Amino acid uptake, content, and metabolism by neuronal and glial enriched cellular fractions from mouse cerebellum.
A series of biochemical determinations was performed on five cellular fractions obtained from the cerebellum of 8- to 14-day-old mice. Cerebellar tissue was dissociated by mild trypsinization and mechanical agitation. The dissociated cellular material was separated into five fractions using a series of continuous density gradients formed with Percoll. Three of the fractions were comprised primarily of cell bodies. One of these was dominated by cells having the size and morphological appearance of granule cells, and based on several criteria the other two were were enriched in astrocyte cell bodies. Morphological analysis indicated that the remaining two fractions were enriched, respectively, in nerve terminals and large nucleated cell bodies. The uptake of 12 amino acids and 4 other metabolites by these cellular fractions was examined, and Km and Vmax values were determined for 10 of the compounds studied. High affinity transport carriers (Km approximately 1 to 20 microM) for most of the compounds studied were evident in neuronal and astrocyte-enriched fractions; however, for glutamate and gamma-aminobutyric acid (GABA) additional carriers with higher substrate affinities (Km approximately 0.1 to 0.3 microM) were evident in the astrocyte-enriched fraction. The fraction enriched in granule cell bodies was distinguished by an exceptionally low uptake of GABA and citrate, and a comparatively low uptake of beta-alanine, taurine, alpha-ketoglutarate, and glutamate. An analysis of the content of nine amino acids in the five fractions revealed that only glutamate, aspartate, and GABA were concentrated in the fraction enriched in nerve terminals. GABA was concentrated also in the fraction enriched in large cell bodies and was present at a low concentration in the fraction enriched in granule cell bodies. The other amino acids measured were distributed nearly evenly among the five fractions. Several differences in metabolic activity among the five fractions were observed. Radiolabel from several precursors was incorporated into GABA preferentially in the fractions enriched in large cell bodies and nerve terminals. In contrast, the accumulation of label in glutamine occurred preferentially in the fractions enriched in astrocytes and granule cell bodies. Labeling of alanine from [14C]pyruvate and of proline from [14C]ornithine was most prominent in the fractions enriched in astrocytes and granule cell bodies.


A human genomic library enriched in transcriptionally active sequences (aDNA library).
Core histone hyperacetylation, in particular of H4, is concentrated in the promoter-upstream regions of active genes and in certain cases is locuswide. Antibodies to hyperacetylated H4 were used to immunoprecipitate dinucleosomal chromatin derived from K562 human erythroleukemic cells by micrococcal nuclease digestion. The extracted DNA was made into a genomic library and was expected to contain sequences from genes active in K562 cells (an active, 'aDNA' library). Clones (180) were randomly selected from the library; 24 of 103 tested (23%) contained highly repeated sequences, as determined by their hybridization to total genomic DNA, and were not analyzed further. An additional 10 clones (6%) were shown to contain no insert DNA. The remaining 146 were sequenced and compared with the nucleic acid databases and in all six frames to the protein databases: Sixeen clones could be assigned to known genes, the majority of which (12) were tissue specific. All but 2 of these 16 corresponded to segments 5' of the coding sequences, as expected if H4 acetylation is concentrated at promoter regions. Thirty-three clones (23%) displayed high sequence identity to cDNAs in the expressed sequence tag database (dbEST). Northern blots and reverse transcription (RT)-PCR were used to determine the proportion of clones representing sequences expressed in K562 cells: Although only 1 of 34 tested clones showed a band in Northern hybridization, RT-PCR demonstrated that at least 12 of 40 tested clones (30%) were present in the mRNA population. Because a further 8 of these 40 clones were identified as gene fragments by database sequence comparisons, it follows that about half of this subset of 40 clones is derived from genes. The aDNA library is thus very gene rich and not skewed toward the most highly expressed sequences, as in mRNA libraries. The aDNA library is also rich in promoters and could be a valuable source of such sequences, particularly those that lack CpG islands or other features that allow their specific selection.


Receptive fields for flexible face categorizations.
Abstract-Examining the receptive fields of brain signals can elucidate how information impinging on the former modulates the latter. We applied this time-honored approach in early vision to the higher-level brain processes underlying face categorizations. Electroencephalograms in response to face-information samples were recorded while observers resolved two different categorizations (gender, expressive or not). Using a method with low bias and low variance, we compared, in a common space of information states, the information determining behavior (accuracy and reaction time) with the information that modulates emergent brain signals associated with early face encoding and later category decision. Our results provide a time line for face processing in which selective attention to diagnostic information for categorizing stimuli (the eyes and their second-order relationships in gender categorization; the mouth in expressive-or-not categorization) correlates with late electrophysiological (P300) activity, whereas early face-sensitive occipito-temporal (N170) activity is mainly driven by the contralateral eye, irrespective of the categorization task.


Acute rejection of orthotopic corneal xenografts in mice depends on CD4(+) T cells and self-antigen-presenting cells.
Guinea pig corneal xenografts have been reported to be rejected acutely in eyes of normal adult mice. Rejection of this type is independent of xenoreactive antibodies, and mice deficient in CD8(+) and NK T cells are unable to reject guinea pig corneal grafts acutely. Therefore, a study was conducted to determine the extent and manner by which CD4(+) T cells are responsible for rejection of orthotopic corneal xenografts. Xenogeneic corneas were prepared from eyes of normal guinea pigs and grafted orthotopically into normal eyes of C57BL/6 mice, class II major histocompatibility complex (MHC) knockout (KO) mice, and class II MHC KO mice reconstituted with syngeneic (C57BL/6) CD4(+) T cells and/or bone marrow cells. Graft survival was assessed clinically, and success of cellular reconstitution was assayed using flow cytometric analysis of peripheral blood leukocytes. T cells from rejector mice were analyzed for proliferative responses to guinea pig xenoantigens in vitro. Median survival times (MST) of corneal xenografts in MHC class II KO mice was significantly delayed (31 days) compared with grafts in wild-type C57BL/6 eyes (9 days). Acute rejection was restored almost completely when MHC class II KO mice were reconstituted simultaneously with C57BL/6 bone marrow and CD4(+) T cells, but not when the KO mice were reconstituted with either CD4(+) T cells or bone marrow cells alone. Mice that rejected guinea pig corneas possessed only CD4(+) T cells capable of responding to guinea pig xenoantigens in vitro. Acute rejection of orthotopic corneal xenografts in mice is mediated by CD4(+) T cells that detect guinea pig xenoantigens that are presented on MHC class II(+) syngeneic antigen-presenting cells. These results strongly suggest that rejection occurs exclusively through the indirect pathway of T-cell activation.


Predicting strike susceptibility and collision patterns of the common buzzard at wind turbine structures in the federal state of Brandenburg, Germany.
With the increase in wind turbines, bird collisions have developed as a potential hazard. In the federal state of Brandenburg, Germany, despite the on-going mitigation efforts of increasing the distances of wind turbines from the breeding areas of the more severely affected populations of red kites (Milvus milvus), the additional detrimental influences on the buzzard populations (Buteo buteo) have added to the challenges for wind power expansion. Using data on the regional distribution of the buzzards, along with their carcass detections around the wind turbines (WTs), we aimed to better understand their collision distribution patterns in relation to their habitat use patterns to predict their exposure to collision risk using boosted regression trees (BRTs). Additionally, we integrated the developed collision potential map with the regional density map of buzzards to identify areas of increased strike susceptibility in turbine installations. Our study showed that the buzzard collisions were primarily concentrated at the turbines situated at sensitive distances from the edges of watercourses (>1000 metres), as well as those along the edges of grasslands (>750 metres), in the green open areas around/areas with minimal settlements (750 metres-1750 metres), and along the edges of bushlands (>1500 metres), together explaining 58% of the variance in their collision distribution. Conclusively, our study is applicable to conservation because it demonstrates the identification of potential collision areas along with the causes of the collisions, in addition to demonstrating the benefits of incorporating a species collision dataset as a proxy for species presence into species distribution models to make informed management decisions to eventually combat biodiversity loss.


Separable enhancer sequences regulate the expression of the neural bHLH transcription factor neurogenin 1.
Ngn1 is a basic helix-loop-helix (bHLH) transcription factor expressed in specific regions within the developing brain and spinal cord, sensory ganglia, and olfactory epithelium. We have identified sequences both 5' and 3' of the mouse ngn1 gene that function in regulating ngn1 expression, and each of these sequences contains distinct regulatory cassettes for different subregions of the expression domain. Enhancers for expression in ngn1 domains of the midbrain, hindbrain, trigeminal ganglia, and ventral-neural tube appear redundant and are spread both 5' and 3' of the ngn1 coding sequence. In contrast, a single discrete dorsal-neural tube enhancer was located in the 5' sequence that is conserved among mouse, human, chick, and zebrafish ngn1 genes. Functionally, this enhancer is both necessary and sufficient for driving expression of a heterologous reporter in transgenic mice specifically to the dorsal domain of ngn1 expression in the spinal neural tube. Thus, sequences are identified that can be used to direct temporally and spatially restricted expression of heterologous genes to the developing neural tube.


SARS-CoV-2 Variants, Vaccines, and Host Immunity.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new beta coronavirus that emerged at the end of 2019 in the Hubei province of China. SARS-CoV-2 causes coronavirus disease 2019 (COVID-19) and was declared a pandemic by the World Health Organization (WHO) on 11 March 2020. Herd or community immunity has been proposed as a strategy to protect the vulnerable, and can be established through immunity from past infection or vaccination. Whether SARS-CoV-2 infection results in the development of a reservoir of resilient memory cells is under investigation. Vaccines have been developed at an unprecedented rate and 7 408 870 760 vaccine doses have been administered worldwide. Recently emerged SARS-CoV-2 variants are more transmissible with a reduced sensitivity to immune mechanisms. This is due to the presence of amino acid substitutions in the spike protein, which confer a selective advantage. The emergence of variants therefore poses a risk for vaccine effectiveness and long-term immunity, and it is crucial therefore to determine the effectiveness of vaccines against currently circulating variants. Here we review both SARS-CoV-2-induced host immune activation and vaccine-induced immune responses, highlighting the responses of immune memory cells that are key indicators of host immunity. We further discuss how variants emerge and the currently circulating variants of concern (VOC), with particular focus on implications for vaccine effectiveness. Finally, we describe new antibody treatments and future vaccine approaches that will be important as we navigate through the COVID-19 pandemic.


Diagnostic value of S100B protein in the differential diagnosis of acute vertigo in the emergency department.
Vertigo is a common presenting complaint resulting from central or peripheral etiologies. Because central causes may be life-threatening, ascertaining the nature of the vertigo is crucial in the emergency department (ED). With a broad range of potential etiologies, distinguishing central causes from benign peripheral causes is a diagnostic challenge. Cranial magnetic resonance imaging (MRI) is the recommended neuroimaging method when clinical findings are ambiguous. However, MRI scanning for every patient with an uncertain diagnosis may not be efficient or possible. Therefore, to improve ED resource utilization for patients with vertigo, there is a need to identify the subset most likely to have MRI abnormalities. It has previously been shown that S100B protein provides a useful serum marker of stroke, subarachnoid hemorrhage, and traumatic brain injury. This study evaluated whether S100B levels could predict central causes of vertigo as identified by cranial MRI in the ED. This prospective, observational study was conducted with adult patients with acute-onset vertigo (within 6 hours) in the ED of a teaching hospital in Kocaeli, Turkey. Patients with nausea or dizziness complaints without previously known vertigo or cranial pathology, and who agreed to participate in the study, were included. Patients with trauma or with neurologic findings that developed concurrent with their symptoms were excluded. Serum levels of S100B were measured with an electrochemiluminescence immunoassay kit. All subjects underwent cranial MRI. The predictors of positive MRI results were evaluated using logistic regression analysis. Sensitivity and specificity of S100B levels for identifying subjects with central causes of vertigo on MRI were calculated with receiver operating characteristic (ROC) curve. Of the 82 subjects included in the study, 48 (58.5%) were female, and the mean (±SD) age was 51 (±16) years. Thirty-one (37.8%) subjects had positive MRI results. Median (with interquartile range [IQR]) serum S100B levels were significantly different between MRI-negative and MRI-positive groups (median = 27.00 pg/mL, IQR = 10.00 to 44.60 vs. median = 60.94 pg/mL, IQR = 38.25 to 77.95, respectively; p = 0.04). In logistic regression analysis, subjective "he or she is spinning" (p = 0.030, odds ratio [OR] = 1.63, 95% confidence interval [CI] = 1.38 to 2.49), systolic blood pressure (sBP; p = 0.045, OR = 1.044, 95% CI = 1.021 to 1.080), and serum S100B level (p = 0.042, OR = 1.22, 95% CI = 1.018 to 1.445) were found to be independent predictors of MRI abnormalities. In the ROC analysis, S100B > 30 pg/mL predicted the clinical outcome with 83.9% sensitivity (95% CI = 66.3% to 94.5%) and 51.0% specificity (95% CI = 36.6% to 65.2%). The area under the ROC curve was 0.774 (95% CI = 0.666 to 0.881). To the best of our knowledge this is the first study assessing the utility of serum S100B levels for diagnosis of acute-onset vertigo. Serum S100B levels are associated with the presence of central causes of vertigo on cranial MRI. However, serum S100B levels are not sufficiently sensitive to exclude candidates from cranial MRI.


Analysis of the intestinal microbiome of a recovered Clostridium difficile patient after fecal transplantation.
Clostridium difficile infections upon antibiotic disruption of the gut microbiota are potentially lethal. Fecal microbiota transplantation (FMT) is a promising treatment option for recurrent C. difficile-associated disease (CDAD). Here, we present a patient with recurrent CDAD that received FMT, leading to full recovery for what has now been 3 years. We performed metagenomic sequencing on stool samples to assess if there are indications for recolonization with C. difficile and changes in the gut microbiota after FMT. DNA from the stool of the donor and recipient was subjected to illumina sequencing. Obtained read sets were assembled to contiguous sequences and open reading frames were predicted. Deduced proteins were taxonomically assigned. We detected complex and apparently healthy microbiomes in the donor's and recipient's intestines after FMT, but no indications for C. difficile colonization. Metagenomic analysis proved suitable to analyze the intestinal microbiome after FMT. Discussion of our evaluation procedure and data management may be helpful for future studies. We demonstrated restoration of a healthy and diverse gut microbiome with chimeric composition from donor and recipient, and long-lasting clearance of C. difficile. The procedure is simple, cheap, caused no side effects, and was stable over 3 years.


The Baltic haline conveyor belt or the overturning circulation and mixing in the Baltic.
A study of the water-mass circulation of the Baltic has been undertaken by making use of a three dimensional Baltic Sea model simulation. The saline water from the North Atlantic is traced through the Danish Sounds into the Baltic where it upwells and mixes with the fresh water inflow from the rivers forming a Baltic haline conveyor belt. The mixing of the saline water from the Great Belt and Oresund with the fresh water is investigated making use of overturning stream functions and Lagrangian trajectories. The overturning stream function was calculated as a function of four different vertical coordinates (depth, salinity, temperature and density) in order to understand the path of the water and where it upwells and mixes. Evidence of a fictive depth overturning cell similar to the Deacon Cell in the Southern Ocean was found in the Baltic proper corresponding to the gyre circulation around Gotland, which vanishes when the overturning stream function is projected on density layers. A Lagrangian trajectory study was performed to obtain a better view of the circulation and mixing of the saline and fresh waters. The residence time of the water masses in the Baltic is calculated to be 26-29 years and the Lagrangian dispersion reaches basin saturation after 5 years.


Tumor uptake of 68Ga-DOTA-Tyr3-octreotate: animal PET studies of tumor flow and acute somatostatin receptor modulation in the CA20948 rat model.
Factors determining the in vivo uptake of radiolabeled somatostatin analogs by neuroendocrine tumors are poorly known. The aim is to evaluate in vivo tumor perfusion and regulation of somatostatin receptors (sstr) following acute exposure to octreotide, in an animal model of neuroendocrine tumor. H(2)(15)O flow studies were performed in 8 CA20948 tumor-bearing rats and another 36 rats underwent three [(68)Ga]-DOTA-Tyr(3)-octreotate imaging sessions at 24-h intervals. After baseline (Day 0) imaging, scanning was repeated on Day 1 after octreotide injection (175 microg/kg), with a variable delay: no injection (controls, n=7), coinjection (n=6), and octreotide injection 20 min (n=7), 2 h (n=8) and 4 h (n=8) before imaging. Repeat images without octreotide was performed at Day 2 followed by sacrifice and tumor counting. H(2)(15)O studies failed to measure quantitative tumor perfusion in this model. On Day 1, ratio of tumor uptake to Day 0 was 1.2+/-0.3 in controls; 0.6+/-0.2 in the coinjection group; 0.9+/-0.2, 1.1+/-0.1 and 1.2+/-0.2 in the other groups, respectively. Uptake in the coinjection group showed a statistically significant reduction of tumor uptake (P<.0001). All groups showed increased uptake on Day 2, without statistical differences between groups. In vivo tumor counts showed good correlation with ex vivo countings (R(2)=0.946). Acute exposure to unlabeled octreotide in this neuroendocrine tumor model results in a rapid recycling or resynthesis of sstr. Positron emission tomography (PET) allowed to reliably assess quantitative uptake of [(68)Ga]-DOTA-Tyr(3)-octreotate over time in the same animal, but failed in this model to measure tumor perfusion.


Wavevector-resolved monochromatic spectral imaging of extraordinary optical transmission through subwavelength aperture arrays.
A technique for wavevector-resolved spectroscopic imaging of extraordinary optical transmission (EOT) is developed and tested. The approach allows a large fraction of the first Brillouin zone to be mapped at a single wavelength, thereby greatly increasing the efficiency of sensitivity mapping experiments. An axially opposed, matched pair of microscope objectives constitutes the core of the apparatus. The condensing lens defines a broad range of wavevectors incident upon the sample, while the second objective with a higher numerical aperture collects all of the light transmitted through the sample. In this way, information related to transmission efficiency over a broad range of in-plane wavevectors is preserved at different spatial coordinates in the final image. A periodically structured gold film, consisting of a square array of cylindrical pores, measuring 90 x 90 pores, 100 nm in diameter, with a lattice constant of 1.1 microm, was chosen for detailed study. Direct imaging of the EOT efficiency simultaneously across the range 0 < kx < 0.001 nm(-1), or 20% of the first Brillouin zone, was accomplished, although this was not the limit of the instrument. The experiment was repeated across 21 values of the wavelength and 7 values of the refractive index, to construct a 4-dimensional data set of transmission efficiency with respect to lambda, kx, and n. This technique is compatible with any of the subwavelength aperture array-based chemical sensing methods reported in the literature, however it offers faster transduction of the full spectrum of plasmonic resonant shifts.


Polymeric Nanocarrier Formulations of Biologics Using Inverse Flash NanoPrecipitation.
The encapsulation of water-soluble therapeutics and biologics into nanocarriers to produce novel therapeutics has been envisioned for decades, but clinical translation has been hampered by complex synthesis strategies. The methods that have been developed are often limited by poor encapsulation efficiency/loading or complex processing to achieve therapeutic loadings high enough to be medically relevant. To address this unmet need, we introduce a solubility-driven self-assembly process to form polymeric nanocarriers comprising a biologic in a hydrophilic core, encapsulated by a poly(lactic acid) shell, and stabilized by a poly(ethylene glycol) brush. Called "inverse Flash NanoPrecipitation (iFNP)," the technique achieves biologic loadings (wt% of total formulation) that are 5-15× higher than typical values (9-27% versus < 2%). In contrast to liposomes and polymersomes, we sequentially assemble the polymer layers to form the final nanocarrier. Installation of the poly(lactic acid) shell before water exposure sequesters the biologic in the core and results in the improved loadings that are achieved. We demonstrate the broad applicability of the process and illustrate its implementation by formulating over a dozen different oligosaccharides, antibiotics, peptides, proteins, and RNA into nanocarriers with narrow size distributions, at high loadings, and with high reproducibility. Lysozyme and horseradish peroxidase are shown to retain 99% activity after processing. These results demonstrate the potential for commercial implementation of this technology, enabling the translation of novel treatments in immunology, oncology, or enzyme therapies.


Cluster wild bootstrapping to handle dependent effect sizes in meta-analysis with a small number of studies.
The most common and well-known meta-regression models work under the assumption that there is only one effect size estimate per study and that the estimates are independent. However, meta-analytic reviews of social science research often include multiple effect size estimates per primary study, leading to dependence in the estimates. Some meta-analyses also include multiple studies conducted by the same lab or investigator, creating another potential source of dependence. An increasingly popular method to handle dependence is robust variance estimation (RVE), but this method can result in inflated Type I error rates when the number of studies is small. Small-sample correction methods for RVE have been shown to control Type I error rates adequately but may be overly conservative, especially for tests of multiple-contrast hypotheses. We evaluated an alternative method for handling dependence, cluster wild bootstrapping, which has been examined in the econometrics literature but not in the context of meta-analysis. Results from two simulation studies indicate that cluster wild bootstrapping maintains adequate Type I error rates and provides more power than extant small-sample correction methods, particularly for multiple-contrast hypothesis tests. We recommend using cluster wild bootstrapping to conduct hypothesis tests for meta-analyses with a small number of studies. We have also created an R package that implements such tests.


Long-acting neuraminidase inhibitor laninamivir octanoate versus oseltamivir for treatment of influenza: A double-blind, randomized, noninferiority clinical trial.
A single administration of laninamivir octanoate, a long-acting neuraminidase inhibitor, against influenza infection has been proven effective in nonclinical studies. This study evaluated the clinical efficacy of laninamivir octanoate for the treatment of adult influenza patients. A double-blind, randomized controlled trial examined whether laninamivir octanoate was noninferior to oseltamivir. A total of 1003 patients aged ≥ 20 years with febrile influenza symptoms for no more than 36 h were randomized to receive either 40 mg of laninamivir octanoate, 20 mg of laninamivir octanoate, or oseltamivir. Laninamivir octanoate was inhaled once on day 1, and oseltamivir (75 mg) was administered orally twice daily for 5 days. The primary end point was the time to illness alleviation. A total of 996 patients were included in the primary analysis (40-mg laninamivir octanoate, n = 334; 20-mg laninamivir octanoate, n = 326; and oseltamivir, n = 336). The median time to illness alleviation in the 40-mg laninamivir octanoate, 20-mg laninamivir octanoate, and oseltamivir groups was 73.0, 85.8, and 73.6 h, respectively. The difference between laninamivir octanoate and oseltamivir was -0.6 h (95% confidence interval, -9.9 to 6.9 h) for the 40-mg group and 12.2 h (95% confidence interval, -1.5 to 17.2 h) for the 20-mg group. The upper limits of the 95% confidence intervals were less than the prespecified noninferiority margin (18 h). The proportion of patients shedding virus at day 3 was significantly lower in the 40-mg laninamivir octanoate group than in the oseltamivir group (P = .006). A single inhalation of laninamivir octanoate is effective for the treatment of seasonal influenza, including that caused by oseltamivir-resistant virus, in adults. NCT00803595.


T2D-Db: an integrated platform to study the molecular basis of Type 2 diabetes.
Type 2 Diabetes Mellitus (T2DM) is a non insulin dependent, complex trait disease that develops due to genetic predisposition and environmental factors. The advanced stage in type 2 diabetes mellitus leads to several micro and macro vascular complications like nephropathy, neuropathy, retinopathy, heart related problems etc. Studies performed on the genetics, biochemistry and molecular biology of this disease to understand the pathophysiology of type 2 diabetes mellitus has led to the generation of a surfeit of data on candidate genes and related aspects. The research is highly progressive towards defining the exact etiology of this disease. T2D-Db (Type 2 diabetes Database) is a comprehensive web resource, which provides integrated and curated information on almost all known molecular components involved in the pathogenesis of type 2 diabetes mellitus in the three widely studied mammals namely human, mouse and rat. Information on candidate genes, SNPs (Single Nucleotide Polymorphism) in candidate genes or candidate regions, genome wide association studies (GWA), tissue specific gene expression patterns, EST (Expressed Sequence Tag) data, expression information from microarray data, pathways, protein-protein interactions and disease associated risk factors or complications have been structured in this on line resource. Information available in T2D-Db provides an integrated platform for the better molecular level understanding of type 2 diabetes mellitus and its pathogenesis. Importantly, the resource facilitates graphical presentation of the gene/genome wide map of SNP markers and protein-protein interaction networks, besides providing the heat map diagram of the selected gene(s) in an organism across microarray expression experiments from either single or multiple studies. These features aid to the data interpretation in an integrative way. T2D-Db is to our knowledge the first publicly available resource that can cater to the needs of researchers working on different aspects of type 2 diabetes mellitus.


Cell response to nano-islands produced by polymer demixing: a brief review.
This review looks at the present literature available regarding cell response to nano-islands produced by nanotopography. Polymer demixing is a chemical method of fabricating large areas of nanotopography quickly and cheaply, making it ideal for cell testing and thus allowing it to be one of the first well-researched methods in cell engineering. The review shows that cells respond strongly to the islands (cell types observed include endothelial cells, fibroblasts, osteoblasts, leucocytes and platelets). Such changes include differences in adhesion, growth, gene expression and morphology.


Restructuring higher taxonomy using broad-scale phylogenomics: The living Ophiuroidea.
The power and throughput of next-generation sequencing is instigating a major transformation in our understanding of evolution and classification of life on our planet. The new trees of life are robust and comprehensive. Here we provide a landmark phylogeny of the living ophiuroids and use it as the basis for a major revision of the higher classification of this class of marine invertebrates. We used an exon-capture system to generate a 1484 exon (273kbp) data-matrix from DNA extracted from ethanol-preserved museum samples. We successfully obtained an average of 90% of our target sequence from 576 species spread across the known taxonomic diversity. The topology of the major lineages was robust to taxon sampling, exon-sampling, models and methods. However, estimates of node age were much less precise, varying by about a quarter of mean age. We used a combination of phylogenetic distinctiveness and temporal-banding to guide our revision of the family-level classification. Empirically, we determined that limiting family crown age to 110±10Ma (mid Cretaceous) selected phylogenetically distinct nodes while minimising disruption to the existing taxonomy. The resulting scheme of 32 families and six orders considerably expands the number of higher taxa. The families are generally longitudinally widespread across the world's oceans, although 17 are largely confined to temperate and equatorial latitudes and six to relatively shallow water (less than 1000m depth).


Measurement of hemodynamic energy at different vessels in an adult swine model.
Extra hemodynamic energy is one of the major benefits of pulsatile flow, improving blood flow to vital organs. But most (80%) of the hemodynamic energy generated from pulsatile flow is damped by the extracorporeal circuit. Most models devised to minimize hemodynamic energy loss have been in vitro pediatric models. The purpose of this study was to measure hemodynamic energy in different vessels of different organs with an in vivo adult swine model. An extracorporeal circuit was constructed for seven Yorkshire swine using a pulsatile pump (Twin-Pulse Life Support). The mean arterial pressure (MAP), energy equivalent pressure (EEP), and surplus hemodynamic energy (SHE) at the renal artery, carotid artery, aortic cannula site, and postoxygenator site were measured simultaneously before starting the pump and at the pump rates of 25, 35, and 45 bpm. The MAP of the renal or carotid artery was 40.0%-51.2% of the postoxygenator site. The EEP and SHE of both arteries were 11.6%-13.0% and 5.5%-7.4% of the postoxygenator site, respectively. The MAP and EEP of both arteries after starting the pump were lower than at baseline. The SHE of the renal artery after starting the pump was significantly higher than at baseline. The SHE of the carotid artery increased substantially after starting the pump although not statistically significantly. There was a significant hemodynamic energy loss in both arterial sites compared with the postoxygenator site. Also, a difference in hemodynamic energy loss was observed in vessel-to-vessel or vessel-to-circuit site comparison. This difference creates a bias in studying pulsatility and its effects. Therefore, the measurement method of hemodynamic energy must be standardized and the measurement site clarified to yield accurate study results.


Clinical effectiveness of bee venom acupuncture and physiotherapy in the treatment of adhesive capsulitis: a randomized controlled trial.
Bee venom acupuncture (BVA) has been used in the treatment of adhesive capsulitis (AC) in the clinical field. This study aimed to investigate whether the addition of BVA to physiotherapy (PT) would be more effective in the management of AC, and whether BVA would have a dose-dependent effect. Sixty-eight patients diagnosed with AC were recruited into 3 groups; BV 1 (1:10,000 BVA plus PT), BV 2 (1:30,000 BVA plus PT), and group 3 (normal saline (NS) injection, as a control, plus PT). PT was composed of 15 minutes of transcutaneous electrical nerve stimulation (TENS), transcutaneous infrared thermotherapy (TDP), and manual PT. Treatments were given in 16 sessions within 2 months. Shoulder pain and disability index (SPADI), pain visual analogue scale (VAS), and 3) active/passive range of motion (ROM) were measured before treatment and at 2, 4, 8, and 12 weeks after the treatment. All 3 groups showed statistically significant improvements in SPADI, pain VAS scores, and active/passive ROM. The BV 1 group showed significantly better outcomes in SPADI at 8 and 12 weeks, in pain VAS (at rest) at 8 weeks, and in pain VAS (during exercise) at 12 weeks than the NS group. No significant differences were found in active/passive ROM among all the groups. BVA in combination with PT can be more effective in improving pain and function than PT alone in AC. However, the effectiveness of BVA was not shown in a dose-dependent manner.


Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair.
Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.


Women with synchronous primary cancers of the endometrium and ovary: do they have Lynch syndrome?
Lynch syndrome (hereditary nonpolyposis colorectal cancer; HNPCC) is an autosomal-dominant cancer predisposition syndrome that increases risk for multiple cancers, including colon, endometrial, and ovarian cancer. Revised Bethesda Criteria recommend that patients with two HNPCC-associated cancers undergo molecular evaluation to determine whether they have a mismatch repair (MMR) defect associated with HNPCC. The purpose of our study was to determine the likelihood of MMR defects (MSH2, MSH6, MLH1) in women with synchronous endometrial and ovarian cancer. Between 1989 and 2004, 102 women with synchronous endometrial and ovarian cancers were identified; 59 patients had tumor blocks available for analysis. Patients were divided into risk groups based on family history: high (met Amsterdam criteria), medium (personal history or first-degree relative with an HNPCC-associated cancer), and low (all others). Protein expression for MSH2, MSH6, and MLH1 was evaluated by immunohistochemistry. Microsatellite instability and MLH1 promoter methylation analyses were performed on a subset of cases. Median age was 50 years. Two patients met Amsterdam criteria for HNPCC. Five additional patients, all medium-risk, had molecular findings consistent with a germline mutation of either MSH2 or MLH1. None of the low-risk patients had molecular results consistent with a germline mutation. Overall, 7% of women in our cohort met either clinical or molecular criteria for Lynch syndrome. All of these women had a prior history or a first-degree relative with an HNPCC-associated cancer. Limiting genetic evaluation to women with synchronous endometrial and ovarian cancer who have a family history suggestive of HNPCC may appropriately identify women with Lynch syndrome.


Assessment of the cortisol awakening response: Expert consensus guidelines.
The cortisol awakening response (CAR), the marked increase in cortisol secretion over the first 30-45 min after morning awakening, has been related to a wide range of psychosocial, physical and mental health parameters, making it a key variable for psychoneuroendocrinological research. The CAR is typically assessed from self-collection of saliva samples within the domestic setting. While this confers ecological validity, it lacks direct researcher oversight which can be problematic as the validity of CAR measurement critically relies on participants closely following a timed sampling schedule, beginning with the moment of awakening. Researchers assessing the CAR thus need to take important steps to maximize and monitor saliva sampling accuracy as well as consider a range of other relevant methodological factors. To promote best practice of future research in this field, the International Society of Psychoneuroendocrinology initiated an expert panel charged with (i) summarizing relevant evidence and collective experience on methodological factors affecting CAR assessment and (ii) formulating clear consensus guidelines for future research. The present report summarizes the results of this undertaking. Consensus guidelines are presented on central aspects of CAR assessment, including objective control of sampling accuracy/adherence, participant instructions, covariate accounting, sampling protocols, quantification strategies as well as reporting and interpreting of CAR data. Meeting these methodological standards in future research will create more powerful research designs, thus yielding more reliable and reproducible results and helping to further advance understanding in this evolving field of research.


A Randomised, Double-blind, Placebo-controlled Trial of Trichuris suis ova in Active Crohn's Disease.
To investigate the efficacy and safety of three different dosages of embryonated, viable eggs of Trichuris suis [TSO] versus placebo for induction of remission in mildly-to-moderately active ileocolonic, uncomplicated Crohn's disease [CD]. Adults with active CD [n = 252] randomly received six fortnightly doses of 250, 2500, or 7500 TSO/15 ml suspension/day [TSO 250, TSO 2500, TSO 7500], or 15 ml placebo solution/day, in a double-blind fashion, with 4 weeks' follow-up. Primary endpoint was the rate of clinical remission [Crohn's Disease Activity Index [CDAI] < 150] at end of treatment, ie at Week 12 or withdrawal. Secondary endpoints included the course of clinical remission, rate of clinical response, change in CDAI, change in markers of inflammation, mucosal healing, and Physician's Global Assessment. Clinical remission at Week 12 occurred in 38.5%, 35.2%, and 47.2% of TSO 250, TSO 2500, and TSO 7500 patients, respectively, and in 42.9% of placebo recipients. TSO induced a dose-dependent immunological response. There was no response regarding laboratory markers of inflammation. Other secondary efficacy variables also showed no advantage of TSO over placebo for treatment of active CD. Administration of TSO did not result in any serious adverse drug reaction. Review of non-serious suspected adverse drug reactions following TSO did not reveal any safety concerns. Administration of 250-7500 TSO fortnightly over 12 weeks was safe and showed a dose-dependent immunological response, but no TSO dose showed a clinically relevant effect over placebo for induction of clinical remission or response in mildly-to-moderately active, ileocolonic CD.


Identification and Morphogenesis of Vestibular Atrial Septal Defects.
Background: The vestibular atrial septal defect is an interatrial communication located in the antero-inferior portion of the atrial septum. Reflecting either inadequate muscularization of the vestibular spine and mesenchymal cap during development, or excessive apoptosis within the developing antero-inferior septal component, the vestibular defect represents an infrequently recognized true deficiency of the atrial septum. We reviewed necropsy specimens from three separate archives to establish the frequency of such vestibular defects and their associated cardiac findings, providing additional analysis from developing mouse hearts to illustrate their potential morphogenesis. Materials and methods: We analyzed the hearts in the Farouk S. Idriss Cardiac Registry at Ann and Robert H. Lurie Children's Hospital in Chicago, IL, the Van Mierop Archive at the University of Florida in Gainesville, Florida, and the archive at Johns Hopkins All Children's Heart Institute in St. Petersburg, Florida, identifying all those exhibiting a vestibular atrial septal defect, along with the associated intracardiac malformations. We then assessed potential mechanisms for the existence of such defects, based on the assessment of 450 datasets of developing mouse hearts prepared using the technique of episcopic microscopy. Results: We analyzed a total of 2100 specimens. Of these, 68 (3%) were found to have a vestibular atrial septal defect. Comparable defects were identified in 10 developing mouse embryos sacrificed at embryonic data 15.5, by which stage the antero-inferior component of the atrial septum is usually normally formed. Conclusion: The vestibular defect is a true septal defect located in the muscular antero-inferior rim of the oval fossa. Our retrospective review of autopsied hearts suggests that the defect may be more common than previously thought. Increased awareness of the location of the defect should optimize its future clinical identification. We suggest that the defect exists because of failure, during embryonic development, of union of the components that bind the leading edge of the primary atrial septum to the atrioventricular junctions, either because of inadequate muscularisation or excessive apoptosis.


Studies on the short range spreading of the male specific lethal (MSL) complex on the X chromosome in Drosophila.
A series of autosomal insertions of chromosomal fragments derived from around the X linked white eye color locus have been examined for male specific lethal (MSL) complex binding using both immunostaining and fluorescence in situ hybridization (FISH) techniques. The results show that the transposing elements (TEs) composed of several genes in the white region (3C2-3C5) do not recruit the MSL complex when inserted into an autosome. The same result is found for the Tp(1:3)wzh insertion, a fragment of the X chromosome inserted into the third chromosome. Two other insertions, Dp(1:2)w70h (3A7-3C2-3) and Dp(1:2)51b (3C2-3D6), which extend more distally or proximally beyond the TE insertion, respectively, display a binding pattern of the MSL complex at the autosomal location. These insertions were also examined in females ectopically expressing MSL-2 and show similar binding activity. In addition, the Tp(3:1)O5 transposition strain containing an autosomal segment in the X chromosome was examined for spreading of the MSL complex. Limited spreading of the MSL complex into autosomal regions was indicated by immunostaining and FISH. This spreading was further confirmed by chromatin immunoprecipitation of the MSL complex covering the autosomal sequences.


Clofibrate in combination with phototherapy for unconjugated neonatal hyperbilirubinaemia.
There are many pathological conditions leading to an elevated unconjugated bilirubin level (hyperbilirubinaemia) in neonates. Currently the standard therapies for unconjugated hyperbilirubinaemia include phototherapy and exchange transfusion. In addition to phototherapy, clofibrate has been studied as a treatment for hyperbilirubinaemia in several countries. To determine the efficacy and safety of clofibrate in combination with phototherapy versus phototherapy alone in unconjugated neonatal hyperbilirubinaemia. Randomised controlled trials were identified by searching MEDLINE (1950 to April 2012) before being translated for use in The Cochrane Library, EMBASE 1980 to April 2012 and CINAHL databases. All searches were re-run on 2 April 2012. We included trials where neonates with hyperbilirubinaemia received either clofibrate in combination with phototherapy or phototherapy alone or placebo in combination with phototherapy. Data were extracted and analysed independently by two review authors (MG and HM). Treatment effects on the following outcomes were determined: mean change in bilirubin levels, mean duration of treatment with phototherapy, number of exchange transfusions needed, adverse effects of clofibrate, bilirubin encephalopathy and neonatal mortality. Study authors were contacted for additional information. Studies were analysed for methodological quality in a 'Risk of bias' table. Fifteen studies (two including preterm neonates and 13 including term neonates) were included in this review. All but one of the included studies were conducted in Iran. For preterm neonates, there was a significantly lower bilirubin level in the 100 mg/kg clofibrate group compared to the control group with a mean difference of -1.37 mg/dL (95% CI -2.19 mg/dL to -0.55 mg/dL) (-23 µmol/L; 95% CI -36 µmol/L to -9 µmol/L) after 48 hours. For the term neonates, there were significantly lower bilirubin levels in the clofibrate group compared to the control group after both 24 and 48 hours of treatment with a weighted mean difference of -2.14 mg/dL (95% CI -2.53 mg/dL to -1.75 mg/dL) (-37 µmol/L; 95% CI -43 µmol/L to -30 µmol/L] and -1.82 mg/dL (95% CI -2.25 mg/dL to -1.38 mg/dL) (-31 µmol/L; 95% CI -38 µmol/L to -24 µmol/L), respectively.There was a significantly lower duration of phototherapy in the clofibrate group compared to the control group for both preterm and term neonates with a weighted mean difference of -23.82 hours (95% CI -30.46 hours to -17.18 hours) and -25.40 hours (95% CI -28.94 hours to -21.86 hours), respectively.None of the studies reported on bilirubin encephalopathy rates, neonatal mortality rates, or the levels of parental or staff satisfactions with the interventions. There are insufficient data from different countries on the use of clofibrate in combination with phototherapy for hyperbilirubinaemia to make recommendations for practice. There is a need for larger trials to determine how effective clofibrate is in reducing the need for, and duration of, phototherapy in term and preterm infants with hyperbilirubinaemia.


A genealogical and clinical study of the phenotypical variation within the Swedish transthyretin His88Arg (p. His108Arg) amyloidosis family.
In 2005 we reported the first case of transthyretin His88Arg (p. His108Arg) amyloidosis, a mutation characterised by cardiomyopathy. Six additional gene carriers of whom five have clinical symptoms of disease have now been identified in Sweden, and we have been able to identify a possible founder and to characterise the Swedish phenotype of the transthyretin (TTR) His88Arg mutation. Genealogical studies of church records were used to identify the individuals with the disease and their families. Routine clinical investigations of neurological and heart manifestation of the disease were utilised. We found that genealogically all seven individuals were related and originated from the same region in Sweden. Amyloid deposits were demonstrated in biopsies and the TTR His88Arg mutation was identified in all patients. Patients had a late onset disease (≥ 50 years of age) and all exhibited a severe amyloid cardiomyopathy. A pronounced peripheral axonal neuropathy was with certainty demonstrated in one patient only, who also was operated for a magnetic resonance confirmed spinal stenosis, however, without any effect on his neurological symptoms. Five of the patients had carpal tunnel syndrome. The first reported case is now deceased from cardiac failure. One patient has had a sequential heart and liver transplantation. One gene carrier had no symptoms or findings of disease on latest evaluation at the age of 44. the Swedish TTRHis88Arg patients all have a common Swedish founder. Cardiomyopathy with heart failure, as well as carpal tunnel syndrome and spinal stenosis were early signs of disease; but peripheral neuropathy was present in one patient before symptoms of cardiomyopathy so the phenotypical presentation of this mutation is variable.


Getting somewhere with the Red Queen: chasing a biologically modern definition of the hypothesis.
The Red Queen hypothesis (RQH) is both familiar and murky, with a scope and range that has broadened beyond its original focus. Although originally developed in the palaeontological arena, it now encompasses many evolutionary theories that champion biotic interactions as significant mechanisms for evolutionary change. As such it de-emphasizes the important role of abiotic drivers in evolution, even though such a role is frequently posited to be pivotal. Concomitant with this shift in focus, several studies challenged the validity of the RQH and downplayed its propriety. Herein, we examine in detail the assumptions that underpin the RQH in the hopes of furthering conceptual understanding and promoting appropriate application of the hypothesis. We identify issues and inconsistencies with the assumptions of the RQH, and propose a redefinition where the Red Queen's reign is restricted to certain types of biotic interactions and evolutionary patterns occurring at the population level.


Interactions of prefrontal cortex in relation to awareness in sensory learning.
In an associative learning paradigm, human subjects could be divided based on whether they were aware that one tone predicted a visual event and another did not. Only aware subjects acquired a differential behavioral response to the tones. Regional cerebral blood flow in left prefrontal cortex showed learning-related changes only in aware subjects. Left prefrontal cortex also showed changes in functional connectivity with contralateral prefrontal cortex, sensory association cortices, and cerebellum. Several of the interacting areas correlated with aware subjects' behavior. These results suggest cerebral processes underlying awareness are mediated through interactions of large-scale neurocognitive systems.


The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis.
The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription. Here we identify the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. We show that genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. We provide evidence that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. We also present an initial characterization of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes. This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development.


Characterization and regulation of the human ML1A melatonin receptor stably expressed in Chinese hamster ovary cells.
The human ML1A melatonin receptor is expressed in the suprachiasmatic nucleus of the hypothalamus and is believed to regulate circadian rhythms. We report the kinetic characteristics and pharmacological profile of 2-[125I]iodomelatonin binding and the signaling pathway and agonist regulation of the human ML1A melatonin receptor stably expressed in Chinese hamster ovary cells. Association of 2-[125I]iodomelatonin binding was maximal by 1.5 hr at 37 degrees and fully dissociated on the addition of 1 microM melatonin. The binding of 2-[125I]iodomelatonin was saturable and of high affinity (KD = 74 +/- 14 PM, Bmax = 679 +/- 88 fmol/mg protein; three experiments). The pharmacological profile of various melatonin analogues revealed a profile (2-iodomelatonin > or = melatonin > N-acetyl serotonin > luzindole) characteristic of an ML1 subtype. Competition of melatonin for 2-[125I]iodomelatonin binding to the human ML1A receptor in lysed or intact cells resulted in biphasic curves revealing the existence of super high (approximately 20%) and high (approximately 80%) affinity states of the receptor. Guanosine-5'-0-(3-thio)triphosphate (100 PM-30 microM) when added alone inhibited 2-[125I]iodomelatonin binding (IC50 = 0.87 +/- 0.12 microM; three experiments), suggesting uncoupling of the receptor from G proteins. In addition, guanosine-5'-O-(3-thio)triphosphate (3 microM) produced a right-ward shift in both the super high and high binding melatonin affinities for 2-[125I]iodomelatonin resulting in monophasic curves. Melatonin (0.1 fM-1 nM) inhibited forskolin-induced cAMP formation in a concentration-dependent and biphasic manner. Low concentrations of melatonin (0.01 fM-1 PM) inhibited forskolin (100 microM)-stimulated cAMP formation with an IC50 of 0.1 +/- 0.05 PM (four experiments) and a maximal inhibitory effect (26%) at 1 PM. Higher concentrations of melatonin (1 PM-1 nM) inhibited forskolin-induced cAMP formation with an IC50 of 64 +/- 1.8 PM (four experiments) and a maximal inhibition (74%) at 1 nM. Luzindole (1 microM), a competitive melatonin receptor antagonist, antagonized the effect of melatonin at the higher concentrations only (IC50 = 1.5 +/- 0.22 nM, pKB = -7.3; three experiments). Pretreatment with pertussis toxin completely abolished melatonin-mediated inhibition of forskolin-induced cAMP formation through these receptors. Pretreatment with various concentrations of melatonin (0.1 PM-1 microM) for different periods of time (1, 6, 18, and 24 hr) did not decrease 2-[125I]iodomelatonin binding. However, competition by melatonin for 2-[125I]iodomelatonin binding to cells pretreated with melatonin and washed was only to a single population of super high affinity sites (IC50 = 1.1 +/- 0.28 nM; three experiments) as revealed by monophasic curves. Cells pretreated with melatonin revealed a persistent inhibition (approximately 20%) of forskolin-induced cAMP formation that was not reversed by extensive washes (up to 1 hr) or when luzindole (1 microM) was added together with melatonin during pretreatment. These results suggest that tight binding of melatonin to the super high affinity state of the human ML1A melatonin receptor may be the mechanism by which low concentrations of circulating hormone in vivo regulates signaling in the suprachiasmatic nucleus of the hypothalamus.


No evidence for association between a functional promoter variant of the Norepinephrine Transporter gene SLC6A2 and ADHD in a family-based sample.
Noradrenergic neurotransmission influences executive functions, attentional performance, and general alertness, involving neuronal networks affected in attention deficit/hyperactivity disorder (ADHD). The norepinephrine transporter facilitates the reuptake of norepinephrine and dopamine in the prefrontal cortex and represents the main target of atomoxetine, an effective drug in the treatment of ADHD. Due to its influence on catecholaminergic signaling, variants of the coding gene (SLC6A2) have been widely investigated in ADHD. Several previous studies report an association between single nucleotide polymorphisms located in SLC6A2 and ADHD; however, the findings are inconsistent. The variant A-3081T (rs28386840) has been shown to have major influence on the expression levels of SLC6A2 due to sequence alteration at a repressor binding site, with the T-allele being associated with ADHD. We tested this potential association of A-3081T in a German family-based ADHD sample of 235 children from 162 families, which has a power >99% based on the previously reported odds ratios. There was no evidence for an overtransmission of the risk allele T (transmission rate: 48.5%, P = 0.55). We conclude that A-3081T is not a major risk variant in our ADHD sample, though SLC6A2 remains an interesting candidate gene in ADHD, especially for the inattentive subtype.


Pharmacokinetics and metabolism of the antimalarial piperaquine after intravenous and oral single doses to the rat.
This study aimed to evaluate the pharmacokinetic properties of piperaquine in the rat after intravenous and oral administration, and to identify and characterize the main piperaquine metabolites in rat plasma, urine, faeces and bile after intravenous administration. Male Sprague-Dawley rats were administered piperaquine as an emulsion orally or as a short-term intravenous infusion. Venous blood for pharmacokinetic evaluation was frequently withdrawn up to 90 h after dose. Urine, bile and faeces were collected after an infusion in rats kept in metabolic cages or in anesthetized rats. Pharmacokinetic characterization was done by compartmental modeling and non-compartmental analysis using WinNonlin. Piperaquine disposition was best described by a 3-compartment model with a rapid initial distribution phase after intravenous administration. The pharmacokinetics of piperaquine was characterized by a low clearance, a large volume of distribution and a long terminal half-life. Piperaquine displayed a low biliary clearance and less than 1% of the total dose was recovered in urine. The absolute oral bioavailability was approximately 50%. The main metabolite after intravenous administration of piperaquine was a carboxylic acid product identical to that reported in humans. The similarity with results in humans indicates the rat to be a suitable species for nonclinical in vivo piperaquine studies.


Revisiting GMOs: Are There Differences in European Consumers' Acceptance and Valuation for Cisgenically vs Transgenically Bred Rice?
Both cisgenesis and transgenesis are plant breeding techniques that can be used to introduce new genes into plant genomes. However, transgenesis uses gene(s) from a non-plant organism or from a donor plant that is sexually incompatible with the recipient plant while cisgenesis involves the introduction of gene(s) from a crossable--sexually compatible--plant. Traditional breeding techniques could possibly achieve the same results as those from cisgenesis, but would require a much larger timeframe. Cisgenesis allows plant breeders to enhance an existing cultivar more quickly and with little to no genetic drag. The current regulation in the European Union (EU) on genetically modified organisms (GMOs) treats cisgenic plants the same as transgenic plants and both are mandatorily labeled as GMOs. This study estimates European consumers' willingness-to-pay (WTP) for rice labeled as GM, cisgenic, with environmental benefits (which cisgenesis could provide), or any combination of these three attributes. Data were collected from 3,002 participants through an online survey administered in Belgium, France, the Netherlands, Spain and the United Kingdom in 2013. Censored regression models were used to model consumers' WTP in each country. Model estimates highlight significant differences in WTP across countries. In all five countries, consumers are willing-to-pay a premium to avoid purchasing rice labeled as GM. In all countries except Spain, consumers have a significantly higher WTP to avoid consuming rice labeled as GM compared to rice labeled as cisgenic, suggesting that inserting genes from the plant's own gene pool is more acceptable to consumers. Additionally, French consumers are willing-to-pay a premium for rice labeled as having environmental benefits compared to conventional rice. These findings suggest that not all GMOs are the same in consumers' eyes and thus, from a consumer preference perspective, the differences between transgenic and cisgenic products are recommended to be reflected in GMO labeling and trade policies.


Plasminogen Activator Inhibitor-2 Plays a Leading Prognostic Role among Protease Families in Non-Small Cell Lung Cancer.
In lung cancer, uPA, its receptor (uPAR), and the inhibitors PAI-1 and PAI-2 of the plasminogen activator family interact with MMP-2 and MMP-9 of the MMP family to promote cancer progression. However, it remains undetermined which of these markers plays the most important role and may be the most useful indicator to stratify the patients by risk. We determined the individual prognostic value of these 6 markers by analyzing a derivation cohort with 98 non-small cell lung cancer patients by immunohistochemical staining. The correlation between the IHC expression levels of these markers and disease prognosis was investigated, and an immunohistochemical panel for prognostic prediction was subsequently generated through prognostic model analysis. The value of the immunohistochemical panel was then verified by a validation cohort with 91 lung cancer patients. In derivation cohort, PAI-2 is the most powerful prognostic factor (HR = 2.30; P = 0.001), followed by MMP-9 (HR = 2.09; P = 0.019) according to multivariate analysis. When combining PAI-2 and MMP-9, the most unfavorable prognostic group (low PAI-2 and high MMP-9 IHC expression levels) showed a 6.40-fold increased risk of a poor prognosis compared to the most favorable prognostic group (high PAI-2 and low MMP-9 IHC expression levels). PAI-2 and MMP-9 IHC panel could more precisely identify high risk patients in both derivation and validation cohort. We revealed PAI-2 as the most powerful prognostic marker among PA and MMP protease family even after considering their close relationships with each other. By utilizing a combination of PAI-2 and MMP-9, more precise prognostic information than merely using pathological stage alone can be obtained for lung cancer patients.


Promoter hypermethylation profile of kidney cancer.
Promoter hypermethylation is an important mechanism of inactivation of tumor suppressor genes in cancer cells. Kidney tumors are heterogeneous in their histology, genetics, and clinical behavior. To gain insight into the role of epigenetic silencing of tumor suppressor and cancer genes in kidney tumorigenesis, we determined a hypermethylation profile of kidney cancer. We examined the promoter methylation status of 10 biologically significant tumor suppressor and cancer genes in 100 kidney tumors (50 clear cell, 20 papillary, 6 chromophobe, 5 collecting duct, 5 renal cell unclassified, 7 oncocytoma, 6 transitional cell carcinomas of the renal pelvis, and 1 Wilms' tumor) by methylation-specific PCR. The hypermethylation profile was examined with regard to clinicopathological characteristics of the kidney cancer patients. Hypermethylation of one or more genes was found in 93 (93%) of 100 tumors. A total of 33% of kidney tumors had one gene, 35% two genes, 14% three genes, and 11% four or more genes hypermethylated. The frequency of hypermethylation of the 10 genes in the 100 tumor DNAs was VHL 8% (all clear cell), p16(INK4a) 10%, p14(ARF) 17%, APC 14%, MGMT 7%, GSTP1 12%, RARbeta2 12%, RASSF1A 45%, E-cadherin 11%, and Timp-3 58%. Hypermethylation was observed in all of the histological cell types and grades and stages examined. No hypermethylation was observed in specimens of normal kidney or ureteral tissue from 15 patients. Hypermethylation of VHL was specific to clear cell tumors. RASSF1A methylation was detected at a significantly higher frequency in papillary renal cell tumors and in high-grade tumors of all cell types. MGMT methylation was more frequent in nonsmokers. Simultaneous methylation of five or more genes was observed in 3 (3%) of 100 tumors and may indicate a methylator phenotype in kidney cancer. In addition, the CpG island in the promoter of the fumarate hydratase (FH) tumor suppressor gene was bisulfite sequenced and was found to be unmethylated in 15 papillary renal tumors. Promoter hypermethylation is common, can occur relatively early, may disrupt critical pathways, and, thus, likely plays an important role in kidney tumorigenesis. A hypermethylation profile may be useful in predicting a patient's clinical outcome and provide molecular markers for diagnostic and prognostic approaches to kidney cancer.


Platelet mitochondrial membrane depolarization reflects disease severity in patients with sepsis and correlates with clinical outcome.
Sepsis is still a leading cause of morbidity and mortality, even in modern times, and thrombocytopenia has been closely associated with unfavorable disease outcome. Decreases in mitochondrial membrane potential (depolarization) were found in different tissues during sepsis. Previous work suggests that mitochondrial dysfunction of platelets correlates with clinical disease activity in sepsis. However, platelet mitochondrial membrane potential (Mmp) has not been investigated in a clinical follow-up design and not with regard to disease outcome. In this study, platelet mitochondrial membrane depolarization was assessed by means of a fluorescent Mmp-Index with flow cytometry in 26 patients with sepsis compared with control patients. Platelet Mmp-Index on admission was correlated with the clinical disease scores Acute Physiology and Chronic Health Evaluation Score II (APACHE II), Sequential Organ Failure Score (SOFA), and Simplified Acute Physiology Score II (SAPS II). Finally, platelet Mmp-Index on admission and follow-up were compared in the group of sepsis survivors and nonsurvivors. Expression of the prosurvival protein Bcl-xL in platelets was quantified by immunoblotting. Platelet mitochondrial membrane depolarization correlated significantly with the simultaneously assessed clinical disease severity by APACHE II (r = -0.867; P < 0.0001), SOFA (r = -0.857; P <0.0001), and SAPS II score (r = -0.839; P < 0.0001). Patients with severe sepsis showed a significant reduction in platelet Mmp-Index compared with sepsis without organ failure (0.18 (0.12 to 0.25) versus 0.79 (0.49 to 0.85), P < 0.0006) or with the control group (0.18 (0.12 to 0.25) versus 0.89 (0.68 to 1.00), P < 0.0001). Platelet Mmp-Index remained persistently low in sepsis nonsurvivors (0.269 (0.230 to 0.305)), whereas we observed recovery of platelet Mmp-Index in the survivor group (0.9 (0.713 to 1.017)). Furthermore, the level of prosurvival protein Bcl-xL decreased in platelets during severe sepsis. In this study, we demonstrated that mitochondrial membrane depolarization in platelets correlates with clinical disease severity in patients with sepsis during the disease course and may be a valuable adjunct parameter to aid in the assessment of disease severity, risk stratification, and clinical outcome.


Probiotic Lactobacillus rhamnosus GG mono-association suppresses human rotavirus-induced autophagy in the gnotobiotic piglet intestine.
Human rotavirus (HRV) is the most important cause of severe diarrhea in infants and young children. Probiotic Lactobacillus rhamnosus GG (LGG) reduces rotavirus infection and diarrhea. However, the molecular mechanisms of LGG-mediated protection from rotavirus infection are poorly understood. Autophagy plays an essential role in responses to microbial pathogens. However, the role of autophagy in HRV infection and LGG treatment is unknown. We hypothesize that rotavirus gastroenteritis activates autophagy and that LGG suppresses virus-induced autophagy and prevents intestinal damage in infected piglets. We used LGG feeding to combat viral gastroenteritis in the gnotobiotic pig model of virulent HRV infection. We found that LGG feeding did not increase autophagy, whereas virus infection induced autophagy in the piglet intestine. Virus infection increased the protein levels of the autophagy markers ATG16L1 and Beclin-1 and the autophagy regulator mTOR. LGG treatment during viral gastroenteritis reduced autophagy marker expression to normal levels, induced apoptosis and partially prevented virus-induced tissue damage. Our study provides new insights into virus-induced autophagy and LGG suppression of uncontrolled autophagy and intestinal injury. A better understanding of the antiviral activity of LGG will lead to novel therapeutic strategies for infant infectious diseases.


Recent advances in controlling the internal and external properties of self-assembling cyclic peptide nanotubes and dimers.
One of the most powerful strategies for the preparation of nanotubes is based on the stacking of flat-shaped cyclic peptide components. This strategy allows precise control of the nanotube internal diameter, the external properties and, more recently, the structural characterisitics of the internal cavity. The recent advances in these technologies and the applications of the resulting materials are described.


Teleconsultation program for deployed soldiers and healthcare professionals in remote and austere environments.
In April 2004, the US Army Medical Department approved the use of the Army Knowledge Online (AKO) electronic e-mail system as a teleconsultation service for remote teledermatology consultations from healthcare providers in Iraq, Kuwait, and Afghanistan to medical subspecialists in the United States. The success of the system has resulted in expansion of the telemedicine program to include 11 additional clinical specialty services: (1) burn-trauma; (2) cardiology; (3) dermatology; (4) infectious disease; (5) nephrology; (6) ophthalmology; (7) pediatric intensive care; (8) preventive and occupational medicine; (9) neurology; (10) rheumatology; and (11) toxicology. The goal of the program is to provide a mechanism for enhanced diagnosis of remote cases resulting in a better evacuation system (i.e., only evacuation of appropriate cases). The service provides a standard practice for managing acute and emergent care requests between remote medical providers in austere environments and rear-based specialists in a timely and consistent manner. Consults are generated using the AKO e-mail system routed through a contact group composed of volunteer, on-call consults. The project manager receives and monitors all teleconsultations to ensure Health Insurance Portability and Accountability Act compliance and consultant's recommendations are transmitted within a 24-hour mandated time period. A subspecialty "clinical champion" is responsible for recruiting consultants to answer teleconsultations and developing a call schedule for each specialty. Subspecialties may have individual consultants on call for specific days (e.g., dermatology and toxicology) or place entire groups on-call for a designated period of time (e.g., ophthalmology). As of May 2007, 2,337 consults were performed during 36 months, with an average reply time of five hours from receipt of the teleconsultation until a recommendation was sent to the referring physician. Most consultations have been for dermatology (66%), followed by infectious disease (10%). A total of 51 known evacuations were prevented from use of the program, while 63 known evacuations have resulted following receipt of the consultants' recommendation. A total of 313 teleconsultations also have been performed for non-US patients, The teleconsultation program has proven to be a valuable resource for physicians deployed in austere and remote locations. Furthermore, use of such a system for physicians in austere environments may prevent unnecessary evacuations or result in appropriate evacuations for patients who initially may have been "underdiagnosed."


Validation, replication, and sensitivity testing of Heckman-type selection models to adjust estimates of HIV prevalence.
A recent study using Heckman-type selection models to adjust for non-response in the Zambia 2007 Demographic and Health Survey (DHS) found a large correction in HIV prevalence for males. We aim to validate this finding, replicate the adjustment approach in other DHSs, apply the adjustment approach in an external empirical context, and assess the robustness of the technique to different adjustment approaches. We used 6 DHSs, and an HIV prevalence study from rural South Africa to validate and replicate the adjustment approach. We also developed an alternative, systematic model of selection processes and applied it to all surveys. We decomposed corrections from both approaches into rate change and age-structure change components. We are able to reproduce the adjustment approach for the 2007 Zambia DHS and derive results comparable with the original findings. We are able to replicate applying the approach in several other DHSs. The approach also yields reasonable adjustments for a survey in rural South Africa. The technique is relatively robust to how the adjustment approach is specified. The Heckman selection model is a useful tool for assessing the possibility and extent of selection bias in HIV prevalence estimates from sample surveys.


Amphiphilic diblock copolymer and polycaprolactone blends to produce new vesicular nanocarriers.
New Melatonin-loaded vesicular nanocarriers were prepared by interfacial deposition using a blend of an amphiphilic diblock copolymer, poly(methyl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate), PMMA-b-PDMAEMA, with poly(epsilon-caprolactone), PCL. Particle size and morphology of the nanocarriers was evaluated. Dynamic light scattering shows that the nanocarriers have hydrodynamic radii between 100 and 180 nm, with unimodal particle size distribution for each formulation. Shape and structure were visualized by transmission electron microscopy (TEM), cryogenic TEM and scanning electron microscopy. Standard TEM for nanocapsules showed an oily core surrounded by a thin layer composed by PCL/PMMA-b-PDMAEMA. Cryo-TEM also indicated the presence of spherical nano-objects with a diffuse polymer corona. Encapsulation efficiencies were determined assaying the nanoparticles by HPLC and higher values of ca. 25% are shown by the nanocapsules. We could successfully incorporate platinum nanoparticles into the nanocarrier as evidenced by TEM, which opens up the possibility for promising applications like monitoring the encapsulated drug in the body.


Epigenetic modulation of β cells by interferon-α via PNPT1/mir-26a/TET2 triggers autoimmune diabetes.
Type 1 diabetes (T1D) is caused by autoimmune destruction of pancreatic β cells. Mounting evidence supports a central role for β cell alterations in triggering the activation of self-reactive T cells in T1D. However, the early deleterious events that occur in β cells, underpinning islet autoimmunity, are not known. We hypothesized that epigenetic modifications induced in β cells by inflammatory mediators play a key role in initiating the autoimmune response. We analyzed DNA methylation (DNAm) patterns and gene expression in human islets exposed to IFN-α, a cytokine associated with T1D development. We found that IFN-α triggers DNA demethylation and increases expression of genes controlling inflammatory and immune pathways. We then demonstrated that DNA demethylation was caused by upregulation of the exoribonuclease, PNPase old-35 (PNPT1), which caused degradation of miR-26a. This in turn promoted the upregulation of ten-eleven translocation 2 (TET2) enzyme and increased 5-hydroxymethylcytosine levels in human islets and pancreatic β cells. Moreover, we showed that specific IFN-α expression in the β cells of IFNα-INS1CreERT2 transgenic mice led to development of T1D that was preceded by increased islet DNA hydroxymethylation through a PNPT1/TET2-dependent mechanism. Our results suggest a new mechanism through which IFN-α regulates DNAm in β cells, leading to changes in expression of genes in inflammatory and immune pathways that can initiate islet autoimmunity in T1D.


Locus-specific contig assembly in highly-duplicated genomes, using the BAC-RF method.
Polyploidy, the presence of multiple sets of chromosomes that are similar but not identical, complicates both chromosome walking and assembly of sequence-ready contigs for many plant taxa including a large number of economically-significant crops. Traditional 'dot-blot hybridization' or PCR-based assays for identifying BAC clones corresponding to a mapped DNA landmark usually do not provide sufficient information to distinguish between allelic and non-allelic loci. A restriction fragment matching method using pools of BAC DNA in combination with dot-blots reveals the locus specificity of individual BACs that correspond to multi-locus DNA probes, in a manner that can efficiently be applied on a large scale. This approach also provides an alternative means of mapping DNA loci that exploits many advantages of 'radiation hybrid' mapping in taxa for which such hybrids are not available. The BAC-RF method is a practical and reliable approach for using high-density RFLP maps to anchor sequence-ready BAC contigs in highly-duplicated genomes, provides an alternative to high-density robotic gridding for screening BAC libraries when the necessary equipment is not available, and permits the expedient isolation of individual members of multigene or repetitive DNA families for a wide range of genetic and evolutionary investigations.


Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas.
Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.


Requirement of glycosylation of West Nile virus envelope protein for infection of, but not spread within, Culex quinquefasciatus mosquito vectors.
Most of sequenced West Nile virus (WNV) genomes encode a single N-linked glycosylation site on their envelope (E) proteins. We previously found that WNV lacking the E protein glycan was severely inhibited in its ability to replicate and spread within two important mosquito vector species, Culex pipiens and Cx. tarsalis. However, recent work with a closely related species, Cx. pipiens pallens, found no association between E protein glycosylation and either replication or dissemination. To examine this finding further, we expanded upon our previous studies to include an additional Culex species, Cx. quinquefasciatus. The non-glycosylated WNV-N154I virus replicated less efficiently in mosquito tissues after intrathoracic inoculation, but there was little difference in replication efficiency in the midgut after peroral infection. Interestingly, although infectivity was inhibited when WNV lacked the E protein glycan, there was little difference in viral spread throughout the mosquito. These data indicate that E protein glycosylation affects WNV-vector interactions in a species-specific manner.


The desert ant odometer: a stride integrator that accounts for stride length and walking speed.
Desert ants, Cataglyphis, use path integration as a major means of navigation. Path integration requires measurement of two parameters, namely, direction and distance of travel. Directional information is provided by a celestial compass, whereas distance measurement is accomplished by a stride integrator, or pedometer. Here we examine the recently demonstrated pedometer function in more detail. By manipulating leg lengths in foraging desert ants we could also change their stride lengths. Ants with elongated legs ('stilts') or shortened legs ('stumps') take larger or shorter strides, respectively, and misgauge travel distance. Travel distance is overestimated by experimental animals walking on stilts, and underestimated by animals walking on stumps - strongly indicative of stride integrator function in distance measurement. High-speed video analysis was used to examine the actual changes in stride length, stride frequency and walking speed caused by the manipulations of leg length. Unexpectedly, quantitative characteristics of walking behaviour remained almost unaffected by imposed changes in leg length, demonstrating remarkable robustness of leg coordination and walking performance. These data further allowed normalisation of homing distances displayed by manipulated animals with regard to scaling and speed effects. The predicted changes in homing distance are in quantitative agreement with the experimental data, further supporting the pedometer hypothesis.


Stiffening thermal membranes by cutting.
Two-dimensional crystalline membranes have recently been realized experimentally in systems such as graphene and molybdenum disulfide, sparking a resurgence in interest in their statistical properties. Thermal fluctuations can significantly affect the effective mechanical properties of properly thermalized membranes, renormalizing both bending rigidity and elastic moduli so that in particular they become stiffer to bending than their bare bending rigidity would suggest. We use molecular dynamics simulations to examine how the mechanical behavior of thermalized two-dimensional clamped ribbons (cantilevers) depends on their precise topology and geometry. We find that a simple slit smooths roughness as measured by the variance of height fluctuations. This counterintuitive effect may be due to the counterposed coupling of the lips of the slit to twist in the intact regions of the ribbon.


Youth empowerment and high school Gay-Straight Alliances.
In the field of positive youth development programs, "empowerment" is used interchangeably with youth activism, leadership, civic participation and self-efficacy. However, few studies have captured what empowerment means to young people in diverse contexts. This article explores how youth define and experience empowerment in youth-led organizations characterized by social justice goals: high school Gay-Straight Alliances (GSAs). Through focus group interviews, fifteen youth leaders of GSAs from different regions of California explain what they think empowerment means and how they became empowered through their involvement with the GSA. Youth describe three inter-related dimensions of empowerment: personal empowerment, relational empowerment, and strategic empowerment through having and using knowledge. When these three dimensions are experienced in combination, GSA leaders have the potential for individual and collective empowerment as agents of social change at school. By understanding these youth's perspectives on the meanings of empowerment, this article clarifies the conceptual arena for future studies of socially marginalized youth and of positive youth development.


Uroplakins Ia and Ib, two major differentiation products of bladder epithelium, belong to a family of four transmembrane domain (4TM) proteins.
The mammalian bladder epithelium elaborates, as a terminal differentiation product, a specialized plasma membrane called asymmetric unit membrane (AUM) which is believed to play a role in strengthening and stabilizing the urothelial apical surface through its interactions with an underlying cytoskeleton. Previous studies indicate that the outer leaflet of AUM is composed of crystalline patches of 12-nm protein particles, and that bovine AUMs contain three major proteins: the 27- to 28-kD uroplakin I, the 15-kD uroplakin II and the 47-kD uroplakin III. As a step towards elucidating the AUM structure and function, we have cloned the cDNAs of bovine uroplakin I (UPI). Our results established the existence of two isoforms of bovine uroplakin I: a 27-kD uroplakin Ia and a 28-kD uroplakin Ib. These two glycoproteins are closely related with 39% identity in their amino acid sequences. Hydropathy plot revealed that both have four potential transmembrane domains (TMDs) with connecting loops of similar length. Proteolytic digestion of UPIa inserted in vitro into microsomal vesicles suggested that its two main hydrophilic loops are exposed to the luminal space, possibly involved in interacting with the luminal domains of other uroplakins to form the 12-nm protein particles. The larger loop connecting TMD3 and TMD4 of both UPIa and UPIb contains six highly conserved cysteine residues; at least one centrally located cysteine doublet in UPIa is involved in forming intramolecular disulfide bridges. The sequences of UPIa and UPIb (the latter is almost identical to a hypothetical, TGF beta-inducible, TI-1 protein of mink lung epithelial cells) are homologous to members of a recently described family all possessing four transmembrane domains (the "4TM family"); members of this family include many important leukocyte differentiation markers such as CD9, CD37, CD53, and CD63. The tissue-specific and differentiation-dependent expression as well as the naturally occurring crystalline state of uroplakin I molecules make them uniquely suitable, as prototype members of the 4TM family, for studying the structure and function of these integral membrane proteins.


A proof of concept infant-microbiota associated rat model for studying the role of gut microbiota and alleviation potential of Cutibacterium avidum in infant colic.
Establishing the relationship between gut microbiota and host health has become a main target of research in the last decade. Human gut microbiota-associated animal models represent one alternative to human research, allowing for intervention studies to investigate causality. Recent cohort and in vitro studies proposed an altered gut microbiota and lactate metabolism with excessive H2 production as the main causes of infant colic. To evaluate H2 production by infant gut microbiota and to test modulation of gut colonizer lactose- and lactate-utilizer non-H2-producer, Cutibacterium avidum P279, we established and validated a gnotobiotic model using young germ-free rats inoculated with fecal slurries from infants younger than 3 months. Here, we show that infant microbiota-associated (IMA) rats inoculated with fresh feces from healthy (n = 2) and colic infants (n = 2) and fed infant formula acquired and maintained similar quantitative and qualitative fecal microbiota composition compared to the individual donor's profile. We observed that IMA rats excreted high levels of H2, which were linked to a high abundance of lactate-utilizer H2-producer Veillonella. Supplementation of C. avidum P279 to colic IMA rats reduced H2 levels compared to animals receiving a placebo. Taken together, we report high H2 production by infant gut microbiota, which might be a contributing factor for infant colic, and suggest the potential of C. avidum P279 in reducing the abdominal H2 production, bloating, and pain associated with excessive crying in colic infants.


Country, regional, and global estimates for lactose malabsorption in adults: a systematic review and meta-analysis.
Studies have shown wide variation in the prevalence of lactose malabsorption across the world, but no systematic reviews or meta-analyses have recently assessed the prevalence of lactose malabsorption in different geographical areas. We aimed to present an updated systematic review and meta-analysis on the prevalence of lactose malabsorption in adults, by countries and regions, and to assess the variation between different testing methods. Studies reporting on prevalence of lactose malabsorption and lactase persistence were identified by searching MEDLINE and Embase from database inception to Nov 2, 2016. We evaluated studies presenting lactose malabsorption or lactase persistence prevalence data in adults and children aged 10 years or older, including cross-sectional and prospective studies, using genotyping, hydrogen breath tests, lactose tolerance tests, and other testing methods. We excluded studies in children younger than 10 years, studies using self-reported data, and studies including inpatients and outpatients at gastroenterological wards. Studies were screened by two authors (CLS and SKF) and data values were extracted by two authors (CLS and SKF) independently. The primary outcome was the prevalence of lactose malabsorption. This study is registered with PROSPERO, number CRD42017064802. We screened 2665 records, and 306 study populations from 116 full-text articles were included (primary sources); data for 144 additional study populations from 59 articles were obtained from review articles, because full-text primary articles could not be obtained (secondary sources). Of the 450 study populations included, 231 were assessed by genotyping, 83 by hydrogen breath tests, 101 by lactose tolerance tests, and 35 by other methods or methods that were not described sufficiently. The studies included 62 910 participants from 89 countries (covering 84% of the world's population). When standardising for country size, the global prevalence estimate of lactose malabsorption was 68% (95% CI 64-72), ranging from 28% (19-37) in western, southern, and northern Europe to 70% (57-83) in the Middle East. When assessing the global prevalence using genotyping data only, the estimate was 74% (69-80), whereas prevalence was 55% (46-65) using lactose tolerance test data, and 57% (46-67) using hydrogen breath test data. Risk of bias was assessed based on ten indicators; 12 of the articles had a score of ten, indicating low risk of bias, 76 had a score of nine, 26 a score of eight, and two articles a score of seven (indicating higher risk of bias). There was substantial heterogeneity between studies within most of the assessed countries. Lactose malabsorption is widespread in most of the world, with wide variation between different regions and an overall frequency of around two-thirds of the world's population. Acknowledging regional patterns of lactose malabsorption is important to guide management of gastrointestinal symptoms. None.


Epizootiologic survey of Mycobacterium bovis in wildlife and farm environments in northern Michigan.
Bovine tuberculosis (bovine TB), caused by Mycobacterium bovis, has reemerged in northern Michigan, USA, with detections in white-tailed deer (Odocoileus virginianus) in 1994 and in cattle in 1998. Since then, significant efforts have been directed toward reducing deer densities in the area in the hopes of reducing the bovine TB prevalence rate in deer and eliminating spillover of the disease into cattle. Despite the success of the efforts to reduce deer densities, additional cattle herds have become infected. Other mammals can be infected with M. bovis, and some carnivores and omnivores had been found to be infected with the disease in northern Michigan, USA. We conducted a multiyear surveillance effort to detect bovine TB in wild species of mammals in the Michigan, USA, outbreak area. From 2002 to 2004, tissue samples from 1,031 individual animals of 32 species were collected, processed, and cultured for M. bovis. Only 10 (1.0%) were culture-positive for M. bovis (five raccoons [Procyon lotor], four opossums [Didelphis virginiana], and one grey fox [Urocyon cinereoargenteus]). We also found two raccoons and four opossums to be positive for Mycobacterium avium. We collected 503 environmental samples from cattle farms recently identified as bovine TB positive; none yielded positive M. bovis culture results. Finally, we used infrared cameras to document wildlife use of four barns in the area. Many avian and mammalian species of wildlife were observed, with raccoons being the most commonly observed species. This surveillance study identified no new wildlife species that should be considered significant reservoirs of bovine TB in the outbreak area in northern Michigan, USA. However, the relatively high, apparent bovine TB prevalence rates in some carnivorous and omnivorous species, their relatively long life spans, and their frequent use of barns, suggests that removal of raccoons, opossums, foxes, and coyotes (Canis latrans) should be considered when a newly infected farm is depopulated of cattle.


Three-dimensional matrix stiffness and adhesive ligands affect cancer cell response to toxins.
There is an immediate need to develop highly predictive in vitro cell-based assays that provide reliable information on cancer drug efficacy and toxicity. Development of biomaterial-based three-dimensional (3D) cell culture models as drug screening platforms has recently gained much scientific interest as 3D cultures of cancer cells have been shown to more adequately mimic the in vivo tumor conditions. Moreover, it has been recognized that the biophysical and biochemical properties of the 3D microenvironment can play key roles in regulating various cancer cell fates, including their response to chemicals. In this study, we employed alginate-based scaffolds of varying mechanical stiffness and adhesive ligand presentation to further explore the role of 3D microenvironmental cues on glioblastoma cell response to cytotoxic compounds. Our experiments suggested the ability of both matrix stiffness and cell-matrix adhesions to strongly influence cell responses to toxins. Cells were found to be more susceptible to the toxins when cultured in softer matrices that emulated the stiffness of brain tissue. Furthermore, the effect of matrix stiffness on differential cell responses to toxins was negated by the presence of the adhesive ligand RGD, but regained when integrin-based cell-matrix interactions were inhibited. This study therefore indicates that both 3D matrix stiffness and cell-matrix adhesions are important parameters in the design of more predictive in vitro platforms for drug development and toxicity screening.


Investigation of the chromosome regions with significant affinity for the nuclear envelope in fruit fly--a model based approach.
Three dimensional nuclear architecture is important for genome function, but is still poorly understood. In particular, little is known about the role of the "boundary conditions"--points of attachment between chromosomes and the nuclear envelope. We describe a method for modeling the 3D organization of the interphase nucleus, and its application to analysis of chromosome-nuclear envelope (Chr-NE) attachments of polytene (giant) chromosomes in Drosophila melanogaster salivary glands. The model represents chromosomes as self-avoiding polymer chains confined within the nucleus; parameters of the model are taken directly from experiment, no fitting parameters are introduced. Methods are developed to objectively quantify chromosome territories and intertwining, which are discussed in the context of corresponding experimental observations. In particular, a mathematically rigorous definition of a territory based on convex hull is proposed. The self-avoiding polymer model is used to re-analyze previous experimental data; the analysis suggests 33 additional Chr-NE attachments in addition to the 15 already explored Chr-NE attachments. Most of these new Chr-NE attachments correspond to intercalary heterochromatin--gene poor, dark staining, late replicating regions of the genome; however, three correspond to euchromatin--gene rich, light staining, early replicating regions of the genome. The analysis also suggests 5 regions of anti-contact, characterized by aversion for the NE, only two of these correspond to euchromatin. This composition of chromatin suggests that heterochromatin may not be necessary or sufficient for the formation of a Chr-NE attachment. To the extent that the proposed model represents reality, the confinement of the polytene chromosomes in a spherical nucleus alone does not favor the positioning of specific chromosome regions at the NE as seen in experiment; consequently, the 15 experimentally known Chr-NE attachment positions do not appear to arise due to non-specific (entropic) forces. Robustness of the key conclusions to model assumptions is thoroughly checked.