateau pressure strategy (1C) for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure in acute lung injury (1C); head of bed elevation in mechanically ventilated patients unless contraindicated (1B); avoiding routine use of pulmonary artery catheters in ALI/ARDS (1A); to decrease days of mechanical ventilation and ICU length of stay, a conservative fluid strategy for patients with established ALI/ARDS who are not in shock (1C); protocols for weaning and sedation/analgesia (1B); using either intermittent bolus sedation or continuous infusion sedation with daily interruptions or lightening (1B); avoidance of neuromuscular blockers, if at all possible (1B); institution of glycemic control (1B), targeting a blood glucose < 150 mg/dL after initial stabilization (2C); equivalency of continuous veno-veno hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1A); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding using H2 blockers (1A) or proton pump inhibitors (1B); and consideration of limitation of support where appropriate (1D). Recommendations specific to pediatric severe sepsis include greater use of physical examination therapeutic end points (2C); dopamine as the first drug of choice for hypotension (2C); steroids only in children with suspected or proven adrenal insufficiency (2C); and a recommendation against the use of recombinant activated protein C in children (1B). There was strong agreement among a large cohort of international experts regarding many level 1 recommendations for the best current care of patients with severe sepsis. Evidenced-based recommendations regarding the acute management of sepsis and septic shock are the first step toward improved outcomes for this important group of critically ill patients.


Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes.
Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276-281]. The protein (lectin-trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein (M(r) approximately 32,500). The recombinant protein (Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the alpha subunit of the lectin-trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.


Lysine methylation-dependent binding of 53BP1 to the pRb tumor suppressor.
The retinoblastoma tumor suppressor protein pRb is a key regulator of cell cycle progression and mediator of the DNA damage response. Lysine methylation at K810, which occurs within a critical Cdk phosphorylation motif, holds pRb in the hypophosphorylated growth-suppressing state. We show here that methyl K810 is read by the tandem tudor domain containing tumor protein p53 binding protein 1 (53BP1). Structural elucidation of 53BP1 in complex with a methylated K810 pRb peptide emphasized the role of the 53BP1 tandem tudor domain in recognition of the methylated lysine and surrounding residues. Significantly, binding of 53BP1 to methyl K810 occurs on E2 promoter binding factor target genes and allows pRb activity to be effectively integrated with the DNA damage response. Our results widen the repertoire of cellular targets for 53BP1 and suggest a previously unidentified role for 53BP1 in regulating pRb tumor suppressor activity.