Vibrio spp. Using historical environmental data, combined with three different statistical models of Vibrio vulnificus probability, we explore the relationship between environmental change and predicted Vibrio vulnificus presence in the upper Chesapeake Bay. We find that the predicted response of V. vulnificus probability to high temperatures in the Bay differs systematically between models of differing structure. As existing publicly available datasets are inadequate to determine which model structure is most appropriate, the impact of climatic change on the probability of V. vulnificus presence in the Chesapeake Bay remains uncertain. This result points to the challenge of characterizing climate sensitivity of ecological systems in which data are sparse and only statistical models of ecological sensitivity exist.The effect that climate change and variability will have on waterborne bacteria is a topic of increasing concern for coastal ecosystems, including the Chesapeake Bay. Surface water temperature trends in the Bay indicate a warming pattern of roughly 0.3–0.4°C per decade over the past 30 years. It is unclear what impact future warming will have on pathogens currently found in the Bay, including  Vibrio spp. bacteria are a threat in many coastal aquatic ecosystems around the world Vibrio cases of infection has nearly doubled in the past decade Vibrio spp. is frequently detected in shellfish harvested for human consumption during the warm summer months Vibrio disease caused by Vibrio spp. bacteria in many coastal regions Vibrio spp. in the Bay varies spatially and seasonally, and researchers have modeled these probability patterns as a statistical function of surface water temperature and salinity Vibrio models have demonstrated skill for available datasets in the Bay and structurally similar statistical models have been applied to predictions of V. cholerae, V. vulnificus, and V. parahaemolyticus in other regions V. vulnificus can vary by region, but in general the bacteria are found in waters with salinity between 5 and 25  and temperature above 15°C V. vulnificus growth: the onset of spring time temperatures (>15°C) has advanced by nearly three weeks V. vulnificus growth season. The consensus of climate models is that there will be a rise in winter and spring precipitation in the northern portion of the watershed Recent studies show that surface water temperatures in the Chesapeake Bay have warmed by 0.3–0.4°C per decade over the past 30 years V. vulnificus might respond to observed and projected trends in these environmental parameters. Here we examine three statistical models of V. vulnificus probability of presence that demonstrate skill in predicting V. vulnificus probability of presence in Chesapeake Bay. All three models use water surface temperature and salinity as the only predictors, but they differ in their structure and/or in the data used for training and evaluation. One model is the generalized linear model (GLM) of Jacobs et al.   =  β|SalnOpt|, where 0β is the intercept, nβ is the regression coefficient for the independent covariates, Temp is daily surface temperature, and |SalnOpt| is the absolute distance from optimal salinity of 11.5], which was trained using 235 V. vulnificus samples collected during the months of July and October of 2007, and April, July, and October of 2008 and were analyzed by the NOAA Chesapeake Bay Office.NOAA_GLM: The generalized linear model (GLM) of Jacobs et al. 2010; : [z(V.v)0 + β1Temp + β3z(V.v)  =  β|SalnOpt|] trained using 148 V. vulnificus, surface temperature, and surface salinity samples collected in the upper Chesapeake Bay during the months of July and September of 2011 and March through June of 2012 + s2z(V.v)  =  β, where i(xi)s is a parameter of the smoothing function, and Saln is the salinity value].JHU_GAM: A generalized additive model GAM;  trained V. vulnificus presence was calculated using p = ze/(1+ze). Diagnostics for each model were performed using Akaike's Information Criterion (AIC) and accuracy (ACC) in an out-of-bag (OOB) cross validation We included a GAM in addition to the two structurally identical GLMs because GAM models allow a more flexible regression modeling of the transformed response that combine the predictor variables in a nonparametric manner V. vulnificus models to temperature and salinity, we used a range of surface water temperature (0–40°C) and surface salinity (0–13) values as independent model input. Here the range of model input extends past the range of the in situ temperature (8–31°C) observations, and is constrained to the range of the surface salinity (0–14) observations that were used in the training of the two JHU models. Additionally, historical temperature and salinity data were tested as model input, enabling identification of V. vulnificus climatology and seasonal trends. To further assess the geographic distribution of the predicted V. vulnificus probability for each method, geospatially-interpolated satellite-derived surface temperature and surface salinity To explore sensitivity of the All statistical computations were carried out in the R Statistical Environment version 2.14, using the ‘mgcv’ and ‘stats’ packages, on an Intel Xeon W3580 Processor, 3.33 GHz machine with 12 GB RAM. Computation time for all statistical models was less than one minute.V. vulnificus. To determine the prediction skill of each model, ACC was calculated using the JHU validation dataset . The AIC and ACC values indicated that the JHU models performed significantly better than a null model that only included seasonality as a predictor.For model evaluation, goodness of fit and predictive skill for the JHU models were determined using AIC and ACC indices. AIC results indicated that the JHU GAM (145.9) offered better model fit than the JHU GLM (160.4), but performance differences between models were small relative to measurement uncertainty. NOAA GLM model fit using the NOAA training dataset yielded an AIC of 164.3 V. vulnificus probability for each of the tested models, with likelihood levels plotted as contour curves. NOAA GLM . Similarly, JHU GLM Click here for additional data file.


For these 2D materials, atoms within their monolayer are covalently bonded. An interesting question arises: Can molecules form a 2D monolayer crystal via van der Waals interactions? Here, we first study the structural stability of a free-standing infinite C60 molecular monolayer using molecular dynamic simulations, and find that the monolayer is stable up to 600 K. We further study the mechanical properties of the monolayer, and find that the elastic modulus, ultimate tensile stress and failure strain are 55–100 GPa, 90–155 MPa, and 1.5–2.3%, respectively, depending on the stretching orientation. The monolayer fails due to shearing and cavitation under uniaxial tensile loading. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the monolayer are found to be delocalized and as a result, the band gap is reduced to only 60% of the isolated C60 molecule. Interestingly, this band gap can be tuned up to ±30% using strain engineering. Owing to its thermal stability, low density, strain-tunable semi-conducting characteristics and large bending flexibility, this van der Waals molecular monolayer crystal presents aplenty opportunities for developing novel applications in nanoelectronics.Two-dimensional (2D) atomic crystals, such as graphene and transition metal dichalcogenides  Two-dimensional 2D) atomic materials, such as graphene, hexagonal boron nitride, silicene, phosphorene, transition metal dichalcogenides and layered oxides, have drawn considerable attention recently due to their unique crystalline structures and fascinating properties1D atomic 345On the other hand, in molecular solids, molecules are also held together in bulk form by weak vdW forces791011121314910111260 molecules have been used as a basic building block in many recent applications, for example, in organic photovoltaics1415181920212260 molecules have been self-assembled into a stable monolayer thin film on substrates,1160 molecules form a stable free-standing 2D molecular monolayer crystal? If the answer is yes, then, up to what temperature? What are the mechanical and failure behaviour of the monolayer? What are its electronic characteristics? Clearly, answers to these questions are not only of scientific significance but also of technological impact. Thus, studying the stability of the C60 molecular monolayer and revealing their mechanical and electronic properties constitute the subject of the present study.Recently, 2D organic molecular crystals have been synthesized and several interesting applications in novel devices have been demonstrated1415161718192060 solid, the C60 molecules adhere together by weak vdW forces and exhibit the face-centered cubic (FCC) structure at room temperature1360 molecules in the hexagonal structure was reported to be in the range of 9.5 to 10 Å26In the crystalline C60 molecular monolayer crystal. All C60 molecules are arranged in a perfect hexagonal pattern as shown in 60 molecule performs random thermal fluctuations in all the degrees of freedom, including both translational and rotational ones. After thermal equilibration and subsequent energy minimization, the C60 molecules adopt a hexagonal lattice structure, but their orientations are randomly distributed  or 5 (for Z2 molecule), depending on the location of the molecule on the edge. The coordination number is 4 and 6 for a C60 at the straight edge (S) and in the centre (C), respectively . At room temperature, we find that the nanotubes with a diameter greater than 90 Å (the curvature below 0.022 Å−1) are stable. For nanotubes with a curvature greater than 0.022 Å−1, we find that thermal vibrations of C60 molecules destabilize the nanotubes and transform the C60 nanotubes into C60 nanowires at room temperature. We have added the C60 nanotube and nanowire structures in the In order to demonstrate that the 2D C60 monolayer on graphene/SiC substrateTo investigate the elastic properties of the molecular monolayer, we apply uniaxial tensile strain and measure the tensile stress of the system. 60 monolayer, it is found that the HOMO and LUMO cover the carbon-carbon bonds, which may act as an electron transport pathway and result in high electron mobility. For comparison, we also investigate the HOMO and LUMO of an isolated C60 fullerene. It is found that the HOMO and LUMO of the isolated C60 fullerene are located between the carbon-carbon bonds and are highly localized. The calculated band gap of the C60 monolayer and isolated C60 molecule are 0.66 eV and 1.63 eV, respectively. These results are consistent with the understanding that localized HOMO and LUMO may result in a large band gap, while delocalized HOMO and LUMO may result in a small band gap. In addition, the calculated band gap of the isolated C60 molecule is consistent with the previously reported value of 1.7–1.9 eVBased on the optimized lattice constants and atomic coordinates obtained by using molecule dynamics calculations, we build a super cell of the 2D monolayer with an inter molecular distance of 9.50 Å as shown in 60 monolayer by applying biaxial strain in the range from −2% to 2%. The negative values represent the compressive strain and the positive values represent the tensile strain. We further explore the strain engineering on the band gap of the C60 molecular monolayer. Due to its thermal stability, low density, strain-tunable semiconducting characteristics, moderate elastic modulus and high bending flexibility, it is envisioned that this monolayer material can be used in many novel applications. For example, this monolayer may be used for storage devices for a wide range of molecules in bio- and energy applications201115In the present study, we have computationally shown the existence of stable CIn summary, we have computationally investigated the structure, mechanical and electronic properties of the free-standing van der Waals monolayer crystal using C60 molecules. Using molecular dynamics simulations, we have shown that the monolayer infinite structure is stable up to 600 K; the thermal expansion coefficient is about 8 × 10−5 K−1; and the stress-strain curve of the monolayer follows an approximately bilinear behaviour. Due to the molecular anisotropic arrangement, the failure strain is orientation-dependent with 1.5 and 2.3% being in the zigzag and straight directions, respectively. Density functional simulations were used to calculate the HOMO and LUMO of the C60 monolayer and an isolated C60 molecule. It is found that the monolayer exhibits a band gap of 0.6 eV, which is much lower than that of isolated C60 molecular band gap of 1.6 eV. The band gap can be tuned by strain engineering from 0.4 to 0.8 eV (±30%). This molecular monolayer is expected to find aplenty opportunities in the future electronic, biomedical and energy storage applications.60 monolayer crystal, C60 molecules are arranged in a hexagonal network as shown in 3739−5 eV for the total energy. To prevent spurious interaction between isolated C60, a large vacuum spacing (at least 15 Å) is introduced. By using the conjugate gradient method, we optimize the atomic positions and lattice constants until the atomic forces are less than 0.01 eV/Å.In order to study the structure and stability of the CHow to cite this article: Reddy, C.D. et al. Two-dimensional van der Waals C60 molecular crystal. Sci. Rep.5, 12221; doi: 10.1038/srep12221 (2015).


Everyday, there are several millions of people that are increasingly unable to combat their frustrating and even fatal romance with getting high and/or experiencing “normal” feelings of well-being. In the USA, the FDA has approved pharmaceuticals for drug and alcohol abuse: tobacco and nicotine replacement therapy. The National Institute on Drug Abuse (NIDA) and the National Institute on Alcohol Abuse and Alcoholism (NIAAA) remarkably continue to provide an increasing understanding of the intricate functions of brain reward circuitry through sophisticated neuroimaging and molecular genetic applied technology. Similar work is intensely investigated on a worldwide basis with enhanced clarity and increased interaction between not only individual scientists but across many disciplines. However, while it is universally agreed that dopamine is a major neurotransmitter in terms of reward dependence, there remains controversy regarding how to modulate its role clinically to treat and prevent relapse for both substance and non-substance-related addictive behaviors. While the existing FDA-approved medications promote blocking dopamine, we argue that a more prudent paradigm shift should be biphasic—short-term blockade and long-term upregulation, enhancing functional connectivity of brain reward circuits. Mille viae ducunt homines per saecula Romam (A thousand roads lead men forever to Rome) in Liber Parabolarum, 591 (1175), by Alain de Lille.Scientific explorations from around the globe agree that substance and non-substance-seeking behaviors are considered an endemic societal problem affecting multimillions. Certainly, we have come a long way since Bill Wilson and Dr. Bob Smith began their crusade in 1933 embracing the “Cambridge” theories and doctrines, resulting in the most powerful anti-alcohol/drug program and fellowship in the world—Alcoholics/Narcotic Anonymous. Many years after Jelnick’s famous article in 1956 providing the medical profession with the “concept of alcoholism as a disease,” the American Society of Addiction Medicine (ASAM) espoused a new definition of addiction indicating that “Addiction is a primary, chronic disease involving brain reward, motivation, memory and related circuitry”; it can lead to relapse, progressive development, and the potential for fatality if not treated. While pathological use of alcohol and, more recently, psychoactive substances has been accepted as an addictive disease, developing brain science has set the stage for inclusion of the process addictions, including food, sex, shopping, and gambling problems, in a broader definition of addiction as set forth by the American Society of Addiction Medicine in 2011 [dopamine agonistic therapy for addiction—Cochrane Systematic Reviews 0, DARE 0, PubMed Central Clinical Queries 9, National Guideline Clearinghouse 0, PsychINFO 0, ACP PIER 83, PsychSage 15, PubMed/MEDLINE 501; dopamine agonist for addiction—Cochrane Systematic Reviews 3, DARE 3, PubMed Central Clinical Queries 10, National Guideline Clearinghouse 0, ACP PIER 0, PsychSage 15, PubMed/MEDLINE 13; dopamine agonistic therapy for reward dependence—Cochrane Systematic Reviews 0, DARE 0, PubMed Central Clinical Queries 1, National Guideline Clearinghouse 0, PsychINFO 0, ACP PIER 0, PsychSage 0, PubMed/MEDLINE 62; dopamine agonist for reward dependence—Cochrane Systematic Reviews 0, DARE 0, PubMed Central Clinical Queries 337, National Guideline Clearinghouse 0, PsychINFO 1, ACP PIER 0, PsychSage 0, PubMed/MEDLINE 120  [denervation supersensitivity. This concept is in agreement with the work of Dahlgren et al. [Taq A1 allele of the dopamine D2 receptor gene compared to Taq A2 allele carriers significantly relapse at a higher rate, suggesting a hypodopaminergic trait. Unfortunately, Taq A1 allele alcoholics have a significant enhanced mortality rate [dopamine depletion hypothesis espoused by Dackis and Gold [Taq A2 allele have a reduced risk for all RDS behaviors including glucose craving and as such is protective [We are cognizant that risk for relapse has now been linked to hypodopaminergic genetics. Dani et al.  suggestey (DART) . We propn et al.  who repoity rate  as well ity rate . Historiand Gold  to explaotective , 28.Over the last decade, a number of scientists pioneered the controversial concept that food addiction and drug addiction constitute common neurobiological mechanisms , 30. AveHomo sapiens reward circuitry of the brain. Based on numerous experiments in the scientific literature, well over 20,000 entries, the major reward neurotransmitter pathway (road) indeed is DA [In earlier days, it was true that all roads did lead to Rome. This simple truth is not too dissimilar when we consider the ed is DA .The role of dopamine and mind function has been fraught with controversy but arguably very interesting and mind expanding. There are many unanswered questions related to what makes us human and what drives our unique behaviors. While many brain theories have focused on the role of brain size and genetic adaptations, Fred Previc, a scientist living in San Antonio, TX, explored the provocative and very speculative concept of a “dopaminergic society” .dopaminergic mind hypothesis seeks to explain the differences between modern humans and their hominid relatives by focusing on changes in dopamine. It theorizes that increased levels of dopamine were part of a general physiological adaptation due to an increased consumption of meat around two million years ago in Homo habilis and later enhanced by changes in diet and other environmental and social factors beginning approximately 80,000 years ago. Under this theory, the “high-dopamine” personality is characterized by high intelligence, a sense of personal destiny, a religious/cosmic preoccupation, an obsession with achieving goals and conquests, an emotional detachment that in many cases leads to ruthlessness, and a risk-taking mentality. High levels of dopamine are proposed to underlie increased psychological disorders in industrialized societies. According to this hypothesis, a dopaminergic society is an extremely goal-oriented, fast-paced, and even manic society, “given that dopamine is known to increase activity levels, speed up our internal clocks, and create a preference for novel over unchanging environments.” In the same way that high-dopamine individuals lack empathy and exhibit a more masculine behavioral style, dopaminergic societies are “typified by more conquest, competition, and aggression than nurturance and communality.”According to Previc, the In our view, the lack of brain dopamine function (dopamine deficiency) either due to genetics and/or environmental elements (epigenetics) could lead to a “hypodopaminergic society” whereby the individual may have a reduced cognition and memory, poor executive function, lack of motivation or achieving goals, low spirituality, liberal non-partisan views, selfishness, high novelty seeking, substance and behavioral reward seeking, lack of attention span, violent behavior, compromised well-being and blunted ability to achieve pleasure states, erotic love styles (less romantic in nature), enhanced immature defense style (lying), lowered pain tolerance, higher percent body fat, less energy, and basically unhappy , support a dopaminergic expansion in humans , accordil-tyrosine found in meat is produced in several areas of the brain, including the “brain reward” (nucleus accumbens) site located in the reptilian old brain region called the mesolimbic system. Scientists now know that there are two major variant forms of the human gene, for example, the dopamine D2 receptor gene (DRD2) that regulates the synthesis of D2 receptors and the most widely studied gene that accounts for major aspects of modern human behavior [H. habilis increased their meat consumption, feeding the brain with the needed l-tyrosine to synthesize more dopamine required to overcome the D2 receptor deficit, a new society was born—the “high-dopamine society” carrying the DRD2 A2 form of this gene. The differences between carriers of the low-dopamine-expressing gene form A1 compared to the normal or higher expression of the dopamine D2 receptor gene A2 have been shown to be protective against ADHD, a subtype of RDS behavior [The possibility does exist that prehistoric ancestral species over two million years ago carried the low dopamine brain function due to low dopamine receptors. It should be noted that dopamine functions as a neurotransmitter activating the five known types of dopamine receptors—D1–D5—and their variants. Dopamine from behavior . Specifibehavior . It is pbehavior .Understanding that low dopamine function leads to impulsive, compulsive, and addictive behaviors paves the way to defining addiction as a brain disorder involving impairments in the so-called reward circuitry. This new definition of addiction has been now adopted by the American Society of Addiction Medicine (ASAM) founded by the San Franciscan visionary David E. Smith . This neWhile having any brain genetic deficit in the reward site may predispose an individual to a higher risk for RDS, it is always the combination of our genes and the interaction with environmental elements that predict not only addictive behaviors in general but specificity of the type of drug or behavior of choice. Using a special mathematical formulation (Bayesian—developed by a sixteenth-century monk) to predict the value of carrying for example the DRD2 A1 (low D2 receptors) at birth and subsequent lifetime, the risk of any RDS behavior has been determined to be as high as 74 % .Thus, our provocative hypodopaminergic RDS theory is highly impacted by what scientists like Steve Sussman of Southern California University call epigenetic factors  rather than just genetic factors and translation to preventive strategies  for schoThe FDA-approved medications for alcohol, drugs, and food are at best only moderately effective and in some cases counter to promoting “dopamine homeostasis” including narcotic antagonists and acamprosate, a NMDA antagonist, for example, utilized to reduce drug-induced euphoria via attenuated neuronal dopamine release at the NAc , 48. We Our laboratory extensively published on the concept of dopamine agonist therapy to treat iatrogenic opioids and even prevent RDS behaviors . A majorMoreover, Radke and Gewirtz  showed tIt is also known that cocaine reinstatement occurs only with D1 receptor agonists, not D2 receptor agonists, and as such Graham et al.  provide In humans, Schmidt et al.  reportedThe work of Ng and George  firmly sl-DOPA at a dose that replenished dopamine signaling in the VMS reversed escalation, thereby demonstrating a causal relationship between diminished dopamine transmission and excessive drug use. While this work is intriguing, it may not morph other theories such as the surfeit rather than deficit dopamine function; however, it does seem to support dopamine-based agonistic modalities as suggested by Willuhn et al. [Understanding the true role of dopamine in escalating, for example, cocaine has been the recent subject of investigation by a number of investigators. Weiss’s group provided strong evidence that attenuated dopamine actions in the core (but not the shell) of accumbens result in greater cocaine intake—shorter interresponse times—during periods of self-administration . Conversn et al. . Along tn et al.  correctln et al. . While wn et al. ), it doeEpigenetics, an emerging area in the neuroscience field, has provided important clues about how drugs of abuse including alcohol interact with epigenome and modulate the genetic functions and regulate addictive endophenotypes , 78. EpiDopamine for Dinner [One important outcome of this concept has been adopted by a new cook book entitled r Dinner  respondi


The Funding Statement is missing an acknowledgment to the Advanced Molecular Detection Initiative at the CDC. The correct Funding Statement is provided here:ET is supported by an American Society for Microbiology (ASM) and Centers for Disease Control and Prevention Postdoctoral Fellowship. We acknowledge support from the Advanced Molecular Detection Initiative at the CDC, the Atlanta Research and Education Foundation, Atlanta, GA, and the CDC Antimicrobial Resistance Working Group. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


However, Opn5m2 does not bind all-trans-retinal and exhibits exclusive binding to 11-cis-retinal, whereas many bistable opsins, including fish Opn5m, can bind directly to all-trans-retinal as well as 11-cis-retinal. Because medaka fish has lost the Opn5m2 gene from its genome, we compared the tissue distribution patterns of Opn5m in medaka fish, zebrafish, and spotted gar, in addition to the distribution patterns of Opn5m2 in zebrafish and spotted gar. Opn5m expression levels showed a gradient along the dorsal–ventral axis of the retina, and preferential expression was observed in the ventral retina in the three fishes. The levels of Opn5m2 showed a similar gradient with preferential expression observed in the dorsal retina. Opn5m expression was relatively abundant in the inner region of the inner nuclear layer, while Opn5m2 was expressed in the outer edge of the inner nuclear layer. Additionally, we could detect Opn5m expression in several brain regions, including the hypothalamus, of these fish species. Opn5m2 expression could not be detected in zebrafish brain, but was clearly observed in limited brain regions of spotted gar. These results suggest that ray-finned fishes can generally utilize UV light information for non-image-forming photoreception in a wide range of cells in the retina and brain.Opn5 is a group within the opsin family of proteins that is responsible for visual and non-visual photoreception in animals. It consists of several subgroups, including Opn5m, the only subgroup containing members found in most vertebrates, including mammals. In addition, recent genomic information has revealed that some ray-finned fishes carry paralogous genes of Opn5m while other fishes have no such genes. Here, we report the molecular properties of the opsin now called Opn5m2 and its distributions in both the retina and brain. Like Opn5m, Opn5m2 exhibits UV light-sensitivity when binding to 11- Opsins constitute a photoreceptive G protein-coupled receptor family responsible not only for visual photoreception, such as image-forming and color vision, but also for non-visual (non-image-forming) photoreception such as photoentrainment of circadian rhythm, photoperiodism, and pupillary light reflex in animals . Most vei-coupled UV-sensitive pigment and that this property is common to Opn5m from various vertebrate species [Opsins identified so far are classified into several groups based on amino acid sequence. Opsin classification correlates well with the diversity of their molecular properties. Among opsin groups, the Opn5 (neuropsin) group has members originally found by using a bioinformatics approach on mouse and human genomes and forms one independent opsin group . Mammals species –10. In a species –11. Ther species . In thisgapdh) and intermediate filament family orphan 1b (iffo1b) genes. We could not find the Opn5m2 gene from medaka fish, platyfish, amazon molly, or tilapia, although the genomes of these species have a similar arrangement of natriuretic peptide C-like (nppcl), gapdh, and iffo1 genes. In the phylogenetic relationship of teleosts, the Amazon molly, platyfish, and medaka fish are classified into one monophyletic group, the cladus Atherinomorpha, while the Nile tilapia is also closely related to them. Hence, these data imply an evolutional scenario in which the common ancestor of ray-finned fishes had already acquired the Opn5m2 gene before the whole genome duplication event in Teleostei, and the common ancestor of Nile tilapia, Amazon molly, platyfish, and medaka fish subsequently lost it  of the UV light-absorbing form (360 nm) is the same as that of zebrafish Opn5m, whereas λmax of the visible light-absorbing form (462 nm) is ~10 nm blue-shifted compared with that of zebrafish Opn5m  during the course of molecular evolution [trans-retinal binding.In the present study, we investigated the molecular properties and the expression patterns of an additional Opn5 paralog, Opn5m2, found exclusively in ray-finned fishes and closely related to Opn5m. Opn5m2 can form a UV light-sensitive pigment by the incorporation of 11-volution . HoweverOur analysis of the distribution patterns of Opn5m and Opn5m2 showed that these opsins are localized in retinal interneurons and ganglion cells with a dorsal-ventral gradient of expression levels. It was reported that two cone pigments, UV- and green-sensitive ones, were expressed in a dorsal-ventral gradient in mouse retina ,21. Simicis-retinal, which may lead to quite a small number of Opn5m2-positive cells in the brain due to a shortage of 11-cis-retinal.The analysis of expression patterns in the brain showed that Opn5m-positive cells are localized not only in the hypothalamus but also in other brain regions of ray-finned fishes, some of which are responsible for reproductive activities in vertebrates. In the medaka fish and spotted gar brains, Opn5m was distributed in the preoptic area, which is consistent with the expression pattern of Opn5m in the mammalian brain . The expIn conclusion, we showed that Opn5m and Opn5m2 diverged by gene duplication early in the evolution of ray-finned fishes, exhibit common UV light-sensitivity, and exhibit different affinities to retinal isomers. In addition, the distribution patterns of Opn5m and Opn5m2 in three ray-finned fishes revealed that these opsins are localized in multiple retinal interneurons and ganglion cells with a dorsal-ventral gradient of expression levels, and also in several brain regions, including the hypothalamus. Therefore, in contrast to only a small number of UV light-sensing visual cells for visual photoreception, a wide range of cells in the retina and brain have the potential to respond to UV light. This finding suggests the importance of UV light information for various non-visual photoreceptions in ray-finned fishes.Danio rerio: ~3 cm) and juvenile spotted gars (Lepisosteus oculatus: ~7 cm) were purchased from Shimizu Laboratory Supplies Co., Ltd., Kyoto, Japan, and Nippon Aquarium Co., Ltd., Tokyo, Japan, respectively. They were euthanized and dissected immediately after they were brought into our laboratory. The medaka fish (Oryzias latipes) strain d-rR was maintained and bred at Kyoto University. They were kept under a light/dark cycle of 14/10 h at 25°C. Adult medaka fishes (~3 cm) were chosen for the experiment. Fishes were euthanized by immobilization using MS222 and immediately decapitated thereafter. The use of animals in these experiments was in accordance with guidelines established by the Ministry of Education, Culture, Sports, Science and Technology of Japan. The protocols in this paper were approved by the Animal Care and Use Committee of Kyoto University (permit number: 26–71).Adult zebrafish  were cloned from total RNA of eyes and brain of medaka fish, zebrafish, and spotted gar.The clones of ray-finned fish Opn5m and Opn5m2 were subject to BLAST searches against Ensemble or NCBI databases by using mammalian Opn5m sequences. Although tetraodon Opn5m2 was not annotated in these databases, exons of orthologous genes were found between the gapdh and iffo1b genes in tetraodon genome through blast search using zebrafish Opn5m2 as bait. Accordingly, cDNA sequence of tetraodon Opn5m2 was manually organized from predicted exons. To express recombinant proteins and conduct Multiple amino acid sequences were aligned using MAFFT . The phycis-retinal or all-trans-retinal was added to the medium . After additional incubation for 1 day in the dark, opsin-expressing cells were collected. The pigments were extracted with a buffer containing dodecyl maltoside and purified using Rho1D4-conjugated agarose.Detailed procedures to prepare recombinant Opn5 proteins were previously reported . In shorAbsorption spectra were recorded using a Shimadzu UV2450 spectrophotometer and an optical cell  according to the previous study . An optiAfter eyes and brains were dissected from juvenile spotted gars (~7 cm), adult zebrafish (~3 cm) and adult medaka fish (~3 cm), they were fixed overnight in PBS-buffered 4% (zebrafish and medaka fish) or 8% (spotted gar) PFA. Tissues were subsequently immersed in 20% sucrose in PBS overnight for cryoprotection and were frozen in a deep freezer in OCT compound (tissue tech). Frozen tissues were sliced into 16 μm sections and were attached to glass slides . Slides were stored in a dry chamber at -20°C.In situ hybridization on tissue sections was performed according to the previously described protocol [2, 100 mM NaCl, 0.1% Tween 20, pH 9.5). Finally, they were immersed in alkaline phosphatase reaction buffer containing NBT and BCIP for color development.protocol  with a sS1 Fig(TIF)Click here for additional data file.S2 Fig(A) Medaka fish Opn5m in the INL of the ventral retina. (B) Medaka fish Opn5m in the GCL of the ventral retina. (C) Medaka fish Opn5m in the dorsal retina. (D) Zebrafish Opn5m in the ventral retina. (E) Zebrafish Opn5m2 in the ventral retina. (F) Zebrafish Opn5m2 in the dorsal retina. (G) Spotted gar Opn5m in the ventral retina. (H) Spotted gar Opn5m2 in the dorsal retina. Scale bar: 10 μm.(TIF)Click here for additional data file.S3 Fig(A) Medaka fish Opn5m in preoptic area. (B) Medaka fish Opn5m in tuberal nucleus. (C) Medaka fish Opn5m in pituitary gland. (D) Medaka fish Opn5m in right habenula. (E) Zebrafish Opn5m in entopeduncular nucleus. (F) Zebrafish Opn5m in optic tectum. (G) Zebrafish Opn5m in pretectal nucleus. (H) Zebrafish Opn5m in paraventricular organ. (I) Zebrafish Opn5m in periventricular nucleus. (J) Spotted gar Opn5m in preoptic area. (K) Spotted gar Opn5m2 in tuberal nucleus. Scale bar: 10 μm.(TIF)Click here for additional data file.


The increasingly high incidence of ischemic stroke caused by thrombosis of the arterial vessels is one of the major factors that threaten people’s health and lives in the world. The present treatments for thrombosis are still unsatisfactory. Herbal preparations have been used since ancient times for the treatment of several diseases. The aim of this study was to investigate whether herbal preparations possess thrombolytic activity or not.in vitro thrombolytic model was used to check the clot lysis effect of the crude extracts and fractions of five Bangladeshi plant viz., Trema orientalis L., Bacopa monnieri L., Capsicum frutescens L., Brassica oleracea L. and Urena sinuata L. using streptokinase as a positive control and water as a negative control. Briefly, venous blood drawn from twenty healthy volunteers was allowed to form clots which were weighed and treated with the test plant materials to disrupt the clots. Weight of clot after and before treatment provided a percentage of clot lysis.An in vitro thrombolytic model, different fractions of five Bangladeshi medicinal plants namely T. orientalis, B. monnieri, C. frutescens, B. oleracea and U. sinuata showed various range of clot lysis activity. Chloroform fractions of T. orientalis, B. monnieri, C. frutescens, B. oleracea and U. sinuata showed highest significant (P < 0.05 and P < 0.001) clot lysis activity viz., 46.44 ± 2.44%, 48.39 ± 10.12%, 36.87 ± 1.27%, 30.24 ± 0.95% and 47.89 ± 6.83% respectively compared with positive control standard streptokinase (80.77 ± 1.12%) and negative control sterile distilled water (5.69 ± 3.09%). Other fractions showed moderate to low clot lysis activity. Order of clot lysis activity was found to be: Streptokinase > Chloroform fractions > Methanol (crude) extract > Hydro-methanol fractions > Ethyl acetate fractions > n-hexane fractions > Water.Using an T. orientalis, B. monnieri and U. sinuata could be considered as very promising and beneficial for the Bangladeshi traditional medicine. Lower effects of other extracts might suggest the lack of bio-active components and/or insufficient quantities in the extract. In vivo clot dissolving property and active component(s) of T. orientalis and B. monnieri for clot lysis could lead the plants for their therapeutic uses. However, further work will establish whether or not, chloroform soluble phytochemicals from these plants could be incorporated as a thrombolytic agent for the improvement of the patients suffering from atherothrombotic diseases.Our study suggests that thrombolytic activity of  Thrombosis, the blockage of blood vessels with clots, can lead to acute myocardial infarction and ischemic stroke, the leading causes of death. Other than surgical interventions to remove or by pass the blockage, or the generation of collateral vessels to provide a new blood supply, the only treatment available is the administration of thrombolytic agents to dissolve the blood clot . ThromboTrema orientalis (Chikan), Bacopa monnieri (Brahmi), Capsicum frutescens (Lanka-marich or Marich), Brassica oleracea (Cauliflower), Urena sinuata  are native to Bangladesh. They are used as traditional medicines for cardiac diseases and blood purification. Trema orientalis has recently been focused due to therapeutic activity on hypoglycemic, analgesic, anti-inflammatory, anti-plasmodial, diuretic activity, laxativity, anti-convulsant, anti-helmintic, anti-sickling, antioxidant and antibacterial activity [Bacopa monnieri possesses neuroprotective properties, nootropic activity with therapeutic implications for patients with memory loss [Capsicum frutescens is a cardiovascular stimulant, Capsicum assists in lowering blood pressure and breaking down cholesterol build-up [Urena sinuata has not been subjected yet.Day-by-day the context, concept and methods of the uses of natural products in treatment of human have undergone remarkable changes. Such changes occurred due to the fact that natural medicine or traditional medicine made a revolutionary come-back with renewed strength and vigour to play a more significant role in the management of human health . Signifiactivity . Bacopa ory loss . The frubuild-up . Brassicbuild-up . The bioTrema orientalis , Bacopa monnieri (B. monnieri), Capsicum frutescens (C. frutescens), Brassica oleracea (B. oleracea), Urena sinuata (U. sinuata) for their clot lysis (thrombolytic activity) by using in vitro models.This study aims to investigate the different organic extracts of the aforementioned five Bangladeshi medicinal plants viz., T. orientalis (Accession No. 30816), leaf of B. monnieri (Accession No. 32216), fruits of C. frutescens (Accession No. 33018), fruits of B. oleracea (Accession No. 30072) and leaf of U. sinuata (Accession No. 36155) were collected from different parts of Chittagong region . The plants were identified by Bangladesh Forest Research Institute (BFRI), Chittagong-4211, Bangladesh. The sample specimens of the identified plants have been preserved in the national herbarium with the mentioned accession numbers.Leaf of T. orientalis 7.74 g (yield 3.87% w/w), B. monnieri 18 g (yield 3.60% w/w), C. frutescens 23 g (yield 5.50% w/w), B. oleracea 48 g (yield 6.40% w/w) and U. sinuata 16 g (yield 3.4% w/w) of dried crude extracts. All of the extracts were placed in glass Petri dishes .Plant materials were dried and ground  into powder  to soak in 2.5 L of methanol for 14 days at room temperature (23 ± 0.5°C). Filtrate obtained through cheesecloth and Whatman filter paper No. 1 was concentrated under reduced pressure at the temperature below 45°C using rotary evaporator . The solvent was completely removed by using rotary evaporator  and obtained Crude extracts were undertaken for solvent-solvent partitioning by using the protocol designed by Kupchan and Tsou  and modiin vitro thrombolysis.To the commercially available lyophilized SK vial  of 15,00,000 I.U., 5 mL sterile distilled water was added and mixed properly. This suspension was used as a stock from which 100 μL  was used for n = 20) without a history of oral contraceptive or anticoagulant therapy using a protocol approved by the Institutional Ethics Committee of Chittagong Medical College, Chittagong-4218, Bangladesh. An earlier consent, approval number ME-CMC 2012/05, was taken from the Chittagong Medical College, Chittagong-4218, for collection of blood samples from human volunteers. Blood collection and preservation were conducted by Dr. Shafiqul Islam . A 500 μL of blood was transferred to each of the eight previously weighed microcentrifuge tubes to form clots.Whole blood  was drawn from healthy human volunteers  of investigators was provided in case of any questions of the donors about this study. The consent form was concluded with major questions on above disclosures in Yes/NO form followed by the signature (with date) of the donor.T. orientalis, B. monnieri, C. frutescens, B. oleracea and U. sinuata) were added separately. As a positive control, 100 μL of SK and as a negative non-thrombolytic control, 100 μL of distilled water were separately added to the control tubes numbered. All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, released fluid was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis. The experiment was repeated with the blood samples of the twenty (20) healthy volunteers.Experiments for clot lysis were carried as reported previously . BrieflyThe significance between % clot lysis by SK and plant extracts was tested by the paired t-test analysis using the software SPSS, version 18.0 . Data are expressed as mean ± standard deviation. The mean difference between positive and negative control was considered significant at P values < 0.05 and 0.001.B. monnieri showed the highest (48.39%) significant  clot lysis activity among the other extracts. Chloroform fractions of U. sinuata (47.89%) and T. orientalis (46.44%) also gave significant  clot lysis which is almost similar to that of B. monnieri chloroform fractions. Chloroform fractions of C. frutiescens and B. oleracea have the moderate but significant  clot lysis activity and the values were respectively 36.87% and 30.24%. However, hydro-methanol fractions of T. orientalis, C. frutiescens and B. oleracea have significant clot lysis activity viz. 45.78%, 43.70%, 40.29% respectively . n-hexane fractions of only B. monnieri showed moderate also significant  clot lysis 32.88% whereas ethyl acetate fractions of T. orientalis (43.29%) and U. sinuata (38.29%)  had significant clot lysis activity. Other ethyl acetate fractions showed very lower clot lyses which were insignificant compared to negative control. Crude methanol extract of all plants except C. frutiescens had significant  clot lysis activity. Percent clot lysis obtained after treating the clots with different organic extracts and appropriate controls is shown in Table Addition of 100 μL SK (positive control) to the clots along with 90 minutes of incubation at 37°C, showed 80.77 ± 1.12% clot lysis. Sterile distilled water (negative control) treated-clots showed only 5.69 ± 3.09% clot lysis which is very negligible. The mean differences in clot lysis percentage between positive and negative control was very significant . Chloroform fractions of T. orientalis and B. monnieri, had the significant activity than the other plants. B. oleracea and U. sinuata have moderate thrombolytic activity. The maximum clot lysis activity was mostly observed in chloroform fractions that mean chloroform soluble compounds are mainly responsible for the thrombolytic activity. Hydro-methanol fractions are next to the chloroform fractions in clot lysis effect. It is evident that there are bacterial contaminants of plants which have plasminogen receptors that bind plasminogen. Cell surface bound plasminogen is easily activated to plasmin, which could lead to fibrinolysis [T. orientalis showed antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis [B. monnieri was also found to have very strong antibacterial effect against Staphylococcus aureus [C. frutescens methanol extracts against Staphyloccus aureus. Rahman et al. [in vitro antibacterial activities of Broccoli (B. oleracea) against food borne bacteria. Individual chemical component-activity relationship, which can explore the other new clue for the observed thrombolytic effects of these plants, will be the next step of the research follow-up of our continuous study.Numbers of pharmaceuticals approved by the Food and Drug Administration (FDA) currently have origins to plant sources. Based on the reported immunomodulatory effects, the most important role for plant-derived compounds has emerged in recent times and has led to the precise scientific examination to determine efficacy and safety ,21. A nuinolysis  althoughinolysis ,27. Coindermidis . B. monns aureus . Koffi-Ns aureus  noticed n et al.  has alson et al. . Sibi etn et al.  have alsT. orientalis, B. monnieri, C. frutescens, B. oleracea and U. sinuata, which are beneficial in the Bangladeshi traditional medicine, validated in this study by in vitro blood clots lysis activity of their different organic fractions. However, T. orientalis and B. monnieri showed the promising thrombolytic effects to be studied further for therapeutic applications. Identification of their bioactive components and a dose–response relationship study in in vivo model is claimed to use these indigenous sources for pharmaceutical preparations. Chloroform soluble phytochemicals from these plants will be focused especially whether the fractions could account for the incorporation as a thrombolytic agent to the improvement of the patients suffering from atherothrombotic diseases.We have described the thrombolytic activity of


The Partnership for Transforming Health Systems Phase 2 (PATHS2) project introduced and supports community based interventions such as safe motherhood initiative demand side (SMID) and an emergency transport scheme within northern Nigeria. This study evaluates the effect of PATHS2’s community based interventions in addressing transportation as a barrier to accessing skilled birth delivery services in health facilities.The PATHS2 project conducted baseline and midline surveys in 2009 and 2012 respectively to evaluate the impact of its health care interventions. Structured questionnaires were applied to respondents in randomly selected households within PATHS2’s intervention communities in Kano and Jigawa States during the baseline and midline surveys. The study respondents were categorized into five wealth quintiles using principal component analysis. Data analysis was undertaken using SPSS version 20.Within intervention communities in Kano and Jigawa States, the proportion of respondents in the poorest and second poorest wealth quintiles at baseline relative to midline who indicated that transportation is a barrier to accessing skilled birth delivery services decreased by 28% and 24% (p<0.001) for Kano State as well as 21% and 25% (p<0.001) for Jigawa State respectively. There were no significant decreases in the proportion of respondents who indicated that transportation is a barrier to accessing skilled birth delivery services among the middle to richest wealth quintiles in both States. Within rural intervention communities in Kano State, the proportion of respondents at baseline relative to midline who indicated that transportation is a barrier to accessing skilled birth delivery services decreased by 11.9% (p<0.001). According to multivariate logistic regression analysis, respondents within intervention communities in the poorest wealth quintiles  and second poorest wealth quintiles  in Jigawa State are about four times less likely to indicate that transportation is a barrier to accessing skilled birth delivery services. While respondents from rural intervention communities in Kano State are four times less likely to indicate that transportation is a barrier to accessing skilled birth delivery services  following the implementation of PATHS2’s community based interventions.These findings demonstrate that PATHS2 is addressing the ‘transportation challenge’ as a barrier to accessing skilled birth delivery services, particularly for the poor. The results from PATHS2’s end-line survey in 2014 are expected to substantiate this evidence more strongly.


To conceive and realize a practical device to help the diagnosis of molecular allergology which I named "the astrolabe of molecular allergology" nine allergological parameters incorporate this idea. The progress realized in the domain of recombined allergens (RA) has led to the appearance of a new concept in the diagnosis of allergy, the molecular diagnosis. It permits to identify molecules potentially responsible of the blossoming of diseases, and to think of molecular families rather than biological ones, allergenic constituents now classified in families of proteins based on their structure and function: A new diagnosis step came to enrich the research in Allergology. The targeted dosage of RA appeals to the data of the molecular diagnosis that the allergist is under the obligation to master. The optimization of the sensibility, the standardisation of allergens and the examination of the possible strategies of prevention of allergic diseases becomes possible: The allergist can manipulate this new data after the dosage of specific IgE, before starting up the desensitization, the oral provocation test or proposing an eviction. The objective of this work is to furnish an adequate pedagogical support, pertinent and perfectible, which, validated, may accompany the allergist in his diagnosis quest. Thanks to the data well listed of molecular allergology, this tool will permit to better delimit the crossed reactions, the hard cases of multi sensibilizations, to take the appropriate desensitization decision and to adapt supplanting diets. The molecular diagnosis of allergy is a new vision that each allergist must know and tame perfectly. Our aim is to accompany him in this progress by proposing this humble practical device. The astrolabe of molecular allergology shows us the way.


Female BALB/c mice were injected subcutaneously with 1×107 or 1×108 cfu of V. vulnificus. Antibiotic therapy was initiated at 2 h after inoculation in the following four therapy groups: cefotaxime; ciprofloxacin; cefotaxime-plus-ciprofloxacin; and cefotaxime-plus-minocycline. The cytotoxicity of V. vulnificus for HeLa cells was measured using the lactate dehydrogenase assay; rtxA1 transcription was measured in a transcriptional reporter strain using a β-galactosidase assay.In vitro time-kill assays exhibited synergism between cefotaxime and ciprofloxacin. In the animal experiments, the 96-h survival rate for the cefotaxime-plus-ciprofloxacin group  was significantly higher than that of the cefotaxime-plus-minocycline  and cefotaxime alone  groups (P<0.05 for both). Bacterial counts in the liver and spleen were significantly lower in the cefotaxime-plus-ciprofloxacin group 24 and 48 h after treatment, relative to the other groups. At sub-inhibitory concentrations, ciprofloxacin inhibited more effectively rtxA1 transcription and mammalian cell cytotoxicity than either minocycline or cefotaxime (P<0.05 for both).rtxA1 transcription and subsequent cytotoxicity than either minocycline or cefotaxime, and the combination of ciprofloxacin and cefotaxime was more effective in clearing V. vulnificus invivo than previously used regimens. These data suggest that the combination of ciprofloxacin and cefotaxime is an effective option for the treatment of V. vulnificus sepsis in humans.Ciprofloxacin is more effective at reducing  Vibrio vulnificus is an opportunistic human pathogen that causes rapidly fatal sepsis, as well as skin and soft tissue infections, including necrotizing fasciitis. This pathogen is transmitted to humans through the exposure of wounds to seawater or the ingestion of seafood, and causes septicemia, especially in patients with immunosuppression and iron overload, as is the case in chronic liver disease V. vulnificus septicemia is ∼70% V. vulnificus infections V. vulnificus sepsis invitroinvivo research Immediate surgical intervention and the administration of effective antibiotics are essential to reduce the mortality rate for Escherichia coliSalmonella species invitro bactericidal activity against V. vulnificus than currently recommended regimens, such as ciprofloxacin alone or cefotaxime plus doxycycline invivo efficacy of this combination remains unclear. Moreover, although RtxA1 plays a key role in the virulence of V. vulnificus and mortality in V. vulnificus-related sepsis, the effects of commonly used therapeutic antibiotics at sub-inhibitory concentrations on rtxA1 expression have not been evaluated. To address these issues, we evaluated the invitro synergism and invivo efficacy of the combination of ciprofloxacin and cefotaxime against V. vulnificus sepsis, as well as the effect of commonly used antibiotics on rtxA1 expression.A combination of cefotaxime and a quinolone has a synergistic effect on enteric Gram-negative pathogens, including All animal experiments were carried out in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of Chonnam National University and the guidelines for animal experiments set forth by the Korean Food and Drug Administration (KFDA) V. vulnificus CMCP6, which is a clinical isolate from Chonnam National University Hospital for which full genome sequence is available (GenBank accession nos. AEO16795 and AEO16796) V. vulnificus strains MO6-24/O and CMM770 (MO6-24/O background with a deletion mutation in the rtxA1 gene) In vitro time-kill studies were performed to evaluate synergy, as described previously 10 cfu/mL increase in killing at 24 h using the combination therapy compared with the level of killing achieved with the most active single drug.The minimal inhibitory concentrations (MICs) of cefotaxime, minocycline, and ciprofloxacin were determined by the microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute 7 and 1×108 cfu as the initial inocula, based on previous studies V. vulnificus and more closely represents the iron-overloaded condition seen clinically, 900 µg ferric ammonium citrate was administered intra-peritoneally (i.p.) 30 min before V. vulnificus inoculation 7 or 1×108 cfu V. vulnificus were injected subcutaneously into the area over the right thigh Female, specific pathogen free, 8-week-old BALB/c mice  with an average weight of 20 g were used throughout the study. The inocula were prepared as described previously Each experiment consisted of five groups: a control group and groups treated with cefotaxime, ciprofloxacin, cefotaxime-plus-ciprofloxacin, and cefotaxime-plus-minocycline. All antibiotics were initially given i.p. beginning 2 h after the animal was infected. Cefotaxime (30 mg/kg body weight [BW] i.p.) was given every 6 h. Minocycline  was given every 12 h. Ciprofloxacin  was given every 12 h, as described previously 7 cfu. Mice were humanely euthanized using ether at 24 and 48 h, and the livers and spleens were homogenized. Then, the homogenized tissues were serially diluted and plated onto Brain-Heart infusion agar, to quantify the bacteria.In addition, we counted the numbers of viable bacteria in the livers and spleens 24 and 48 h of infected mice after initiation of antibiotic treatment using an initial inoculum of 1×10V. vulnificus cytotoxicity for HeLa cells was measured using the CytoTox96 non-radioactive cytotoxicity assay kit , as described elsewhere 2. After incubation for 24 h, the cells were washed twice with pre-warmed serum-free Dulbecco’s modified Eagle’s medium (DMEM). Overnight-cultured V. vulnificus bacteria were incubated in fresh 2.5% NaCl heart infusion (HI) broth for 3 h. The logarithmically growing culture was harvested by centrifugation, washed with PBS, and re-suspended in PBS. Then, 2×105 HeLa cells/mL in 250 µL of DMEM were infected with 5×105 cfu/mL V. vulnificus with or without 1/4 MICs of antibiotics for 120 min. Lactate dehydrogenase (LDH) released into the supernatant fluid was assayed as a marker of cytotoxicity, in accordance with the manufacturer’s protocol.rtxA1::lacZ transcriptional reporter strain of V. vulnificus MO6–24/O was constructed as described previously 5 cfu/mL with or without 1/4 MICs of antibiotics for 120 min. The culture was lysed in lysis buffer, and β-galactosidase activity was assayed using 2×β-galactosidase substrate , following the method described previously A chromosomal Pt-test. Survival analysis was performed using the Kaplan–Meier method and log-rank test. All tests of significance were two-tailed, and P-values≤0.05 were deemed to indicate statistical significance. Statistical analyses of the data were performed using the SPSS ver. 19.0 software  and the GraphPad Prism ver. 5.0 program .The Kolmogorov–Smirnov goodness-of-fit test was used to determine the distribution of each set of data for normality before analysis, and continuous variables were compared using Student’s V. vulnificus CMCP6 were 0.0625, 0.0625, and 0.03 mg/L, respectively. In the time-kill assay using 3/4 MICs of each antibiotic, the cefotaxime-ciprofloxacin combination was found to have synergistic activity against V. vulnificus CMCP6, and the bacterial colony counts after 24 h of invitro treatment were lower for cefotaxime-plus-ciprofloxacin than for cefotaxime, ciprofloxacin, minocycline or cefotaxime-plus-minocycline  compared with the cefotaxime  and cefotaxime-plus-minocycline groups  . The 96-h survival rate in the cefotaxime-plus-ciprofloxacin group was also higher than that in the ciprofloxacin group , although the difference was not statistically significant (P = 0.148).7 cfu V. vulnificus CMCP6 was used as the initial inoculum, all infected control mice (n = 6) died within 12 h, whereas all 48 of the infected mice treated with cefotaxime (n = 12), ciprofloxacin (n = 12), cefotaxime-plus-ciprofloxacin (n = 12), or cefotaxime-plus-minocycline (n = 12) were still alive after 96 h.In experiments where 1×10P<0.001 at 24 h and 48 h, each), ciprofloxacin alone  and cefotaxime-plus-minocycline . The viable bacterial counts in spleen were lower in mice treated with cefotaxime-plus-ciprofloxacin than those treated with cefotaxime alone (P<0.001) and ciprofloxacin alone (P = 0.003) at 24 h and those treated with cefotaxime alone (P<0.001) and cefotaxime-plus-minocycline (P = 0.008) at 48 h , as compared with MO6–24/O (WT), indicates that the cytotoxicity noted at 120 min was mainly due to RtxA1 (V. vulnificus-induced cytotoxicity was inhibited more effectively by 1/4 MIC of ciprofloxacin than by 1/4 MIC of cefotaxime or 1/4 MIC of minocycline (P<0.05 for each). Similarly, the transcription of rtxA1 was inhibited more efficiently by 1/4 MIC of ciprofloxacin than by 1/4 MIC of cefotaxime or 1/4 MIC of minocycline , as a subsequent study using quinolones 7 cfu) of V. vulnificus. Here, the survival rate was higher among mice treated with ciprofloxacin-based regimens than among mice treated with the cefotaxime-minocycline combination, although the invivo V. vulnificus clearance rates for ciprofloxacin monotherapy and cefotaxime-plus-minocycline therapy were similar .We performed a survival analysis using 107 cfu V. vulnificus. Although all infected control mice died rapidly, all of the mice in the treatment groups survived, regardless of the regimen used, including cefotaxime monotherapy. Similar results were reported by Tang et al. 7 cfu V. vulnificus; they found that the survival rates of the mice were excellent at >80%, regardless of the administered regimen. Kim et al. 8 cfu V. vulnificus as the initial inoculum 7 cfu V. vulnificus and treated with antibiotics died, regardless of the regimen . We considered it unfeasible to compare the survival rates for different antibiotic regimens when <108 cfu/mL V. vulnificus were used as the initial inoculum, given that many samples would be needed to show a statistically significant difference if the survival rate was >80% for all regimens used. Therefore, we enumerated the bacteria in the organs of the inoculated and treated mice and found that cefotaxime-ciprofloxacin was the most effective regimen in terms of clearing V. vulnificus. These data provide further evidence for the superior efficacy of cefotaxime-plus-ciprofloxacin regimens in clearing V. vulnificus invivo.To compare the efficacies of the treatment regimens on host survival and bacterial numbers in the organs, we infected mice with 1×10V. vulnificusrtxA1 or its regulators decrease the cytotoxicity and increase the LD50 significantly in mice V. vulnificus, and V. vulnificus causes rapidly fatal necrotizing skin and soft tissue infections, the effects of sub-inhibitory concentration of antibiotics that are frequently used in therapy on RtxA1 production have not been evaluated to date. In the present study, we showed that ciprofloxacin more effectively suppressed rtxA1 transcription and protected host cells than either minocycline or cefotaxime. In a previous study, suppressing RtxA1 production per se without direct killing of bacteria was shown to reduce the mortality rate of mice infected with V. vulnificusSub-inhibitory concentrations of antibiotics interfere with the processes of host-parasite interactions, such as phagocytosis, adherence, and toxin production. For this reason, the anti-toxin effects of sub-inhibitory levels of antibiotics have been studied specifically in pathogens that cause rapidly fatal toxin-related diseases, such as necrotizing skin and soft tissue infections, in which anti-toxin efficacy is therapeutically important Certain conclusions that we can draw from this study are limited, as we examined only the combination of ciprofloxacin-plus-cefotaxime. Further studies will be needed to evaluate the efficacy of combinations of ciprofloxacin or minocycline and other third-generation cephalosporins, including ceftazidime and cefrtriaxone.rtxA1 transcription and subsequent cytotoxicity than either minocycline or cefotaxime. Moreover, we demonstrated that the combination of ciprofloxacin and cefotaxime is more effective in clearing V. vulnificus invivo than commonly used regimens, which suggests that this combination is a good candidate for the treatment of V. vulnificus sepsis in humans.In summary, we found that, at sub-inhibitory concentrations, ciprofloxacin is more effective at reducing


Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.A series of genetically related lines of common bean ( Storage protein deficiency in crops is compensated through a mechanism of proteome rebalancing, whereby seed protein concentration is maintained at its normal level . This prPhaseolus vulgaris) is the most important food legume for direct human consumption. A set of genetically related lines integrating a progressive deficiency in major storage proteins has been described  genotypes were grown in a growth cabinet  under 16 h light (300–400 μmol photons m-2 s-1) and a temperature cycling between 18 and 24°C .Quantitative proteomic data  were re-D-mannose agarose and eluted competitively with methyl α-D-mannopyranoside  as described by 1.Mannose lectin FRIL was purified by affinity chromatography on Mature seed tissue (100 mg) was extracted and protein quantified as described above. Equal amount (2 μg) separated by SDS-PAGE on a 15% polyacrylamide gel was transferred to a nitrocellulose membrane (9 cm × 6 cm) at 15 V for 20 min using a semi-dry transfer apparatus . The membrane was blocked with Odyssey Blocking Buffer  at room temperature for 1 h. The membrane was incubated with 1:2000 dilution of anti-α-amylase inhibitor antibodies  for 1 h,ARC1, F: 5′-AGCAACGACGCCTCCTTCAACG-3′ and R: 5′-CCTTTAAGTTTGGGCCGAGAGCCG-3′; for arc3-II, F: 5′-ACTAGCTTCCACCAAGGCGATCC-3′ and R: 5′-TTCTGTCATAGCGGAGGGTGTAGC-3′; for arc4-I, F: 5′-AGTATCCGCCCATACAGTAACAATG-3′ and R: 5′-CACGCTGCTGGTAGAGAAGTTG-3′; for pha-E, F: 5′-CGCACACACTTGCAACATCCC-3′ and R: 5′-GGTTTGGGGTCCCAGTGAACGT-3′; for α-amylase inhibitor 1, F: 5′-GAAACCTCCTTCAACATCGATGG-3′ and R: 5′-CCCTCACCCAGTCGTAAACTTCT-3′; and for mannose lectin FRIL, F: 5′-GTGGAGGAAACCCTGTGGGTGC-3′ and R: 5′-CGGCTCCTTCACCTCGTTGTTCT-3′. The following primers were used to confirm the presence of pdlec1 in SMARC1N-PN1 and Great Northern 1140: PhaL-F165, 5′-CTCCTCTTCTCACTATGACAC-3′ and PhaL-R838, 5′-GACTCCAAACTCCACCTTCC-3′. The following primers were used to amplify the β-phaseolin promoter: pBetaPhsF, 5′-CCTTTCTTGGTATGTAAGTCCG-3′ and pAlphaBetaPhsR, 5′-AGTAGAGTAGTATTGAATATGAGTTG-3′. PCR products were sequenced using a 3130XL Genetic Analyzer .Leaf tissue was frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Genomic DNA was isolated using the GenElute Plant Genomic DNA Miniprep Kit (Sigma–Aldrich) following the manufacturer’s protocol. PCR was carried out using 50 ng of genomic DNA as template for 35 cycles with Taq DNA polymerase and the following gene specific primers: for DNA was isolated using a Qiagen DNeasy Plant Mini Kit . Care was taken to isolate intact genomic DNA. To minimize shearing, the samples were not vortexed and wide-bore pipette tips were used for handling. After the final wash, the samples were eluted in 100 μl of 10 mM Tris-HCl pH 8.0. DNA samples were visualized on a 1% agarose gel. DNA concentration and purity was determined using a Nanodrop 1000 . Genomic DNA samples from Sanilac, SARC1, SMARC1-PN1 and SMARC1N-PN1 were submitted for paired-end read sequencing on an Illumina HiSeq 2000/2500 platform  at the Clinical Genomics Centre, Toronto, ON, Canada, following recommended guidelines. Low quality reads were filtered out, resulting in approximately 925–1025 million reads per sample. In addition to paired-end read sequencing, three mate-pair libraries were prepared, short (3.5–4.5 kb), medium (5–7 kb), and large (8–11 kb) using Illumina’s protocol to obtain 50 base pair reads. Samples from each size were multiplexed and run on a single lane. Sequencing data can be found in the short read archive at the National Center for Biotechnology Information, with the following accession numbers: for Sanilac, SRP055506; for SARC1, SRP055509; for SMARC1N-PN1, SRP055510; and for SMARC1-PN1, SRP055511.P. vulgaris G19833 genome sequence (2), a BAC clone for the APA locus of an arcelin-5 genotype  present in SMARC1N-PN1. In prior analyses, the presence of β-phaseolin in SMARC1N-PN1 had been inferred from the results of two-dimensional gel electrophoresis based proteomics . Three bands were observed having apparent molecular masses of 20, 17, and 16 kDa, respectively. The first one constitutes the N-terminal subunit and the other two the C-terminal subunit (Table 2).For lectins detected at relatively low levels by spectral counting, it was not clear whether they are truly present or whether they are detected based on their high sequence similarity with other lectins. This was further investigated for mannose lectin FRIL and α-amylase inhibitor 1. Mannose lectin FRIL was affinity purified from mature seed of Sanilac, SARC1, SMARC1-PN1 and SMARC1N-PN1 on mannose-agarose and the purified protein analyzed by SDS-PAGE. Protein bands corresponding to mannose lectin FRIL were uniquely present in SMARC1N-PN1 , corresponding to chain 2 and chain 1 of α-amylase inhibitor 1, respectively . In addition, no amplification of pha-E, encoding erythroagglutinating phytohemagglutinin, was observed in SMARC1N-PN1 and in Great Northern 1140, the source of lectin deficiency. The genomic PCR data confirmed the presence of the α-amylase inhibitor 1 gene in SMARC1N-PN1 and its absence in SARC1, SMARC1-PN1 and G12882. The α-amylase inhibitor 1 gene was also detected in Sanilac. Mannose lectin is encoded on chromosome 7 and not in the APA locus which is situated chromosome 4. The mannose lectin gene was found to be present in SMARC1N-PN1, G12882 and the P. coccineus genotype. Alignment of conceptual translations of PCR products indicated that mannose lectin FRIL originates from G12882 in SMARC1N-PN1, and was likely lost during crossing and propagation of the lines that led to SARC1 and SMARC1-PN1 .Based on the above results, analysis of lectin gene composition was conducted by genomic PCR using primers complementary to the coding sequence or, where possible, to the 5′-untranslated region. Samples included the three genetically related lines as well as the parental background Sanilac, the two other parents, G12882 and Great Northern 1140 and a de novo assembly, with a sequence read coverage of the estimated genome size greater than 150-fold . However, the composition of APA genes varied. For the phytohemagglutinin gene located between pha-E and the α-amylase inhibitor like protein gene , different alleles were found to be present. G19833 and OAC-Rex have pha-L, as do Sanilac, SARC1 and SMARC1-PN1. BAT93 and SMARC1N-PN1 have the pdlec1 pseudogene, previously characterized from Pinto UI111 . It was not possible to verify the presence of PDLEC2 by genomic PCR due to high degree of sequence identity between leucoagglutinating phytohemagglutinin genes.For analysis of the APA locus, BAT-93, a Mesoamerican genotype, was most similar to SMARC1N-PN1. to UI111 . The preto UI111 . G02771,to UI111 . ArcelinFigure 3A). Promoter sequences were aligned. Polymorphic sites were searched for differences in cis regulatory motifs between SMARC1N-PN1 versus SARC1 and SMARC1-PN1 using the PLACE database . These include a CAAACAC element characterized in the napin promoter of Brassica napus  . Deletion analysis studies previously showed that the G-box, RY motifs, E-box and CAAT box are required for high level expression of a reporter in transgenic Arabidopsis seed   . Analysis of paired end read alignments to the reference genome G19833 revealed complete coverage of the β-phaseolin gene in the four genotypes. Focusing on the proximal promoter, all of the cis-regulatory elements described above except one were conserved . Sanilac and SARC1 have a second G-box downstream from the first element. A single nucleotide polymorphism converts the ACGT motif present in SMARC1-PN1 and SMARC1N-PN1 into this second G-box motif . This polymorphism was confirmed by genomic PCR and sequencing of the PCR products. In addition, the same fragment was amplified from phaseolin-containing and phaseolin-deficient P. coccineus genotypes. Both had the ACGT motif present. These results suggest that the single nucleotide polymorphism was introduced from P. coccineus into SMARC1-PN1 and SMARC1N-PN1, abrogating the second G-box motif. The present study associates this single nucleotide polymorphism with the genotypic difference in β-phaseolin accumulation.In SMARC1N-PN1, β-phaseolin accumulates at lower levels than in SARC1, by approximately 20-fold . Surprisingly, some polymorphisms were observed between the alignment of reads from Sanilac and the reference sequences. Polymorphisms in phaseolin exons and introns clustered in pairs between Sanilac/SARC1 and SMARC1-PN1/SMARC1N-PN1, as expected. The presence of polymorphisms in SMARC1-PN1/SMARC1N-PN1 did not introduce premature stop codons, or affect intron splicing as predicted by GeneSeqer  . They arn allele . Althougto UI111 . The preto UI111 . These tto UI111 . While tto UI111 . The refde novo genome assemblies was insufficient to reach a definitive conclusion regarding this gene. The high degree of sequence identity between phaseolin or lectin coding sequences hampers the assembly process. In future, the addition of long reads may facilitate gap closing and scaffold joining in the assemblies. This may help to clarify the status of the α-phaseolin gene in SMARC1-PN1 and SMARC1N-PN1. The large decrease in β-phaseolin accumulation in SMARC1N-PN1 as compared with SARC1, of approximately 20-fold, was associated with a single nucleotide polymorphism converting a G-box motif into an ACGT motif in the proximal promoter. The originally characterized sequence from Tendergreen (Arabidopsis seeds (P. coccineus accession, as it was present in P. coccineus genotypes examined in this study.The deficiency in α-phaseolin is likely to be due to the partial or complete absence of the gene in SMARC1-PN1 and SMARC1N-PN1. Notably, the promoter sequence was poorly covered in read alignments. The high degree of sequence identity between phaseolin genes precluded the design of primers specific to α-phaseolin. The quality of the dergreen , an Andeis seeds . Howevercis-regulatory elements related to seed expression specific to SMARC1N-PN1, including a CAAT box in the right location, important for high level expression of tissue-specific genes. For legumin, the major storage protein in SMARC1N-PN1, accounting for close to 20% of total protein, the proximal promoters of 670 bp in size were identical between SARC1 and SMARC1N-PN1. While legumin levels are raised by close to 7-fold in SMARC1N-PN1 relative to SARC1, its transcripts levels were elevated by approximately 2-fold during seed development (The results of this study also shed light on the mechanisms leading to compensation by residual lectins. Mannose lectin FRIL and α-amylase inhibitor 1 genes are absent from SARC1 and present in SMARC1N-PN1. The levels of α-amylase inhibitor 1 are slightly higher in SMARC1N-PN1 than in Sanilac, by 2.5-fold, the gene being present in a different genomic context. α-Amylase inhibitor-like protein is of particular interest. Unlike β-phaseolin, the large difference in protein accumulation, of approximately 50-fold, is associated with multiple polymorphisms in the proximal promoter, introducing eight potential positive elopment . These relopment . The autelopment . The ideSP, FY, BC, SH, WC, KP, and FM designed the research; SP, MD, FY, SM, FM analyzed data; GP contributed data; FS and RB contributed germplasm and reagents; all authors contributed to writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


M1) and of 4’-mercapto--4-carbonitrile (M2) between gold electrodes. As a function of electrode separation, characterized by the conductance close to 0 V, we found several plateaus of relative stability, with those close to 0.01G0 being the most pronounced. The overall conductance of symmetric and asymmetric molecules were surprisingly similar, only the range of stability was smaller for M2. While M1 yielded symmetric I–V-curves, only small asymmetries were detected for M2. These are also reflected in the comparable values for coupling parameters using the single level resonance model. The high conductance for the asymmetric molecule is interpreted as a result of coherent coupling of electronic states through the whole molecule, so that the outcome cannot be predicted just by adding conductive properties of individual molecular groups.The contacts and the chemical bonds formed between metallic electrodes and molecules determine to a large extent the conductive properties of single molecular junctions, which represent the smallest possible active elements in an electronic circuit. We therefore investigated in a comparative study, using the break junction technique (MCBJ), the conductive properties of -4,4’-dithiol ( The ultimate goal of molecular electronics is to implement single molecules as active functional elements in future electronic devices such as amplifiers, rectifiers, diodes and logic switches . Two decEven in the simplest case with a single molecule and well defined atomic contacts, the junction consists of the metal electrodes, the molecule and, most important, the molecular endgroups that form the bonds with the electrodes. While typically electrodes consisting of noble –8 and tr2), carboxyls (COOH), dimethylphosphines (PMe2) and selenols (SeH) -4-carbonitrile (M2) in toluene. The chemical structures are shown in = X-ray photoelectron spectroscopy) was measured in a UHV chamber after adsorption of molecules in the solvent for 24 h.The molecules were purchased from Sigma–Aldrich and dissolved to 1 mM concentration in toluene for -4,4’-dithiol  -4,4’-dithiol in comparison with 4’-mercapto--4-carbonitrile, using the break junction technique, shows high conductance for both molecules with only slight asymmetries in the File 1Supporting information features details of the setup, XPS data, and details of the data evaluation.