Diabetes Distress and Glycemic Control: The Buffering Effect of Autonomy Support From Important Family Members and Friends.
To examine whether autonomy support (defined as social support for an individual's personal agency) for diabetes management from informal health supporters (family/friends) reduces the detrimental effects of diabetes distress on glycemic control. Three hundred eight veterans with type 2 diabetes and one or more risk factors for diabetes complications completed a survey that included measures of diabetes distress and perceived autonomy support from their main informal health supporter. Hemoglobin A1c (HbA1c) data from 12 months before and after the survey were extracted from electronic medical records. Linear mixed modeling examined the main effects and interaction of autonomy support and diabetes distress on repeated measures of HbA1c over the 12 months after the survey, controlling for mean prior 12-month HbA1c, time, insulin use, age, and race/ethnicity. Diabetes distress (B = 0.12 [SE 0.05]; P = 0.023) was associated with higher and autonomy support (B = -0.16 [SE 0.07]; P = 0.032) with lower subsequent HbA1c levels. Autonomy support moderated the relationship between diabetes distress and HbA1c (B = -0.13 [SE 0.06]; P = 0.027). Greater diabetes distress was associated with higher HbA1c at low (B = 0.21 [SE 07]; P = 0.002) but not high (B = 0.01 [SE 0.07]; P = 0.890) levels of autonomy support. Autonomy support from main health supporters may contribute to better glycemic control by ameliorating the effects of diabetes distress. Interventions that reduce diabetes distress and enhance the autonomy supportiveness of informal supporters may be effective approaches to improving glycemic control.


Fibrillin-2b regulates endocardial morphogenesis in zebrafish.
scotch tape (sco) is a zebrafish cardiac mutant initially proposed to exhibit a reduced amount of cardiac jelly, the extracellular matrix between the myocardial and endocardial layers. We analyzed sco(te382) mutant hearts in detail using both selective plane illumination microscopy (SPIM) and transmission electron microscopy (TEM), and observed a fascinating endocardial defect. Time-lapse SPIM imaging of wild-type and mutant embryos revealed significant and dynamic gaps between endocardial cells during development. Although these gaps close in wild-type animals, they fail to close in the mutants, ultimately leading to a near complete absence of endocardial cells in the atrial chamber by the heart looping stage. TEM analyses confirm the presence of gaps between endocardial cells in sco mutants, allowing the apparent leakage of cardiac jelly into the lumen. High-resolution mapping places the sco(te382) mutation within the fbn2b locus, which encodes the extracellular matrix protein Fibrillin 2b (OMIM ID: 121050). Complementation and further phenotypic analyses confirm that sco is allelic to puff daddy(gw1) (pfd(gw1)), a null mutant in fbn2b, and that sco(te382) is a hypomorphic allele of fbn2b. fbn2b belongs to a family of genes responsible for the assembly of microfibrils throughout development, and is essential for microfibril structural integrity. In sco(te382) mutants, Fbn2b is disabled by a missense mutation in a highly conserved cbEGF domain, which likely interferes with protein folding. Integrating data obtained from microscopy and molecular biology, we posit that this mutation impacts the rigidity of Fbn2b, imparting a structural defect that weakens endocardial adhesion thereby resulting in perforated endocardium.


Elastomeric high-mineral content hydrogel-hydroxyapatite composites for orthopedic applications.
The design of synthetic bone grafts that mimic the structure and composition of bone and possess good surgical handling characteristics remains a major challenge. We report the development of poly(2-hydroxyethyl methacrylate) (pHEMA)-hydroxyapatite (HA) composites termed "FlexBone" that possess osteoconductive mineral content approximating that of human bone yet exhibit elastomeric properties enabling the press-fitting into a defect site. The approach involves crosslinking pHEMA hydrogel in the presence of HA using viscous ethylene glycol as a solvent. The composites exhibit excellent structural integration between the apatite mineral component and the hydroxylated hydrogel matrix. The stiffness of the composite and the ability to withstand compressive stress correlate with the microstructure and content of the mineral component. The incorporation of porous aggregates of HA nanocrystals rather than compact micrometer-sized calcined HA effectively improved the resistance of the composite to crack propagation under compression. Freeze-dried FlexBone containing 50 wt % porous HA nanocrystals could withstand hundreds-of-megapascals compressive stress and >80% compressive strain without exhibiting brittle fractures. Upon equilibration with water, FlexBone retained good structural integration and withstood repetitive moderate (megapascals) compressive stress at body temperature. When subcutaneously implanted in rats, FlexBone supported osteoblastic differentiation of the bone marrow stromal cells pre-seeded on FlexBone. Taken together, the combination of high osteoconductive mineral content, excellent organic-inorganic structural integration, elasticity, and the ability to support osteoblastic differentiation in vivo makes FlexBone a promising candidate for orthopedic applications.


Fitness for transport of cull dairy cows at livestock markets.
Cows are regularly removed from dairy herds and sold at livestock markets. Many cows are removed because of health problems, and their fitness for transport may vary because of seasonal variation, delayed or poor on-farm culling decisions, injuries during transport, and other factors. However, many dairy producers lack feedback about the condition of their cows during the marketing process and how cow condition influences sale price. This study evaluated the condition of cull dairy cows sold at livestock markets, tested how changing demand for milk influenced fitness for transport, and quantified how cow condition affected the price paid. For 12 mo, 2 livestock markets in British Columbia, Canada, were visited during 137 auction events when cull dairy cows were sold; 3 trained assessors observed 6,263 cull dairy cows while they were marketed in a sale ring. Observers recorded the cows' body condition score (BCS), locomotion score (LS), udder condition, quality defects (e.g., injuries, illness), and price. Logistic regression was used to test how month-to-month changes in demand for milk affected cows' fitness for transport, and a linear mixed model assessed how the animals' condition influenced the price. About 10% of the cows were thin (BCS ≤2), 7% were severely lame (locomotion score ≥4), 13% had engorged or inflamed udders, and 6% had other quality defects including abscesses, injuries, and signs of sickness (e.g., pneumonia). Cows culled during months with increased milk demand had much higher odds of poor fitness for transport (odds ratio 8.6, 95% confidence interval: 4.02-18.22). The price was most reduced if cows were thin (BCS ≤2) or visibly sick (-$0.63 ± 0.01/kg and -$0.56 ± 0.02/kg, respectively). Prices were reduced to a lesser degree by locomotion score ≥4 (-$0.35 ± 0.02/kg) and by udder condition (udder inflammation; -$0.30 ± 0.02/kg). Overall fitness for transport reduced the price by $0.51 ± 0.01/kg. In summary, about 30% of the cows sold at livestock markets had poor fitness for transport, which was partially influenced by increased milk demand and resulted in reduced market prices.


mTORC1 Signaling in oocytes is dispensable for the survival of primordial follicles and for female fertility.
The molecular mechanisms underlying reproductive aging and menopausal age in female mammals are poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) is a central controller of cell growth and proliferation. To determine whether mTORC1 signaling in oocytes plays a direct role in physiological follicular development and fertility in female mice, we conditionally deleted the specific and essential mTORC1 component Rptor (regulatory-associated protein of mTORC1) from the oocytes of primordial follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We provide in vivo evidence that deletion of Rptor in the oocytes of both primordial and further-developed follicles leads to the loss of mTORC1 signaling in oocytes as indicated by loss of phosphorylation of S6K1 and 4e-bp1 at T389 and S65, respectively. However, the follicular development and fertility of mice lacking Rptor in oocytes were not affected. Mechanistically, the loss of mTORC1 signaling in Rptor-deleted mouse oocytes led to the elevation of phosphatidylinositol 3-kinase (PI3K) signaling that maintained normal follicular development and fertility. Therefore, this study shows that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K signaling cascade that maintains normal ovarian follicular development and fertility.


Revisiting the cost-effectiveness of the COMBINE study for alcohol dependent patients: the patient perspective.
Most cost and cost-effectiveness studies of substance abuse treatments focus on the costs to the provider/payer. Although this perspective is important, the costs incurred by patients should also be considered when evaluating treatment. This article presents estimates of patients' costs associated with the Combined Pharmacotherapies and Behavioral Interventions (COMBINE) alcohol treatments and evaluates the treatments' cost-effectiveness from the patient perspective. A prospective cost-effectiveness study of patients in COMBINE, a randomized controlled clinical trial of 9 alternative alcohol treatment regimens involving 1383 patients with diagnoses of primary alcohol dependence across 11 US clinic sites. We followed a microcosting approach that allowed estimation of patients' costs for specific COMBINE treatment activities. The primary clinical outcomes from COMBINE are used as indicators of treatment effectiveness. The average total patient time devoted to treatment ranged from about 30 hours to 46 hours. Time spent traveling to and from treatment sessions and participation in self-help meetings accounted for the largest portion of patient time costs. The cost-effectiveness results indicate that 6 of the 9 treatments were economically dominated and only 3 treatments are potentially cost-effective depending on patient's willingness to pay for the considered outcomes: medical management (MM) + placebo, MM + naltrexone, and MM + naltrexone + acamprosate. Few studies consider the patient's perspective in estimating costs and cost-effectiveness even though these costs may have a substantial impact on a patient's treatment choice, ability to access treatment, or treatment adherence. For this study, the choice of the most cost-effective treatment depends on the value placed on the outcomes by the patient, and the conclusions drawn by the patient may differ from that of the provider/payer.


SimBac: simulation of whole bacterial genomes with homologous recombination.
Bacteria can exchange genetic material, or acquire genes found in the environment. This process, generally known as bacterial recombination, can have a strong impact on the evolution and phenotype of bacteria, for example causing the spread of antibiotic resistance across clades and species, but can also disrupt phylogenetic and transmission inferences. With the increasing affordability of whole genome sequencing, the need has emerged for an efficient simulator of bacterial evolution to test and compare methods for phylogenetic and population genetic inference, and for simulation-based estimation. We present SimBac, a whole-genome bacterial evolution simulator that is roughly two orders of magnitude faster than previous software and includes a more general model of bacterial evolution, allowing both within- and between-species homologous recombination. Since methods modelling bacterial recombination generally focus on only one of these two modes of recombination, the possibility to simulate both allows for a general and fair benchmarking. SimBac is available from https://github.com/tbrown91/SimBac and is distributed as open source under the terms of the GNU General Public Licence.


Discerning the role of topography and ion exchange in cell response of bioactive tissue engineering scaffolds.
Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.


Linking molecular and population stress responses in Daphnia magna exposed to cadmium.
DNA microarrays can be used to measure environmental stress responses. If they are to be predictive of environmental impact, we need to determine if altered gene expression translates into negative impacts on individuals and populations. A large cDNA microarray (14000 spots) was created to measure molecular stress responses to cadmium in Daphnia magna,the mostwidely used aquatic indicator species, and relate responses to population growth rate (pgr). We used the array to detect differences in the transcription of genes in juvenile D. magna (24 h old) after 24 h exposure to a control and three cadmium concentrations (6, 20, and 37 microg Cd2+ L(-1)). Stress responses at the population level were estimated following a further 8 days exposure. Pgr was approximately linear negative with increasing cadmium concentration over this range. The microarray profile of gene expression in response to acute cadmium exposure begins to provide an overview of the molecular responses of D. magna, especially in relation to growth and development. Of the responding genes, 29% were involved with metabolism including carbohydrate, fat and peptide metabolism, and energy production, 31% were involved with transcription/translation, while 40% of responding genes were associated with cellular processes like growth and moulting, ion transport, and general stress responses (which included oxidative stress). Our production and application of a large Daphnia magna microarray has shown that measured gene responses can be logically linked to the impact of a toxicant such as cadmium on somatic growth and development, and consequently pgr.


"Appropriate" diagnostic testing: supporting diagnostics with evidence-based medicine and shared decision making.
Evidence-based medicine is an important approach to avoiding care that is unlikely to benefit patients in both the treatment and the diagnostic context. The medical evidence alone may not determine the most appropriate care decision. Patient interests are best served when the advantages and risks of a diagnostic test are viewed through the lens of the patient's values. That is, the paradigm of evidence-based medicine should be complemented by the paradigm of shared decision making. Diagnostic testing may offer physiological and psychological benefits. Clinicians should also discuss the potential harms, however, which may be physiological (e.g. radiation or scarring), psychological (e.g. anxiety), and financial (e.g. cost-sharing burdens). All three of these concerns are compounded by the risk of false positives or incidental findings that are not serious, but which require decisions about further testing or treatment. We suggest that patient-centered decision making around diagnostic testing involves a two-step inquiry:(1) Is the test medically appropriate? Does the available evidence documenting short- and long-term risk and benefits support the test for its intended use, given the patient's characteristics and symptoms?(2) Is the test appropriate for this patient? Has the provider initiated a conversation about tradeoffs that helps the patient evaluate whether the balance of risks and benefits is consonant with the patient's own values and preferences? Potential benefits and harms to consider include the physiological, the psychological, and the financial.


Can Ebola virus evolve to be less virulent in humans?
Understanding Ebola virus (EBOV) virulence evolution not only is timely but also raises specific questions because it causes one of the most virulent human infections and it is capable of transmission after the death of its host. Using a compartmental epidemiological model that captures three transmission routes (by regular contact, via dead bodies and by sexual contact), we infer the evolutionary dynamics of case fatality ratio on the scale of an outbreak and in the long term. Our major finding is that the virus's specific life cycle imposes selection for high levels of virulence and that this pattern is robust to parameter variations in biological ranges. In addition to shedding a new light on the ultimate causes of EBOV's high virulence, these results generate testable predictions and contribute to informing public health policies. In particular, burial management stands out as the most appropriate intervention since it decreases the R0 of the epidemics, while imposing selection for less virulent strains.


Stimulated Raman spectroscopy of analytes evanescently probed by a silicon nitride photonic integrated waveguide.
We report, to the best of our knowledge, the first demonstration of stimulated Raman spectroscopy enhanced by a nanophotonic integrated circuit. The Raman response of low-concentration dimethyl sulfoxide is evanescently probed via centimeter-long wire waveguides. A signal enhancement of close to five orders of magnitude, as compared to the case of on-chip spontaneous Raman scattering, is demonstrated. This significant enhancement factor allows for the use of continuous-wave lasers with milliwatt-level power and uncooled detectors and, therefore, sets the basis of future all-on-a-chip Raman spectrometers suitable for both gas and liquid detection.


Percutaneous needle biopsy of the lung and its impact on patient management.
Percutaneous needle biopsy (PNB) of the lung is a commonly performed procedure, mainly used for the investigation of solitary pulmonary nodules. Developments in imaging, particularly computed tomography (CT), have enable accurate preliminary assessment and targeting of lesions. Improvements in needle design ensure the provision of diagnostic samples for both cytologic and histologic assessment; and the development of immunocytochemistry and immunohistochemistry have allowed improved accuracy in diagnosis. A significant improvement in diagnostic accuracy for benign lesions has been associated with the use of cutting needles that provide cores for histologic diagnosis, in contrast to cytologic analysis from fine-needle aspiration. The complications of PNB are well recorded and have not changed significantly with the newer imaging techniques and needles. The preliminary assessment of solitary pulmonary nodules, and the pretest likelihood of malignancy, has improved using contrast-enhanced CT and positron emission tomography; the latter modality is increasingly having a major impact on the investigation of patients with suspected malignancy. The performance of PNB must always be determined on an individual case basis and when the result is likely to affect management. The complementary roles of PNB, bronchoscopic biopsy, and video-assisted thoracoscopic biopsy continue to evolve.


Exploring the use of little cigars by students at a historically black university.
Considerable evidence exists that little cigars are popular among African American adolescents and young adults who smoke. However, few studies have been published on the use of this tobacco product by young blacks in the United States. This research investigated little-cigar use among students at a historically black university in the southeastern United States. As a follow-up to a survey on tobacco use among freshmen that revealed unexpectedly high rates of little-cigar use, 3 focus groups were conducted with current or former smokers of little cigars. Topics included preferred brands of little cigars, preference for little cigars over cigarettes, social contexts for smoking little cigars, perceived health risks of smoking little cigars relative to smoking cigarettes, and thoughts about quitting. Focus group participants preferred little cigars to cigarettes for various reasons, among them taste, smell, a better "buzz," social purposes, status, and perceptions that smoking little cigars is less addictive and less harmful than smoking cigarettes. Opinions on health risks varied; some participants believed that health risks can be reduced by removing the inner liner of little cigars. Use of little cigars should be addressed in tobacco research, use prevention, and use cessation efforts, targeting students at historically black colleges and perhaps other young African Americans. Results also suggest that clear distinctions should be made among cigarettes, little cigars, and cigars, and that tobacco use prevention and cessation programs should debunk myths that little cigars are a safe alternative to cigarettes. Study findings should be confirmed and elucidated through additional research.


Maintenance of immune tolerance to a neo-self acetylcholine receptor antigen with aging: implications for late-onset autoimmunity.
Age-related changes in immune regulation are likely to account for the age-associated increase in serum autoantibody levels and in certain autoimmune disorders, such as myasthenia gravis (MG). To demonstrate directly a loss of immune tolerance in older individuals, responses to the acetylcholine receptor, the autoantigen in MG, were assessed in transgenic mice expressing the Torpedo californica acetylcholine receptor (TAChR) alpha-chain as a neo-self Ag. T cells from young transgenic mice had been shown to be tolerant to p146-162, the TAChR alpha-chain peptide that dominated young nontransgenic T cell responses in vitro. The immunodominance of p146-162 was not lost with age; fine specificity was preserved. Moreover, T cell tolerance to p146-162, as well as to other epitopes of the TAChR alpha-chain extracellular domain, was maintained in old transgenic mice. Even multiple TAChR immunizations coupled with the MG-enhancing cytokine, IL-12, did not break tolerance. In addition, T cells exhibiting CD4 upregulation, an early activation marker, were reduced in frequency equivalently in old and young transgenic animals, suggesting that immune regulation in this model was not impacted by aging. Moreover, B cell tolerance was also maintained with age. The persistence of immune tolerance was accompanied by an increase in the proportion of T regulatory cells; it is speculated that this may compensate for deficiencies in central tolerance that occur owing to thymic involution. In summary, our study reveals, for the first time, that some immune tolerance mechanisms do survive aging; this suggests that certain late-onset autoimmune disorders may be induced by a specific insult that disrupts immune homeostasis.


Complete genome sequence of plant growth-promoting bacterium Leifsonia xyli SE134, a possible gibberellin and auxin producer.
Leifsonia xyli SE134 is a potential plant growth-promoting bacterium isolated from a soil in Daegu, Republic of Korea, which produces large amounts of gibberellin (GA) and indole acetic acid (IAA). In this study, we sequenced the complete genome of L. xyli SE134 by the Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. The genome of L. xyli SE134 contains a single chromosome that is 3,596,761bp in length, with 70.2% G+C content. The genome contains 3466 protein-coding genes (CDSs) and 51 rRNA- and 46 tRNA-coding genes. By genomic analysis, we identified genes that are potentially involved in plant growth promotion such as genes participating in indole-3-acetic acid (IAA) biosynthesis, siderophore, and trehalose production. L. xyli SE134 also harbours genes for central carbohydrate metabolism, indicating that it can utilise the root exudates with other organic materials as an energy source. Furthermore, the SE134 genome is equipped with various kinds of genes for adaptation to plant surfaces, e.g. defence against desiccation, nutrient deficiencies, and oxidative stress, and a large proportion of genes related to secretion mechanisms and signalling. The genetic information provided here may help to expand this bacterium's biotechnological potential and to further improve its plant growth-promoting characteristics.


A facile method for the deposition of volatile natural compound-based nanoparticles on biodegradable polymer surfaces.
In the current work, stable nanoparticles (NPs) of vanillin are formed in situ from an aqueous/ethanol solution and deposited on the surface of chitosan, a natural polymer, using a high-intensity ultrasonic method. The spectroscopic, physical, mechanical and morphological properties of the coated chitosan films are examined by helium-ion microscopy (HIM), atomic-force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR), UV-vis spectroscopy, X-ray diffractometry (XRD), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC) and texture analysis, and compared with the original film properties. Vanillin NPs were detected on the film surface. It was also found that the sonochemical deposition method does not affect the bulk properties of the chitosan films. All the chitosan films demonstrated antimicrobial activity against Gram-negative and Gram-positive bacteria. The deposition of vanillin NPs on the chitosan film also leads to significant antibiofilm activity, especially against the biofilm formation of Escherichia coli bacteria. The in vivo antimicrobial effect of the modified chitosan films was examined on fresh-cut watermelon, melon and strawberry. Vanillin NP-coated chitosan films led to inhibition of total microbial growth and the substantial inhibition of mold and yeast on the fruit. This research can serve as a platform for the development of a mild and effective method for the activation and modification of natural polymers for their future application in biomedical devices and biodegradable active packaging materials.


Correlation of early dynamic CT perfusion imaging with whole-brain MR diffusion and perfusion imaging in acute hemispheric stroke.
Compared with MR imaging, dynamic CT perfusion imaging covers only a fraction of the whole brain. An important assumption is that CT perfusion abnormalities correlate with total ischemic volume. The purpose of our study was to measure the degree of correlation between abnormalities seen on CT perfusion scans and the volumes of abnormality seen on MR diffusion and perfusion images in patients with acute large-vessel stroke. Fourteen patients with acute hemispheric stroke symptoms less than 12 hours in duration were studied with single-slice CT perfusion imaging and multislice MR diffusion and perfusion imaging. CT and MR perfusion studies were completed within 2.5 hours of one another (mean, 77 minutes) and were reviewed independently by two neuroradiologists. Hemodynamic parameters included cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT). Extents of abnormality on images were compared by using Kendall correlation. Statistically significant correlation was found between CT-CBF and MR-CBF abnormalities (tau = 0.60, P =.003) and CT-MTT and MR-MTT abnormalities (tau = 0.65, P =.001). Correlation of CT-CBV with MR-CBV approached significance (tau = 0.39, P =.06). Extent of initial hyperintensity on diffusion-weighted images correlated best with extent of MR-CBV abnormality (tau = 0.69, P =.001), extent of MR-MTT abnormality (tau = 0.67, P =.002), and extent of CT-CBV abnormality (tau = 0.47, P =.02). Good correlation was seen between CT and MR for CBF and MTT abnormalities. It remains uncertain whether CT perfusion CBV abnormalities correspond well to whole-brain abnormalities.


Polymer gate dielectric surface viscoelasticity modulates pentacene transistor performance.
Nanoscopically confined polymer films are known to exhibit substantially depressed glass transition temperatures (Lg's) as compared to the corresponding bulk materials. We report here that pentacene thin films grown on polymer gate dielectrics at temperatures well below their bulk Tg's exhibit distinctive and abrupt morphological and microstructural transitions and thin-film transistor (TFT) performance discontinuities at well-defined growth temperatures. The changes reflect the higher chain mobility of the dielectric in its rubbery state and are independent of dielectric film thickness. Optimization of organic TFT performance must recognize this fundamental buried interface viscoelasticity effect, which is detectable in the current-voltage response.


Complex epidemiology of a zoonotic disease in a culturally diverse region: phylogeography of rabies virus in the Middle East.
The Middle East is a culturally and politically diverse region at the gateway between Europe, Africa and Asia. Spatial dynamics of the fatal zoonotic disease rabies among countries of the Middle East and surrounding regions is poorly understood. An improved understanding of virus distribution is necessary to direct control methods. Previous studies have suggested regular trans-boundary movement, but have been unable to infer direction. Here we address these issues, by investigating the evolution of 183 rabies virus isolates collected from over 20 countries between 1972 and 2014. We have undertaken a discrete phylogeographic analysis on a subset of 139 samples to infer where and when movements of rabies have occurred. We provide evidence for four genetically distinct clades with separate origins currently circulating in the Middle East and surrounding countries. Introductions of these viruses have been followed by regular and multidirectional trans-boundary movements in some parts of the region, but relative isolation in others. There is evidence for minimal regular incursion of rabies from Central and Eastern Asia. These data support current initiatives for regional collaboration that are essential for rabies elimination.


Plantar sensation, plantar pressure, and postural stability alterations and effects of visual status in older adults.
Ageing leads to plantar sensation and pressure alterations and poor postural control. The aim of this study was to compare the plantar sensation and static plantar pressure distribution between young and older adults. A secondary aim was to investigate the effect of ageing and visual status on postural stability. Forty older subjects and 43 young adult individuals participated in the study. Plantar light touch sensation was evaluated using Semmes-Weinstein monofilaments. Static plantar pressure and postural stability were assessed with the WinTrack® Pedobarography device. Plantar sensation thresholds of the older individuals were higher compared to the young in all plantar regions (p < 0.001). The plantar contact area was greater in older individuals (p < 0.001). Maximum plantar pressure of midfoot was higher and maximum plantar pressure of the rearfoot and whole foot was less in older individuals during quiet stance (p < 0.05). The main effects of group and visual condition were significant for mean latero-lateral and antero-posterior sway speed with large effect sizes (p < 0.05). The sensation of all plantar regions reduced, the rearfoot plantar pressure decreased, and the midfoot plantar pressure increased in older individuals compared to young. Postural stability was reduced in the older individuals, and their postural control was more affected by the eliminated visual information compared to the young. Increased plantar contact area and midfoot plantar pressure may be related to decreased MLA height in older individuals. Older individuals may need visual information more to maintain postural control because of reduced plantar sensation.


Type I IFNs and CD8 T cells increase intestinal barrier permeability after chronic viral infection.
Intestinal barrier leakage constitutes a potential therapeutic target for many inflammatory diseases and represents a disease progression marker during chronic viral infections. However, the causes of altered gut barrier remain mostly unknown. Using murine infection with lymphocytic choriomeningitis virus, we demonstrate that, in contrast to an acute viral strain, a persistent viral isolate leads to long-term viral replication in hematopoietic and mesenchymal cells, but not epithelial cells (IECs), in the intestine. Viral persistence drove sustained intestinal epithelial barrier leakage, which was characterized by increased paracellular flux of small molecules and was associated with enhanced colitis susceptibility. Type I IFN signaling caused tight junction dysregulation in IECs, promoted gut microbiome shifts and enhanced intestinal CD8 T cell responses. Notably, both type I IFN receptor blockade and CD8 T cell depletion prevented infection-induced barrier leakage. Our study demonstrates that infection with a virus that persistently replicates in the intestinal mucosa increases epithelial barrier permeability and reveals type I IFNs and CD8 T cells as causative factors of intestinal leakage during chronic infections.


SMAD4-dependent polysome RNA recruitment in human pancreatic cancer cells.
Pancreatic cancer is the fourth leading cause of cancer death in the United States because most patients are diagnosed too late in the course of the disease to be treated effectively. Thus, there is a pressing need to more clearly understand how gene expression is regulated in cancer cells and to identify new biomarkers and therapeutic targets. Translational regulation is thought to occur primarily through non-SMAD directed signaling pathways. We tested the hypothesis that SMAD4-dependent signaling does play a role in the regulation of mRNA entry into polysomes and that novel candidate genes in pancreatic cancer could be identified using polysome RNA from the human pancreatic cancer cell line BxPC3 with or without a functional SMAD4 gene. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for most of the remaining polysome RNA, levels are regulated via a "tagging" of the RNAs in the nucleus for rapid entry into the polysomes; (iv) a SMAD4-dependent pathway appears to indeed play a role in regulating mRNA entry into polysomes; and (v) a gene list derived from differentially expressed polysome RNA in BxPC3 cells generated new candidate genes and cell pathways potentially related to pancreatic cancer.


CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses.
Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials.


Interactions of C-reactive protein with the complement system. II. C-reactive protein-mediated consumption of complement by poly-L-lysine polymers and other polycations.
Cationic homopolymers of poly-L-lysine were found to activate complement (C) via C-reactive protein (CRP) and deplete C3 and C5 as well as early-acting C components. Maximum C consumption was obtained with polymers of 2,000-8,000 daltons; polymers of 1,700, 11,000, and 23,000 daltons were intermediate in reactivity, while L-lysine, lysyl-L-lysine, tetra-L-lysine, and polymers of 70,000-400,000 daltons lacked significant C-consuming activity. Naturally occurring polycations which consumed C in the presence of CRP included myelin basic proteins, cationic proteins of rabbit leukocytes, and both lysine- and arginine-rich histones; poly-L-arginine polymers of 17,000 but not 65,000 daltons also were C-consuming. Polycations without such reactivity included poly-L-orithine (5,000 and 165,000 daltons), egg white and human lysozymes, and Polybrene. The polycations which failed to induce C consumption via CRP, inhibited its consumption by both active polycations and by C-polysaccharide (CPS). The relative inhibitory capacity of phosphorylcholine and polycations in CPS- and polycations-CRP systems was consistent with the concept that phosphate esters and polycations react at the same or an overlapping combining site. The ability of certain polycations to activate C via CRP increases the potential for initiation of host reactions via C. The capacity of other polycations to inhibit C activation via CRP introduces a potential for physiologic or pharmacologic manipulation. These considerations would seem to expand the potential role of CRP in the initiation and modulation of the inflammatory response.


Perinatal exposure of pregnant rats to cypermethrin delays testicular descent, impairs fertility in F1 male progeny leading to developmental defects in F2 generation.
Cypermethrin (CYP) is a widely used synthetic pyrethroid insecticide and regarded as a potential endocrine disruptor. CYP exposure may pose a great risk to human health including adverse effect on their reproductive functions. This study aimed to delineate the effects of perinatal exposure of rats to CYP on the sexual maturation and fertility of F1 male progeny. Pregnant rats (F0) were gavaged daily with CYP (0, 1, 10, 25 mg/kg BW/day) and Diethylestilbestrol (DES, 10 μg/kg BW/day), as positive control from gestation day 6 to postnatal day 21. The effects of CYP on body weight gain and reproductive functions were evaluated at the Juvenile (PND 22), peri-pubertal (PND 45) and adult (PND 75) stages of development. A significant delay in the age of testicular descent and prepuce separation was observed at 1 and 25 mg/kg doses of CYP. At the same dose level, reproductive organ development and their functions were also affected. A significant alteration in testicular histology, expression of steroid hormone receptors, and circulatory steroid hormones was observed throughout development. Reduced sperm count and motility were observed at PND 75 leading to subfertility and reduced litter size. These adult male rats when cohabitated with unexposed normal cycling females, the F2 fetuses exhibited developmental defects. Taken together, CYP perinatal exposure caused significant long lasting effects of the reproductive functions of F1 generation male rats, which were vertically transmitted to F2 generation leading to developmental defects. The mechanism of transgenerational effects needs to be explored in details.


Phosphorylation Modulates Aspartyl-(Asparaginyl)-β Hydroxylase Protein Expression, Catalytic Activity and Migration in Human Immature Neuronal Cerebellar Cells.
Abundant aspartyl-asparaginyl-β-hydroxylase (ASPH) expression supports robust neuronal migration during development, and reduced ASPH expression and function, as occur in fetal alcohol spectrum disorder, impair cerebellar neuron migration. ASPH mediates its effects on cell migration via hydroxylation-dependent activation of Notch signaling networks. Insulin and Insulin-like growth factor (IGF-1) stimulate ASPH mRNA transcription and enhance ASPH protein expression by inhibiting Glycogen Synthase Kinase-3β (GSK-3β). This study examines the role of direct GSK-3β phosphorylation as a modulator of ASPH protein expression and function in human cerebellar-derived PNET2 cells. Predicted phosphorylation sites encoded by human ASPH were ablated by S/T→A site-directed mutagenesis of an N-Myc-tagged wildtype (WT) cDNA regulated by a CMV promoter. Phenotypic and functional features were assessed in transiently transfected PNET2 cells. Cells transfected with WT ASPH had increased ASPH protein expression, directional motility, Notch-1 and Jagged-1 expression, and catalytic activity relative to control. Although most single- and multi-point ASPH mutants also had increased ASPH protein expression, their effects on Notch and Jagged expression, directional motility and adhesion, and catalytic activity varied such that only a few of the cDNA constructs conferred functional advantages over WT. Immunofluorescence studies showed that ASPH phosphorylation site deletions can alter the subcellular distribution of ASPH and therefore its potential interactions with Notch/Jagged at the cell surface. Inhibition of ASPH phosphorylation enhances ASPH protein expression, but attendant alterations in intra-cellular trafficking may govern the functional consequences in relation to neuronal migration, adhesion and Notch activated signaling.


Vitamin A deficiency and anemia among children 12-71 months old in Honduras.
Vitamin A deficiency (VAD) and iron deficiency anemia (IDA) have been recognized as public health problems in Honduras for over 30 years. This paper, based on the 1996 National Micronutrient Survey on 1678 children 12-71 months of age, presents the results for vitamin A status and anemia prevalence, as well as the level of vitamin A in sugar at the household level. The results showed that 14% of the children were subclinically vitamin A deficient (plasma retinol < 20 micrograms/dL) and 32% were at risk of VAD (plasma retinol 20-30 micrograms/dL). These data indicate that VAD is a moderate public health problem in Honduras. Logistic regression analysis showed that children 12-23 months old living in areas other than the rural south of the country were at greatest risk of subclinical VAD. Infection, indicated by an elevated alpha-1-acid-glycoprotein level, increased the risk of subclinical VAD more than three-fold. Children from households that obtained water from a river, stream, or lake were at twice the risk of subclinical VAD compared with other children. That same doubled risk was found for children from a household with an outside toilet. VAD can be controlled by fortifying sugar. Retinol levels in sugar at the household level were about 50% of those mandated by Honduran law. There appears to be significant leakage of unfortified sugar into the market. This is particularly true in the rural north, where 33% of samples contained no retinol. Overall, 30% of children were anemic (Hb < 11 g/dL). Logistic regression analysis showed that children whose fathers lived with them but who had not attended at least grade 4 of primary school were at 33% greater risk of being anemic. Infection and being underweight increased the risk of being anemic by 51% and 21%, respectively. Many of the anemic children had not been given iron supplements, suggesting health care providers may not be aware that anemia is widespread among young children and/or know how to diagnose it.


Humic acid decreases acute toxicity and ventilation frequency in eastern rainbowfish (Melanotaenia splendida splendida) exposed to acid mine drainage.
Acid mine drainage (AMD) is a global problem leading to the acidification of freshwaters, as well as contamination by heavy metals. The ability of humic substances (HS) such as humic acid (HA) to decrease toxicity of heavy metals is widely known, whereas limited studies have examined the ability of HS to decrease toxicity linked with multiple stressors such as those associated with AMD. This study investigated the ability of HA to decrease acute toxicity defined as morbidity and ventilation frequency (measured via the time elapsed for ten operculum movements) in eastern rainbowfish (Melanotaenia splendida splendida) exposed to the multiple stressors of AMD-driven heavy metal concentrations, together with low pH. Water from the Mount Morgan open pit (a now closed gold and copper mine site), located at Mount Morgan, Central Queensland, Australia, was used as the AMD source. Fish were exposed to zero per cent (pH 7.3), two per cent (pH 6.7), three per cent (pH 5.7) and four per cent (pH 4.6) AMD in the presence of 0, 10 and 20mg/L Aldrich Humic Acid (AHA) over 96h. HA was shown to significantly decrease the acute toxicity of AMD and its adverse effects on ventilation frequency. These results are important in showing that HA can influence toxicity of metal mixtures and low pH, thus indicating a potential role for HA in decreasing toxicity of multiple environmental stressors more widely, and possible value as a rehabilitation aid.


Local Health Departments' Partners and Challenges in Electronic Exchange of Health Information.
Unprecedented amounts of data are produced by the health care and other sectors, presenting opportunities for local health departments (LHDs) to access these data. LHDs will need to participate in health information exchange (HIE) with a number of partners in order to benefit from these data resources. LHDs' participation in HIEs with specific partners has not been studied. To describe the level of and challenges in LHD participation in HIE with other partners, and variation by LHD population size and governance type. This research uses data from the 2015 Informatics Capacity and Needs Assessment Survey, with a target population of all LHDs in the United States. A representative sample of 650 LHDs was drawn using a stratified random sampling design. A total of 324 completed responses were received with a 50% response rate. Survey data were cleaned, and bivariate comparisons were conducted using χ and Somer's D. Substantial variation existed in LHDs' participation in HIE by type of exchange partner. Although 71% participated in HIE with the state departments of health, only 12% with jail/correctional health, 14% with health or county-based purchasing plans, and 15% with home health agencies. Compared with large LHDs (jurisdiction populations of ≥500 000), smaller LHDs were more likely to participate in HIE with state departments of health, but less likely with other exchange partners. The challenges to HIE participation were technological, and organizational/interorganizational in nature and variation existed by LHDs' population size and governance structure with respect to state authority. Local public health agencies more commonly participate in HIE with some partners, but may need to improve HIE with many others. National strategies targeting an increase in HIE of LHDs may use our findings to focus those initiatives.


Outcomes following prehospital airway management in severe traumatic brain injury.
Prevention of hypoxia and thus secondary brain injury in traumatic brain injury (TBI) is critical. However there is controversy regarding the role of endotracheal intubation in the prehospital management of TBI. To describe the outcome of TBI with various airway management methods employed in the prehospital setting in the Cape Town Metropole. The study was a cohort descriptive observational analysis of 124 consecutively injured adult patients who were admitted for severe TBI (Glasgow Coma Score ≤8) to Groote Schuur and Tygerberg hospitals between 1 January 2009 and 31 August 2011. Patients were categorised by their method of airway management: rapid sequence intubation (RSI), sedation-assisted intubation, failed intubation, basic airway management, and intubated without drugs. Good outcomes were defined by a Glasgow Outcome Score of 4 - 5. There was a statistically significant association between airway management and outcome (p=0.013). Patients who underwent basic airway management had a higher proportion of a good outcome (72.9%) than patients who were intubated in the prehospital setting. A good outcome was observed with 61.8% and 38.4% of patients who experienced sedation-assisted intubation and RSI, respectively. Patients intubated without drugs had the poorest outcome (88%), followed by rapid sequence intubation (61.5%) and by the sedation assisted group (38.2%). Prehospital intubation did not demonstrate improved outcomes over basic airway management in patients with severe TBI. A large prospective, randomised trial is warranted to yield some insight into how these airway interventions influence outcome in severe TBI.


Probing the excited-state chemical shifts and exchange parameters by nitrogen-decoupled amide proton chemical exchange saturation transfer (HNdec-CEST).
CEST-NMR spectroscopy is a powerful tool for probing the conformational dynamics of macromolecules. We present a HNdec-CEST experiment that simplifies the relaxation matrix, reduces fitting parameters, and enhances signal resolution. Importantly, fitting of HNdec-CEST profiles enables robust extraction of exchange rates as well as excited-state chemical shifts and populations.


MR imaging of infectious spondylitis.
MR images of 14 patients with pyogenic and three patients with tuberculous infectious spondylitis were studied to develop criteria for diagnosis. T1-weighted scans, 800/20 (TR/TE), were obtained in 17 patients and T2-weighted scans, greater than 2000/30,80, were obtained in 14. In seven patients, T2*-weighted scans (gradient-recalled acquisition into steady state, 25/15/5-7 degrees [TR/TE/flip angle]) and short-T1 inversion-recovery scans (STIR), 1400/150/40 (TR/TI/TE), as well as fat and water images (using a suppression technique), were obtained. Unenhanced and gadopentetate-dimeglumine-enhanced scans were obtained in four patients. In all but two patients with pyogenic infectious spondylitis, the T1-weighted sagittal scan showed characteristic findings: narrowed disk space, low signal intensity in the marrow of at least two adjacent vertebrae, subligamentous or epidural soft-tissue masses, and erosion of cortical bone. In one patient the T1-weighted scan was normal and abnormalities could be detected only on the T2-weighted scan. The remaining patient had abnormal marrow signal on the T1-weighted scan but only in one vertebral body. On T2-weighted images the major findings were a narrowed disk space with variable signal changes, abnormal high signal in marrow of at least two adjacent vertebrae, high-signal subligamentous or epidural masses, and cortical bone erosion. The findings in the three patients with tuberculous spondylitis included areas of increased and decreased signal intensity in vertebrae on T1-weighted images. Disk spaces were relatively spared given the extent of disease. Extraosseous soft-tissue components could be large. Bone erosion was best seen on the first echo of a T2-weighted sequence and on a water image; the latter was most reliable since it had no chemical-shift artifact. The use of gadopentetate dimeglumine could obscure or clarify MR findings, depending on the situation. T1- and T2-weighted MR images should be obtained for assessment of infectious spondylitis. STIR scans are particularly helpful. Fat images can be useful in subtle presentations, since they are very sensitive to marrow replacement, and gadopentetate dimeglumine may be helpful for epidural delineation of disease.


Climate, demography and lek stability in an Amazonian bird.
Lekking is a rare, but iconic mating system where polygynous males aggregate and perform group displays to attract females. Existing theory postulates that demographic and environmental stability are required for lekking to be an evolutionarily viable reproductive strategy. However, we lack empirical tests for the hypotheses that lek stability is facilitated by age-specific variation in demographic rates, and by predictable, abundant resources. To address this knowledge gap, we use multistate models to examine how two demographic elements of lek stability-male survival and recruitment-vary with age, social status and phase of the El Niño Southern Oscillation (ENSO) in a Neotropical frugivorous bird, the wire-tailed manakin (Pipra filicauda). Our results show that demographic and environmental conditions were related to lek stability in the Ecuadorean Amazon. Apparent annual survival probability of territorial males was higher than that of non-territorial floaters, and recruitment probability increased as males progressed in an age-graded queue. Moreover, annual survival of territorial males and body condition of both floaters and territory holders were higher following years with El Niño conditions, associated with reduced rainfall and probably higher fruit production in the northern Neotropics, and lower after years with wet, La Niña conditions that predominated our study. Recruitment probabilities varied annually, independent of ENSO phase, and increased over our study period, but the annual mean number of territorial males per lek declined. Our results provide empirical support for hypothesized demographic and environmental drivers of lek dynamics. This study also suggests that climate-mediated changes in resource availability can affect demography and subsequent lek stability in a relatively buffered, lowland rainforest.


Increased jejunal permeability in human obesity is revealed by a lipid challenge and is linked to inflammation and type 2 diabetes.
Obesity and its metabolic complications are characterized by subclinical systemic and tissue inflammation. In rodent models of obesity, inflammation and metabolic impairments are linked with intestinal barrier damage. However, whether intestinal permeability is altered in human obesity remains to be investigated. In a cohort of 122 severely obese and non-obese patients, we analyzed intestinal barrier function combining in vivo and ex vivo investigations. We found tight junction impairments in the jejunal epithelium of obese patients, evidenced by a reduction of occludin and tricellulin. Serum levels of zonulin and LPS binding protein, two markers usually associated with intestinal barrier alterations, were also increased in obese patients. Intestinal permeability per se was assessed in vivo by quantification of urinary lactitol/mannitol (L/M) and measured directly ex vivo on jejunal samples in Ussing chambers. In the fasting condition, L/M ratio and jejunal permeability were not significantly different between obese and non-obese patients, but high jejunal permeability to small molecules (0.4 kDa) was associated with systemic inflammation within the obese cohort. Altogether, these results suggest that intestinal barrier function is subtly compromised in obese patients. We thus tested whether this barrier impairment could be exacerbated by dietary lipids. To this end, we challenged jejunal samples with lipid micelles and showed that a single exposure increased permeability to macromolecules (4 kDa). Jejunal permeability after the lipid load was two-fold higher in obese patients compared to non-obese controls and correlated with systemic and intestinal inflammation. Moreover, lipid-induced permeability was an explicative variable of type 2 diabetes. In conclusion, intestinal barrier defects are present in human severe obesity and exacerbated by a lipid challenge. This paves the way to the development of novel therapeutic approaches to modulate intestinal barrier function or personalize nutrition therapy to decrease lipid-induced jejunal leakage in metabolic diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Haptoglobin Administration in Cardiovascular Surgery Patients: Its Association With the Risk of Postoperative Acute Kidney Injury.
Acute kidney injury (AKI) often occurs after cardiac surgery. During cardiac surgery, plasma free hemoglobin (fHb) would increase due to hemolysis. Since plasma fHb is thought to be nephrotoxic, haptoglobin, which is an fHb scavenger, may have the potential to prevent postoperative AKI (pAKI). However, there have been few studies in which the association of intraoperative administration of haptoglobin with the incidence of AKI after cardiac surgery was assessed. This study was a retrospective observational study to assess the independent association of intraoperative administration of haptoglobin with the incidence pAKI in cardiac surgery patients. We screened cardiac surgery patients who required cardiopulmonary bypass from 2008 to 2015. We excluded patients who required renal replacement therapy preoperatively. We also excluded patients in whom descending aortic replacement was performed. pAKI was defined according to AKI Network criteria. A propensity score-matched model was used to adjust confounders. For sensitive analysis, we further developed a logistic regression model. We included 1326 patients in this study. The incidence of AKI in the total cohort was 25.5% (338 patients). Haptoglobin was administered in 260 patients (19.6%). In the crude cohort, the incidence of AKI in patients with haptoglobin administration was 24.6%, which was not significantly different from the incidence of 25.7% in those without haptoglobin administration (P = .72; odds ratio, 0.94 [95% confidence interval, 0.69-1.29]). After propensity score matching, we had 249 patients in each group (for a total of 498 patients). In this propensity score-matched cohort, the incidence of AKI in patients with haptoglobin administration was 22.5%, which was significantly lower than the incidence of 30.9% in those without haptoglobin administration (P = .033; odds ratio, 0.65 [0.43-0.97]). In our logistic regression model for the risk of pAKI, haptoglobin administration was independently associated with decreased risk of AKI (P = .029; adjusted odds ratio, 0.54 [0.31, 0.93]). In this hypothesis-generating, single-center retrospective observational study, intraoperative administration of haptoglobin was independently associated with lower risk of AKI after cardiovascular surgery.


Unsupervised correction of gene-independent cell responses to CRISPR-Cas9 targeting.
Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes. Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries. CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.


Occurrence of plasmids in pathogenic strains of Nocardia.
The purpose of the present study was to determine the relative distribution of plasmids in 87 clinical isolates of Nocardia, belonging to the five major pathogenic species. A correlation between plasmid content and the site of infection within the host, resistance to antibiotics and enzymic profiles was also investigated. The plasmid extraction procedure of Kado and Liu was used. Electrophoretic analysis revealed one-to-four plasmid bands, ranging in size from <8 to >50 kb, in 27 strains (31%). Based on the number of isolates tested, the incidence of plasmid-bearing strains was significantly higher among N. farcinica than N. asteroides strains. Within N. farcinica, the incidence of plasmids was higher among strains isolated in the Paris area than in strains isolated elsewhere, such as in the French provinces or outside France. A statistically significant correlation was demonstrated between the cutaneous localisation of infections and the incidence of plasmid-bearing strains. The presence of plasmids in nocardiae could not be associated with specific phenotypic traits such as resistance to antibiotics or enzymic activity. The fact that the majority of Nocardia clinical isolates (60 of 87) did not contain plasmids suggests that plasmids are not involved directly in virulence and that there is no selective pressure for plasmid acquisition.


Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin.
The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.


In vivo evaluation of optic nerve aging in adult rhesus monkey by diffusion tensor imaging.
Aging of the optic nerve can result in reduced visual sensitivity or vision loss. Normal optic nerve aging has been investigated previously in tissue specimens but poorly explored in vivo. In the present study, the normal aging of optic nerve was evaluated by diffusion tensor imaging (DTI) in non-human primates. Adult female rhesus monkeys at the ages of 9 to 13 years old (young group, n=8) and 21 to 27 years old (old group, n=7) were studied using parallel-imaging-based DTI on a clinical 3T scanner. Compared to young adults, the old monkeys showed 26% lower fractional anisotropy (P<0.01), and 44% greater radial diffusivity, although the latter difference was of marginal statistical significance (P=0.058). These MRI findings are largely consistent with published results of light and electron microscopic studies of optic nerve aging in macaque monkeys, which indicate a loss of fibers and degenerative changes in myelin sheaths.


X-ray microtomographic confirmation of the reliability of CBCT in identifying the scalar location of cochlear implant electrode after round window insertion.
Cone-beam computed tomography (CBCT) plays a key role in cochlear implantation in both planning implantation before surgery and quality control during surgery due to the high spatial resolution and convenience of application in the operation theater. We recently designed a novel, highresolution cone-beam acquisition system that has been tested in temporal bones with cochlear implantation to identify the scalar localization of the electrode arrays. The current study aimed to verify the reliability of the experimental CBCT set-up using high-resolution in vitro X-ray microtomography (μCT) imaging as a reference. Nine human temporal bones were studied by inserting a straight electrode of a cochlear implant using the round window approach followed by sequential imaging using experimental CBCT and μCT with and without 1% iodine as the contrast agent. In the CBCT images, the electrodes were located in the scala tympani and near the lateral wall in all temporal bones. In the μCT images, the cochlear fine structures, including Reissner's membrane, stria vascularis, spiral ligament, basilar membrane, spiral limbus, osseous spiral lamina, and Rosenthal's canal that hosts the spiral ganglion cells, were clearly delineated; the electrode array avoided the lateral wall of the scala tympani in the hook region and then ran along the lateral wall of the scala tympani without any exception, a feature that was also detected in a temporal bone with ruptures in the basilar and Reissner's membranes. In conclusion, the current in vitro μCT imaging system produced high-quality images that could demonstrate the fine cochlear structures faithfully and verify the reliability of a novel experimental CBCT set-up aimed for clinical application in identifying the scalar localization of the electrode array. The straight electrode is safe for cochlear structures with low risk of translocation and is suitable for atraumatic implantation, although a large gap between the contacts and the modiolus exists.


Injuries sustained by passengers travelling in the cargo area of light delivery vehicles.
Despite its inherently dangerous nature, the practice of transporting passengers in the cargo area of light delivery vehicles (LDVs) is widespread in South Africa. To review the patterns and outcome of injuries associated with events involving LDVs transporting passengers. All patients presenting to the Pietermaritzburg Metropolitan Trauma Service in KwaZulu-Natal Province following an event in which they had been travelling in the cargo area of an LDV between January 2011 and December 2012 were included in the audit. A total of 66 patients were treated during the study period; 35% were children under the age of 18, and 90% were ejected from the LDV during the incident. The mean injury severity score (ISS) was 23. Collision events were associated with a higher mean ISS (33) than non-collision events (15) (p=0.008). The region most commonly injured was the head and neck, and 11% of victims sustained a permanent disability. The patients collectively spent 873 days in hospital and 70 days in an intensive care unit, and underwent 17 operations. Transporting passengers in the cargo area of an LDV is dangerous, as ejection from the vehicle resulting from a collision is associated with significant morbidity and mortality. Legislative initiatives to prevent this practice are required as part of an ongoing comprehensive injury prevention programme.


[Digital psychiatry].
The digitization of society has an increasing impact on healthcare in general and, therefore, also on psychiatry.
 AIM: To provide an overview of digital developments and their influence on the design of future professional psychiatric care.
 METHOD: With the help of examples from literature, show how digitization will influence diagnostic procedures as well as psychiatric treatment.
 RESULTS: Digitization will have a major impact on psychiatric diagnostics and treatment. For example, psychiatric diagnostics will be strengthened by continuous monitoring of behaviour with digital wearables and the collection of large amounts of personal data. How we deal with these new sources of information needs to be developed in everyday practice. Psychiatric treatments with E-health, online therapies, apps and virtual reality are being developed rapidly. There is increasing evidence concerning the efficacy of these treatments in a variety of patient groups.
 CONCLUSION: The digital revolution in psychiatric health services has just begun. To maximise the benefits of digitization for psychiatry, it is necessary to connect technological possibilities with well-founded scientific knowledge, professional expertise, expectations and needs of patients, and clear legal instructions.


Antarctic ice shelf disintegration triggered by sea ice loss and ocean swell.
Understanding the causes of recent catastrophic ice shelf disintegrations is a crucial step towards improving coupled models of the Antarctic Ice Sheet and predicting its future state and contribution to sea-level rise. An overlooked climate-related causal factor is regional sea ice loss. Here we show that for the disintegration events observed (the collapse of the Larsen A and B and Wilkins ice shelves), the increased seasonal absence of a protective sea ice buffer enabled increased flexure of vulnerable outer ice shelf margins by ocean swells that probably weakened them to the point of calving. This outer-margin calving triggered wider-scale disintegration of ice shelves compromised by multiple factors in preceding years, with key prerequisites being extensive flooding and outer-margin fracturing. Wave-induced flexure is particularly effective in outermost ice shelf regions thinned by bottom crevassing. Our analysis of satellite and ocean-wave data and modelling of combined ice shelf, sea ice and wave properties highlights the need for ice sheet models to account for sea ice and ocean waves.


Development of a core outcome set for open lower limb fracture.
Open lower limb fracture is life-changing, resulting in substantial morbidity and resource demand, while inconsistent outcome-reporting hampers systematic review and meta-analysis. A core outcome set establishes consensus among key stakeholders for the recommendation of a minimum set of outcomes. This study aims to define a core outcome set for adult open lower limb fracture. Candidate outcomes were identified from a previously published systematic review and a secondary thematic analysis of 25 patient interviews exploring the lived experience of recovery from open lower limb fracture. Outcomes were categorized and sequentially refined using healthcare professional and patient structured discussion groups. Consensus methods included a multi-stakeholder two-round online Delphi survey and a consensus meeting attended by a purposive sample of stakeholders, facilitated discussion, and voting using a nominal group technique. Thematic analysis and systematic review identified 121 unique outcomes, reduced to 68 outcomes following structured discussion groups. Outcomes were presented to 136 participants who completed a two-round online Delphi survey. The Delphi survey resulted in 11 outcomes identified as consensus 'in' only. All outcomes were discussed at a consensus meeting attended by 15 patients, 14 healthcare professionals, 11 researchers, and one patient-carer. Consensus was achieved for a four-core outcome set: 'Walking, gait and mobility', 'Being able to return to life roles', 'Pain or discomfort', and 'Quality of life'. This study used robust consensus methods to establish a core outcome set that should be measured in all future research studies and audits of clinical practice without precluding the measurement of additional outcomes.


The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: quantification of steroid intermediates and end products in H295R cells.
The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.


Increased Col10a1 expression is not causative for the phenotype of Phex-deficient Hyp mice.
X-linked hypophosphatemic rickets (XLHR) is a severe disorder of phosphate homeostasis and skeletal mineralization caused by mutations of PHEX, encoding a bone-specific endopeptidase. Phex-deficient Hyp mice have been extensively studied to understand the molecular bases of XLHR, and here it was found that Fgf23, encoding a major phosphaturic hormone, was transcriptionally activated in bone-forming osteoblasts. We and others could additionally show that Col10a1 expression is increased in Hyp osteoblasts and bones, thereby raising the possibility that ectopic production of type X collagen could contribute to the impaired mineralization of the Hyp bone matrix. Here we show that an additional deficiency of the Col10a1 gene does not overtly affect the skeletal phenotype of Hyp mice. More specifically, Col10a1-deficient Hyp mice displayed severe disturbances of skeletal growth, bone mass acquisition and bone matrix mineralization, and they were essentially indistinguishable from Hyp littermates. This was confirmed by non-decalcified histology and bone-specific histomorphometry quantifying all relevant parameters of growth plate maturation, trabecular bone architecture and osteoid accumulation. Taken together, our results show that increased Col10a1 expression in Phex-deficient osteoblasts is not a major cause of the XLHR phenotype, which was an important issue to address based on the previous findings.


Experimental therapeutics for refractory obsessive-compulsive disorder: translational approaches and new somatic developments.
Significant advances over the past 20 years in our understanding of the phenomenology and pathophysiology of obsessive-compulsive disorder, made in part from structural and functional neuroimaging and genetics research, can guide treatments that target brain regions, circuits, and neurotransmitter systems specific to obsessive-compulsive disorder, the disruption of which may alleviate obsessive-compulsive disorder symptoms. We discuss here our current understanding of the underlying neurobiology and heritability of obsessive-compulsive disorder and integrate that understanding with a review of the current pharmacological, neurosurgical, and brain stimulation treatments of refractory obsessive-compulsive disorder. Expanding on these studies, we hope that new pharmacological and psychological treatment strategies and research-driven targets for lesioning, stimulation, or other types of focal neuromodulation can be identified that could lead to future research directions. Cross-species translational research and neuroimaging of the physiological and anatomical pathways implicated in the pathophysiology and treatment response in obsessive-compulsive disorder will advance our understanding of the neural basis of obsessive-compulsive disorder and lead to more targeted and effective treatment options.


Molybdenum isotope evidence for widespread anoxia in mid-Proterozoic oceans.
How much dissolved oxygen was present in the mid-Proterozoic oceans between 1.8 and 1.0 billion years ago is debated vigorously. One model argues for oxygenation of the oceans soon after the initial rise of atmospheric oxygen approximately 2.3 billion years ago. Recent evidence for H(2)S in some mid-Proterozoic marine basins suggests, however, that the deep ocean remained anoxic until much later. New molybdenum isotope data from modern and ancient sediments indicate expanded anoxia during the mid-Proterozoic compared to the present-day ocean. Consequently, oxygenation of the deep oceans may have lagged that of the atmosphere by over a billion years.


Isolated gallbladder perforation following blunt abdominal trauma.
A 32-year-old man sustained an isolated perforation of the gallbladder following blunt abdominal trauma. A preoperative diagnosis was made on an ultrasound scan, which showed a pericholecystic fluid collection only. At laparotomy, a gallbladder perforation at the infundibulum was identified with a localised bile collection, warranting a cholecystectomy. Isolated gallbladder injury from blunt abdominal trauma is rare, and a high index of suspicion is required to establish the diagnosis. Cholecystectomy is the treatment of choice.


Water fluoridation: a critical review of the physiological effects of ingested fluoride as a public health intervention.
Fluorine is the world's 13th most abundant element and constitutes 0.08% of the Earth crust. It has the highest electronegativity of all elements. Fluoride is widely distributed in the environment, occurring in the air, soils, rocks, and water. Although fluoride is used industrially in a fluorine compound, the manufacture of ceramics, pesticides, aerosol propellants, refrigerants, glassware, and Teflon cookware, it is a generally unwanted byproduct of aluminium, fertilizer, and iron ore manufacture. The medicinal use of fluorides for the prevention of dental caries began in January 1945 when community water supplies in Grand Rapids, United States, were fluoridated to a level of 1 ppm as a dental caries prevention measure. However, water fluoridation remains a controversial public health measure. This paper reviews the human health effects of fluoride. The authors conclude that available evidence suggests that fluoride has a potential to cause major adverse human health problems, while having only a modest dental caries prevention effect. As part of efforts to reduce hazardous fluoride ingestion, the practice of artificial water fluoridation should be reconsidered globally, while industrial safety measures need to be tightened in order to reduce unethical discharge of fluoride compounds into the environment. Public health approaches for global dental caries reduction that do not involve systemic ingestion of fluoride are urgently needed.


Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death.
Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations. Reaction-diffusion modelling suggested that even if strong spatial anisotropies existed during mitochondrial cytochrome c release, these would be eliminated by free diffusion of the cytosolic proteins that instantiate the apoptosis execution network. Experimentally, rapid sampling of mitochondrial permeabilisation and effector caspase activity in individual HeLa cervical cancer cells confirmed predictions of the reaction-diffusion model and demonstrated that the signalling network of apoptosis execution could efficiently translate spatial anisotropies in mitochondrial permeabilisation into a homogeneous effector caspase response throughout the cytosol. Further systems modelling suggested that a more than 10,000-fold impaired diffusivity would be required to maintain spatial anisotropies as observed during mitochondrial permeabilisation until the time effector caspases become activated. Multi-protein diffusion efficiently contributes to eliminating spatial asynchronies which are present during the initiation of apoptosis execution and thereby ensures homogeneous apoptosis execution throughout the entire cell body. For previously reported biological scenarios in which effector caspase activity was shown to be targeted selectively to specific subcellular regions additional mechanisms must exist that limit or spatially coordinate caspase activation and/or protect diffusing soluble caspase substrates from unwanted proteolysis.


Hypernatremia is associated with increased risk of mortality in pediatric severe traumatic brain injury.
Acquired hypernatremia in hospitalized patients is often associated with poorer outcomes. Our aim was to evaluate the relationship between acquired hypernatremia and outcome in children with severe traumatic brain injury (sTBI). We performed a retrospective cohort study of all severely injured trauma patients (Injury Severity Score ≥12) with sTBI (Glasgow Coma Scale [GCS] ≤8 and Maximum Abbreviated Injury Scale [MAIS] ≥4) admitted to a Pediatric Critical Care Unit ([PCCU]; 2000-2009). In a cohort of 165 patients, 76% had normonatremia (135-150 mmol/L), 18% had hypernatremia (151-160 mmol/L), and 6% had severe hypernatremia (>160 mmol/L). The groups were similar except for lower GCS (p=0.002) and increased incidence of fixed pupil(s) on admission in both hypernatremia groups (p<0.001). Mortality rate was four-fold and six-fold greater with hypernatremia and severe hypernatremia, respectively (p<0.001), and mortality rates were unchanged when patients with fixed pupils or those with central diabetes insipidus were excluded (p<0.001). Hypernatremic patients had fewer ventilator-free days (p<0.001). Survivors with hypernatremia had greater PCCU (p=0.001) and hospital (p=0.031) lengths of stays and were less frequently discharged home (p=0.008). Logistic regression analyses of patient characteristics and sTBI interventions demonstrated that hypernatremia was independently associated with the presence of fixed pupil(s) on admission (odds ratio [OR] 5.38; p=0.003); administration of thiopental (OR 8.64; p=0.014), and development of central diabetes insipidus (OR 5.66; p=0.005). Additional logistic regression analyses demonstrated a significant association between hypernatremia and mortality (OR 6.660; p=0.034). In summary, acquired hypernatremia appears to signal higher risk of mortality in pediatric sTBI and is associated with a higher discharge level of care in sTBI survivors.


Complexities of Next-Generation Sequencing in Solid Tumors: Case Studies.
With the promise and potential of clinical next-generation sequencing for tumor and germline testing to impact treatment and outcomes of patients with cancer, there are also risks of oversimplification, misinterpretation, and missed opportunities. These issues risk limiting clinical benefit and, at worst, perpetuating false conclusions that could lead to inappropriate treatment selection, avoidable toxicity, and harm to patients. This report presents 5 case studies illustrating challenges and opportunities in clinical next-generation sequencing interpretation and clinical application in solid tumor oncologic care. First is a case that dissects the origin of an ATM mutation as originating from a hematopoietic clone rather than the tumor. Second is a case illustrating the potential for tumor sequencing to suggest germline variants associated with a hereditary cancer syndrome. Third are 2 cases showing the potential for variant reclassification of a germline variant of uncertain significance when considered alongside family history and tumor sequencing results. Finally, we describe a case illustrating challenges with using microsatellite instability for predicting tumor response to immune checkpoint inhibitors. The common theme of the case studies is the importance of examining clinical context alongside expert review and interpretation, which together highlight an expanding role for contextual examination and multidisciplinary expert review through molecular tumor boards.


Enabling research in care homes: an evaluation of a national network of research ready care homes.
In the UK care homes are one of the main providers of long term care for older people with dementia. Despite the recent increase in care home research, residents with dementia are often excluded from studies. Care home research networks have been recommended by the Ministerial Advisory Group on Dementia Research (MAGDR) as a way of increasing research opportunities for residents with dementia. This paper reports on an evaluation of the feasibility and early impact of an initiative to increase care home participation in research. A two phase, mixed methods approach was used; phase 1 established a baseline of current and recent studies including the National Institute for Health Research portfolio. To explore the experiences of recruiting care homes and research participation, interviews were conducted with researchers working for the Dementia and Neurodegenerative Diseases Research Network (DeNDRoN) and care home managers. In phase 2, four DeNDRoN area offices recruited care homes to a care home network for their region. The care home networks were separate from the DeNDRoN research network. Diaries were used to document and cost recruitment; DeNDRoN staff were interviewed to understand the barriers, facilitators and impact of the care home networks. Thirty three current or recent studies were identified as involving care homes as care home specific studies or those which included residents. Further details of care home recruitment were obtained on 20 studies by contacting study teams. Care home managers were keen to be involved in research that provided staff support, benefits for residents and with minimal disruption. In phase 2, 141 care homes were recruited to the care home research networks, through corporate engagement and individual invitation. Pre-existing relationships with care homes facilitated recruitment. Sites with minimal experience of working with care homes identified the need for care home training for researchers. Phase 1 review revealed a small but increasing number of studies involving care homes. Phase 2 demonstrated the feasibility of care home research networks, their potential to increase recruitment to research and develop partnerships between health services and care homes, but highlighted the need for care home training for researchers.


Sodium laurate, a novel protease- and mass spectrometry-compatible detergent for mass spectrometry-based membrane proteomics.
The hydrophobic nature of most membrane proteins severely complicates their extraction, proteolysis and identification. Although detergents can be used to enhance the solubility of the membrane proteins, it is often difficult for a detergent not only to have a strong ability to extract membrane proteins, but also to be compatible with the subsequent proteolysis and mass spectrometric analysis. In this study, we made evaluation on a novel application of sodium laurate (SL) to the shotgun analysis of membrane proteomes. SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin. Furthermore, SL could be efficiently removed by phase transfer method from samples after acidification, thus ensuring not to interfere with the subsequent CapLC-MS/MS analysis of the proteolytic peptides of proteins. When SL was applied to assist the digestion and identification of a standard protein mixture containing bacteriorhodoposin and the proteins in rat liver plasma membrane-enriched fractions, it was found that, compared with other two representative enzyme- and MS-compatible detergents RapiGest SF (RGS) and sodium deoxycholate (SDC), SL exhibited obvious superiority in the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.


Adult human pancreatic duct and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf-1.
The homeodomain transcription factor encoded by the pancreatic and duodenal homeobox gene-1 (Ipf-1) is essential for pancreatic ontogenesis. Whether Ipf-1 is also involved in the neogenesis of beta-cells in the adult pancreas is unknown. We examined whether Ipf-1 is expressed in adult human pancreatic ducts, which are thought to generate new beta-cells. In tissue sections, virtually all duct cells were immunopositive for Ipf-1, as were the islet beta-cells but not the acinar cells. After isolation and culture, both duct and islet cell preparations contained the Ipf-1 immunoreactive proteins p42 and p45 (42 and 45 kDa, respectively) in similar proportions, but the expression levels were twofold lower in duct cells. After 4 h of labeling, the endocrine cells exhibited a sevenfold higher phosphorylation of p42 than the duct cells, whereas p45 was phosphorylated only in endocrine cells. Homeobox binding transcription factor complexes with Ipf-1 in duct cells differed from those in endocrine cells in terms of gel mobility, sequence specificity, and affinity. The observed similarities in Ipf-1 expression by adult human pancreatic duct cells and endocrine cells may reflect their common ontogenic origin, whereas the differences in Ipf-1 phosphorylation and complex formation may correlate with their divergent differentiation.


Vibrationally bond-selected chemisorption of methane isotopologues on Pt(111) studied by reflection absorption infrared spectroscopy.
Reflection absorption infrared spectroscopy (RAIRS) was used to probe for vibrational bond-selectivity in the dissociative chemisorption of three partially deuterated methane isotopologues on a Pt(111) surface. While a combination of incident translational energy and thermal vibrational excitation produces a nearly statistical distribution of C-H and C-D bond cleavage products, we observe that laser excitation of an infrared active C-H stretch normal mode leads to highly selective dissociation of a C-H bond for CHD3, CH2D2, and CH3D. Our results show that vibrational energy redistribution between C-H and C-D stretch modes due to methane/surface interactions is negligible during the sub-picosecond collision time which indicates that vibrational bond-selectivity may be the rule rather than the exception in heterogeneous reactions of small polyatomic molecules.


Systematic reappraisal of Coniella and Pilidiella, with specific reference to species occurring on Eucalyptus and Vitis in South Africa.
The genus Pilidiella, including its teleomorphs in Schizoparme, has a cosmopolitan distribution and is associated with disease symptoms on many plants. In the past, conidial pigmentation has been used as a character to separate Pilidiella (hyaline to pale brown conidia) from Coniella (dark brown conidia). In recent years, however, the two genera have been regarded as synonymous, the older name Coniella having priority. To address the generic question, sequences of the internal transcribed spacer region (ITS1, ITS2), 5.8S gene, large subunit (LSU) and elongation factor 1-alpha gene (EF 1-alpha) were analysed to compare the type species of Pilidiella and Coniella. All three gene regions supported the separation of Coniella from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by having species with hyaline to pale brown conidia (avg. length:width > 1.5), in contrast to the dark brown conidia of Coniella (avg. length:width < or = 1.5). Pilidiella diplodiella, which is a pathogen associated with white rot of grapevines, was shown to be an older name for C. petrakii. To delineate species in the P. diplodiella species complex, isolates were also compared based on histone (H3) gene sequences. Analyses derived from these sequence data separated P. diplodiella from a newly described species, P. diplodiopsis. The new species P. eucalyptorum sp. nov. is proposed for isolates formerly treated as C. fragariae and associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens sp. nov. is newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. A key based on morphological characteristics is provided to separate the taxa treated in this study.


Hox gene expression in limbs: colinearity by opposite regulatory controls.
Genes of the HoxD complex have a crucial role in the morphogenesis of vertebrate limbs. During development, their functional domains are colinear with their genomic positions within the HoxD cluster such that Hoxd13 and Hoxd12 are necessary for digit development, whereas Hoxd11 and Hoxd10 are involved in making forearms. Mutational analyses of these genes have demonstrated their importance and illustrated the requirement for a precise control of their expression during early limb morphogenesis. To study the nature of this control, we have scanned the posterior part of the HoxD complex with a targeted reporter transgene and analyzed the response of this foreign promoter to limb regulatory influences. The results suggest that this regulation is achieved through the opposite effects of two enhancer elements which would compete with each other for interacting with nearby-located promoters. The physical position of a given gene within this genomic interval of opposite regulations might thus determine its final expression pattern. This model provides a conceptual link between the morphology of the future limb and the genetic organization of the Hox gene cluster, a translation of a genomic context into a morphogenetic topology.


Batrachotoxin: chemistry and pharmacology.
Batrachotoxin has been shown to be a pyrrolecarboxylic ester of a novel steroidal base with unique and selective actions on a variety of electrogenic membranes. The effects of batrachotoxin in neuromuscular preparations both pre- and postsynaptically, in nerve axons, in superior cervical ganglion, in heart Purkinje fibers, and in brain slices appear to be due to the selective and irreversible increase in permeability of membranes to sodium ions. The subsequent effects of this increase in Na(+) permeability evoked by batrachotoxin-such as membrane depolarization, enhanced spontaneous transmitter release, muscle contracture, and enhanced formation of cyclic AMP in brain slices-may be blocked reversibly by tetrodotoxin.


Neuronally produced versican V2 renders C-fiber nociceptors IB4 -positive.
A subpopulation of nociceptors, the glial cell line-derived neurotrophic factor (GDNF)-dependent, non-peptidergic C-fibers, expresses a cell-surface glycoconjugate that can be selectively labeled with isolectin B4 (IB4 ), a homotetrameric plant lectin from Griffonia simplicifolia. We show that versican is an IB4 -binding molecule in rat dorsal root ganglion neurons. Using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunofluorescence experiments on rat lumbar dorsal root ganglion, we provide the first demonstration that versican is produced by neurons. In addition, by probing Western blots with splice variant-specific antibodies we show that the IB4 -binding versican contains only the glycosaminoglycan alpha domain. Our data support V2 as the versican isoform that renders this subpopulation of nociceptors IB4 -positive (+). A subset of nociceptors, the GDNF-dependent non-peptidergic C-fibers can be characterized by its reactivity for isolectin B4 (IB4), a plant lectin from Griffonia simplicifolia. We have previously demonstrated that versican V2 binds IB4 in a Ca2 + -dependent manner. However, given that versican is thought to be the product of glial cells, it was questionable whether versican V2 can be accountable for the IB4-reactivity of this subset of nociceptors. The results presented here prove - for the first time - a neuronal origin of versican and suggest that versican V2 is the molecule that renders GDNF-dependent non-peptidergic C-fibers IB4-positive.