An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.     

Live non-invasive Shigella dysenteriae 1 strain induces homologous protective immunity in a guinea pig colitis model

A non-invasive live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed from a Shiga toxin gene deleted mutant of Shigella dysenteriae 1 by introducing a plasmid vector pPR1347 that carried a lipopolysaccharide biosynthesis gene (rfb and rfc) of Salmonella typhimurium. In guinea pigs, four successive oral administrations of LTSH Δstx showed complete protection against rectal challenge with wild type S. dysenteriae 1 strain. Exponential increase of the serum IgG and IgA titer against lipopolysaccharide of LTSH Δstx was observed during immunization, peaked on day 28 and remained at that level until day 35 after the initiation of the immunization. In intestinal lavage of the immunized animals, significant increase of IgA titer against lipopolysaccharide of LTSH Δstx was also observed. These data suggested that LTSH Δstx could be a useful candidate to induce protective immunity against S. dysenteriae 1 infection.     

WIP1 Phosphatase as a Potential Therapeutic Target in Neuroblastoma

The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with current chemotherapeutic approaches.     

Porcine insulin-like growth factor (IGF) binding protein blocks IGF-I action on porcine adipose tissue

A growth hormone-dependent insulin-like growth factor (IGF) binding protein (IGFBP) purified from porcine serum specifically blocked the acute insulin-like effects of IGF-I on lipogenesis and glucose oxidation in porcine adipose tissue. This inhibition was dose dependent with half-maximal effective concentrations of IGFBP of 530 ng/ml for lipogenesis and 590 ng/ml for glucose oxidation in the presence of 10(-8) M IGF-I. The IGFBP also caused decreased rates of lipogenesis following a 1-hr preincubation of tissue with IGF-I (10(-8) M). The IGFBP had no effect on insulin action on porcine adipose tissue. These findings demonstrate the inhibitory effects of a highly purified porcine serum IGFBP on the biologic effects of IGF-I in vitro, and provide evidence that the growth hormone-dependent IGFBP blocks the acute insulin-like actions of IGF-I in vivo.     

A Comparison of Health Outcomes in Older versus Younger Adults following a Road Traffic Crash Injury: A Cohort Study

Background

Given the aging demographics of most developed countries, understanding the public health impact of mild/moderate road traffic crash injuries in older adults is important. We aimed to determine whether health outcomes (pain severity and quality of life measures) over 24 months differ significantly between older (65+) and younger adults (18–64).

Methods

Prospective cohort study of 364, 284 and 252 participants with mild/moderate injury following a vehicle collision at baseline, 12 and 24 months, respectively. A telephone-administered questionnaire obtained information on socio-economic, pre- and post-injury psychological and heath characteristics.

Results

At baseline, there were 55 (15.1%) and 309 (84.9%) participants aged ≥65 and 18–64 years, respectively. At 12- and 24-month follow-up, older compared to younger participants who had sustained a mild/moderate musculoskeletal injury had lower physical functioning (3.9-units lower Short Form-12 Physical Composite Score, multivariable-adjusted p = 0.03 at both examinations). After multivariable adjustment, older (n = 45) versus younger (n = 207) participants had lower self-perceived health status (8.1-units lower European Quality of Life-5 Dimensions Visual Acuity Scale scores at 24 months, p = 0.03), 24 months later.

Conclusions

Older compared to younger participants who sustained a mild/moderate injury following a road-traffic crash demonstrated poorer physical functioning and general health at 24 months.     

Comprehensive DNA copy number profile and BAC library construction of an Indian individual

Bacterial artificial chromosomes (BACs) are used in genomic variation studies due to their capacity to carry a large insert, their high clonal stability, low rate of chimerism and ease of manipulation. In the present study, an attempt was made to create the first genomic BAC library of an anonymous Indian male (IMBL4) consisting of 100,224 clones covering the human genome more than three times. Restriction mapping of 255 BAC clones by pulse field gel electrophoresis confirmed an average insert size of 120 kb. The library was screened by PCR using SHANK3 (SH3 and multiple ankyrin repeat domains 3) and OLFM3 (olfactomedin 3) specific primers. A selection of clones was analyzed by fluorescent in situ hybridization (FISH) and sequencing. Fine mapping of copy number variable regions by array based comparative genomic hybridization identified 467 CNVRs in the IMBL4 genome. The IMBL4 BAC library represents the first cataloged Indian genome resource for applications in basic and clinical research.     

Shedding light on Salmonella carriers

Host-to-host transmission in most Salmonella serovars occurs primarily via the fecal-oral route. Salmonella enterica serovar Typhi is a human host-adapted pathogen and some S. Typhi patients become asymptomatic carriers. These individuals excrete large numbers of the bacteria in their feces and transmit the pathogen by contaminating water or food sources. The carrier state has also been described in livestock animals and is responsible for food-borne epidemics. Identification and treatment of carriers are crucial for the control of disease outbreaks. In this review, we describe recent advances in molecular profiling of human carriers and the use of animal models to identify potential host and bacterial genes involved in the establishment of the carrier state.