Prevalence and predictors of vitamin D insufficiency in children: a Great Britain population based study

ObjectivesTo evaluate the prevalence and predictors of vitamin D insufficiency (VDI) in children In Great Britain.DesignA nationally representative cross-sectional study survey of children (1102) aged 4–18 years (999 white, 570 male) living in private households (January 1997–1998). Interventions provided information about dietary habits, physical activity, socio-demographics, and blood sample. Outcome measures were vitamin D insufficiency (<50 nmol/L).ResultsVitamin D levels (mean = 62.1 nmol/L, 95%CI 60.4–63.7) were insufficient in 35%, and decreased with age in both sexes (p<0.001). Young People living between 53–59 degrees latitude had lower levels (compared with 50–53 degrees, p = 0.045). Dietary intake and gender had no effect on vitamin D status. A logistic regression model showed increased risk of VDI in the following: adolescents (14–18 years old), odds ratio (OR) = 3.6 (95%CI 1.8–7.2) compared with younger children (4–8 years); non white children (OR = 37 [95%CI 15–90]); blood levels taken December-May (OR = 6.5 [95%CI 4.3–10.1]); on income support (OR = 2.2 [95%CI 1.3–3.9]); not taking vitamin D supplementation (OR = 3.7 [95%CI 1.4–9.8]); being overweight (OR 1.6 [95%CI 1.0–2.5]); <1/2 hour outdoor exercise/day/week (OR = 1.5 [95%CI 1.0–2.3]); watched >2.5 hours of TV/day/week (OR = 1.6[95%CI 1.0–2.4]).ConclusionWe confirm a previously under-recognised risk of VDI in adolescents. The marked higher risk for VDI in non-white children suggests they should be targeted in any preventative strategies. The association of higher risk of VDI among children who exercised less outdoors, watched more TV and were overweight highlights potentially modifiable risk factors. Clearer guidelines and an increased awareness especially in adolescents are needed, as there are no recommendations for vitamin D supplementation in older children.     

Levosulpiride, (S)-(-)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl) methyl]-2-methoxybenzamide, enhances the transduction efficiency of PEP-1-ribosomal protein S3 in vitro and in vivo

Many proteins with poor transduction efficiency were reported to be delivered to cells by fusion with protein transduction domains (PTDs). In this study, we investigated the effect of levosulpiride on the transduction of PEP-1 ribosomal protein S3 (PEP-1-rpS3), and examined its influence on the stimulation of the therapeutic properties of PEP-1-rpS3. PEP-1-rpS3 transduction into HaCaT human keratinocytes and mouse skin was stimulated by levosulpiride in a manner that did not directly affect the cell viability. Following 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced inflammation in mice, levosulpiride alone was ineffective in reducing TPA-induced edema and in inhibiting the elevated productions of inflammatory mediators and cytokines, such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor- α. Anti-inflammatory activity by PEP-1-rpS3 + levosulpiride was significantly more potent than by PEP-1-rpS3 alone. These results suggest that levosulpiride may be useful for enhancing the therapeutic effect of PEP-1-rpS3 against various inflammatory diseases. [BMB reports 2011; 44(5): 329-334].     

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

The KIT gene has been shown to have multiple functions in hematopoiesis, melanogenesis, and gametogenesis. In addition, mutations of this gene cause pigmentation disorders in humans and mice and are responsible for coat color differences in pigs. While characterizing polymorphisms in the porcine KIT gene, we detected alternative splicing (AS) of the NAGNAG splice acceptor site at the boundary of intron 4 and exon 5. This AS event generated the E and I isoforms, characterized by insertion or deletion, respectively, of CAG at the borders of coding sequence. AS patterns measured in tissue samples from two randomly selected animals did not identified any tissue-specific outcomes. Analysis of AS patterns using three breeds demonstrated that Landrace and Large White pigs expressed both the E and I isoforms. In contrast, a subset of specimens from Korean Native Pigs (KNP) yielded a single I isoform. Alignment of the sequence from several species revealed that the region between the branch point sequence (BPS) and 3′ acceptor site is conserved. However, it is appeared that the selection of either the proximal or distal splice site varied between species. To test the breed specificity the NAGNAG splice acceptor site, we constructed two lineages of minigenes from KNP and Landrace pigs harboring breed-specific mutations. The minigene splicing assay demonstrated that both types of minigenes expressed both the E and I isoforms in two host cell lines, and no differences were detected in the AS pattern between the two breeds. We conclude that the AS at the NAGNAG splice acceptor site on intron 4/exon 5 in the porcine KIT gene is the result of noise selection at the splice site by the splicing machinery. Therefore, this AS event in the porcine KIT gene is unlikely to have any relationship with the coat color variations of Landrace and KNP breeds.     

Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

AIM:To characterize single-cell-derived mouse clonal mesenchymal stem cells (mcMSCs) established with bone marrow samples from three different mouse strains. METHODS:We established mcMSC lines using subfractionation culturing method from bone marrow samples obtained from long bones.These lines were characterized by measuring cell growth, cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. Nonclonal MSCs isolated by the conventional gradient centrifugation method were used as controls. RESULTS:All mcMSC lines showed typical nonclonal MSC-like spindle shape morphology. Lines differed inoptimal growth density requirement.Cell surface epitope prof iles of these mcMSC lines were similar to those of nonclonal MSCs. However, some lines exhibited different expression levels in a few epitopes, such as CD44 and CD105. Differentiation assays showed that 90% of the mcMSC lines were capable of differentiating into adipogenic and/or chondrogenic lineages, but only 20% showed osteogenic lineage differentiation. T-cell suppression analysis showed that 75% of the lines exhibited T-cell suppression capability. CONCLUSION:mcMSC lines have similar cell morphology and cell growth rate but exhibit variations in their cell surface epitopes, differentiation potential, lineage-specifi c gene expression and T-cell suppression capability.     

Attenuated Bordetella pertussis BPZE1 as a live vehicle for heterologous vaccine antigens delivery through the nasal route

Whereas the great majority of the current vaccines are delivered through the parenteral route, mucosal administration has been increasingly considered for controlling infection and preventing disease. Mucosal vaccination can trigger both humoral and cell-mediated protection, not only at the targeted mucosal surface, but also systemically. In this regard, nasal vaccination has shown great potential. The live attenuated strain of Bordetella pertussis, BPZE1, is particularly attractive and promising as a nasal vaccine delivery vector of heterologous antigen vaccine candidates. BPZE1 was originally developed as a live nasal pertussis vaccine candidate, and is currently undergoing phase I clinical trial in human (http://www.child-innovac.org). Highly adapted to the human respiratory tract and offering several potential protein carriers for presentation of the heterologous antigen vaccine candidates, BPZE1 represents an appealing platform for the development of live recombinant vaccines delivered via the nasal route that would confer simultaneous protection against pertussis and the targeted infectious disease(s).     

Nocturnal hypoxia and loss of kidney function

Background

Although obstructive sleep apnea (OSA) is more common in patients with kidney disease, whether nocturnal hypoxia affects kidney function is unknown.

Methods

We studied all adult subjects referred for diagnostic testing of sleep apnea between July 2005 and December 31 2007 who had serial measurement of their kidney function. Nocturnal hypoxia was defined as oxygen saturation (SaO2) below 90% for ≥12% of the nocturnal monitoring time. The primary outcome, accelerated loss of kidney function, was defined as a decline in estimated glomerular filtration rate (eGFR) ≥4 ml/min/1.73 m² per year.

Results

858 participants were included and followed for a mean study period of 2.1 years. Overall 374 (44%) had nocturnal hypoxia, and 49 (5.7%) had accelerated loss of kidney function. Compared to controls without hypoxia, patients with nocturnal hypoxia had a significant increase in the adjusted risk of accelerated kidney function loss (odds ratio (OR) 2.89, 95% confidence interval [CI] 1.25, 6.67).

Conclusion

Nocturnal hypoxia was independently associated with an increased risk of accelerated kidney function loss. Further studies are required to determine whether treatment and correction of nocturnal hypoxia reduces loss of kidney function.     

Proline-rich tyrosine kinase 2 (Pyk2) promotes cell motility of hepatocellular carcinoma through induction of epithelial to mesenchymal transition

Aims

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells.

Methodology/Principal Findings

We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization.

Conclusion

These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT.     

Biophysics of malarial parasite exit from infected erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.     

Proteomic identification of IPSE/alpha-1 as a major hepatotoxin secreted by Schistosoma mansoni eggs

Background

Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage.

Methodology/Principal Findings

Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter''s toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively.

Conclusions

We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs.     

In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.