Ten gigahertz microwave radiation impairs spatial memory, enzymes activity, and histopathology of developing mice brain.
For decades, there has been an increasing concern about the potential hazards of non-ionizing electromagnetic fields that are present in the environment and alarming as a major pollutant or electro-pollutant for health risk and neuronal diseases. Therefore, the objective of the present study was to explore the effects of 10 GHz microwave radiation on developing mice brain. Two weeks old mice were selected and divided into two groups (i) sham-exposed and (ii) microwave-exposed groups. Animals were exposed for 2 h/day for 15 consecutive days. After the completion of exposure, within an hour, half of the animals were autopsied immediately and others were allowed to attain 6 weeks of age for the follow-up study. Thereafter results were recorded in terms of various biochemical, behavioral, and histopathological parameters. Body weight result showed significant changes immediately after treatment, whereas non-significant changes were observed in mice attaining 6 weeks of age. Several other endpoints like brain weight, lipid peroxidation, glutathione, protein, catalase, and superoxide dismutase were also found significantly (p < 0.05) altered in mice whole brain. These significant differences were found immediately after exposure and also in follow-up on attaining 6 weeks of age in microwave exposure group. Moreover, statistically significant (p < 0.001) effect was investigated in spatial memory of the animals, in learning to locate the position of platform in Morris water maze test. Although in probe trial test, sham-exposed animals spent more time in searching for platform into the target quadrant than in opposite or other quadrants. Significant alteration in histopathological parameters (qualitative and quantitative) was also observed in CA1 region of the hippocampus, cerebral cortex, and ansiform lobule of cerebellum. Results from the present study concludes that the brain of 2 weeks aged mice was very sensitive to microwave exposure as observed immediately after exposure and during follow-up study at 6 weeks of age.


Calibration of the spring constant of cantilevers of arbitrary shape using the phase signal in an atomic force microscope.
The measurement of cantilever parameters is an essential part of performing a calibrated measurement with an atomic force microscope (AFM). The thermal motion method is a widely used technique for calibrating the spring constant of an AFM cantilever, which can be applied to non-rectangular cantilevers. Given the trend towards high frequency scanning, calibration of non-rectangular cantilevers is of increasing importance. This paper presents two results relevant to cantilever calibration via the thermal motion method. We demonstrate the possibility of using the AFM's phase signal to acquire the thermal motion. This avoids the challenges associated with connecting the raw photodiode signal to a separate spectrum analyser. We also describe how numerical calculations may be used to calculate the parameters needed in a thermal motion calibration of a non-rectangular cantilever. Only accurate knowledge of the relative size of the in-plane dimensions of the cantilever is needed in this computation. We use this pair of results in the calibration of a variety of rectangular and non-rectangular cantilevers. We observe an average difference between the Sader and thermal motion values of cantilever stiffness of 10%.


Childhood Traumatic Experiences and Receptive Anal Intercourse Among Women.
Receptive anal intercourse (RAI), which has become increasingly common among U.S. heterosexual women, is associated with STDs, including HIV, when it is unprotected and coercive. Childhood traumatic experiences may increase sexual risk behavior, but the relationship between childhood trauma and RAI among women has not been examined. Data from 4,876 female participants in Waves 1 (1994-1995), 3 (2001-2002) and 4 (2007-2008) of the National Longitudinal Study of Adolescent to Adult Health were used to examine associations between nine self-reported childhood traumas (neglect; emotional, physical and sexual abuse; parental binge drinking and incarceration; and witnessing, being threatened with and experiencing violence) and RAI during adulthood using modified Poisson regression analysis. Whether depression, low self-esteem, drug use, relationship characteristics or sex trade involvement mediated the relationship between trauma and RAI was also explored. Forty percent of the sample reported having engaged in receptive anal intercourse. After adjustment for sociodemographic characteristics, eight of the nine childhood traumas were associated with increased risk of RAI (adjusted prevalence ratios, 1.2-1.5); the strongest association was with experience of violence. Each unit increase in the number of traumas yielded a 16% increase in RAI prevalence. In mediation analyses, only drug use and relationship factors slightly attenuated the association between childhood trauma and RAI (1.2 for each). Women with a history of childhood trauma may be at increased risk of engaging in RAI, highlighting the importance of screening and trauma-informed education in sexual health settings. Pathways linking childhood trauma and RAI among women are complex and warrant further research.


Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology.
Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1 The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials.


Nephrogenic systemic fibrosis: A 15-year retrospective study at a single tertiary care center.
Despite multiple therapeutic approaches for nephrogenic systemic fibrosis (NSF), no single treatment has convincingly shown consistent benefit. The most successful outcomes have been associated with recovery of renal function, although evidence remains limited and past studies have been inconclusive. We sought to investigate whether improvement of renal function via successful transplantation or via return of renal function after acute kidney injury correlates with improvement of NSF, and to further characterize the clinical features and progression of NSF. A retrospective medical chart review led to the identification of patients (n = 8) diagnosed with NSF who presented to a single academic tertiary referral center over a 15-year period. These 8 patients were contacted by phone to obtain information related to treatment and clinical course of their NSF and renal function. Statistical analysis was performed using Fisher's exact test. There is a significant correlation (P = .0286) of improved renal function with improvement of NSF. All 4 patients who had improvement of renal function also had improvement of NSF. Two of these patients had end-stage renal disease and a successful kidney transplant, and two had acute kidney injury that resolved. No improvement in NSF was observed without kidney function resolution. Our study is limited by a small sample size (n = 8) and a retrospective study design, which increased its potential for selection and recall bias. Improvement of renal function through either transplantation or resolution of acute kidney injury with medical management is significantly associated with improvement of NSF.


Th17 cells upregulate polymeric Ig receptor and intestinal IgA and contribute to intestinal homeostasis.
Although CD4(+) Th17 cells are enriched in normal intestines, their role in regulation of the host response to microbiota, and whether and how they contribute to intestinal homeostasis, is still largely unknown. It is also unclear whether Th17 cells regulate intestinal IgA production, which is also abundant in the intestinal lumen and has a crucial role as the first defense line in host response to microbiota. In this study, we found that intestinal polymeric Ig receptor (pIgR) and IgA production was impaired in T cell-deficient TCR-βxδ(-/-) mice. Repletion of TCR-βxδ(-/-) mice with Th17 cells from CBir1 flagellin TCR transgenic mice, which are specific for a commensal Ag, increased intestinal pIgR and IgA. The levels of intestinal pIgR and IgA in B6.IL-17R (IL-17R(-/-)) mice were lower than wild type mice. Treatment of colonic epithelial HT-29 cells with IL-17 increased pIgR expression. IL-17R(-/-) mice demonstrated systemic antimicroflora Ab response. Consistently, administering dextran sulfate sodium (DSS) to C57BL/6 mice after treatment with IL-17-neutralizing Ab resulted in more severe intestinal inflammation compared with control Ab. Administering DSS to IL-17R(-/-) mice resulted in increased weight loss and more severe intestinal inflammation compared with wild type mice, indicating a protective role of Th17 cells in intestinal inflammation. Individual mice with lower levels of pIgR and intestinal-secreted IgA correlated with increased weight loss at the end of DSS administration. Collectively, our data reveal that microbiota-specific Th17 cells contribute to intestinal homeostasis by regulating intestinal pIgR expression and IgA secretion.


Infrared thermography--a non-invasive tool to evaluate thermal status of neonatal pigs based on surface temperature.
Hypothermia is a major cause of mortality in neonatal pigs. Infrared (IR) thermography is a promising non-invasive method to assess thermal status, but has not been evaluated for use on neonatal pigs from birth. The aim of this study was to evaluate the application of IR thermography as a non-invasive tool to estimate body temperature and assess the thermal status in newborn pigs by (1) estimating the relationship between surface temperature and rectal temperature (RT) in neonatal pigs; and (2) estimating the influence of air temperature (AT), birth weight and the time from birth on the relationship between surface temperature and RT. The method was evaluated on the basis of 1695 thermograms and 915 RTs on 91 neonatal pigs born in loose farrowing pens with floor heating at 34°C, and three different ATs (15°C, 20°C and 25°C). Full-body thermograms of the back and the side of the pigs and RT were acquired at 11 sampling times between birth and 48 h after birth. The maximum (IRmax), minimum, average of the full body and ear minimum IR surface temperatures were derived from the thermograms. IRmax had the highest correlation with RT (0.82) and was therefore used in the statistical analysis. The relation of RT by IRmax depended on time at: 0 h (slope: 0.20°C, P<0.001), 0.25 h (slope: 0.42°C, P<0.01), and 0.5 and 1 h after birth (slope: 0.68°C, P<0.001). After the 1st hour (1.5 to 48 h) the relation of RT by IRmax was no longer affected by time (slope: 0.63°C, P<0.001). The agreement between RT and IRmax was improved (P<0.001) after the 1st hour (RT-IRmax 0 to 1 h: 2.02 (1.44)°C; 1.5 to 48 h: 0.95 (0.85)°C). IRmax below 30°C was indicative of piglets having RT<32°C (91.3%). The location of IRmax was identified predominantly at the base of the ears (27/50), other sites in the region of the head (12/50) and the axilla area (8/50). There was a small but significant effect of the angle as IRmax_side-IRmax_back: mean 0.20°C (P<0.001). On the basis of the low difference between IRmax from back and side view thermograms, and the location of IRmax, the angle seems less important and thus the method has the potential to be used without the need for manual restraint of the pigs. On the basis of the results of this study, we propose that IRmax temperature from full-body thermograms has implication as a valid tool to assess the thermal status in neonatal piglets but not as an identical substitute for RT.


Internet gambling and pornography: illustrative examples of the psychological consequences of communication anarchy.
Two areas of Internet behavior, gambling and pornography distribution, are examined for what they reveal about the profound social and psychological changes caused by recent advances in communication technology. The overview of these domains clearly shows that there will be an expected increase in people presenting for treatment with Internet behavior related difficulties, and that their treatment will be, at present, uninformed by any specific empirical research. These two domains are used as examples to illustrate how the Internet is creating a paradigm shift in the basic nature of an individual's relationships to local, state, and federal governments. It is no longer possible for people, even minors, to be fully protected by their governments from material deemed harmful by the community one lives in. This is demonstrated by a review of the current availability, in millions of homes nationwide, of the opportunity to experience Internet casino gambling and to acquire pornographic material not sold in United States adult bookstores. The psychological effect of the increased need for individual responsibility in the access to potentially harmful domains is not well understood at this time. This article is a call for the kind of basic research that will delineate the base rates for pathological involvement in online gambling and pornography, as a means of discovering the potential negative psychological consequences of the inability to regulate Internet content.


Adherence to Human Colon Cells by Multidrug Resistant Enterobacterales Strains Isolated From Solid Organ Transplant Recipients With a Focus on Citrobacter freundii.
Enterobacteria species are common causes of hospital-acquired infections, which are associated with high morbidity and mortality rates. Immunocompromised patients such as solid organ transplant (SOT) recipients are especially at risk because they are frequently exposed to antibiotics in the course of their treatments. In this work, we used a collection of 106 Escherichia coli, 78 Klebsiella pneumoniae, 25 Enterobacter spp., and 24 Citrobacter spp. multidrug resistant strains isolated from transplant patients (hepatic, renal or renal/pancreatic) in order to examine their ability to adhere in vitro to HT-29 human colon cells, and to determine if some adhesive characteristics are associated with prevalence and persistence of these strains. A total of 33 E. coli (31%), 21 K. pneumoniae (27%), 7 Enterobacter spp. (28%), and 5 Citrobacter spp. (21%), adhered to the colon epithelial cells. Two main adherence patterns were observed in the four species analyzed, diffuse adherence, and aggregative adherence. Under transmission electronic microscopy (TEM), most bacteria lacked visible fimbria on their surface, despite their strong adherence to epithelial cells. None of the strains studied was able to induce any cytotoxic effect on HT-29 cells although some of them strongly colonizing both cells and glass coverslips at high density. Some of the strains failed to adhere to the epithelial cells but adhered strongly to the cover-slide, which shows that microscopy studies are mandatory to elucidate the adherence of bacteria to epithelial cells in vitro, and that quantitative assays using colony forming unit (CFUs) counting need to be supplemented with pictures to determine definitively if a bacterial strain adheres or not to animal cells in vitro. We report here, for the first time, the aggregative adherence pattern of two multidrug resistant (MDR) Citrobacter freundii strains isolated from human patients; importantly, biofilm formation in Citrobacter is totally dependent on the temperature; strong biofilms were formed at room temperature (RT) but not at 37°C, which can play an important role in the colonization of hospital surfaces. In conclusion, our results show that there is a great variety of adhesion phenotypes in multidrug-resistant strains that colonize transplanted patients.


Analysis of tractor-related deaths.
The aim of the study was to evaluate the properties of tractor-related death cases and discuss the precautions in order to prevent such events. For this aim, reports made between 2006 and 2009 by the First Specialty Board of Council of Forensic Medicine (FSBCFM), Turkey, were evaluated retrospectively. Forty-one tractor-related death cases were included in this study. Thirty-five (85.4%) of the cases were male. The mean age was 42.52 ± 25.1 (with a range between 1 and 84 years old). The average time interval between the incident and death was 13.76 ± 33.53 days (range: 0-176 days) and 20 (48.8%) cases died on the incident spot or during transportation to the hospital. Among the cases, 34.1% (n = 14) were drivers, whereas 39.0% (n = 16) were passengers and the remaining were pedestrians (26.9%, n = 11). The accident was caused by the tractor rolling over in 34.1%, the victim being run over in 22.0%, and the victim falling from the tractor or trailer in 22.0% of the cases. Most of the events (56.1%) occurred on the road. In conclusion, tractors must not be used as a transportation vehicle for passengers. Periodic training of drivers can be useful for reducing tractor-related accidents.


Archaeological evidence for two separate dispersals of Neanderthals into southern Siberia.
Neanderthals were once widespread across Europe and western Asia. They also penetrated into the Altai Mountains of southern Siberia, but the geographical origin of these populations and the timing of their dispersal have remained elusive. Here we describe an archaeological assemblage from Chagyrskaya Cave, situated in the Altai foothills, where around 90,000 Middle Paleolithic artifacts and 74 Neanderthal remains have been recovered from deposits dating to between 59 and 49 thousand years ago (age range at 95.4% probability). Environmental reconstructions suggest that the Chagyrskaya hominins were adapted to the dry steppe and hunted bison. Their distinctive toolkit closely resembles Micoquian assemblages from central and eastern Europe, including the northern Caucasus, more than 3,000 kilometers to the west of Chagyrskaya Cave. At other Altai sites, evidence of earlier Neanderthal populations lacking associated Micoquian-like artifacts implies two or more Neanderthal incursions into this region. We identify eastern Europe as the most probable ancestral source region for the Chagyrskaya toolmakers, supported by DNA results linking the Neanderthal remains with populations in northern Croatia and the northern Caucasus, and providing a rare example of a long-distance, intercontinental population movement associated with a distinctive Paleolithic toolkit.


Radiation-related risk of basal cell carcinoma: a report from the Childhood Cancer Survivor Study.
Basal cell carcinoma (BCC) is the most common malignancy in the United States. Ionizing radiation is an established risk factor in certain populations, including cancer survivors. We quantified the association between ionizing radiation dose and the risk of BCC in childhood cancer survivors. Participants in the Childhood Cancer Survivor Study who reported a BCC (case subjects, n = 199) were matched on age and length of follow-up to three study participants who had not developed a BCC (control subjects, n = 597). The radiation-absorbed dose (in Gy) to the BCC location was calculated based on individual radiotherapy records using a custom-designed dosimetry program. Conditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for associations between demographic and treatment factors, therapeutic radiation dose, and surrogate markers of sun sensitivity (skin and hair color) and the risk of BCC. A linear dose-response model was fitted to evaluate the excess odds ratio per Gy of radiation dose. Among case subjects, 83% developed BCC between the ages of 20 and 39 years. Radiation therapy, either alone or in combination with chemotherapy, was associated with an increased risk of BCC compared with no chemotherapy or radiation. The odds ratio for subjects who received 35 Gy or more to the skin site vs no radiation therapy was 39.8 (95% CI = 8.6 to 185). Results were consistent with a linear dose-response relationship, with an excess odds ratio per Gy of 1.09 (95% CI = 0.49 to 2.64). No other treatment variables were statistically significantly associated with an increased risk of BCC. Radiation doses to the skin of more than 1 Gy are associated with an increased risk of BCC.


Progressive bicytopenia due to persistent parvovirus B19 infection after immunochemotherapy with fludarabine/cyclophosphamide and rituximab for relapsed B cell lymphoma.
Human parvovirus B 19 is known as a virus causing erythema infectiosum, arthropathy, transient aplastic crisis and intrauterine fetal death. Healthy hosts are able to clear the virus within weeks after infection. There are a few reports available in the literature regarding immunocompromised renal transplant recipients with persistent infection without seroconversion. Herein, we describe a 56-year old woman with a relapse of grade II follicular lymphoma who received a combined immunochemotherapy of rituximab, fludarabine and cyclophosphamide and subsequently developed a persistent parvovirus B19 infection. In the absence of serum immunoglobulin antibodies, PCR analysis of peripheral blood and bone marrow aspirate were positive for parvovirus B19. Treatment with IVIG treatment resulted in normalization of peripheral blood counts within 7 weeks.


Postal urine specimens: are they a feasible method for genital chlamydial infection screening?
A United Kingdom (UK) screening programme for Chlamydia trachomatis has recently been announced. Pilot projects involving the opportunistic testing of women attending health facilities are due to commence in several sites. There is a danger that this approach will fail to obtain adequate population coverage. The alternative--true systematic population screening--is generally assumed to be unfeasible. Studies in Denmark using postal urine specimens have challenged this assumption. No such studies have been reported from the UK. To assess the potential of urine specimens sent by post as the basis for a UK population screening strategy for genital chlamydial infection. Two hundred patients (100 men, 100 women) aged 18 to 45 years were randomly sampled from the list of one urban group practice. Subjects were mailed an explanatory letter, a urine sample container, a sexual lifestyle questionnaire, and a prepaid return envelope. Non-responders were contacted by telephone; persistent non-responders were visited at home. Samples were tested for Chlamydia by DNA amplification and enzyme immunoassay. Sixty-four (32%) subjects were no longer living at their GP registered address. Of the remaining 136, 126 (93%) responded to the survey and 113 (83%) accepted the request for a urine sample and completed a questionnaire. Acceptance rates were similar for men and women and across age groups. Four samples (3%) were Chlamydia positive. Home mailed urine specimen collection in conjunction with a self-completed postal questionnaire is feasible. This could provide a viable basis both for determining population Chlamydia prevalence and for a UK Chlamydia population screening strategy. Overall cost effectiveness of such a strategy will depend on the cost of the test used. Comparative performance characteristics of the different currently available tests in this setting have yet to be fully determined.


Refractory ascites after liver transplantation: an analysis of 1058 liver transplant patients at a single center.
A retrospective study of 1058 liver transplant recipients was performed to determine: (i) the incidence, etiology, timing, clinical features and treatment of refractory ascites (RA), (ii) risk factors for RA development, (iii) predictors of RA disappearance, (iv) predictors of survival following RA and (v) the impact of RA on patient survival. Sixty-two patients (5.9%) developed RA and its disappearance occurred in 27/62 cases. Patients having hepatitis C virus (HCV) had a significantly higher hazard rate of developing RA (p < 0.00001). No other baseline characteristic was associated with RA. Cox stepwise regression analysis of the hazard rate of RA disappearance found two significant factors: HCV recurrence as the reason for developing RA implied a poorer outcome (p = 0.006), whereas an unknown reason implied a favorable outcome (p = 0.02). In addition, survival following RA was significantly poorer among patients having bacterial peritonitis or HCV recurrence. Finally, the mortality rate was significantly (nearly 8.6 times) higher in patients following RA development while it was ongoing (p < 0.00001); however, if the RA disappeared, then the additional risk of death also disappeared. This study illustrates the importance of developing an optimal treatment strategy to (i) effectively treat RA if it develops and (ii) prevent hepatitis C recurrence.


Acute effects of hydrocortisone on the metabolic response to a glucose load: increase in the first-phase insulin secretion.
Several basic science studies support the existence of non-genomic glucocorticoid signaling in pancreas, liver, and adipocytes, but its clinical relevance has not yet been elucidated. This study aimed at investigating the rapid effects of hydrocortisone on the human metabolic response to glucose. In a randomized placebo-controlled crossover study, ten healthy men received an i.v. bolus of 0.6 mg/kg hydrocortisone once and placebo once 4 min before the administration of 330 mg/kg glucose. Cortisol, glucose, insulin, C-peptide, ghrelin, and peptide YY (PYY) levels were measured during the following 3 h. Minimal model analysis was performed for evaluating the metabolic response. Hydrocortisone attenuated the rise in plasma glucose during the initial 15 min following glucose administration (P=0.039), and it led to lower glucose levels during the first 2 h (P=0.017). This was accompanied by enhanced circulating insulin (P=0.02) and C-peptide (P=0.03) levels during the initial 15 min, and a 35% increase in the first-phase beta-cell function (P=0.003). Hydrocortisone decreased PYY concentrations during the initial 30 min (P=0.014), but it did not affect the ghrelin response to glucose. One i.v. bolus of hydrocortisone induces rapid effects on carbohydrate metabolism increasing the first-phase beta-cell function. The modulation of PYY plasma levels suggests the possible non-genomic effects of glucocorticoids on appetite-regulatory hormones.


Fast membrane hemifusion via dewetting between lipid bilayers.
The behavior of lipid bilayers is important to understand the functionality of cells like the trafficking of ions. Standard procedures to explore the properties of lipid bilayers and hemifused states typically use supported membranes or vesicles. Both techniques have several shortcomings in terms of bio-relevance or accessibility for measurements. In this article, the formation of individual free standing hemifused states between model cell membranes is studied using an optimized microfluidic scheme which allows for simultaneous optical and electrophysiological measurements. In the first step, two model membranes are formed at a desired location within a microfluidic device using a variation of the droplet interface bilayer (DiB) technique. In the second step, the two model membranes are brought into contact forming a single hemifused state. For all tested lipids, the hemifused state between free standing membranes forms within hundreds of milliseconds, i.e. several orders of magnitude faster than those reported in literature. The formation of a hemifused state is observed as a two stage process, whereas the second stage can be explained as a dewetting process under no-slip boundary conditions. The formed hemifusion states have a long lifetime and a single fusion event can be observed when triggered by an applied electric field as demonstrated for monoolein.


Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva.
The discovery of disease-specific biomarkers in oral fluids has revealed a new dimension in molecular diagnostics. Recent studies have reported the mechanistic involvement of tumor cells derived mediators, such as exosomes, in the development of saliva-based mRNA biomarkers. To further our understanding of the origins of disease-induced salivary biomarkers, we here evaluated the hypothesis that tumor-shed secretory lipidic vesicles called exosome-like microvesicles (ELMs) that serve as protective carriers of tissue-specific information, mRNAs, and proteins, throughout the vasculature and bodily fluids. RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva. Our data confirmed that the majority of extracellular RNAs (exRNAs) in saliva were encapsulated within ELMs. Nude mice implanted with human lung cancer H460 cells expressing hCD63-GFP were used to follow the circulation of tumor cell specific protein and mRNA in the form of ELMs in vivo. We were able to identify human GAPDH mRNA in ELMs of blood and saliva of tumor bearing mice using nested RT-qPCR. ELMs positive for hCD63-GFP were detected in the saliva and blood of tumor bearing mice as well as using electric field-induced release and measurement (EFIRM). Altogether, our results demonstrate that ELMs carry tumor cell-specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer. These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.


LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions.
The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.


Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of trans- endoplasmic reticulum N-acetylglucosamine-1-phosphate transferase.
A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.


The influence of stress on the adrenals of the springbok (Antidorcas marsupialis).
The absolute and relative adrenal mass as well as histological sections of the adrenals of springbok (Antidorcas marsupialis) (n = 76; 36 ewes and 39 rams) were studied over a period of 12 months. No significant difference was found in the absolute mass of the adrenals of rams and ewes. However, a significant difference (p < 0.01) was found in the relative masses (adrenal mass in terms of carcass mass), in that relative masses in males were greater than females. The zona fasciculata was found to be enlarged during the dry season (February and March), and showed increased activity in both sexes. The zona fasciculata of the ewes showed an additional increase in activity during September (late pregnancy) and October (lactation). In rams increased activity of the zona fasciculata was seen during the mating season in April and May. It appears that stressful events during reproduction and during the dry season cause an increase in adrenal mass and activity.


Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity.
Pseudomonas aeruginosa is a persistent pathogen in the airways of patients with cystic fibrosis or bronchiectasis from other causes and appears to have evolved strategies to survive the inflammatory response of the host. We hypothesized that the secreted hemolytic phospholipase C (PLC) of P. aeruginosa (PlcHR) would decrease neutrophil respiratory burst activity. We found that while intact wild-type P. aeruginosa cells stimulated moderate respiratory burst activity from human neutrophils, an isogenic mutant pseudomonas (DeltaHR strain) containing a targeted deletion of the plcHR operon induced a much more robust oxidative burst from neutrophils. In contrast, a second pseudomonas mutant (DeltaN) containing a disruption in the gene encoding the nonhemolytic PLC (PlcN) was not different from the wild type in stimulating neutrophil O2.- production. Readdition of purified PlcHR to the DeltaHR strain suppressed neutrophil O2.- production to levels stimulated by wild-type bacteria. Interestingly, purified PlcHR decreased phorbol myristate acetate (PMA)- but not formyl methionyl-leucyl-proline (fMLP)-induced respiratory burst activity, suggesting interference by PlcHR with a protein kinase C (PKC)-specific signaling pathway. Accordingly, the PKC inhibitor bisindolylmaleimide inhibited the oxidative burst induced by either PMA or intact pseudomonas, but not by fMLP, whereas the p38 kinase inhibitor SB-203580 fully inhibited the respiratory burst induced by fMLP or the PlcHR-replete wild-type bacteria, but not PMA or the PlcHR-deficient DeltaHR bacterial mutant. We conclude that expression of PlcHR by P. aeruginosa suppresses bacterium-induced neutrophil respiratory burst by interfering with a PKC-dependent, non-p38 kinase-dependent pathway.


Simple, high-yield purification of xanthine oxidase from bovine milk.
Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.


Neurophysiological responses after short-term strength training of the biceps brachii muscle.
The neural adaptations that mediate the increase in strength in the early phase of a strength training program are not well understood; however, changes in neural drive and corticospinal excitability have been hypothesized. To determine the neural adaptations to strength training, we used transcranial magnetic stimulation (TMS) to compare the effect of strength training of the right elbow flexor muscles on the functional properties of the corticospinal pathway. Motor-evoked potentials (MEPs) were recorded from the right biceps brachii (BB) muscle from 23 individuals (training group; n = 13 and control group; n = 10) before and after 4 weeks of progressive overload strength training at 80% of 1-repetition maximum (1RM). The TMS was delivered at 10% of the root mean square electromyographic signal (rmsEMG) obtained from a maximal voluntary contraction (MVC) at intensities of 5% of stimulator output below active motor threshold (AMT) until saturation of the MEP (MEPmax). Strength training resulted in a 28% (p = 0.0001) increase in 1RM strength, and this was accompanied by a 53% increase (p = 0.05) in the amplitude of the MEP at AMT, 33% (p = 0.05) increase in MEP at 20% above AMT, and a 38% increase at MEPmax (p = 0.04). There were no significant differences in the estimated slope (p = 0.47) or peak slope of the stimulus-response curve for the left primary motor cortex (M1) after strength training (p = 0.61). These results demonstrate that heavy-load isotonic strength training alters neural transmission via the corticospinal pathway projecting to the motoneurons controlling BB and in part underpin the strength changes observed in this study.


Role of the wrist cuff in forearm plethysmography.
1. To determine whether a wrist cuff is necessary to measure the forearm blood flow correctly, we studied the effects of wrist cuff inflation to supra-venous and supra-systolic pressure values over a large range of forearm blood flow values: in the basal state, during post-occlusive hyperaemia of the hand, and during heating of the hand with warm air. Eleven healthy men participated, and the study was carried out at two different ambient temperatures of 20 and 25 degrees C. 2. In the basal state, the measured forearm blood flow was lowest with the wrist cuff at supra-systolic pressure. With the wrist cuff at supra-venous pressure the forearm blood flow was also lower than with an uninflated cuff, but only significantly so when the basal forearm blood flow was higher (at a room temperature of 25 degrees C). 3. During post-occlusive hyperaemia, inflating the wrist cuff to supra-systolic pressure produced the lowest forearm blood flow value at both room temperatures. In addition, with the wrist cuff at supra-venous pressure, forearm blood flow values were lower than with the uninflated cuff, but the supra-venous cuff pressure was clearly less efficient in excluding the hand blood flow than the supra-systolic cuff pressure. 4. During heating of the hand, both supra-systolic and supra-venous cuff pressures were effective in excluding the hand blood flow at both room temperatures. The forearm blood flow measured with the wrist cuff at supra-systolic pressure was lower than that measured with the wrist cuff at supra-venous pressure, but the difference was only significant at a room temperature of 20 degrees C. 5. In conclusion, we have demonstrated that a wrist cuff at supra-systolic pressure is most appropriate for the exclusion of the hand circulation in order to measure the forearm blood flow correctly.


A comparison of hip guidance with reciprocating gait orthoses in children with spinal paraplegia: results of a ten-year prospective study.
Twenty-two children with spinal paraplegia were entered into a prospective randomised study to assess the efficacy of two reciprocating orthoses and to identify any prognostic factors that might affect continuing use of the devices. Thirteen received a hip guidance orthosis (HGO) and nine a reciprocating gait orthosis (RGO). They were followed for a mean of ten years. At one year follow-up there were three statistically significant differences between the two groups at the 5% level: repairs were commoner in the RGO group, the RGO group improved in their ability to walk over difficult outdoor surfaces and the HGO group improved more in their ability to rise from a sitting to standing position. At one year follow-up there was a positive parental and child's view of the benefits of the orthoses, but by ten years only 24% of the patients were still using the orthoses. We were not able to show any definite advantage of one device over the other or any statistically significant prognostic factors for walking in the longer term with a reciprocating orthosis. We question whether or not the routine provision of these types of orthosis is justifiable when it appears that, in the longer term, the patients we studied preferred wheelchair mobility.


Controlled damage in thick specimens by multiphoton excitation.
Controlled damage by light energy has been a valuable tool in studies of cell function. Here, we show that the Ti:Sapphire laser in a multiphoton microscope can be used to cause localized damage within unlabeled cells or tissues at greater depths than previously possible. We show that the damage is due to a multiphoton process and made wounds as small as 1 microm in diameter 20 microm from the surface. A characteristic fluorescent scar allows monitoring of the damage and identifies the wound site in later observations. We were able to lesion a single axon within a bundle of nerves, locally interrupt organelle transport within one axon, cut dendrites in a zebrafish embryo, ablate a mitotic pole in a sea urchin egg, and wound the plasma membrane and nuclear envelope in starfish oocytes. The starfish nucleus collapsed approximately 1 h after wounding, indicating that loss of compartmentation barrier makes the structure unstable; surprisingly, the oocyte still completed meiotic divisions when exposed to maturation hormone, indicating that the compartmentalization and translocation of cdk1 and its regulators is not required for this process. Multiphoton excitation provides a new means for producing controlled damage deep within tissues or living organisms.


In vivo photothermal optical coherence tomography of gold nanorod contrast agents.
Photothermal optical coherence tomography (PT-OCT) is a potentially powerful tool for molecular imaging. Here, we characterize PT-OCT imaging of gold nanorod (GNR) contrast agents in phantoms, and we apply these techniques for in vivo GNR imaging. The PT-OCT signal was compared to the bio-heat equation in phantoms, and in vivo PT-OCT images were acquired from subcutaneous 400 pM GNR Matrigel injections into mice. Experiments revealed that PT-OCT signals varied as predicted by the bio-heat equation, with significant PT-OCT signal increases at 7.5 pM GNR compared to a scattering control (p < 0.01) while imaging in common path configuration. In vivo PT-OCT images demonstrated an appreciable increase in signal in the presence of GNRs compared to controls. Additionally, in vivo PT-OCT GNR signals were spatially distinct from blood vessels imaged with Doppler OCT. We anticipate that the demonstrated in vivo PT-OCT sensitivity to GNR contrast agents is sufficient to image molecular expression in vivo. Therefore, this work demonstrates the translation of PT-OCT to in vivo imaging and represents the next step towards its use as an in vivo molecular imaging tool.


The rare Costello variant HRAS c.173C>T (p.T58I) with severe neonatal hypertrophic cardiomyopathy.
We report a 10-year-old girl presenting with severe neonatal hypertrophic cardiomyopathy (HCM), feeding difficulties, mildly abnormal facial features, and progressive skeletal muscle symptoms but with normal cognitive development. Targeted oligonucleotide-selective sequencing of 101 cardiomyopathy genes revealed the genetic diagnosis, and the mutation was verified by Sanger sequencing in the patient and her parents. To offer insights into the potential mechanism of patient mutation, protein structural analysis was performed using the resolved structure of human activated HRAS protein with bound GTP analogue (PDB id 5P21) in Discovery Studio 4.5 (Dassault Systèmes Biovia, San Diego, CA). The patient with hypertrophic cardiomyopathy and normal cognitive development was diagnosed with an HRAS mutation c.173C>T (p.T58I), a milder variant of Costello syndrome affecting a highly conserved amino acid, threonine 58. Our analysis suggests that the p.G12 mutations slow GTP hydrolysis rendering HRAS unresponsive to GTPase activating proteins, and resulting in permanently active state. The p.T58I mutation likely affects binding of guanidine-nucleotide-exchange factors, thereby promoting the active state but also allowing for slow inactivation. Patients with the HRAS mutation c.173C>T (p.T58I) might go undiagnosed because of the milder phenotype compared with other mutations causing Costello syndrome. We expand the clinical and molecular picture of the rare HRAS mutation by reporting the first case in Europe and the fourth case in the literature. Our protein structure analysis offers insights into the mechanism of the mildly activating p.T58I mutation. © 2016 Wiley Periodicals, Inc.


Suppression by thimerosal of ex-vivo CD4+ T cell response to influenza vaccine and induction of apoptosis in primary memory T cells.
Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.


Epidemiology of schistosomiasis in Gezira area Central Sudan and analysis of cytokine profiles.
To determine and compare anti-schistosoma IgG, interleukin-10 (IL-10) and interferon-γ(IFN-γ) levels in the serum of patients and endemic controls and to investigate the epidemiological situation of Al-Hebaika village in the northern part of Gezira Agricultural Irrigation Scheme in 2005. During 2005 survey, serum were collected from 118 villagers. Sixty eight were parasitological positive (patients), and 50 were negative (endemic controls). Indirect ELISA was used to measure and compare the levels of immunoglobulin G (IgG) against Schistsoma mansoni (S. mansoni) soluble worm antigen (SWA) in the patients and endemic control groups from the village and compared with 20 healthy non endemic controls. Sandwich ELISA was also used to measure and compare IL-10 and IFN-γ in the serum of the selected groups. The overall prevalence of S. haematobium was 20.0% and 0.9% in the first and the second surveys respectively, while the intensity of infection was the same in the two surveys 1.38 [geometric mean egg count (GMFC)]. The overall prevalence of S. mansoni infection was 68.5% and 15.4%, while the intensity of infection was 2.75 (GMEC) and 1.70 (GMEC) in the two surveys respectively. IgG reactivity against SWA showed no significant difference between Schistosoma positive patients and endemic controls. However, there were high significant differences between each of these two groups and the non endemic control group (P= 0,000). Schistosoma patients and exposed controls had significantly higher IL-10 concentration compared with non endemic controls. While endemic controls showed significantly higher IFN-γ concentration than patients (P = 0.000). Also there was very significant difference between IFN-γ levels of each of patients endemic controls and that of the non endemic controls (P = 0.003). The study concluded that IFN-γ has a role in the natural resistant to schistosoma mansoni infection. The prevalence and intensity of S. mansoni in the Gezira Irrigation Scheme was greatly reduced. S. haematobium has disappeared from the area.


A phase II trial on alpha-interferon (alpha IFN) effect in patients with monoclonal IgM gammopathy.
Waldenström's macroglobulinemia (WM) is an incurable disorder of B cells. Following occasional reports of response to alpha interferon (IFN) and in view of its effectiveness in hairy cell leukemia, we tested this agent in a relatively large group (n = 88) of patients who had an IgM monoclonal component (MC) greater than 10 g/l. Thirty eight patients had a MC > 30 g/l and were classified as Waldenström's macroglobulinemia (WM), while fifty had either WM in an early stage or an IgM monoclonal gammopathy of undeterminated significance (all of them operationally classified as IgM-MGUS). All patients received IFN 3 MU/day for one month and then 3 times/week. Response to treatment was mainly based on MC reduction in two consecutive determinations (> 50%: major response; 25-50%: minor response). Of 36 evaluable WM patients, 12 had a major and 6 a minor response; of 41 evaluable IgM-MGUS patients, 2 had a major and 6 a minor response. In WM patients with a major response, MC reduction was associated with disappearance of hyperviscosity symptoms, raised Hb level and reduced bone marrow lymphoplasmacytosis. At the dose used, tolerance was excellent in the majority of patients; only 15% withdrew from the study due to side effects. Although single cases and very small series have already been reported, no large study collecting quantitative data on the effects of alpha IFN in WM has been published so far. Our results suggest that IFN treatment is not indicated for patients with a low monoclonal component, while it is of clinical benefit in about 50% of patients with IgM > 30 g/l.


Levofloxacin for the treatment of respiratory tract infections.
Fluoroquinolone use has dramatically increased since the introduction of the first respiratory fluoroquinolone in the late 1990s. Levofloxacin , like other fluoquinolones, is a potent antibiotic, due to high levels of susceptibility among Gram-negative, Gram-positive (including penicillin-resistant strains of Streptococcus pneumonia) and atypical pathogens. Levofloxacin is recommended for the treatment of community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP) and in the management of acute exacerbations of chronic bronchitis (AECB). Levofloxacin demonstrates good safety, bioavailability and tissue penetration, thus maintaining adequate concentrations at the site of infection. High-dose (750 mg), short-course (5 days) therapy regimens may offer improved treatment, especially in HAP, due to higher drug concentrations, increased adherence and the potential to reduce the development of resistance. This article covers medical literature published in any language since 1990 until November 2011, on 'levofloxacin', identified using PubMed and MEDLINE. The search terms used were 'levofloxacin' and 'community acquired pneumonia', 'hospital pneumonia' or 'AECB'. Levofloxacin is a valuable antimicrobial agent and an optimal treatment option for AECB, CAP (as a monotherapy) and HAP (as combination therapy at a high-dose regimen). Its improved bioavailability and safety profile makes the possibility of shorter hospital stays a reality.


Juxta-caval hepatic angiomyolipoma masquerading as hepatocellular carcinoma.
Hepatic angiomyolipoma is an uncommon primary benign epithelial liver neoplasm. Most patients with angiomyolipomas are middle-aged women who commonly present with epigastric fullness or pain. Angiomyolipomas are tumors consisting of three tissue types: blood vessels, smooth muscle, and fat. Hepatic angiomyolipomas may be difficult to differentiate from other liver neoplasms by noninvasive imaging. We report a 58-year-old asymptomatic woman with a mass in the right lobe of the liver, found incidentally on routine abdominal sonography. Preoperative radiographic evaluation revealed a 6.5-cm hypervascular lesion abutting the inferior vena cava. Preoperative histologic study demonstrated an epithelial neoplasm suspicious for hepatocellular carcinoma. Metastatic workup was negative. At resection, the tumor was found to be an angiomyolipoma composed of lipoid, vascular and smooth muscle cells. Further staining was positive for HMB-45. Resection margins were negative. The woman had an uneventful recovery and was discharged on postoperative day seven. She currently remains well several months after her right hepatectomy.


Obstacles in provision of anti-retroviral treatment to drug users in Central and Eastern Europe and Central Asia: a regional overview.
Central and Eastern Europe and Central Asia is currently the region with the fastest growing HIV epidemic, mainly among injecting drug users (IDUs). This study explored access to anti-retroviral (ARV) treatment among IDUs and evaluated obstacles to gaining access to treatment. Semi-structured questionnaires were collected from 21 countries from agencies which deliver services to IDUs (N=55), including AIDS centres, drug treatment institutions and Non-governmental Organisations. Results showed that there was poor access to ARV treatment for IDUs. The major obstacles reported were: limited range of institutions for the provision of ARVs, lack of treatment due to high cost of ARVs, lack of clear policies and regulations in providing treatment for IDUs, lack of infrastructure and trained staff to provide treatment, and in some countries, absence of mechanisms such as methadone substitution programmes to support IDUs receiving ARV. There is a need for human and capital resources to bring ARV treatment to IDU populations in the region. Regulations and treatment protocols need to be developed to address this particular group of HIV positive clients to insure better adherence and monitoring of clients with HCV co-infection. Integration of provision of ARV treatment with drug treatment and low-threshold services is advised. Substitution therapy should be advocated for in countries where it is not available or where access is limited. Finally, more research needs to be conducted to understand what will work best in each country, region or setting.


p53 Modulates the exonuclease activity of Werner syndrome protein.
Werner syndrome (WS) is characterized by the early onset of symptoms of premature aging, cancer, and genomic instability. The molecular basis of the defects is not understood but presumably relates to the DNA helicase and exonuclease activities of the protein encoded by the WRN gene that is mutated in the disease. The attenuation of p53-mediated apoptosis in WS cells and reported physical interaction between WRN and the tumor suppressor p53 suggest that p53 and WRN functionally interact in a pathway necessary for the normal cellular response. In this study, we have demonstrated that p53 inhibits the exonuclease activity of the purified full-length recombinant WRN protein. p53 did not have an effect on a truncated amino-terminal WRN fragment that retains exonuclease activity but lacks the physical interaction domain for p53 located in the carboxyl terminus. Two naturally occurring p53 mutants found in human cancer displayed a reduced ability to inhibit WRN exonuclease activity. In cells arrested in S phase with hydroxyurea, WRN exits the nucleolus and colocalizes with p53 in the nucleoplasm. The regulation of WRN function by p53 is likely to play an important role in the maintenance of genomic integrity and prevention of cancer and other clinical symptoms associated with WS.


Telomere structure and shortening in telomerase-deficient Trypanosoma brucei.
Telomerase consists of a reverse transcriptase (TERT) and an RNA that contains a template for telomere-repeat extension. Telomerase is required to prevent telomere erosion and its activity or lack thereof is important for tumorigenesis and ageing. Telomerase has been identified in numerous organisms but it has not been studied in kinetoplastid protozoa. Trypanosoma brucei, the causative agent of African sleeping sickness, evades the host immune response by frequently changing its variant surface glycoprotein (VSG). The single expressed VSG is transcribed from one of approximately 20 subtelomeric 'Expression Sites', but the role telomeres might play in regulating VSG transcription and switching is unknown. We identified and sequenced the T.brucei TERT gene. Deleting TERT resulted in progressive telomere shortening of 3-6 bp per generation. In other organisms, the rate of telomere shortening is proportional to the length of the terminal 3' single-strand overhang. In T.brucei, G-overhangs were undetectable (<30 nt) by in-gel hybridization. The rate of telomere shortening therefore, agrees with the predicted shortening due to the end replication problem, and is consistent with our observation that G-overhangs are short. Trypanosomes whose telomere length can be manipulated provide a new tool to investigate the role of telomeres in antigenic variation.


Continuously tunable microdroplet-laser in a microfluidic channel.
This paper describes the generation and optical characterization of a series of dye-doped droplet-based optical microcavities with continuously decreasing radius in a microfluidic channel. A flow-focusing nozzle generated the droplets (~21 μm in radius) using benzyl alcohol as the disperse phase and water as the continuous phase. As these drops moved down the channel, they dissolved, and their size decreased. The emission characteristics from the drops could be matched to the whispering gallery modes from spherical micro-cavities. The wavelength of emission from the drops changed from 700 to 620 nm as the radius of the drops decreased from 21 μm to 7 μm. This range of tunability in wavelengths was larger than that reported in previous work on droplet-based cavities.


Infant manual performance during reaching and grasping for objects moving in depth.
Few studies have investigated manual performance in infants when reaching and grasping for objects moving in directions other than across the fronto-parallel plane. The present preliminary study explored object-oriented behavioral strategies and side preference in 8- and 10-month-old infants during reaching and grasping for objects approaching in depth from three positions (midline, and 27° diagonally from the left and right). Effects of task constraint by using objects of three different types and two sizes were further examined for behavioral strategies and hand opening prior to grasping. Additionally, assessments of hand preference by a dedicated handedness test were performed. Regardless of object starting position, the 8-month-old infants predominantly displayed right-handed reaches for objects approaching in depth. In contrast, the older infants showed more varied strategies and performed more ipsilateral reaches in correspondence with the side of the approaching object. Conversely, 10-month-old infants were more successful than the younger infants in grasping the objects, independent of object starting position. The findings regarding infant hand use strategies when reaching and grasping for objects moving in depth are similar to those from earlier studies using objects moving along a horizontal path. Still, initiation times of reaching onset were generally long in the present study, indicating that the object motion paths seemingly affected how the infants perceived the intrinsic properties and spatial locations of the objects, possibly with an effect on motor planning. Findings are further discussed in relation to future investigations of infant reaching and grasping for objects approaching in depth.


Effects of dietary glutamine on the homeostasis of CD4+ T cells in mice with dextran sulfate sodium-induced acute colitis.
This study investigated the effects of dietary glutamine (Gln) on T-helper (Th) and T regulatory (Treg) cell homeostasis and colonic inflammatory mediator expression in mice with dextran sulfate sodium (DSS)-induced colitis. Mice were randomly assigned to 4 groups with 2 normal control (C and G) and 2 DSS-treated groups (DC and DG). The C and DC groups were fed a common semipurified diet, while the G and DG groups received an identical diet except that part of the casein was replaced by Gln, which provided 25% of the total amino acid nitrogen. Mice were fed the diets for 10 days. On day 6, mice in the normal control groups were given distilled water, while those in the DSS groups were given distilled water containing 1.5% DSS for 5 d. At the end of the experiment, the mice were sacrificed for further examination. Results showed that DC group had higher plasma haptoglobin, colonic weight, immunoglobulin G, inflammatory cytokine and nuclear factor (NF)-κB protein levels. Gln administration lowered inflammatory mediators and NF-κB/IκBα ratio in colitis. Compared with the DC group, the percentages of interleukin-17F and interferon-γ in blood and transcription factors, T-bet and RAR-related orphan receptor-γt, gene expressions in mesenteric lymph nodes were lower, whereas blood Foxp3 was higher in the DG group. Also, DG group had lower colon injury score. These results suggest that Gln administration suppressed Th1/Th17 and Th-associated cytokine expressions and upregulated the expression of Tregs, which may modulate the balance of Th/Treg and reduce inflammatory reactions in DSS-induced colitis.


A new marine reptile from the Triassic of China, with a highly specialized feeding adaptation.
The Luoping fauna (Anisian, Middle Triassic) is probably the oldest of Triassic faunas in Guizhou-Yunnan area, China. The reptilian assemblage is comprised of ichthyosaurs, a number of sauropterygians (pachypleurosaur-like forms), saurosphargids, protorosaurs, and archosauriforms. Here, we report on a peculiar reptile, newly found in this fauna. Its dentition is fence or comb-like and bears more than 175 pleurodont teeth in each ramus of the upper and lower jaws, tooth crown is needle-like distally and blade-shaped proximally; its rostrum strongly bends downward and the anterior end of its mandible expands both dorsally and ventrally to form a shovel-headed structure; and its ungual phalanges are hoof-shaped. The specializations of the jaws and dentition indicate that the reptile may have been adapted to a way of bottom-filter feeding in water. It is obvious that such delicate teeth are not strong enough to catch prey, but were probably used as a barrier to filter microorganisms or benthic invertebrates such as sea worms. These were collected by the specialized jaws, which may have functioned as a shovel or pushdozer (the mandible) and a grasper or scratcher (the rostrum). Our preliminary analysis suggests that the new reptile might be more closely related to the Sauropterygia than to other marine reptiles.


Characterization of an imatinib-sensitive subset of high-grade human glioma cultures.
High-grade gliomas, including glioblastomas, are malignant brain tumors for which improved treatment is urgently needed. Genetic studies have demonstrated the existence of biologically distinct subsets. Preliminary studies have indicated that platelet-derived growth factor (PDGF) receptor signaling contributes to the growth of some of these tumors. In this study, human high-grade glioma primary cultures were analysed for sensitivity to treatment with the PDGF receptor inhibitor imatinib/Glivec/Gleevec/STI571. Six out of 15 cultures displayed more than 40% growth inhibition after imatinib treatment, whereas seven cultures showed less than 20% growth inhibition. In the sensitive cultures, apoptosis contributed to growth inhibition. Platelet-derived growth factor receptor status correlated with imatinib sensitivity. Supervised analyses of gene expression profiles and real-time PCR analyses identified expression of the chemokine CXCL12/SDF-1 (stromal cell-derived factor 1) as a predictor of imatinib sensitivity. Exogenous addition of CXCL12 to imatinib-insensitive cultures conferred some imatinib sensitivity. Finally, coregulation of CXCL12 and PDGF alpha-receptor was observed in glioblastoma biopsies. We have thus defined the characteristics of a novel imatinib-sensitive subset of glioma cultures, and provided evidence for a functional relationship between imatinib sensitivity and chemokine signaling. These findings will assist in the design and evaluation of clinical trials exploring therapeutic effects of imatinib on malignant brain tumors.


Beyond cortical localization in clinico-anatomical correlation.
Last year was the 150th anniversary of Paul Broca's landmark case report on speech disorder that paved the way for subsequent studies of cortical localization of higher cognitive functions. However, many complex functions rely on the activity of distributed networks rather than single cortical areas. Hence, it is important to understand how brain regions are linked within large-scale networks and to map lesions onto connecting white matter tracts. To facilitate this network approach we provide a synopsis of classical neurological syndromes associated with frontal, parietal, occipital, temporal and limbic lesions. A review of tractography studies in a variety of neuropsychiatric disorders is also included. The synopsis is accompanied by a new atlas of the human white matter connections based on diffusion tensor tractography freely downloadable on http://www.natbrainlab.com. Clinicians can use the maps to accurately identify the tract affected by lesions visible on conventional CT or MRI. The atlas will also assist researchers to interpret their group analysis results. We hope that the synopsis and the atlas by allowing a precise localization of white matter lesions and associated symptoms will facilitate future work on the functional correlates of human neural networks as derived from the study of clinical populations. Our goal is to stimulate clinicians to develop a critical approach to clinico-anatomical correlative studies and broaden their view of clinical anatomy beyond the cortical surface in order to encompass the dysfunction related to connecting pathways.


Pathophysiology of severe malaria in children.
Over the past decade there has been a growing recognition that the rationalization of severe malaria in children into the two major syndromes of cerebral malaria and severe malaria anaemia is much too simplistic. Indeed, it has become apparent that death from severe malaria may arise from a wider spectrum of pathophysiological disorders with many features in common with the derangements seen in sepsis syndromes. Amongst these derangements acidosis has emerged as a central feature of severe malaria and the major predictor of a fatal outcome. We review the improved understanding of the pathophysiology of severe malaria through a series of clinical scenarios that reflect more accurately the clinical diversity of severe malaria in African children. Current therapeutic challenges are discussed and research priorities are highlighted.


Uncovering the roles of long non-coding RNAs in cancer stem cells.
Cancer has been a major public health problem that has threatened human life worldwide throughout history. The main causes that contribute to the poor prognosis of cancer are metastasis and recurrence. Cancer stem cells are a group of tumor cells that possess self-renewal and differentiation ability, which is a vital cause of cancer metastasis and recurrence. Long non-coding RNAs refer to a class of RNAs that are longer than 200 nt and have no potential to code proteins, some of which can be specifically expressed in different tissues and different tumors. Long non-coding RNAs have great biological significance in the occurrence and progression of cancers. However, how long non-coding RNAs interact with cancer stem cells and then affect cancer metastasis and recurrence is not yet clear. Therefore, this review aims to summarize recent studies that focus on how long non-coding RNAs impact tumor occurrence and progression by affecting cancer stem cell self-renewal and differentiation in liver cancer, prostate cancer, breast cancer, and glioma.


Effect of alcohol consumption on host release of interleukin-17 during pulmonary infection with Klebsiella pneumoniae.
A link between alcohol abuse and bacterial pneumonia has been recognized for centuries, but mechanisms to explain this relationship are unclarified. Interleukin-17 (IL-17) is a lymphocyte-derived cytokine that is part of the inflammatory cytokine cascade. Previous studies from our laboratory indicated that IL-17 is released in lung tissue in a murine model of bacterial pneumonia caused by Klebsiella pneumoniae. The effects of alcohol consumption on pulmonary release of IL-17 are unknown. Mice were maintained on 20% ethanol in drinking water or on a control diet without alcohol. After 2 weeks, alcohol and control mice were challenged with intratracheal K. pneumoniae. Mice were followed for survival after bacterial challenge, neutrophil recruitment was assayed as myeloperoxidase, and IL-17 was measured in lung lavage fluid by enzyme-linked immunosorbent assay. In additional experiments, splenocytes from control mice were incubated with ethanol in vitro, and release of IL-17 was measured in culture supernatants. Finally, control and alcohol mice received intrapulmonary gene transfer of E-1-deleted adenovirus containing the murine IL-17 gene. These mice were then challenged with K. pneumoniae and followed for survival and neutrophil recruitment. In these studies, we demonstrate that a 2-week history of ethanol consumption in mice suppresses release of IL-17 into lung tissue, decreases neutrophil recruitment, and increases mortality from experimental K. pneumonia. In vitro experiments confirm a direct suppressive effect of ethanol on the release of IL-17 from splenocytes. In vivo administration of the IL-17 gene in an adenoviral vector to alcohol-consuming mice results in release of IL-17 into lavage fluid and normalizes neutrophil recruitment and mortality after bacterial challenge. The results of these experiments strongly implicate IL-17 as an important pathway for the immunosuppression associated with alcohol abuse and support gene therapeutic approaches to augment immune function in the alcoholic host or to treat infections associated with alcoholism.


Ultrasound Measurements of Subcutaneous Fat Thickness Are Robust Against Hydration Changes.
Ultrasound is an appealing tool to assess body composition, combining the portability of a field method with the accuracy of a laboratory method. However, unlike other body composition methods, the effect of hydration status on validity is unknown. This study evaluated the impact of acute hydration changes on ultrasound measurements of subcutaneous fat thickness and estimates of body fat percentage. In a crossover design, 11 adults (27.1 ± 10.5 years) completed dehydration and hyperhydration trials to alter body mass by approximately ±2%. Dehydration was achieved via humid heat (40 °C, 60% relative humidity) with exercise, whereas hyperhydration was via ingestion of lightly salted water. Ultrasound measurements were taken at 11 body sites before and after each treatment. Participants lost 1.56 ± 0.58 kg (-2.0 ± 0.6%) during the dehydration trial and gained 0.90 ± 0.21 kg (1.2 ± 0.2%) during the hyperhydration trial even after urination. The sum of fat thicknesses as measured by ultrasound differed by <0.90 mm across trials (p = .588), and ultrasound estimates of body fat percentage differed by <0.5% body fat. Ultrasound measures of subcutaneous adipose tissue were unaffected by acute changes in hydration status by extents beyond which are rare and overtly self-correcting, suggesting that this method provides reliable and robust body composition results even when subjects are not euhydrated.


Crashes involving cyclists aged 50 and over in the Netherlands: An in-depth study.
The number of seriously injured cyclists is increasing in the Netherlands. The majority of these seriously injured cyclists were involved in single-bicycle or bicycle-bicycle crashes. Little is known about the circumstances in which these crashes occur, as the police only registers 4% of these crashes. Therefore, an in-depth study was carried out to gain insight into the factors and circumstances that influence the occurrence and consequences of these crashes. The focus was on crashes involving cyclists aged 50 and over, as this group has a large share in the number of cyclist-only crashes. Detailed information on 41 single-bicycle and bicycle-bicycle crashes was collected and analysed. This resulted in a description of the course of events for every analysed crash, including a list of factors that had contributed to the occurrence of the crash and possible injuries. Subsequently, crashes with a similar course of events and a comparable combination of contributory factors were grouped into types of crashes. Results showed that cyclists aged 75 and over are more often involved in falls from a bicycle than younger cyclists. Contributory factors that played a role in a large number of crashes were behaviour of another road user, distraction and narrow cycling facilities or traffic lanes. However, which factors played a role in the occurrence of a crash depended on the type of crash. Eight types of crashes were identified. Based on the factors that played a role in the occurrence of these crashes, remedial measures can be developed to prevent similar crashes from occurring in the future.


Leukocytosis after Clozapine Treatment in a Patient with Chronic Schizophrenia.
Clozapine is an atypical antipsychotic drug that is approved by the US Food and Drug Administration (FDA) for the treatment of psychotic disorders. Agranulocytosis is a well-established side effect of clozapine; clozapine has also been associated with other blood dyscrasias like leukocytosis, albeit rarely. In this paper, we aim to report a case of possible clozapine-associated leukocytosis in a 41-year-old woman.


Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development.
The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis.


Women's television watching and reproductive health behavior in Bangladesh.
Bangladesh has made significant social, economic, and health progress in recent decades, yet many reproductive health indicators remain weak. Access to television (TV) is increasing rapidly and provides a potential mechanism for influencing health behavior. We present a conceptual framework for the influence of different types of TV exposure on individual's aspirations and health behavior through the mechanisms of observational learning and ideational change. We analyze data from two large national surveys conducted in 2010 and 2011 to examine the association between women's TV watching and five reproductive health behaviors controlling for the effects of observed confounders. We find that TV watchers are significantly more likely to desire fewer children, are more likely to use contraceptives, and are less likely to have a birth in the two years before the survey. They are more likely to seek at least four antenatal care visits and to utilize a skilled birth attendant. Consequently, continued increase in the reach of TV and associated growth in TV viewing is potentially an important driver of health behaviors in the country.


A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes.
Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the majority of published human faecal metagenomes, which we refer to as crAssphage. Its ~97 kbp genome is six times more abundant in publicly available metagenomes than all other known phages together; it comprises up to 90% and 22% of all reads in virus-like particle (VLP)-derived metagenomes and total community metagenomes, respectively; and it totals 1.68% of all human faecal metagenomic sequencing reads in the public databases. The majority of crAssphage-encoded proteins match no known sequences in the database, which is why it was not detected before. Using a new co-occurrence profiling approach, we predict a Bacteroides host for this phage, consistent with Bacteroides-related protein homologues and a unique carbohydrate-binding domain encoded in the phage genome.


Patients on hemodialysis are better served by a proximal arteriovenous fistula for long-term venous access.
Patients with end-stage renal disease should have arteriovenous fistula (AVF) formation 3 to 6 months prior to commencing hemodialysis (HD). However, this is not always possible with strained health care resources. We aim to compare autologous proximal AVF (PAVF) with distal AVF (DAVF) in patients already on HD. Primary end point is 4-year functional primary. Secondary end point is freedom from major adverse clinical events (MACEs). From January 2003 to June 2009, out of 495 AVF formations, 179 (36%) patients were already on HD. These patients had 200 AVF formations (49 DAVF vs 151 PAVF) in arms in which no previous fistula had been formed. No synthetic graft was used. Four-year primary functional patency significantly improved with PAVF (68.9% ± SD 8.8%) compared to DAVF (7.3% ± SD 4.9%; P < .0001). Five-year freedom from MACE was 85% with PAVF compared to 40% with DAVF (P < .005). Proximal AVF bestows long-term functional access with fewer complications compared to DAVF for patients already on HD.


Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies.
We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM10, all directed against the C3bi receptor of human monocytes and macrophages (M phi). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on M phi adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from M phi lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM10 binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from 125I-surface-labeled M phi. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human M phi is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.


The high Andes, gene flow and a stable hybrid zone shape the genetic structure of a wide-ranging South American parrot.
While the gene flow in some organisms is strongly affected by physical barriers and geographical distance, other highly mobile species are able to overcome such constraints. In southern South America, the Andes (here up to 6,900 m) may constitute a formidable barrier to dispersal. In addition, this region was affected by cycles of intercalating arid/moist periods during the Upper/Late Pleistocene and Holocene. These factors may have been crucial in driving the phylogeographic structure of the vertebrate fauna of the region. Here we test these hypotheses in the burrowing parrot Cyanoliseus patagonus (Aves, Psittaciformes) across its wide distributional range in Chile and Argentina. Our data show a Chilean origin for this species, with a single migration event across the Andes during the Upper/Late Pleistocene, which gave rise to all extant Argentinean mitochondrial lineages. Analyses suggest a complex population structure for burrowing parrots in Argentina, which includes a hybrid zone that has remained stable for several thousand years. Within this zone, introgression by expanding haplotypes has resulted in the evolution of an intermediate phenotype. Multivariate regressions show that present day climatic variables have a strong influence on the distribution of genetic heterogeneity, accounting for almost half of the variation in the data. Here we show how huge barriers like the Andes and the regional environmental conditions imposed constraints on the ability of a parrot species to colonise new habitats, affecting the way in which populations diverged and thus, genetic structure. When contact between divergent populations was re-established, a stable hybrid zone was formed, functioning as a channel for genetic exchange between populations.


Endothelial loss of Fzd5 stimulates PKC/Ets1-mediated transcription of Angpt2 and Flt1.
Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. Using short interference RNA-mediated loss-of-function assays, the function and mechanism of signaling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neovessel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G0/G1 cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of canonical Wnt signaling, non-canonical Wnt/Ca2+-mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2.


Not all Pseudomonas aeruginosa are equal: strains from industrial sources possess uniquely large multireplicon genomes.
Pseudomonas aeruginosa is a highly versatile, antibiotic-resistant Gram-negative bacterium known for causing opportunistic infections and contamination of industrial products. Despite extensive genomic analysis of clinical P. aeruginosa strains, no genomes exist for preservative-tolerant industrial strains. A unique collection of 69 industrial isolates was assembled and compared to clinical and environmental strains; 16 genetically distinct industrial strains were subjected to array tube genotyping, multilocus sequence typing and whole-genome sequencing. The industrial strains possessed high preservative tolerance and were dispersed widely across P. aeruginosa as a species, but recurrence of strains from the same lineage within specific industrial products and locations was identified. The industrial P. aeruginosa genomes (mean=7.0 Mb) were significantly larger than those of previously sequenced environmental (mean=6.5 Mb; n=19) and clinical (mean=6.6 Mb; n=66) strains. Complete sequencing of the P. aeruginosa industrial strain RW109, which encoded the largest genome (7.75 Mb), revealed a multireplicon structure including a megaplasmid (555 265 bp) and large plasmid (151 612 bp). The RW109 megaplasmid represented an emerging plasmid family conserved in seven industrial and two clinical P. aeruginosa strains, and associated with extremely stress-resilient phenotypes, including antimicrobial resistance and solvent tolerance. Here, by defining the detailed phylogenomics of P. aeruginosa industrial strains, we show that they uniquely possess multireplicon, megaplasmid-bearing genomes, and significantly greater genomic content worthy of further study.


Advances in the management of chronic obstructive pulmonary disease.
Chronic obstructive pulmonary disease (COPD), the fourth leading cause of death, seems to be increasing in worldwide prevalence, and carries with it a significant health and economic burden. Smoking cessation is the only available intervention proven to halt disease progression. The authors discuss the role of the newly approved agent, varenicline, in promotion of smoking cessation. The remainder of presently available therapies treat the symptoms of COPD, but do not impact progression of disease. As the understanding of the pathogenesis of COPD improves, new targets for therapies are emerging. Given the large number of potential targets and the results of recent studies, it seems unlikely that a single new agent will result in a cure. Rather, management of COPD should involve a multi-pronged approach including smoking cessation, bronchodilators, treatment of infection, and eventual targeting of inflammatory pathways and genetic predispositions. In this article, the authors discuss presently available therapies as well as agents under development.


Diversity of the parB and repA genes of the Burkholderia cepacia complex and their utility for rapid identification of Burkholderia cenocepacia.
Burkholderia cenocepacia is the most prominent species of the B. cepacia complex (Bcc), a group of nine closely related and difficult to identify bacteria that cause serious infections in patients with cystic fibrosis. Despite its clinical relevance, identification of B. cenocepacia as a single species is unavailable, as it splits by a widely used recA gene-based PCR identification method into discrete phylogenetic subgroups IIIA, IIIB, IIIC and IIID. With the aim of identifying gene targets suitable for unified detection of B. cenocepacia strains, we examined sequence polymorphisms in the repA and parB genes. These essential genes are involved in the replication and partitioning of bacterial replicons, hence we also had the opportunity for the first time to investigate the evolution of the multireplicon (three chromosome) structure of Bcc genomes. Alignment of the repA and parB genes from publicly available Bcc genome sequences enabled the design of primers for their amplification and sequence analysis. Multilocus sequencing typing, a highly discriminatory method for Bcc species and strain discrimination, was used to select strains of unique sequence types (STs) that spanned the known Bcc genetic diversity. Sequence datasets of repA (83 isolates, 67 STs) and parB (120 isolates, 95 STs) genes from the second chromosome were aligned and examined phylogenetically to identify polymorphisms suitable for identification of B. cenocepacia. In contrast to parB, the Bcc repA sequences demonstrated distinct clustering of B. cenocepacia from other species, which enabled the design a species-specific multiplex PCR. The novel single-reaction B. cenocepacia detection method was tested on a panel of 142 different Bcc strains (142 STs) and distinguished recA groups IIIA, IIIB and IIID, from all other Bcc members with 100% sensitivity and 93% specificity. The repA-based multiplex PCR is a useful aid to the rapid identification of the most clinically relevant B. cenocepacia recA subgroups IIIA, IIIB and IIID. Phylogenetic analysis of repA and parB genes demonstrated that acquisition of the second and third replicons of Bcc genomes occurred prior to their differentiation into discrete species and that the sharing of replicons across species had not occurred.


Stimulant treatment for attention-deficit hyperactivity disorder and risk of developing substance use disorder.
Attention-deficit hyperactivity disorder (ADHD) is linked to increased risk for substance use disorders and nicotine dependence. To examine the effects of stimulant treatment on subsequent risk for substance use disorder and nicotine dependence in a prospective longitudinal ADHD case-control study. At baseline we assessed ADHD, conduct disorder and oppositional defiant disorder. Substance use disorders, nicotine dependence and stimulant treatment were assessed retrospectively after a mean follow-up of 4.4 years, at a mean age of 16.4 years. Stimulant treatment of ADHD was linked to a reduced risk for substance use disorders compared with no stimulant treatment, even after controlling for conduct disorder and oppositional defiant disorder (hazard ratio (HR) = 1.91, 95% CI 1.10-3.36), but not to nicotine dependence (HR = 1.12, 95% CI 0.45-2.96). Within the stimulant-treated group, a protective effect of age at first stimulant use on substance use disorder development was found, which diminished with age, and seemed to reverse around the age of 18. Stimulant treatment appears to lower the risk of developing substance use disorders and does not have an impact on the development of nicotine dependence in adolescents with ADHD.


The importance of clinician, patient and researcher collaborations in Alport syndrome.
Alport syndrome is caused by mutations in the genes COL4A3, COL4A4 or COL4A5 and is characterised by progressive glomerular disease, sensorineural hearing loss and ocular defects. Occurring in less than 1:5000, Alport syndrome is a rare genetic disorder but still accounts for > 1% of the prevalent population receiving renal replacement therapy. There is also increasing awareness about the risk of chronic kidney disease in individuals with heterozygous mutations in Alport syndrome genes. The mainstay of current therapy is the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, yet potential new therapies are now entering clinical trials. The 2017 International Workshop on Alport Syndrome in Glasgow was a pre-conference workshop ahead of the 50th anniversary meeting of the European Society for Pediatric Nephrology. It focussed on updates in clinical practice, genetics and basic science and also incorporated patient perspectives. More than 80 international experts including clinicians, geneticists, researchers from academia and industry, and patient representatives took part in panel discussions and breakout groups. This report summarises the workshop proceedings and the relevant contemporary literature. It highlights the unique clinician, patient and researcher collaborations achieved by regular engagement between the groups.


Oncologic doses of zoledronic acid induce osteonecrosis of the jaw-like lesions in rice rats (Oryzomys palustris) with periodontitis.
Though osteonecrosis of the jaw (ONJ) is temporally-associated with the use of nitrogen-containing bisphosphonates (N-BPs), a cause-and-effect relationship has not yet been established. We hypothesize that ONJ is a two-stage process in which: (1) risk factors initiate pathologic processes in the oral cavity that lead to a supranormal rate of hard tissue necrosis; and (2) powerful antiresorptives reduce the rate of removal of necrotic bone sufficiently to allow its net accumulation in the jaw. To test this hypothesis, we used the rice rat model of periodontitis. At age 28 days, rats (n = 15/group) were placed on a high-sucrose and casein diet to exacerbate the development of periodontitis. Animals were injected subcutaneously (SC) biweekly with vehicle or alendronate (ALN, 15 µg/kg), or IV once monthly with vehicle, a low dose (LD) of zoledronic acid (ZOL), or a high dose (HD) of ZOL and sacrificed after 6, 12, 18, and 24 weeks. Mandibles and maxillae were analyzed to determine the effects on the: (1) progression of periodontitis; (2) integrity of alveolar bone; (3) status of bone resorption and formation; (4) vascularity; and (5) osteocyte viability. We found that only HD-ZOL induced ONJ-like lesions in mandibles of rice rats after 18 and 24 weeks of treatment. These lesions were characterized by areas of exposed necrotic alveolar bone, osteolysis, a honeycomb-like appearance of the alveolar bone, presence of bacterial colonies, and periodontal tissue destruction. In addition, inhibition of bone formation, a paradoxical abolition of the antiresorptive effect of only HD-ZOL, increased osteocyte necrosis/apoptosis, and decreased blood vessel number were found after 18 and/or 24 weeks. Our study suggests that only HD-ZOL exacerbates the inflammatory response and periodontal tissue damage in rice rats, inducing bone lesions that resemble ONJ.