Is there indirect selection on female extra-pair reproduction through cross-sex genetic correlations with male reproductive fitness?
One key hypothesis explaining the evolution and persistence of polyandry, and resulting female extra-pair reproduction in socially monogamous systems, is that female propensity for extra-pair reproduction is positively genetically correlated with male reproductive fitness and consequently experiences positive cross-sex indirect selection. However, key genetic correlations have rarely been estimated, especially in free-living populations experiencing natural (co)variation in reproductive strategies and fitness. We used long-term life-history and pedigree data from song sparrows (Melospiza melodia) to estimate the cross-sex genetic correlation between female propensity for extra-pair reproduction and adult male lifetime reproductive success, and thereby test a key hypothesis regarding mating system evolution. There was substantial additive genetic variance in both traits, providing substantial potential for indirect selection on female reproductive strategy. However, the cross-sex genetic correlation was estimated to be close to zero. Such small correlations might arise because male reproductive success achieved through extra-pair paternity was strongly positively genetically correlated with success achieved through within-pair paternity, implying that the same successful males commonly sire offspring produced by polyandrous and monogamous females. Cross-sex indirect selection may consequently have limited capacity to drive evolution of female extra-pair reproduction, or hence underlying polyandry, in systems where multiple routes to paternity success exist.


Auditory Brainstem Response Altered in Humans With Noise Exposure Despite Normal Outer Hair Cell Function.
Recent animal studies demonstrated that cochlear synaptopathy, a partial loss of inner hair cell-auditory nerve fiber synapses, can occur in response to noise exposure without any permanent auditory threshold shift. In animal models, this synaptopathy is associated with a reduction in the amplitude of wave I of the auditory brainstem response (ABR). The goal of this study was to determine whether higher lifetime noise exposure histories in young people with clinically normal pure-tone thresholds are associated with lower ABR wave I amplitudes. Twenty-nine young military Veterans and 35 non Veterans (19 to 35 years of age) with normal pure-tone thresholds were assigned to 1 of 4 groups based on their self-reported lifetime noise exposure history and Veteran status. Suprathreshold ABR measurements in response to alternating polarity tone bursts were obtained at 1, 3, 4, and 6 kHz with gold foil tiptrode electrodes placed in the ear canal. Wave I amplitude was calculated from the difference in voltage at the positive peak and the voltage at the following negative trough. Distortion product otoacoustic emission input/output functions were collected in each participant at the same four frequencies to assess outer hair cell function. After controlling for individual differences in sex and distortion product otoacoustic emission amplitude, the groups containing participants with higher reported histories of noise exposure had smaller ABR wave I amplitudes at suprathreshold levels across all four frequencies compared with the groups with less history of noise exposure. Suprathreshold ABR wave I amplitudes were reduced in Veterans reporting high levels of military noise exposure and in non Veterans reporting any history of firearm use as compared with Veterans and non Veterans with lower levels of reported noise exposure history. The reduction in ABR wave I amplitude in the groups with higher levels of noise exposure cannot be accounted for by sex or variability in outer hair cell function. This change is similar to the decreased ABR wave I amplitudes observed in animal models of noise-induced cochlear synaptopathy. However, without post mortem examination of the temporal bone, no direct conclusions can be drawn concerning the presence of synaptopathy in the study groups with higher noise exposure histories.


Lowering blood pressure blocks mesangiolysis and mesangial nodules, but not tubulointerstitial injury, in diabetic eNOS knockout mice.
Recently, we and others reported that diabetic endothelial nitric oxide synthase knockout (eNOSKO) mice develop advanced glomerular lesions that include mesangiolysis and nodular lesions. Interestingly, insulin treatment lowered blood pressure and prevented renal lesions, raising the question as to whether these beneficial effects of insulin were due to its ability to lower either high glucose levels or high blood pressure. We, therefore, examined the effect of lowering blood pressure using hydralazine in this diabetic eNOSKO mouse model. Hydralazine treatment significantly blocked the development of mesangiolysis and microaneurysms, whereas tubulointerstitial injury was not prevented in these mice. Additionally, hydralazine did not reduce expression levels of either tubulointerstitial thrombospondin-1 or transforming growth factor-beta despite controlling blood pressure. On the other hand, the critical role of high glucose levels on the development of tubulointerstitial injury was suggested by the observation that serum glucose levels were correlated with tubulointerstitial injury, as well as with the expression levels of both transforming growth factor-beta and thrombospondin-1. Importantly, controlling blood glucose with insulin completely blocked tubulointerstitial injury in diabetic eNOSKO mice. These data suggest that glomerular injury is dependent on systemic blood pressure, whereas hyperglycemia may have a more important role in tubulointerstitial injury, possibly due to the stimulation of the thrombospondin-1-transforming growth factor-beta pathway in diabetic eNOSKO mice. This study could provide insights into the pathogenesis of advanced diabetic nephropathy in the presence of endothelial dysfunction.


Assessing agreement between multiple raters with missing rating information, applied to breast cancer tumour grading.
We consider the problem of assessing inter-rater agreement when there are missing data and a large number of raters. Previous studies have shown only 'moderate' agreement between pathologists in grading breast cancer tumour specimens. We analyse a large but incomplete data-set consisting of 24,177 grades, on a discrete 1-3 scale, provided by 732 pathologists for 52 samples. We review existing methods for analysing inter-rater agreement for multiple raters and demonstrate two further methods. Firstly, we examine a simple non-chance-corrected agreement score based on the observed proportion of agreements with the consensus for each sample, which makes no allowance for missing data. Secondly, treating grades as lying on a continuous scale representing tumour severity, we use a Bayesian latent trait method to model cumulative probabilities of assigning grade values as functions of the severity and clarity of the tumour and of rater-specific parameters representing boundaries between grades 1-2 and 2-3. We simulate from the fitted model to estimate, for each rater, the probability of agreement with the majority. Both methods suggest that there are differences between raters in terms of rating behaviour, most often caused by consistent over- or under-estimation of the grade boundaries, and also considerable variability in the distribution of grades assigned to many individual samples. The Bayesian model addresses the tendency of the agreement score to be biased upwards for raters who, by chance, see a relatively 'easy' set of samples. Latent trait models can be adapted to provide novel information about the nature of inter-rater agreement when the number of raters is large and there are missing data. In this large study there is substantial variability between pathologists and uncertainty in the identity of the 'true' grade of many of the breast cancer tumours, a fact often ignored in clinical studies.


Involvement of progranulin in hypothalamic glucose sensing and feeding regulation.
Progranulin (PGRN) is a secreted glycoprotein with multiple biological functions, including modulation of wound healing and inflammation. Hypothalamic PGRN has been implicated in the development of sexual dimorphism. In the present study, a potential role for PGRN in the hypothalamic regulation of appetite and body weight was investigated. In adult rodents, PGRN was highly expressed in periventricular tanycytes and in hypothalamic neurons, which are known to contain glucose-sensing machinery. Hypothalamic PGRN expression levels were decreased under low-energy conditions (starvation and 2-deoxy-D-glucose administration) but increased under high-energy condition (postprandially). Intracerebrovetricular administration of PGRN significantly suppressed nocturnal feeding as well as hyperphagia induced by 2-deoxyglucose, neuropeptide Y, and Agouti-related peptide. Moreover, the inhibition of hypothalamic PGRN expression or action increased food intake and promoted weight gain, suggesting that endogenous PGRN functions as an appetite suppressor in the hypothalamus. Investigation of the mechanism of action revealed that PGRN diminished orexigenic neuropeptide Y and Agouti-related peptide production but stimulated anorexigenic proopiomelanocortin production, at least in part through the regulation of hypothalamic AMP-activated protein kinase. Notably, PGRN was also expressed in hypothalamic microglia. In diet-induced obese mice, microglial PGRN expression was increased, and the anorectic response to PGRN was blunted. These findings highlight a physiological role for PGRN in hypothalamic glucose-sensing and appetite regulation. Alterations in hypothalamic PGRN production or action may be linked to appetite dysregulation in obesity.


Cerebellar expression of copper chaperone for superoxide, cytosolic cu/zn-superoxide dismutase, 4-hydroxy-2-nonenal, acrolein and heat shock protein 32 in patients with menkes kinky hair disease: immunohistochemical study.
To clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky hair disease (MD), a disorder of copper absorption, we investigated the morphological and functional abnormalities of residual Purkinje cells in MD patients and the mechanism of cell death. Seven MD patients and 39 neurologically normal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinje cells. In addition, we used immunohistochemistry to detect copper-dependent enzymes [cytosolic Cu/Zn-superoxide dismutase (SOD1) and copper chaperone for superoxide dismutase (CCS)], oxidative stress markers [4-hydroxy-2-nonenal (HNE) and acrolein] and heat shock protein 32 (hsp 32). The surviving MD Purkinje cells showed abnormal development, such as somatic sprouts and heterotopic location. Due to maldevelopment and degeneration, dendrites showed the cactus and weeping willow patterns. Axonal degeneration led to the formation of torpedoes. Quantitative analysis revealed loss of approximately 50% of the Purkinje cells in MD patients. Almost all of the normal Purkinje cells were positive for immunostaining by anti-CCS and anti-SOD1 antibodies, with staining of the cell bodies, dendrites and axons. Normal Purkinje cells were not stained by antibodies for HNE, acrolein or hsp 32. In MD patients, the majority of Purkinje cells were positive for CCS, but the positive rate for SOD1 was only about 23%. Approximately 56%, 42% and 40% of the Purkinje cells of MD patients were positive for HNE, acrolein and hsp 32, respectively. In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment and degeneration. In the residual Purkinje cells, CCS expression seems to be nearly normal as a protective response to decreased SOD1 activity due to copper deficiency. Because oxidative stress is elevated secondary to decreased SOD1 activity, hsp 32 is induced as another protective mechanism.


What is learned from Mindfulness Based Childbirth and Parenting Education? - Participants' experiences.
In the search for effective interventions aiming to prevent perinatal stress, depression and anxiety, we are evaluating a Mindfulness Based Childbirth and Parenting (MBCP) Program. In this study we explore the participants' experiences of the program. This is a descriptive qualitative study with influences of phenomenology. The participants were expectant couples who participated in the program and the pregnant women had an increased risk of perinatal stress, anxiety and depression. Ten mothers and six fathers were interviewed in depth, at four to six months postpartum. Thematic analysis of the transcripts was conducted. The participants' descriptions show a variety in how motivated they were and how much value they ascribed to MBCP. Those who experienced that they benefitted from the intervention described that they did so at an intra-personal level-with deeper self-knowledge and self-compassion; and on an inter-personal level-being helpful in relationships. Furthermore, they perceived that what they had learned from MBCP was helpful during childbirth and early parenting. Our findings demonstrate that most of the parents experienced MBCP as a valuable preparation for the challenges they met when they went through the life-changing events of becoming parents. The phenomenon of participating in the intervention, integrating the teachings and embodying mindfulness seems to develop inner resources that foster the development of wisdom. ClinicalTrials.gov ID: NCT02441595, May 4, 2015.


The distribution of MICA alleles in an Austrian population: evidence for increasing polymorphism.
The Major Histocompatibility Complex Class I Chain-Related Gene A (MICA) is located 46.4 Kb centromeric to HLA-B locus on chromosome 6; 84 alleles have been described so far. To assess the distribution of MICA alleles in an Austrian population, 322 unrelated Austrian blood donors have been typed for MICA by direct sequencing of amplified exons 2-5; sequencing of exon 6 and separating alleles by haplotype specific primers or by cloning was performed to resolve ambiguities. HLA-B was typed at low level resolution and linkage disequilibrium was determined. We observed 20 already known and four novel MICA alleles. MICA*008:01/04 was the most frequent allele (42%), followed by MICA*002:01 (11%) and MICA*009:01 (9%), three alleles (MICA*029, *067 and *068) were observed only once. No deviation from the Hardy Weinberg equilibrium was observed. Linkage disequilibrium between MICA and HLA-B alleles was observed, most extensively between MICA*008:01/04 and HLA-B*07. Our population data are in agreement with other European populations. The fact that four novel alleles have been observed indicates that the polymorphism of MICA is larger than currently estimated.


The cymA gene, encoding a tetraheme c-type cytochrome, is required for arsenate respiration in Shewanella species.
In Shewanella sp. strain ANA-3, utilization of arsenate as a terminal electron acceptor is conferred by a two-gene operon, arrAB, which lacks a gene encoding a membrane-anchoring subunit for the soluble ArrAB protein complex. Analysis of the genome sequence of Shewanella putrefaciens strain CN-32 showed that it also contained the same arrAB operon with 100% nucleotide identity. Here, we report that CN-32 respires arsenate and that this metabolism is dependent on arrA and an additional gene encoding a membrane-associated tetraheme c-type cytochrome, cymA. Deletion of cymA in ANA-3 also eliminated growth on and reduction of arsenate. The DeltacymA strains of CN-32 and ANA-3 negatively affected the reduction of Fe(III) and Mn(IV) but not growth on nitrate. Unlike the CN-32 DeltacymA strain, growth on fumarate was absent in the DeltacymA strain of ANA-3. Both homologous and heterologous complementation of cymA in trans restored growth on arsenate in DeltacymA strains of both CN-32 and ANA-3. Transcription patterns of cymA showed that it was induced under anaerobic conditions in the presence of fumarate and arsenate. Nitrate-grown cells exhibited the greatest level of cymA expression in both wild-type strains. Lastly, site-directed mutagenesis of the first Cys to Ser in each of the four CXXCH c-heme binding motifs of the CN-32 CymA nearly eliminated growth on and reduction of arsenate. Together, these results indicate that the biochemical mechanism of arsenate respiration and reduction requires the interactions of ArrAB with a membrane-associated tetraheme cytochrome, which in the non-arsenate-respiring Shewanella species Shewanella oneidensis strain MR-1, has pleiotropic effects on Fe(III), Mn(IV), dimethyl sulfoxide, nitrate, nitrite, and fumarate respiration.


The effect of advanced access implementation on quality of diabetes care.
The study analyzes the effect of an advanced access program on quality of diabetes care. We conducted this study in a medical group of 240,000 members served by 17 primary care clinics. Seven thousand adult patients older than 18 years of age with diabetes were identified from administrative databases. Two aspects of advanced access - wait time for appointments and continuity of care - were calculated yearly for each patient during 1999 through 2001. We developed three composite measures of glucose and lipid control - process (proportion of patients with appropriate testing rates of hemoglobin A1c [HbA1c] and low-density lipoprotein [LDL]), good control (proportion with HbA1c < 8% and LDL < 130 mg/dL) and excellent control (proportion with HbA1c < 7% and LDL < 100 mg/dL) - and assessed them each year for each patient. We used multilevel logistic regression to predict the measures in 2000 and 2001 (years during and after advanced access implementation) relative to 1999 (year pre-advanced access). After implementation of advanced access, wait time decreased from 21.6 days to 4.2 days, and continuity improved by 6.5% (both P < .01). The percentage of patients with HbA1c < 7% increased from 44.4% to 52.3% and with LDL < 100 mg/dL from 29.8% to 38.7%. Increased continuity predicted improved process (P = .01), good control (P = .033), and excellent control (P < .001). However, wait time did not significantly predict process (P = .62) or quality measures (P = .95). Measures of the quality of diabetes control improved in the year after implementation of advanced access, but better care did not correlate with decreased wait time to see a provider. However, improved continuity of care predicted improvements in both process and quality of diabetes care.


Ultrastructure of lung in surfactant protein B deficiency.
Congenital alveolar proteinosis (CAP), a cause of respiratory failure in fill-term newborns, often leads to death in infancy despite medical therapy. We recently described an inherited deficiency of surfactant protein B (SP-B) (N. Engl. J. Med. 1993; 328:406-410) in two siblings with CAP. The SP-B deficiency was accompanied by marked abnormalities, both quantitative (increase) and qualitative (distribution), of SP-A and SP-C in the lungs of the affected infants. Ultrastructural studies of the lung of one of these infants and of a third affected sibling born in the index family showed abundant alveolar concentric multilamellated structures and membranous vesicles but no typical tubular myelin. In addition, membranous vesicles from type II cells and immunogold labeled SP-A and SP-C were found between type II cells and their basement membrane despite intact interepithelial cell junctions. These findings suggest an important role for SP-B in the directionality of surfactant secretion and in the formation of tubular myelin.


MtDNA haplogroups in the populations of Croatian Adriatic Islands.
The number of previous anthropological studies pointed to very complex ethnohistorical processes that shaped the current genetic structure of Croatian island isolates. The scope of this study was limited to the general insight into their founding populations and the overall level of genetic diversity based on the study mtDNA variation. A total of 444 randomly chosen adult individuals from 32 rural communities of the islands of Krk, Brac, Hvar and Korcula were sampled. MtDNA HVS-I region together with RFLP sites diagnostic for main Eurasian and African mtDNA haplogroups were analysed in order to determine the haplogroup structure. The most frequent haplogroups were "H" (27.8-60.2%), "U" (10.2-24.1%), "J" (6.1-9.0%) and "T" (5.1-13.9%), which is similar to the other European and Near Eastern populations. The genetic drift could have been important aspect in history, as there were examples of excess frequencies of certain haplogroups (11.3% of "I" and 7.5% of "W" in Krk, 10.5% of "HV" in Brac, 13.9% of "J" in Hvar and 60.2% of "H" in Korcula). As the settlements on the islands were formed trough several immigratory episodes of genetically distant populations, this analysis (performed at the level of entire islands) showed greater genetic diversity (0.940-0.972) than expected at the level of particular settlements.


Rapamycin reverses splenomegaly and inhibits tumor development in a transgenic model of Epstein-Barr virus-related Burkitt's lymphoma.
Epstein-Barr virus (EBV) infection and latency has been associated with malignancies, including nasopharyngeal carcinoma and Burkitt's lymphoma. EBV encoded latent membrane protein 2A (LMP2A) is expressed in most EBV-associated malignancies and as such provides a therapeutic target. Burkitt's lymphoma is a hematopoietic cancer associated with the translocation of c-MYC to one of the immunoglobulin gene promoters leading to abnormally high expression of MYC and development of lymphoma. Our laboratory has developed a murine model of EBV-associated Burkitt's lymphoma by crossing LMP2A transgenic mice with MYC transgenic mice. Since LMP2A has been shown to activate the PI3K/Akt/mTOR pathway, we tested the therapeutic efficacy of mTOR inhibitor rapamycin on the tumors and splenomegaly in these double transgenic mice (Tg6/λ-MYC). We found that rapamycin reversed splenomegaly in Tg6/λ-MYC mice prior to tumor formation by targeting B cells. In a tumor transfer model, we also found that rapamycin significantly decreased tumor growth, splenomegaly, and metastasis of tumor cells in the bone marrow of tumor recipients. Our data show that rapamycin may be a valuable candidate for the development of a treatment modality for EBV-positive lymphomas, such as Burkitt's lymphoma, and more importantly, provides a basis to develop inhibitors that specifically target viral gene function in tumor cells that depend on LMP2A signaling for survival and/or growth.


A Transport Model for Estimating the Time Course of ERK Activation in the C. elegans Germline.
The Caenorhabditis elegans germline is a well-studied model system for investigating the control of cell fate by signaling pathways. Cell signals at the distal tip of the germline promote cell proliferation; just before the loop, signals couple cell maturation to organism-level nutrient status; at the proximal end of the germline, signals coordinate oocyte maturation and fertilization in the presence of sperm. The latter two events require dual phosphorylation and activation of ERK, the effector molecule of the Ras/MAPK cascade. In C. elegans, ERK is known as MPK-1. At this point, none of today's methods for real-time monitoring of dually phosphorylated MPK-1 are working in the germline. Consequently, quantitative understanding of the MPK-1-dependent processes during germline development is limited. Here, we make a step toward advancing this understanding using a model-based framework that reconstructs the time course of MPK-1 activation from a snapshot of a fixed germline. Our approach builds on a number of recent studies for estimating temporal dynamics from fixed organisms, but takes advantage of the anatomy of the germline to simplify the analysis. Our model predicts that the MPK-1 signal turns on ∼30 h into germ cell progression and peaks ∼7 h later.


The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane.
Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.


Novel screening techniques for ion channel targeting drugs.
Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.


Effect of hypertension and hypertension-glucose intolerance on myocardial ischemic injury.
Systemic hypertension and type 2 diabetes mellitus are major risk factors for myocardial infarction. Yet, glucose intolerance, a prelude stage to type 2 diabetes, is associated with reduced infarct size. Since chronic hypertension adversely affects the myocardium, we tested the hypothesis that the coexistence of systemic hypertension and glucose intolerance reverses the cardioprotection associated with impaired glucose tolerance. Hearts from 9-month-old animals were subjected to a 40-minute occlusion of the left coronary artery followed by 2 hours of reperfusion. Before ischemia, similar values for the four experimental groups were observed for the coronary flow, heart rate, and maximum ventricular pressure. During the course of the ischemia-reperfusion insult, the two hypertensive groups displayed greater reductions in contractility than their normotensive counterparts. Infarct size was lower in the normotensive glucose-intolerant rat than in the normotensive control rat. Surprisingly, the hypertrophied hearts of the hypertensive and hypertensive glucose-intolerant rats displayed reduced infarct size (P<0.05). However, raising the afterload pressure from 100 to 160 cm H2O increased infarct size in the two hypertensive groups. This narrowed the differential between the hypertensive glucose-intolerant (160 cm H2O) and the normotensive control (100 cm H2O) rats. Nonetheless, at the higher afterload pressure, infarct size was less in the hypertensive glucose-intolerant rats than in their hypertensive counterparts. In conclusion, the impairment in contractile function despite the reduction in infarct size underscores the increased susceptibility of the hypertrophied, hypertensive heart to ischemic injury. Furthermore, exacerbation of cell death at elevated afterload pressure indicates the potential benefit of aggressive antihypertensive therapy.


Heritability and Genome-Wide Association Analyses of Human Gait Suggest Contribution of Common Variants.
Human gait is a complex neurological and musculoskeletal function, of which the genetic basis remains largely unknown. To determine the influence of common genetic variants on gait parameters, we studied 2,946 participants of the Rotterdam Study, a population-based cohort of unrelated elderly individuals. We assessed 30 gait parameters using an electronic walkway, which yielded seven independent gait domains after principal component analysis. Genotypes of participants were imputed to the 1,000 Genomes reference panel for generating genetic relationship matrices to estimate heritability of gait parameters, and for subsequent genome-wide association scans (GWASs) to identify specific variants. Gait domains with the highest age- and sex-adjusted heritability were Variability (h (2) = 61%), Rhythm (37%), and Tandem (32%). For other gait domains, heritability estimates attenuated after adjustment for height and weight. Genome-wide association scans identified a variant on 1p22.3 that was significantly associated with single support time, a variable from the Rhythm domain (rs72953990; N = 2,946; β [SE] = 0.0069 (0.0012), p = 2.30×10(-8)). This variant did not replicate in an independent sample (N = 362; p = .78). In conclusion, human gait has highly heritable components that are explained by common genetic variation, which are partly attributed to height and weight. Collaborative efforts are needed to identify robust single variant associations for the heritable parameters.


Sexual Harassment Among Medical Students: Prevalence, Prediction, and Correlated Outcomes.
Few studies are dedicated to understanding the extent and impact of sexual harassment among medical students. The aim of this study was to use behaviorally specific measures to examine prevalence of sexual harassment toward medical students. Associated mental health and academic impacts were also studied. A multisite survey was conducted at four medical schools. Sexual harassment was measured using the Sexual Experiences Questionnaire (SEQ), a valid and reliable instrument. Students were also surveyed about depressive and post-traumatic stress disorder (PTSD) symptoms and their level of academic engagement. We also assessed their perceptions of institutional response and whether they felt safe at their institution. The final sample included 524 medical students (response rate = 13%). Findings revealed that 36.6% reported sexual harassment by a faculty/staff member and 38.5% reported harassment by a fellow student. The odds of harassment by faculty/staff, as well as peers, were significantly higher for women with an adjusted odds ratio (AOR) = 9.83, 95% confidence interval (CI) = [3.74, 25.80] and multiracial students with an AOR: 2.93, 95% CI: [1.16, 7.39]. Those who experienced sexual harassment were more likely to report academic disengagement and symptoms of depression and PTSD. Sexual harassment in medical schools can potentially limit a student's academic success and negatively impact their mental health. Supportive services and efforts to address peer and professional cultures that promote harassment are needed. Experiences of harassment require swift and competent responses by medical school leadership in collaboration with occupational and/or student health services to mitigate detrimental impacts and support medical students throughout their training.


Gene networks inference using dynamic Bayesian networks.
This article deals with the identification of gene regulatory networks from experimental data using a statistical machine learning approach. A stochastic model of gene interactions capable of handling missing variables is proposed. It can be described as a dynamic Bayesian network particularly well suited to tackle the stochastic nature of gene regulation and gene expression measurement. Parameters of the model are learned through a penalized likelihood maximization implemented through an extended version of EM algorithm. Our approach is tested against experimental data relative to the S.O.S. DNA Repair network of the Escherichia coli bacterium. It appears to be able to extract the main regulations between the genes involved in this network. An added missing variable is found to model the main protein of the network. Good prediction abilities on unlearned data are observed. These first results are very promising: they show the power of the learning algorithm and the ability of the model to capture gene interactions.


Photo-induced dynamic processes in perovskite solar cells: the influence of perovskite composition in the charge extraction and the carrier recombination.
We describe in this work how the use of the photo-induced charge extraction (PICE) technique can be useful to study and understand the relationship between perovskite composition, ion migration processes and solar cell performance. Our results show that varying the organic cation and halide composition in perovskite crystals has an effect on the charge extraction process, which is related to the differences in the ion migration process occurring during light irradiation. Moreover, we do also observe that different perovskites also show differences in interfacial carrier recombination kinetics under operando conditions but the dynamics are too fast to be influenced by the ion migration.


Angiography and Embolization in the Management of Bleeding Pelvic Fractures.
The use, timing, and priority of angioembolization in the management of bleeding pelvic fractures remain ambiguous. The most common vessels for angioembolization are, in decreasing order, the internal iliac artery and its branches, the superior gluteal artery, the obturator artery, and the internal pudendal artery. Technical success rates for this treatment option range from 74% to 100%. The fracture patterns most commonly requiring angioembolization are the Young and Burgess lateral compression and anterior-posterior compression types and Tile type C. Mortality rates after angioembolization of 16% to 50% have been reported, but deaths are usually related to concomitant injuries. The sensitivity and specificity of contrast-enhanced CT in detecting the need for angioembolization range from 60% to 90% and 92% to 100%, respectively. Angioembolization can be effective in the management of bleeding pelvic fractures, but as with any treatment, the risks of complications must be considered. Availability of angioembolization and institutional expertise/preference for the alternative strategy of pelvic packing influence its use.


Somatic and germinal recombination of a direct repeat in Arabidopsis.
Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis. The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene. This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed. Selection for active NPT by resistance to kanamycin gave both fully and partly (chimeric) recombinant seedlings. Rates for one transgenic line were estimated at less than 2 x 10(-5) events per division for germinal and greater than 10(-6) events per division for somatic recombination, a much smaller difference than between meiotic and mitotic recombination in yeast. Southern analysis showed that recombinants could be formed by either crossing over or gene conversion. A surprisingly high fraction (at least 2/17) of the recombinants, however, appeared to result from the concerted action of two or more independent simple events. Some evolutionary implications are discussed.


Reference electronic structure calculations in one dimension.
Large strongly correlated systems provide a challenge to modern electronic structure methods, because standard density functionals usually fail and traditional quantum chemical approaches are too demanding. The density-matrix renormalization group method, an extremely powerful tool for solving such systems, has recently been extended to handle long-range interactions on real-space grids, but is most efficient in one dimension where it can provide essentially arbitrary accuracy. Such 1d systems therefore provide a theoretical laboratory for studying strong correlation and developing density functional approximations to handle strong correlation, if they mimic three-dimensional reality sufficiently closely. We demonstrate that this is the case, and provide reference data for exact and standard approximate methods, for future use in this area.


The communicative potential of bat echolocation pulses.
Ecological constraints often shape the echolocation pulses emitted by bat species. Consequently some (but not all) bats emit species-specific echolocation pulses. Because echolocation pulses are often intense and emitted at high rates, they are potential targets for eavesdropping by other bats. Echolocation pulses can also vary within species according to sex, body size, age, social group and geographic location. Whether these features can be recognised by other bats can only be determined reliably by playback experiments, which have shown that echolocation pulses do provide sufficient information for the identification of sex and individual in one species. Playbacks also show that bats can locate conspecifics and heterospecifics at foraging and roost sites by eavesdropping on echolocation pulses. Guilds of echolocating bat species often partition their use of pulse frequencies. Ecology, allometric scaling and phylogeny play roles here, but are not sufficient to explain this partitioning. Evidence is accumulating to support the hypothesis that frequency partitioning evolved to facilitate intraspecific communication. Acoustic character displacement occurs in at least one instance. Future research can relate genetic population structure to regional variation in echolocation pulse features and elucidate those acoustic features that most contribute to discrimination of individuals.


Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III.
DNA polymerase III, the main replicative polymerase of E. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. It was recently discovered that E. coli bacteriophage P1 encodes a theta homolog, named HOT. The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR. The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different. Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT. As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein.


fMRI localizer technique: efficient acquisition and functional properties of single retinotopic positions in the human visual cortex.
Current fMRI retinotopic mapping procedures often use checkerboard stimuli consisting of expanding rings and rotating wedges to measure the topography within human visual areas. Efficient procedures are well described in the literature. For many experimental paradigms, e.g., visuo-spatial attention paradigms, the identification of task-relevant positions is the only mandatory prerequisite. To define these specific "regions-of-interest" (ROIs), spatially defined localizers are used. A precise evaluation of localizer techniques in regard to efficient scanning time, optimal BOLD (blood oxygenic level dependent) response, as well as quantification of the resulting ROIs within each visual area (size, overlap, surround effects) has not been studied to date. Here, we suggest a mapping procedure designed to quantify spatial and functional properties of single positions at close proximity in multiple human visual areas. During a passive viewing task, various stimuli (e.g., checkerboards or colored objects) subtending 1.4 degrees of visual angle were presented at one out of four positions in a randomized block design. We measured the degree of overlap between positions at different hierarchical levels of the visual system (V1-V4v) and quantified modulatory effects on a specific position by stimulation at neighboring (1.7 degrees spacing) or distant positions (5.1 degrees or 8.5 degrees spacing). Within each visual area, "mexican-hat" distributions of local signal intensity changes, which describe a particular combination of facilitatory and suppressive effects, were found. Cubic fitting revealed the most localized tuning effect in V1, which gradually decreased throughout the higher visual areas. Colored objects were most efficient in localizing circumscribed retinotopic positions in both early and higher areas.


Proteomic Analysis and Functional Validation of a Brassica oleracea Endochitinase Involved in Resistance to Xanthomonas campestris.
Black rot is a severe disease caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), which can lead to substantial losses in cruciferous vegetable production worldwide. Although the use of resistant cultivars is the main strategy to control this disease, there are limited sources of resistance. In this study, we used the LC-MS/MS technique to analyze young cabbage leaves and chloroplast-enriched samples at 24 h after infection by Xcc, using both susceptible (Veloce) and resistant (Astrus) cultivars. A comparison between susceptible Xcc-inoculated plants and the control condition, as well as between resistant Xcc-inoculated plants with the control was performed and more than 300 differentially abundant proteins were identified in each comparison. The chloroplast enriched samples contributed with the identification of 600 additional protein species in the resistant interaction and 900 in the susceptible one, which were not detected in total leaf sample. We further determined the expression levels for 30 genes encoding the identified differential proteins by qRT-PCR. CHI-B4 like gene, encoding an endochitinase showing a high increased abundance in resistant Xcc-inoculated leaves, was selected for functional validation by overexpression in Arabidopsis thaliana. Compared to the wild type (Col-0), transgenic plants were highly resistant to Xcc indicating that CHI-B4 like gene could be an interesting candidate to be used in genetic breeding programs aiming at black rot resistance.


Perineuronal Nets, Inhibitory Interneurons, and Anxiety-Related Ventral Hippocampal Neuronal Oscillations Are Altered by Early Life Adversity.
In humans, accumulated adverse experiences during childhood increase the risk of anxiety disorders and attention-deficit/hyperactivity disorder. In rodents, the ventral hippocampus (vHIP) is associated with anxiety regulation, and lesions in this region alter both anxiety-like behavior and activity levels. Neuronal oscillations in the vHIP of the theta frequency range (4-12 Hz) have been implicated in anxious states and derive in part from the activity of inhibitory interneurons in the hippocampus, some of which are enwrapped with perineuronal nets (PNNs), extracellular matrix structures known to regulate plasticity. We sought to investigate the associations among early life stress-induced anxiety and hyperactivity with vHIP neuronal oscillations, inhibitory interneurons, and PNNs in mice. We used repeated maternal separation with early weaning (MSEW) to model accumulated early life adversity in mouse offspring and studied the underlying cellular and electrophysiological changes in the vHIP that are associated with excessive anxiety and hyperactivity. We found increased anxiety-like behavior and activity levels in MSEW adult males, along with increased theta power and enhanced theta-gamma coupling in the vHIP. MSEW mice showed reduced intensity of parvalbumin as well as increased PNN intensity around parvalbumin-positive interneurons in the vHIP. We further observed that MSEW increased orthodenticle homeobox protein 2, a transcription factor promoting PNN development, in the choroid plexus, where it is produced, as well as in parvalbumin-positive interneurons, where it is sequestered. These findings raise the possibility of causal links among parvalbumin-positive interneurons, PNNs, orthodenticle homeobox protein 2, and MSEW-induced anxiety and hyperactivity.


Comparison of p24 measurement by ELISA versus indicator cells for detecting residual HIV infectivity in vitro.
Inactivation of HIV-1 contaminated materials or biological samples is of great importance and requires the use of a reliable assay to detect residual infectivity. In this study we treated cell-free or cell-associated (monocyte-derived macrophages) HIV-1 with two chemicals known for their antiviral activities, beta-propiolactone (beta PL) and formaldehyde (FO), and tested it for the presence of residual infectivity. HIV-1 infected primary monocyte-derived macrophages (MDM) or cell-free HIV-1 were fixed with increasing concentrations of either beta PL or FO for 1 day at 4 degrees C. Then either fresh primary MDM or fresh medium was added, and the supernatant p24 levels were assayed up to 12 days after infection. All the supernatants harvested were added to indicator cells, fresh primary MDM, to assess for residual infectivity. The results show that p24 measurement is not a reliable assay for the detection of residual infectious virions after chemical fixation of HIV-infected primary MDM. In contrast, the use of indicator primary cells (MDM) is a much more sensitive and reliable assay. By performing an indicator cell assay we showed that FO efficiently inactivates cell-associated and cell-free HIV-1 at concentrations as low as 1% v/v. In contrast beta PL is more efficient in inactivating cell-free than cell-associated virus and does not inactivate cell-associated HIV-1 at concentrations as high as 1% v/v.


Selective Small Molecule Recognition of RNA Base Pairs.
Many types of RNAs exist in the human transcriptome, yet only the bacterial ribosome has been exploited as a small molecule drug target. Aside from rRNA, other cellular RNAs such as noncoding RNAs have primarily secondary structure and limited tertiary structure. Within these secondary structures of noncanonically paired and unpaired regions, more than 50% are base paired, with most efforts to target these structures focused on looped regions. A void exists in the availability of small molecules capable of targeting RNA base pairs. Using chemoinformatics, an RNA-focused library enriched for nitrogen-containing heterocycles was developed and tested for binding RNA base pairs, leading to the identification of six selective and previously unknown binders. While all binders were derivatives of benzimidazoles, those with expanded aromatic polycycles bound selectively to AU pairs, while those with flexible urea side chains bound selectively to GC pairs. Two of the three selective GC pair binders can distinguish between two different orientations, 5'GG/3'CC and 5'GC/3'CG pairs. Furthermore, all six molecules showed >50-fold selectivity for RNA over DNA. These studies provide foundational knowledge to better exploit RNA as targets for small molecule chemical probes or lead therapeutics by using modules that target RNA base pairs.


Recombinant toxins for cancer treatment.
Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.


Meta-barcoding of 'dirt' DNA from soil reflects vertebrate biodiversity.
DNA molecules originating from animals and plants can be retrieved directly from sediments and have been used for reconstructing both contemporary and past ecosystems. However, the extent to which such 'dirt' DNA reflects taxonomic richness and structural diversity remains contentious. Here, we couple second generation high-throughput sequencing with 16S mitochondrial DNA (mtDNA) meta-barcoding, to explore the accuracy and sensitivity of 'dirt' DNA as an indicator of vertebrate diversity, from soil sampled at safari parks, zoological gardens and farms with known species compositions. PCR amplification was successful in the full pH range of the investigated soils (6.2 ± 0.2 to 8.3 ± 0.2), but inhibition was detected in extracts from soil of high organic content. DNA movement (leaching) through strata was evident in some sporadic cases and is influenced by soil texture and structure. We find that DNA from the soil surface reflects overall taxonomic richness and relative biomass of individual species. However, one species that was recently introduced was not detected. Furthermore, animal behaviour was shown to influence DNA deposition rates. The approach potentially provides a quick methodological alternative to classical ecological surveys of biodiversity, and most reliable results are obtained with spatial sample replicates, while relative amounts of soil processed per site is of less importance.


Modern-day environmental factors in the pathogenesis of osteoarthritis.
The prevalence of osteoarthritis (OA) is rising for reasons that are not fully understood. In this Opinion article, we review the possibility that OA is an evolutionary mismatch disease, which is a disease more common today than in the past because genes inherited from previous generations are inadequately or imperfectly adapted to modern environmental conditions. We focus on four major environmental factors in OA pathogenesis that have become ubiquitous within the past half-century: obesity, metabolic syndrome, dietary changes and physical inactivity. Because a cure for OA does not yet exist, prevention strategies that target these modifiable environmental factors are needed to curb further increases in OA prevalence.


True nutrient and amino acid digestibility of dog foods made with human-grade ingredients using the precision-fed cecectomized rooster assay.
For a pet diet to be labeled as human-grade, every ingredient and the finished food must be stored, handled, processed, and transported according to the current good manufacturing practices for human edible foods. Human-grade dog foods are now available and increasing in popularity, but little research has been conducted to test the digestibility of these foods. For this reason, the objective of this experiment was to determine the true nutrient and amino acid (AA) digestibilities of dog foods formulated with human-grade ingredients using the precision-fed cecectomized rooster assay. Six commercial dog foods were tested, including the Beef & Russet Potato (BRP), Chicken & White Rice (CWR), Fish & Sweet Potato (FSP), Lamb & Brown Rice (LBR), Turkey & Whole Wheat Macaroni (TWM), and Venison & Squash (VSR) formulas provided by Just Food For Dogs LLC (Irvine, CA). Before analysis, all foods were lyophilized and ground. A precision-fed rooster assay using cecectomized roosters was conducted to determine the true nutrient digestibility and standardized AA digestibilities of the foods tested. Conventional roosters were used to determine the nitrogen-corrected true metabolizable energy (TMEn) of the foods. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation. The substrates and rooster excreta were analyzed for macronutrient and AA composition. All data were analyzed using the Mixed Models procedure of SAS (version 9.4; SAS Institute, Cary, NC). In general, all foods tested were highly digestible. Dry matter digestibility was similar among CWR, LBR, and TWR foods, and greater (P < 0.0001) than that of FSP and VSR foods. Organic matter digestibility was highest (P = 0.0002) for CWR and lowest (P = 0.0002) for VSR. For the majority of indispensable AA, digestibilities were greater than 85%, with some being greater than 90%. TMEn was higher (P < 0.0001) for BRP than the other foods, which were similar to one another. Also, TMEn values were much higher than what would be estimated by using modified Atwater factors and often above the predictive equations for metabolizable energy (ME) recommended by the National Research Council or by using Atwater factors. Although statistical differences were observed among foods, they all performed well and the foods tested had very high AA digestibilities. Additionally, the TMEn data suggest that existing methods and equations for ME prediction underestimate the energy content of the foods tested.


Lsp family proteins regulate antibiotic biosynthesis in Lysobacter enzymogenes OH11.
Ax21 family proteins have been shown to play regulatory roles in plant- and animal-pathogenic species in the bacterial family Xanthomonadaceae, but the protein have not been investigated previously in the non-pathogenic members of this bacterial family. Lysobacter enzymogenes, is a non-pathogenic species known for its capacity as a biocontrol agent of plant pathogens. It is also noted for the production of antimicrobial secondary metabolites, heat stable antifungal factor (HSAF) and WAP-8294A2, that have potential for agricultural and pharmaceutical applications. The species also displays type IV pili-dependent twitching motility and the production of multiple extracellular lytic enzymes as additional biocontrol-related traits. Here, we show that L. enzymogenes strain OH11 possesses three genes widely separated in the OH11 genome that code for unique Ax21-like proteins (Lsp). By comparing the wildtype OH11 with mutant strains having a single lsp gene or a combination of lsp genes deleted, we found that each Lsp protein individually is involved in positive regulation of HSAF and WAP-8294A2 biosynthesis, but the proteins collectively do not exert additive effects in this regulation. None of the Lsp proteins were found to influence twitching motility or the production of three extracellular lytic enzymes. This study is the first to provide evidence linking Ax21-family proteins to antibiotic biosynthesis and, hence, adds new insights into the diversity of regulatory functions of Ax21 family proteins in bacteria.


A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.
Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.


PM-IRRAS Determination of Molecular Orientation of Phosphonic Acid Self-Assembled Monolayers on Indium Zinc Oxide.
Self-assembled monolayers (SAMs) of phosphonic acids (PAs) on transparent conductive oxide (TCO) surfaces can facilitate improvement in TCO/organic semiconductor interface properties. When ordered PA SAMs are formed on oxide substrates, interface dipole and electronic structure are affected by the functional group properties, orientation, and binding modes of the modifiers. Choosing octylphosphonic acid (OPA), F13-octylphosphonic acid (F13OPA), pentafluorophenyl phosphonic acid (F5PPA), benzyl phosphonic acid (BnPA), and pentafluorobenzyl phosphonic acid (F5BnPA) as a representative group of modifiers, we report polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS) of binding and molecular orientation on indium-doped zinc oxide (IZO) substrates. Considerable variability in molecular orientation and binding type is observed with changes in PA functional group. OPA exhibits partially disordered alkyl chains but on average the chain axis is tilted ∼57° from the surface normal. F13OPA tilts 26° with mostly tridentate binding. The F5PPA ring is tilted 23° from the surface normal with a mixture of bidentate and tridentate binding; the BnPA ring tilts 31° from normal with a mixture of bidentate and tridentate binding, and the F5BnPA ring tilts 58° from normal with a majority of bidentate with some tridenate binding. These trends are consistent with what has been observed previously for the effects of fluorination on orientation of phosphonic acid modifiers. These results from PM-IRRAS are correlated with recent results on similar systems from near-edge X-ray absorption fine structure (NEXAFS) and density functional theory (DFT) calculations. Overall, these results indicate that both surface binding geometry and intermolecular interactions play important roles in dictating the orientation of PA modifiers on TCO surfaces. This work also establishes PM-IRRAS as a routine method for SAM orientation determination on complex oxide substrates.


Mixed Adenoneuroendocrine Carcinoma of the Esophagus: A Case Report and Review of the Literature.
The most frequent presentation of esophageal cancer is adenocarcinoma and squamous cell carcinoma. In recent years, the latter has decreased its incidence increasing the adenocarcinoma. Currently, another type of tumor with a much lower incidence has been described, which has a neuroendocrine component along with another exocrine glandular component and has been classified since 2010 as mixed adenoneuroendocrine carcinoma (MANEC). We present the case of a 68-year-old male with a history of dyspepsia and epigastric pain who after performing a gastroscopy, was diagnosed with a malignant neoplasm of the esophagus. The patient underwent a total esophagectomy with reconstruction by tubular gastroplasty with cervical anastomosis. The final result of the piece after immunohistochemistry revealed that the tumor was composed of one component of adenocarcinoma in 60% together with another component compatible with neuroendocrine in 40%. With these findings and according to the World Health Organization classification of 2010 was diagnosed as esophageal MANEC. MANECs are rare tumors, described in other locations of the digestive tract, the esophagus being an infrequent location. Its preoperative diagnosis is difficult, and it is not until the final analysis of the complete piece by means of specific immunohistochemical techniques when its diagnosis can be established. Its treatment is fundamentally surgical, whereas the adjuvant therapeutic schemes with chemotherapy are not well defined at present because of their low incidence.


Age-related susceptibility and resistance to nonlethal Plasmodium yoelii infection in C57BL/6 mice.
In cases of human malaria, children suffer very high rates of morbidity and mortality. To analyse the mechanisms involved in age-dependent protection against malaria, we investigated the characterization of immune responses to Plasmodium yoelii 17XNL (P.y 17XNL) in young (3 weeks) and middle-aged (8 months) C57BL/6 mice. In this study, we found that 100% of young mice succumbed to P.y 17XNL infection with higher parasitemia, while middle-aged mice were able to clear blood parasites and no mortality was observed. These observations suggested that the young C57BL/6 mice were susceptible to P.y 17XNL infection, whereas the middle-aged mice were resistant. Cellular analysis revealed that both the numbers of splenic myeloid dendritic cells (mDCs) as well as the expression of DC maturation markers were higher in middle-aged mice than those in young mice. The numbers of IgG1- or IgG2a-secreting B cells increased markedly in middle-aged mice after infection with P.y 17XNL. The dynamic change of the number of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in mice infected with P.y 17XNL was also different between the two groups. In addition, the levels of IFN-gamma and NO increased in both groups during early parasite infection, while there was also an obvious increase in IL-4 production in the infected middle-aged mice. The change in IL-10 levels following infection was consistent with that of the change in the number of Tregs. The survival of middle-aged mice following P.y 17XNL infection was dependent upon the establishment of effective Thl and Th2 responses and a successful switch between Th1 and Th2 responses, as well as appropriate functioning of Tregs.


The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism.
Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolism by corepressing MYC transcriptional activity. Lastly, Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.


The absence of adult mortality data for sub-Saharan Africa: a practical solution.
Information on cause of death among adults in sub-Saharan Africa is essentially nonexistent. Published sources provide statistics on both cause-specific and overall rates of mortality, but closer examination reveals that these data consist mostly of extrapolations and outright guesses. In the absence of accurate and comprehensive registries of vital events for the majority of the region's inhabitants, longitudinal studies of defined population-based cohorts represent the only realistic strategy to fill this void in basic public health information. The advantage of longitudinal studies is particularly clear for chronic diseases, the category for which the least is known. Noncommunicable diseases account for a significant portion of adult deaths in sub-Saharan Africa, yet the empirical bases for public health policies and interventions are essentially absent. Verbal autopsy has great potential to contribute to understanding about the cause of death among African adults. This method is discussed in the present article, and practical considerations for longitudinal studies using this methodology are reviewed.


Crystal structures of YBHB and YBCL from Escherichia coli, two bacterial homologues to a Raf kinase inhibitor protein.
In rat and human cells, RKIP (previously known as PEBP) was characterized as an inhibitor of the MEK phosphorylation by Raf-1. In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found. The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well. We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E. coli, respectively, after which we determined their crystallographic structures. These structures verify that YBHB and YBCL belong to the same structural family as mammalian RKIP/PEBP proteins. The general fold and the anion binding site of these proteins are extremely well conserved between mammals and bacteria and suggest functional similarities. However, the bacterial proteins also exhibit some specific structural features, like a substrate binding pocket formed by the dimerization interface and the absence of cis peptide bonds. This structural variety should correspond to the recognition of multiple cellular partners.


Irreversible Electroporation for Hepatic Tumors: Protocol Standardization Using the Modified Delphi Technique.
A consensus study of panelists was performed to provide a uniform protocol regarding (contra) indications, procedural parameters, perioperative care, and follow-up of irreversible electroporation (IRE) for the treatment of hepatic malignancies. Interventional radiologists who had 2 or more publications on IRE, reporting at least 1 patient cohort in the field of hepatobiliary IRE, were recruited. The 8 panelists were asked to anonymously complete 3 iterative rounds of IRE-focused questionnaires to collect data according to a modified Delphi technique. Consensus was defined as having reached 80% or greater agreement. Panel members' response rates were 88%, 75%, and 88% in rounds 1, 2, and 3, respectively; consensus was reached on 124 of 136 items (91%). Percutaneous or intraoperative hepatic IRE should be considered for unresectable primary and secondary malignancies that are truly unsuitable for thermal ablation because of proximity to critical structures. Absolute contraindications are ventricular arrhythmias, cardiac stimulation devices, and congestive heart failure of New York Heart Association class 3 or higher. A metal stent outside the ablation zone should not be considered a contraindication. For the only commercially available IRE device, the recommended settings are an inter-electrode distance of 10-20 mm and an exposure length of 20 mm. After 10 test pulses, 90 treatment pulses of 1500 V/cm should be delivered continuously, with a pulse length of 70-90 μs. The first post-procedural follow-up should take place 1 month after IRE and thereafter every 3 months, using cross-sectional imaging plus tumor marker assessment. This article provides recommendations, created by a modified Delphi consensus study, regarding patient selection, workup, procedure, and follow-up of IRE treatment for hepatic malignancies.


Engineering a light-responsive, quorum quenching biofilm to mitigate biofouling on water purification membranes.
Quorum quenching (QQ) has been reported to be a promising approach for membrane biofouling control. Entrapment of QQ bacteria in porous matrices is required to retain them in continuously operated membrane processes and to prevent uncontrollable biofilm formation by the QQ bacteria on membrane surfaces. It would be more desirable if the formation and dispersal of biofilms by QQ bacteria could be controlled so that the QQ bacterial cells are self-immobilized, but the QQ biofilm itself still does not compromise membrane performance. In this study, we engineered a QQ bacterial biofilm whose growth and dispersal can be modulated by light through a dichromatic, optogenetic c-di-GMP gene circuit in which the bacterial cells sense near-infrared (NIR) light and blue light to adjust its biofilm formation by regulating the c-di-GMP level. We also demonstrated the potential application of the engineered light-responsive QQ biofilm in mitigating biofouling of water purification forward osmosis membranes. The c-di-GMP-targeted optogenetic approach for controllable biofilm development we have demonstrated here should prove widely applicable for designing other controllable biofilm-enabled applications such as biofilm-based biocatalysis.


Assessment of exposure to shiftwork mechanisms in the general population: the development of a new job-exposure matrix.
To develop a job-exposure matrix (JEM) that estimates exposure to eight variables representing different aspects of shiftwork among female workers. Occupational history and shiftwork exposure data were obtained from a population-based breast cancer case-control study. Exposure to light at night, phase shift, sleep disturbances, poor diet, lack of physical activity, lack of vitamin D, and graveyard and early morning shifts, was calculated by occupational code. Three threshold values based on the frequency of exposure were considered (10%, 30% and 50%) for use as cut-offs in determining exposure for each occupational code. JEM-based exposure classification was compared with that from the OccIDEAS application (job-specific questionnaires and assessment by rules) by assessing the effect on the OR for phase shift and breast cancer. Using data from the Australian Workplace Exposure Study, the specificity and sensitivity of the threshold values were calculated for each exposure variable. 127 of 413 occupational codes involved exposure to one or more shiftwork variables. Occupations with the highest probability of exposure shiftwork included nurses and midwives. Using the 30% threshold, the OR for the association between phase shift exposure and breast cancer was decreased and no longer statistically significant (OR=1.14, 95% CI 0.92 to 1.42). The 30% cut-off point demonstrated best specificity and sensitivity, although results varied between exposure variables. This JEM provides a set of indicators reflecting biologically plausible mechanisms for the potential impact of shiftwork on health and may provide an alternative method of exposure assessment in the absence of detailed job history and exposure data.


Efficiency control of dietary pesticide intake reduction by human biomonitoring.
In spite of food safety controls for pesticide residues, a conventional diet still leads to a noticeable exposure of the general population to several pesticides. In a pilot study the response of exposure reduction by organic diet intervention on the urinary levels of pesticide metabolites was investigated. In the study two adult individuals were kept on a conventional diet for 11days and morning urine voids were collected at the last four days of the period. Afterwards, the participants switched to exclusively organic food intake for 18days and likewise morning urine samples were collected at the last four days of this period. In the urine samples six pyrethroid metabolites, six dialkylphosphates, four phenolic parameter for organophosphate pesticides and carbamates, 6-chloronicotinic acid (ClNA) as parameter for neonicotinoid insecticides, seven phenoxy herbicides, glyphosate and its metabolite AMPA were quantified using gas chromatographic mass spectrometric methods. Generally, the comparative analyses revealed greater shares as well as higher levels of the parameters in the samples taken during the common diet period compared to the organic diet period. Considerable decrease of the levels was found for almost all pyrethroid metabolites, dialkyphosphates and phenoxy herbicids, as well as for the phenolic metabolites 4-nitrophenol and 3,5,6-trichloropyridinol. In contrast, higher values were found for the organic diet period for ClNA and the metabolite of coumaphos in one of the volunteers. The present study confirms the results of former studies which indicated that an organic diet intervention results in considerable lower exposure to organophosphate pesticides and pyrethroids. It also verifies the former experience that monitoring of urinary parameters for non-persistent pesticides permits a reliable efficiency control of short-time effects by dietary interventions. Additionally to former studies, the results of the present study highlight the need of an extension of the parameter spectrum to all prominent pesticide groups.


The Spatial Profile of Macular Pigments Is Related to the Topological Characteristics of the Foveal Avascular Zone.
Macular pigments are preferentially concentrated in the central fovea, an area devoid of vasculature. We hypothesized that there may be a link between the macular pigment profile and the size and structural characteristics of the foveal avascular zone (FAZ). Two-wavelength autofluorescence method was used to quantify macular pigment optical density (MPOD) and the radius at half peak of MPOD, which was defined as the retinal eccentricity where the MPOD value was 50% of the peak value. Volumetric spectral-domain optical coherence tomography (OCT) images of the macula were obtained from 32 subjects. The equivalent radius of the FAZ was determined using data generated from OCT angiography. Generalized estimating equations were used to test the hypothesis that there are interrelationships among the central foveal thickness, peak MPOD, the radius at half peak of MPOD and the equivalent radius of the FAZ. The equivalent radius of the FAZ was highly correlated with the radius at half peak of MPOD (P < .001). The equivalent radius of the FAZ was a significant predictor for central foveal thickness (P < .001). The significant predictor for peak MPOD was central foveal thickness (P = .004). Eyes with larger FAZs were more likely to have a secondary peak in their MPOD spatial profile in a zone ranging from 0.5 to 1.0 degrees from the foveal center. The spatial distribution of macular pigment is related to the size of the FAZ, in addition to the central foveal thickness. It is possible that xanthophyll pigment accumulation in the macula serves functions, such as attenuation of shorter wavelengths of light, that would have been provided by the light-filtering characteristics of blood vessels.


Linking soil microbial communities to vascular plant abundance along a climate gradient.
The ongoing expansion of shrub cover in response to climate change represents a unique opportunity to explore the link between soil microbial communities and vegetation changes. This link is particularly important in peatlands where shrub expansion is expected to feed back negatively on the carbon sink capacity of these ecosystems. Microbial community structure and function were measured seasonally in four peatlands located along an altitude gradient representing a natural gradient of climate and associated vascular plant abundance. We show that increased soil temperature and reduced water content are associated with greater vascular plant biomass, in particular that of ericoids, and that this, in turn, is correlated with greater microbial biomass. More specifically, microbial community structure is characterized by an increasing dominance of fungi over bacteria with improved soil oxygenation. We also found that the carbon and nitrogen stoichiometry of microbial biomass differs in relation to soil microbial community structure and that this is ultimately associated with a different investment in extracellular enzymatic activity. Our findings highlight the fact that the determination of the structural identity of microbial communities can help to explain the biogeochemical dynamics of organic matter and provide a better understanding of ecosystem response to environmental changes.


Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives.
The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.


RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing.
Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.


Growth mixture models of adaptive behavior in adolescents with autism spectrum disorder.
This study examined growth trajectories of teacher-reported adaptive behavior in a diverse sample of adolescents with autism spectrum disorder. The participants were 244 adolescents between the ages of 14 and 21 years who were assessed at up to four time points across two and a half years of high school. Demographic variables (age, sex, race, maternal education), phenotypic characteristics (intelligence quotient, autism severity) and school factors (location of the school, school quality) were collected. Growth mixture modeling was used to identify distinct classes of growth trajectories in communication, daily living skills, and socialization domains of adaptive behavior. Two distinct classes were identified for each domain. The first class had moderately low adaptive behavior scores and demonstrated growth of adaptive behavior over time and the second class had low adaptive behavior scores and did not demonstrate change over time. Adolescents within the moderately low adaptive behavior classes were younger at enrollment in the study, had higher IQs, and lower autism symptom severity. Logistic regressions were performed, and aspects of school quality predicted the likelihood of being in the moderately low classes above and beyond autism symptoms.


Predictors of full enteral feeding achievement in very low birth weight infants.
To elucidate the role of prenatal, neonatal and early postnatal variables in influencing the achievement of full enteral feeding (FEF) in very low birth weight (VLBW) infants and to determine whether neonatal intensive care units (NICUs) differ in this outcome. Population-based retrospective cohort study using data on 1,864 VLBW infants drawn from the "Emilia-Romagna Perinatal Network" Registry from 2004 to 2009. The outcome of interest was time to FEF achievement. Eleven prenatal, neonatal and early postnatal variables and the study NICUs were selected as potential predictors of time to FEF. Parametric survival analysis was used to model time to FEF as a function of the predictors. Marginal effects were used to obtain adjusted estimates of median time to FEF for specific subgroups of infants. Lower gestational age, exclusive formula feeding, higher CRIB II score, maternal hypertension, cesarean delivery, SGA and PDA predicted delayed FEF. NICUs proved to be heterogeneous in terms of FEF achievement. Newborns with PDA had a 4.2 days longer predicted median time to FEF compared to those without PDA; newborns exclusively formula-fed had a 1.4 days longer time to FEF compared to those fed human milk. The results of our study suggest that time to FEF is influenced by clinical variables and NICU-specific practices. Knowledge of the variables associated with delayed/earlier FEF achievement could help in improving specific aspects of routine clinical management of VLBW infants and to reduce practice variability.


Evaluating the decisional balance construct of the Transtheoretical Model: are two dimensions of pros and cons really enough?
The Transtheoretical Model of behavior change (TTM) postulates that behavior change is a process involving progress through five distinct stages of change (SOC). One of the key components for progress to a later stage is decisional balance (pros and cons of changing to the target behavior). The goal of the present study is to test the two dimensions of decisional balance as postulated in the TTM in the context of exercising behavior. The analyses are based on data from an online survey of 266 freshman students at the University of Zurich; participants self-reported their frequency of exercising and their weighing of the importance of 49 pros and cons of exercising. The results indicate that a two-dimensional solution of decisional balance is insufficient. The analysis of pros and cons of exercising yielded a seven-factor solution with in part different progressions through the SOC. With the subdivision into different pros and cons, intervention programs can be developed that better match the needs of participants in terms of fostering and decreasing the most important pros and cons of exercising.


Variable-temperature NMR spectroscopy for metabolite identification in biological materials.
Nuclear magnetic resonance is a "workhorse technique" used in metabolomics, complementary to mass spectrometry. Unfortunately, only the most basic NMR methods are sensitive enough to allow fast medical screening. The most common of them, a simple 1H NMR, suffers from low dispersion of resonance frequencies, which often hampers the identification of metabolites. In this article we show that 1H NMR spectra contain previously overlooked parameters potentially helpful in metabolite identification, namely the rates of temperature-induced changes of chemical shifts. We prove that they are reproducible between various metabolite mixtures and can be determined quickly when Radon transform is used to process the data.


Audit of the dietetic care of patients with type 1 diabetes at a large Australian paediatric tertiary hospital and comparison with the International Society for Paediatric and Adolescent Diabetes nutrition guidelines.
Optimal glycaemic control is important in children and adolescents with type 1 diabetes (T1D) to reduce the risk of developing microvascular complications. Modest reductions in HbA1c translate to significant risk reductions of these complications. Research indicates that targeted dietetic care using evidence-based guidelines improves HbA1c levels. The aim of this study was to audit the dietetic care provided to patients with T1D at the Women's and Children's Hospital (WCH) and compare the service provided to the International Society for Paediatric and Adolescent Diabetes (ISPAD) nutrition guidelines. A retrospective medical records audit of the dietetic service was conducted on 410 (99% of eligible) patients who attended the WCH Diabetes Outpatient Clinic between 1 January 2010 and 30 June 2013. These data were then compared to the ISPAD nutrition guidelines, with a focus on the key service delivery targets. Of the newly diagnosed patients (n = 128), 100% received initial dietary advice by a paediatric diabetes dietitian; 61% of these patients then received a follow-up appointment within one month of diagnosis. Of those who were >1 year post-diagnosis (n = 389), only 6% were seen annually; 79% of patients were identified with a condition requiring more frequent review, of whom 23% were seen more frequently. This large and comprehensive audit, conducted for a 3.5-year period, has shown that the dietetic care of newly diagnosed children and adolescents is mostly in line with ISPAD guidelines, but there is suboptimal follow-up after diagnosis.


Adaptive choice of the number of bootstrap samples in large scale multiple testing.
It is a common practice to use resampling methods such as the bootstrap for calculating the p-value for each test when performing large scale multiple testing. The precision of the bootstrap p-values and that of the false discovery rate (FDR) relies on the number of bootstraps used for testing each hypothesis. Clearly, the larger the number of bootstraps the better the precision. However, the required number of bootstraps can be computationally burdensome, and it multiplies the number of tests to be performed. Further adding to the computational challenge is that in some applications the calculation of the test statistic itself may require considerable computation time. As technology improves one can expect the dimension of the problem to increase as well. For instance, during the early days of microarray technology, the number of probes on a cDNA chip was less than 10,000. Now the Affymetrix chips come with over 50,000 probes per chip. Motivated by this important need, we developed a simple adaptive bootstrap methodology for large scale multiple testing, which reduces the total number of bootstrap calculations while ensuring the control of the FDR. The proposed algorithm results in a substantial reduction in the number of bootstrap samples. Based on a simulation study we found that, relative to the number of bootstraps required for the Benjamini-Hochberg (BH) procedure, the standard FDR methodology which was the proposed methodology achieved a very substantial reduction in the number of bootstraps. In some cases the new algorithm required as little as 1/6th the number of bootstraps as the conventional BH procedure. Thus, if the conventional BH procedure used 1,000 bootstraps, then the proposed method required only 160 bootstraps. This methodology has been implemented for time-course/dose-response data in our software, ORIOGEN, which is available from the authors upon request.


Unusual heme binding properties of the dissimilative nitrate respiration regulator, a bacterial nitric oxide sensor.
In the opportunistic pathogen Pseudomonas aeruginosa, nitric oxide (NO) triggers the respiration of nitrate (denitrification), thus allowing survival in chronic infection sites as a microaerobic-anaerobic biofilm. The NO-dependent induction of denitrification is mediated by the dissimilative nitrate respiration regulator (DNR), a transcription factor forming a stable complex with heme, which is required to sense the physiological messenger (i.e., NO). The molecular details of NO sensing in DNR and, more in general, in this class of sensors are largely unknown, and a study aimed at integrating microbiology and biochemistry is needed. Here we present a comprehensive study, including in vivo results and spectroscopy, kinetics, and protein engineering, that demonstrates the direct involvement of a histidine residue in heme iron coordination. Moreover, a peculiar phenomenon of ligand switching around heme iron, which hampers the identification of the second heme axial ligand, is also suggested. These results indicate that DNR is characterized by a remarkable flexibility in solution, as observed for other cAMP receptor protein/fumarate and nitrate reductase regulators (CRP-FNR) to which DNR belongs. The present work represents one of the few studies focused on the biochemistry of NO sensing by bacterial transcriptional regulators. The data presented demonstrate that structural plasticity of DNR is crucial for the sensing activity and confers to the protein unusual heme binding properties. Protein flexibility and dynamics is a key structural feature essential to explain the evolutionary success and adaptability of CRP-FNR, and may represent a common strategy employed by heme-based redox sensors, which presents features deeply different from those of canonical hemeproteins.


Immunoscore System for Predicting Clinical Outcome of Metastatic Renal Cell Carcinoma Patients Treated with Tyrosine Kinase Inhibitors.
We conducted this study to determine whether immunoscore system (IS) predicts survival in patients with metastatic renal cell carcinoma (mRCC). A total of 218 mRCC patients treated with sunitinib or sorafenib in Zhongshan Hospital, Fudan University were recruited during 2007-2017, retrospectively. CD8, CD4, Treg, PD-1 and PD-L1 expression were evaluated by immunohistochemical staining of paraffin embedded slide. Kaplan-Meier method and COX regression model were used in survival analyses. Multivariate analyses demonstrated that expressions of CD8, Treg, PD-1 and stromal PD-L1 (sPD-L1) expressions were independent predictive factors for OS, thus IS was established containing these four immunological factors. Subsequent analysis revealed that performance of IS provided good differentiation of OS and PFS. Besides, multivariate analysis identified IS as an independent prognostic factor for OS (p<0.001) and PFS (p=0.002). IS, compared with International mRCC Database Consortium (IMDC) risk model, and provided better prediction ability for OS. Results suggested that IS was a powerful prognostic factor for OS and PFS in patients with mRCC treated with tyrosine kinase inhibitors. And IS can be used as essential supplement to IMDC for outcome prediction in mRCC patients.


Associations of Maternal Vitamin B12 Concentration in Pregnancy With the Risks of Preterm Birth and Low Birth Weight: A Systematic Review and Meta-Analysis of Individual Participant Data.
Vitamin B12 (hereafter referred to as B12) deficiency in pregnancy is prevalent and has been associated with both lower birth weight (birth weight <2,500 g) and preterm birth (length of gestation <37 weeks). Nevertheless, current evidence is contradictory. We performed a systematic review and a meta-analysis of individual participant data to evaluate the associations of maternal serum or plasma B12 concentrations in pregnancy with offspring birth weight and length of gestation. Twenty-two eligible studies were identified (11,993 observations). Eighteen studies were included in the meta-analysis (11,216 observations). No linear association was observed between maternal B12 levels in pregnancy and birth weight, but B12 deficiency (<148 pmol/L) was associated with a higher risk of low birth weight in newborns (adjusted risk ratio = 1.15, 95% confidence interval (CI): 1.01, 1.31). There was a linear association between maternal levels of B12 and preterm birth (per each 1-standard-deviation increase in B12, adjusted risk ratio = 0.89, 95% CI: 0.82, 0.97). Accordingly, B12 deficiency was associated with a higher risk of preterm birth (adjusted risk ratio = 1.21, 95% CI: 0.99, 1.49). This finding supports the need for randomized controlled trials of vitamin B12 supplementation in pregnancy.


Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal.
Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.


Prognostic value of bone marrow involvement by clonal immunoglobulin gene rearrangements in follicular lymphoma.
We aimed to evaluate the prognostic value of routine use of PCR amplification of immunoglobulin gene rearrangements in bone marrow (BM) staging in patients with follicular lymphoma (FL). Clonal rearrangements were assessed by immunoglobulin heavy and light-chain gene rearrangement analysis in BM aspirates from 96 patients diagnosed with FL and related to morphological detection of BM involvement in biopsies. In 71 patients, results were also compared with concurrent flow cytometry analysis. BM involvement was detected by PCR in 34.4% (33/96) of patients. The presence of clonal rearrangements by PCR was associated with advanced clinical stage (I-III vs IV; p<0.001), high FL International Prognostic Index (FLIPI) score (0-1, 2 vs ≥3; p=0.003), and detection of BM involvement by morphology and flow cytometry analysis (p<0.001 for both). PCR-positive patients had a significantly poorer survival than PCR-negative patients (p=0.001, log-rank test). Thirteen patients positive by PCR but without morphologically detectable BM involvement, had significantly poorer survival than patients with negative morphology and negative PCR result (p=0.002). The poor survival associated with BM involvement by PCR was independent of the FLIPI score (p=0.007, Cox regression). BM involvement by morphology or flow cytometry did not show a significant impact on survival. Our results showed that routine use of PCR-based clonality analysis significantly improved the prognostic impact of BM staging in patients with FL. BM involvement by PCR was also an independent adverse prognostic factor.