Combined transgene immortalized urothelial cells capable of reprogramming and hepatic differentiation

Human primary cells, including urine-derived cells (UCs), are an excellent source for generation of pluripotent stem cells (iPSCs) to model disease. However, replicative senescence starts early and shortens the time window for generation of iPSCs. We addressed the question whether combinations of transgenes allows efficient immortalization of UCs, iPSC generation, and differentiation into hepatocyte-like cells (HLCs). Retroviral transfer of three gene cassettes HPVE6E7 (H), hTERT/p53DD (T), cyclinD1/CDK4R24C (C) encoding five genes was established in primary UCs. Long-term cell proliferation was observed in cells carrying transgenes H, HT, HC, and HCT, whereas cells carrying transgenes C, T and CT showed early senescence similar to UCs. iPSCs could be exclusively generated from immortalized UCs transduced with transgenes HCT and HC. iPSC colonies appeared however later and in smaller number as compared to UCs. Using an established hepatic differentiation protocol, HLCs were obtained with high efficacy. Of note, a high expression of individual transgenes was observed in immortalized UCs, which was down-regulated after reprogramming in four out of five genes. One transgene was re-expressed in HLCs as compared to iPSCs. Our data suggest that individual transgene combinations result in advanced growth rates of immortalized cells and do not prevent iPSC formation and HLC differentiation. Retroviral transgene expression is mostly silenced in iPSCs but can be rarely re-expressed after hepatic differentiation. An extended time window for iPSC establishment can be proposed that allows straightforward functional analyses of differentiated cells.     

Extraction and physicochemical characterization of Sargassum vulgare alginate from Brazil

Alginate fractions from Sargassum vulgare brown seaweed were characterized by (1)H NMR and fluorescence spectroscopy and by rheological measurements. The alginate extraction conditions were investigated. In order to carry out the structural and physicochemical characterization, samples extracted for 1 and 5h at 60 degrees C were further purified by re-precipitation with ethanol and denoted as SVLV (S. vulgare low viscosity) and SVHV (S. vulgare high viscosity), respectively. The M/G ratio values for SVLV and SVHV were 1.56 and 1.27, respectively, higher than the ratio for most Sargassum spp. alginates (0.19-0.82). The homopolymeric blocks F(GG) and F(MM) of these fractions characterized by (1)H NMR spectroscopy were 0.43 and 0.55 for SVHV and 0.36 and 0.58 for SVLV samples, respectively, these values typically being within 0.28-0.77 and 0.07-0.41, respectively. Therefore, the alginate samples from S. vulgare are much richer in mannuronic block structures than those from other Sargassum species. Values of M(w) for alginate samples were also calculated using intrinsic viscosity data. The M(w) value for SVLV (1.94 x 10(5)g/mol) was lower than that for SVHV (3.3 x 10(5)g/mol). Newtonian behavior was observed for a solution concentration as high as 0.7% for SVLV, while for SVHV the solutions behaved as a Newtonian fluid up to 0.5%. The optimal conditions for obtaining the alginates from S. vulgare were 60 degrees C and 5h extraction. Under these conditions, a more viscous alginate in higher yield was extracted from the seaweed biomass.     

Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni

Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron-lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre-formed Hz crystals, suggesting that hydrophilic-hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid-water interface of these structures.     

An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.     

Terminal regions of wheat chromosomes select their pairing partners in meiosis

Many plant species, including important crops like wheat, are polyploids that carry more than two sets of genetically related chromosomes capable of meiotic pairing. To safeguard a diploid-like behavior at meiosis, many polyploids evolved genetic loci that suppress incorrect pairing and recombination of homeologues. The Ph1 locus in wheat was proposed to ensure homologous pairing by controlling the specificity of centromere associations that precede chromosome pairing. Using wheat chromosomes that carry rye centromeres, we show that the centromere associations in early meiosis are not based on homology and that the Ph1 locus has no effect on such associations. Although centromeres indeed undergo a switch from nonhomologous to homologous associations in meiosis, this process is driven by the terminally initiated synapsis. The centromere has no effect on metaphase I chiasmate chromosome associations: homologs with identical or different centromeres, in the presence and absence of Ph1, pair the same. A FISH analysis of the behavior of centromeres and distal chromomeres in telocentric and bi-armed chromosomes demonstrates that it is not the centromeric, but rather the subtelomeric, regions that are involved in the correct partner recognition and selection.     

The lactate dehydrogenases encoded by the ldh and ldhB genes in Lactococcus lactis exhibit distinct regulation and catalytic properties - comparative modeling to probe the molecular basis

Lactococcus lactis FI9078, a construct carrying a disruption of the ldh gene, converted approximately 90% of glucose into lactic acid, like the parental strain MG1363. This unexpected lactate dehydrogenase activity was purified, and ldhB was identified as the gene encoding this protein. The activation of ldhB was explained by the insertion of an IS905-like element that created a hybrid promoter in the intergenic region upstream of ldhB. The biochemical and kinetic properties of this alternative lactate dehydrogenase (LDHB) were compared to those of the ldh-encoded enzyme (LDH), purified from the parental strain. In contrast to LDH, the affinity of LDHB for NADH and the activation constant for fructose 1,6-bisphosphate were strongly dependent on pH. The activation constant increased 700-fold, whereas the K(m) for NADH increased more than 10-fold, in the pH range 5.5-7.2. The two enzymes also exhibited different pH profiles for maximal activity. Moreover, inorganic phosphate acted as a strong activator of LDHB. The impact of replacing LDH by LDHB on the physiology of L. lactis was assessed by monitoring the evolution of the pools of glycolytic intermediates and cofactors during the metabolism of glucose by in vivo NMR. Structural analysis by comparative modeling of the two proteins showed that LDH has a slightly larger negative charge than LDHB and a greater concentration of positive charges at the interface between monomers. The calculated pH titration curves of the catalytic histidine residues explain why LDH maintains its activity at low pH as compared to LDHB, the histidines in LDH showing larger pH titration ranges.     

Multiple treatment comparisons in epilepsy monotherapy trials

Background

Previous trials of the RTS, S malaria candidate vaccine have shown that this vaccine is safe, tolerated and immunogenic. The development plan for this vaccine aims at administering it in the first year of life through the Expanded Program on Immunization (EPI). The objective was to evaluate the safety and reactogenicity of RTS, S/AS02D (0.5 ml dose), a pediatric formulation of GlaxoSmithKline Biologicals' current malaria candidate vaccine RTS, S/AS02A (0.25 ml dose). A 0.5 ml dose of AS02D is composed of the same active ingredients in the same quantities as in a 0.25 ml dose of AS02A and has been developed to be easily introduced into routine EPI practices.

Methods

We performed a phase I/IIb randomized double-blind bridging study in a malaria-endemic region of Mozambique, to compare the safety and immunogenicity of both candidate vaccines with the aim of replacing RTS, S/AS02A with RTS, S/AS02D as the candidate pediatric vaccine. 200 Mozambican children aged 3 to 5 years were randomized 1:1 to receive one of the 2 vaccines according to a 0, 1, 2 month schedule.

Results

Both vaccines were safe and had similar reactogenicity profiles. All subjects with paired pre and post-vaccination samples showed a vaccine response with respect to anti-circumsporozoite (CS) antibodies irrespective of initial anti-CS serostatus. Geometric mean titers (GMTs) were 191 EU/ml (95% CI 150–242) in recipients of RTS, S/AS02D compared to 180 EU/ml (95% CI 146–221) in recipients of RTS, S/AS02A. For the anti-hepatitis B surface antigen (HBsAg), all subjects were seroprotected at day 90, and the GMTs were 23978 mIU/ml (95% CI 17896–32127) in RTS, S/AS02D recipients and 17410 mIU/ml (95% CI 13322–22752) in RTS, S/AS02A recipients. There was a decrease in anti-CS GMTs between months 3 and 14 in both groups (191 vs 22 EU/mL in RTS, S/AS02D group and 180 vs 29 EU/mL in RTS, S/AS02A group).

Conclusion

Our data show that the RTS, S/AS02D is safe, well tolerated, and demonstrates non-inferiority (defined as upper limit of the 95% confidence interval of the anti-CS GMT ratio of RTS, S/AS02A to RTS, S/AS02D below 3.0) of the antibody responses to circumsporozoite and HBsAg induced by the RTS, S/AS02D as compared to the RTS, S/AS02A.     

Application and investigation of a bound for outcome reporting bias

Self-care interventions are promoted as effective strategies for improving the quality of life and health outcomes for individuals with long-term health conditions. Outcome measures used in evaluations using Randomised Controlled Trials (RCTs) are not designed to consider patients' prior management strategies and experience of illness. Yet the experience of illness literature suggests that adjusting to living with chronic illness, together with broader contextual influences, are likely to be relevant to understanding responses to self-management initiatives. Using group and individual interview data we attempt to illuminate the transposition of IBS from a condition unsatisfactorily managed by medicine to one successfully managed within the life worlds of individuals. If routine embedding of complex interventions depends on the accomplishment of integration and workability in patients' everyday lives then the design and evaluation of such interventions should view participation as part of a process of continuity as well as change. Responses to formal self-management can be extended beyond psychological and other quantitatively measured outcomes. A useful addendum to trial outcomes for self-management education is an understanding of change as being inextricably linked to people's previous attempts to, and experience of, managing long-term conditions. We suggest that the benefits of understanding the prior experience of managing illness and contact with health services include the acceptability and workability of complex interventions in patients' everyday lives.     

Difficulties implementing a mental health guideline: an exploratory investigation using psychological theory

Background

Monitoring and Messaging Devices (MMDs) are telehealth systems used by patients in their homes, and are designed to promote patient self-management, patient education, and clinical monitoring and follow-up activities. Although these systems have been widely promoted by health care systems, including the Veterans Health Administration, very little information is available on factors that facilitate use of the MMD system, or on barriers to use.

Methods

We conducted in-depth qualitative interviews with clinicians using MMD-based telehealth programs at two Veterans Affairs Medical Centers in the Midwestern United States.

Results

Findings suggest that MMD program enrollment is limited by both clinical and non-clinical factors, and that patients have varying levels of program participation and system use. Telehealth providers see MMDs as a useful tool for monitoring patients who are interested in working on management of their disease, but are concerned with technical challenges and the time commitment required to use MMDs.

Conclusion

Telehealth includes a rapidly evolving and potentially promising range of technologies for meeting the growing number of patients and clinicians who face the challenges of diabetes care, and future research should explore the most effective means of ensuring successful program implementation.