Identification and spatio-temporal expression analysis of barley genes that encode putative modular xylanolytic enzymes.

Arabinoxylans are cell wall polysaccharides whose re-modelling and degradation during plant development are mediated by several classes of xylanolytic enzymes. Here, we present the identification and new annotation of twelve putative (1,4)-ß-xylanase and six ß-xylosidase genes, and their spatio-temporal expression patterns during vegetative and reproductive growth of barley (Hordeum vulgare cv. Navigator). The encoded xylanase proteins are all predicted to contain a conserved carbohydrate-binding module (CBM) and a catalytic glycoside hydrolase (GH) 10 domain. Additional domains in some xylanases define three discrete phylogenetic clades: one clade contains proteins with an additional N-terminal signal sequence, while another clade contains proteins with multiple CBMs. Homology modelling revealed that all fifteen xylanases likely contain a third domain, a ß-sandwich folded from two non-contiguous sequence segments that bracket the catalytic GH domain, which may explain why the full length protein is required for correct folding of the active enzyme. Similarly, predicted xylosidase proteins share a highly conserved domain structure, each with an N-terminal signal peptide, a split GH 3 domain, and a C-terminal fibronectin-like domain. Several genes appear to be ubiquitously expressed during barley growth and development, while four newly annotated xylanase and xylosidase genes are expressed at extremely high levels, which may be of broader interest for industrial applications where cell wall degradation is necessary.

The fibroblast growth factor receptor substrate 3 adapter is a developmentally regulated microtubule-associated protein expressed in migrating and differentiated neurons.

Fibroblast growth factor (FGF) mediated signaling is essential to many aspects of neural development. Activated FGF receptors signal primarily through the FGF receptor substrate (Frs) adapters, which include Frs2/Frs2alpha and Frs3/Frs2beta. While some studies suggest that Frs3 can compensate for the loss of Frs2 in transfected cells, the lack of an effective Frs3 specific antibody has prevented efforts to determine the role(s) of the endogenous protein. To this end, we have generated a Frs3 specific antibody and have characterized the pattern of Frs3 expression in the developing nervous system, its subcellular localization as well as its biochemical properties. We demonstrate that Frs3 is expressed at low levels in the ventricular zone of developing cortex, between E12 and E15, and it co-localizes with nestin and acetylated alpha-tubulin in radial processes in the ventricular/subventricular zones as well as with betaIII tubulin in differentiated cortical neurons. Subcellular fractionation studies demonstrate that endogenous Frs3 is both soluble and plasma membrane associated while Frs3 expressed in 293T cells associates exclusively with lipid rafts. Lastly, we demonstrate that neuronal Frs3 binds microtubules comparable to the microtubule-associated protein, MAP2, while Frs2 does not. Collectively, these data suggest that neuronal Frs3 functions as a novel microtubule binding protein and they provide the first biochemical evidence that neuronal Frs3 is functionally distinct from Frs2/Frs2alpha.

Psychological variables impacting weight gain rapidity in adolescents hospitalized for eating disorders.

OBJECTIVE: This prospective study examined whether psychological variables related to the transdiagnostic theory of eating disorders measured at admission predicted length of time to reach 85 per cent of ideal body weight (IBW) among underweight adolescents hospitalized for an eating disorder. METHOD: Thirty-three girls (aged 12-17) weighing below 85 per cent of IBW, admitted to an inpatient and/or partial hospitalization eating disorder programme completed self-report measures at admission. Cox regression tested whether scores on admission measures predicted time to reach 85 per cent of IBW. RESULTS: After controlling for IBW at admission, higher self-esteem and lower perfectionism predicted shorter time to reach 85 per cent of IBW, with emotion regulation as a marginally significant predictor. CONCLUSION: Self-esteem and perfectionism may be predictors of responsiveness to weight-gain efforts during hospitalization. Further studies are necessary to determine whether these variables might be appropriate targets for intervention to promote weight gain in underweight girls with eating disorders.

Hepatitis B virus and hepatitis C virus interaction in Huh-7 cells.

BACKGROUND/AIMS: Co-infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) increases the risk of development and the severity of chronic liver disease. Although dominant and suppressive effects of each virus over the other have been reported in vivo, in vitro studies of HBV/HCV co-infection have been limited to analysis of the effects of over-expression of HCV proteins on HBV replication. METHODS: We have re-examined HBV/HCV interactions in Huh-7 cells following co-infection with cell culture-propagated HCV (HCVcc; genotype 2a) and a recombinant adenovirus vector capable of delivering a replication-competent HBV genome (AdHBV; genotype A). RESULTS: While intracellular HCV RNA levels were significantly increased when cells were pre-infected with AdHBV, HCV replication and virion secretion were not altered by simultaneous infection with AdHBV or AdHBV superinfection of HCV-infected cells. Likewise intracellular and secreted HBV DNA levels and HBV promoter activities were either unchanged or modestly increased by HCVcc infection. Despite this, HCV E2 and HBsAg proteins colocalized extensively in co-infected cells suggesting shared stages in viral egress. CONCLUSIONS: These studies indicate that there is little direct interaction of HBV and HCV in co-infected hepatocytes and imply that indirect effects of host-viral interactions dictate viral dominance in HBV/HCV co-infected individuals.

Frequency of psychological distress in gynecologic cancer patients seen in a large urban medical center.

Psychological distress in cancer is a well-documented phenomenon, but additional information is needed about demographic and disease correlates in diverse populations with different forms of cancer. This study focused on gynecologic cancers. Using the Distress Thermometer and the Hospital Anxiety and Depression Scale, this study examined distress levels in 94 women with gynecologic cancer who were being treated as outpatients at a large urban medical center. The distress levels in this sample were lower than in comparable studies, raising questions about openness to reporting distress. Those who reported higher levels of distress were more likely to also report a mental health diagnosis or psychiatric medication. This suggests that an alternate form for distress screening may involve inquiring about mental health treatment. In this sample, younger women and those with higher educational achievement or private health insurance had higher levels of distress. Conversely, there were no relations between distress levels and disease characteristics, indicating that, for example, women with early stage disease have just as much risk of distress as those with later-stage disease.

Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.

The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3-14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3-14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testing.

A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins.

The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.

Immunization with recombinant Chaperonin60 (Chp60) outer membrane protein induces a bactericidal antibody response against Neisseria meningitidis.

Sera from individuals colonized with Neisseria meningitidis and from patients with meningococcal disease contain antibodies specific for the neisserial heat-shock/chaperonin (Chp)60 protein. In this study, immunization of mice with recombinant (r)Chp60 in saline; adsorbed to aluminium hydroxide; in liposomes and detergent micelles, with and without the adjuvant MonoPhosphoryl Lipid A (MPLA), induced high and similar (p>0.05) levels of antibodies that recognized Chp60 in outer membranes (OM). FACS analysis and immuno-fluorescence experiments demonstrated that Chp60 was surface-expressed on meningococci. By western blotting, murine anti-rChp60 sera recognized a protein of Mr 60kDa in meningococcal cell lysates. However, cross-reactivity with human HSP60 protein was also observed. By comparing translated protein sequences of strains, 40 different alleles were found in meningococci in the Bacterial Isolate Genome Sequence database with an additional 5 new alleles found in our selection of 13 other strains from colonized individuals and patients. Comparison of the non-redundant translated amino acid sequences from all the strains revealed ≥97% identity between meningococcal Chp60 proteins, and in our 13 strains the protein was expressed to high and similar levels. Bactericidal antibodies (median reciprocal titres of 32-64) against the homologous strain MC58 were induced by immunization with rChp60 in liposomes, detergent micelles and on Al(OH)3. Bactericidal activity was influenced by the addition of MPLA and the delivery formulation used. Moreover, the biological activity of anti-Chp60 antisera did not extend significantly to heterologous meningococcal strains. Thus, in order to provide broad coverage, vaccines based on Chp60 would require multiple proteins and specific bactericidal epitope identification.

Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner.

AIM: To investigate the role of osteopontin (OPN) and its splice variants in the proliferation of hepatocellular carcinoma (HCC). METHODS: The expression of OPN variants in HCC cell lines as well as HCC tissue samples and non-tumour tissue was studied using polymerase chain reaction. OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines. OPN expression was studied in these cells using Western blotting, immunofluoresnce and enzyme linked immunosorbent assay. A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture. Huh-7 cells stably expressing either OPN-A, -B or -C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth. Expression of OPN mRNA and protein in these tumours was examined using reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: OPN is expressed in HCC in 3 forms, the full length OPN-A and 2 splice variants OPN-B and -C. OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue. Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium. Transfer of OPN conditioned media to naïve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner. Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect. OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model, with Huh-7 cells expressing OPN-A showing the greatest proliferative effect. CONCLUSION: This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44. Modulation of this interaction could represent a novel strategy for the control of HCC.

Quality of life after septic illness.

PURPOSE: The study was undertaken to evaluate the quality of life of survivors of septic illness. MATERIALS AND METHODS: A questionnaire survey of survivors of septic illness (experimental group) and acute myocardial infarction (control group) was conducted using information from the Adult Neuropsychological History and the Sickness Impact Profile forms. Eight patients diagnosed with sepsis (using the Bone et al 1992 criteria [Bone RC, Sprung CL, Sibbald WJ. Crit Care Med 1992;20:724-726]) and 15 patients diagnosed with acute myocardial infarction participated in the study. RESULTS: On the Sickness Impact Profile, greater difficulty with work was reported in the sepsis group than in the cardiac control group (P < .04). When retired individuals were excluded from the analysis, individuals in the sepsis group reported more symptoms on the sensory, physical, and behavior sections of the Adult Neuropsychological History form and greater difficulty with sleep and rest, emotional behavior, body care and movement, and physical and psychosocial functioning on the Sickness Impact Profile. As well, more individuals in the sepsis than the control group endorsed symptoms related to problem solving, concentration, memory, sensory, and physical ability. CONCLUSIONS: Individuals surviving sepsis may have problems with physical, sensory, emotional, and cognitive functioning that become most apparent when involved in more challenging activities, such as working.