Workshop to identify critical windows of exposure for children's health: neurobehavioral work group summary.

This paper summarizes the deliberations of a work group charged with addressing specific questions relevant to risk estimation in developmental neurotoxicology. We focused on eight questions. a) Does it make sense to think about discrete windows of vulnerability in the development of the nervous system? If it does, which time periods are of greatest importance? b) Are there cascades of developmental disorders in the nervous system? For example, are there critical points that determine the course of development that can lead to differences in vulnerabilities at later times? c) Can information on critical windows suggest the most susceptible subgroups of children (i.e., age groups, socioeconomic status, geographic areas, race, etc.)? d) What are the gaps in existing data for the nervous system or end points of exposure to it? e) What are the best ways to examine exposure-response relationships and estimate exposures in vulnerable life stages? f) What other exposures that affect development at certain ages may interact with exposures of concern? g) How well do laboratory animal data predict human response? h) How can all of this information be used to improve risk assessment and public health (risk management)? In addressing these questions, we provide a brief overview of brain development from conception through adolescence and emphasize vulnerability to toxic insult throughout this period. Methodological issues focus on major variables that influence exposure or its detection through disruptions of behavior, neuroanatomy, or neurochemical end points. Supportive evidence from studies of major neurotoxicants is provided

Efficient Construction of an Inverted Minimal H1 Promoter Driven siRNA Expression Cassette: Facilitation of Promoter and siRNA Sequence Exchange

RNA interference (RNAi), mediated by small interfering RNA (siRNA), is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.We show here the use of a ligase chain reaction (LCR) to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each). Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette

Control of MRSA infection and colonisation in an intensive care unit by GeneOhm MRSA assay and culture methods

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens. Due to the diffusion of MRSA strains in both hospital and community settings, prevention and control strategies are receiving increased attention. Approximately 25% to 30% of the population is colonised with S. aureus and 0.2% to 7% with MRSA. The BD GeneOhm MRSA real-time PCR assay offers quicker identification of MRSA-colonised patients than do culture methods. Ninety-five patients admitted to the Intensive Care Unit of IRCCS Policlinico San Matteo of Pavia (Italy) for a period > 24 h were screened for MRSA colonisation with both the culture method and the GeneOhm assay. Of the 246 nasal swabs collected from 95 patients, 36 samples were found to be positive by both methods (true-positive). 30% of colonised patients had developed the MRSA infection. Our results show that the GeneOhm MRSA assay is a valuable diagnostic tool for detecting MRSA quickly in nasal swabs. This study confirms that colonisation represents a high risk factor for MRSA infection, and that good MRSA surveillance in an Intensive Care Unit is therefore an excellent way to prevent MRSA infectio

Maf1, a New Player in the Regulation of Human RNA Polymerase III Transcription

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the α-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells

Dexamethasone inhibits the HSV-tk/ ganciclovir bystander effect in malignant glioma cells

BACKGROUND: HSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed. METHODS: Stable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and Bcl(XL). RESULTS: Dexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade. CONCLUSION: The overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment

Recovering probabilities for nucleotide trimming processes for T cell receptor TRA and TRG V-J junctions analyzed with IMGT tools

<p>Abstract</p> <p>Background</p> <p>Nucleotides are trimmed from the ends of variable (V), diversity (D) and joining (J) genes during immunoglobulin (IG) and T cell receptor (TR) rearrangements in B cells and T cells of the immune system. This trimming is followed by addition of nucleotides at random, forming the N regions (N for nucleotides) of the V-J and V-D-J junctions. These processes are crucial for creating diversity in the immune response since the number of trimmed nucleotides and the number of added nucleotides vary in each B or T cell. IMGT^®sequence analysis tools, IMGT/V-QUEST and IMGT/JunctionAnalysis, are able to provide detailed and accurate analysis of the final observed junction nucleotide sequences (tool "output"). However, as trimmed nucleotides can potentially be replaced by identical N region nucleotides during the process, the observed "output" represents a <it>biased </it>estimate of the "true trimming process."</p> <p>Results</p> <p>A probabilistic approach based on an analysis of the standardized tool "output" is proposed to infer the probability distribution of the "true trimmming process" and to provide plausible biological hypotheses explaining this process. We collated a benchmark dataset of TR alpha (TRA) and TR gamma (TRG) V-J rearranged sequences and junctions analysed with IMGT/V-QUEST and IMGT/JunctionAnalysis, the nucleotide sequence analysis tools from IMGT^®, the international ImMunoGeneTics information system^®, <url>http://imgt.cines.fr</url>. The standardized description of the tool output is based on the IMGT-ONTOLOGY axioms and concepts. We propose a simple first-order model that attempts to transform the observed "output" probability distribution into an estimate closer to the "true trimming process" probability distribution. We use this estimate to test the hypothesis that Poisson processes are involved in trimming. This hypothesis was not rejected at standard confidence levels for three of the four trimming processes: TRAV, TRAJ and TRGV.</p> <p>Conclusion</p> <p>By using trimming of rearranged TR genes as a benchmark, we show that a probabilistic approach, applied to IMGT^®standardized tool "outputs" opens the way to plausible hypotheses on the events involved in the "true trimming process" and eventually to an exact quantification of trimming itself. With increasing high-throughput of standardized immunogenetics data, similar probabilistic approaches will improve understanding of processes so far only characterized by the "output" of standardized tools.</p

Modelling the Costs and Effects of Selective and Universal Hospital Admission Screening for Methicillin-Resistant Staphylococcus aureus

Background: Screening at hospital admission for carriage of methicillin-resistant Staphylococcus aureus (MRSA) has been proposed as a strategy to reduce nosocomial infections. The objective of this study was to determine the long-term costs and health benefits of selective and universal screening for MRSA at hospital admission, using both PCR-based and chromogenic media-based tests in various settings. Methodology/Principal Findings: A simulation model of MRSA transmission was used to determine costs and effects over 15 years from a US healthcare perspective. We compared admission screening together with isolation of identified carriers against a baseline policy without screening or isolation. Strategies included selective screening of high risk patients or universal admission screening, with PCR-based or chromogenic media-based tests, in medium (5%) or high nosocomial prevalence (15%) settings. The costs of screening and isolation per averted MRSA infection were lowest using selective chromogenic-based screening in high and medium prevalence settings, at $4,100 and$10,300, respectively. Replacing the chromogenic-based test with a PCR-based test costs $13,000 and$36,200 per additional infection averted, and subsequent extension to universal screening with PCR would cost $131,000 and$232,700 per additional infection averted, in high and medium prevalence settings respectively. Assuming $17,645 benefit per infection averted, the most cost-saving strategies in high and medium prevalence settings were selective screening with PCR and selective screening with chromogenic, respectively. Conclusions/ Significance: Admission screening costs$4,100-$21,200 per infection averted, depending on strategy and setting. Including financial benefits from averted infections, screening could well be cost saving

Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans

Global urban environmental change drives adaptation in white clover.

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale