Acute effects of breaking up prolonged sedentary time on cardiovascular disease risk markers in adults with paraplegia

© 2020 The Authors. Elevated levels of cardiovascular disease (CVD) risk markers are highly prevalent in people with a spinal cord injury (SCI). Breaking up prolonged sedentary time with short, regular bouts of physical activity can reduce postprandial glucose and lipid levels in able-bodied individuals. The effects in people with paraplegia are unknown. The study aims were to examine the acute postprandial glucose (primary aim), lipid, blood pressure, and psychological responses (secondary aims) to breaking up prolonged sedentary time in individuals with paraplegia. This was a randomized crossover design trial. Fourteen participants with paraplegia (age 51 ± 9 years, trunk fat mass 44.3 ± 7.7%) took part in the following two, 5.5-hour conditions: (1) uninterrupted sedentary time (SED), and (2) sedentary time interrupted with 2 minutes of moderate-intensity arm crank ergometer physical activity every 20 minutes (SED-ACT). Standardized breakfast and lunch test meals were consumed during each condition. The outcomes were compared between conditions using linear mixed models. Glucose area under the curve (AUC) was significantly lower during the lunch postprandial period in SED-ACT vs SED (incremental AUC 1.9 [95% CI 1.0, 2.7) and 3.0 [2.1, 3.9] mmol/L∙2.5 hour, respectively, P = .015, f = 0.34). There were no differences between conditions for the breakfast or total 5.5 hours postprandial periods (P > .05). Positive affect was higher in SED-ACT than SED (P = .001). Breaking up prolonged sedentary time acutely attenuates lunch postprandial glucose and improves positive affect in people with paraplegia. This may have clinical relevance for reducing CVD risk and improving psychological well-being in this population.Heart Research UK. Grant Number: RG2655/17/1

The effect on behavior and bone mineral density of individualized bone mineral density feedback and educational interventions in premenopausal women: a randomized controlled trial [NCT00273260]

BACKGROUND: Limited information is available on ways to influence osteoporosis risk in premenopausal women. This study tested four hypotheses regarding the effects of individualized bone density (BMD) feedback and different educational interventions on osteoporosis preventive behavior and BMD in pre-menopausal women, namely: that women are more likely to change calcium intake and physical activity if their BMD is low; that group education will be more efficacious at changing behavior than an information leaflet; that BMD feedback and group education have independent effects on behavior and BMD; and, that women who improve their physical activity or calcium intake will have a change in bone mass over 2 years that is better than those who do not alter their behavior. METHODS: We performed a 2-year randomized controlled trial of BMD feedback according to T-score and either an osteoporosis information leaflet or small group education in a population-based random sample of 470 healthy women aged 25–44 years (response rate 64%). Main outcome measures were dietary calcium intake, calcium supplement use, smoking behavior, physical activity, endurance fitness, lower limb strength and BMD. We used paired t-tests, one-way ANOVA and linear regression techniques for data analysis. RESULTS: Women who had feedback of low BMD had a greater increase in femoral neck BMD than those with normal BMD (1.6% p.a. vs. 0.7% p.a., p = 0.0001), but there was no difference in lumbar spine BMD change between these groups (0.1% p.a. vs. 0.08% p.a., p = 0.9). Both educational interventions had similar increases in femoral neck BMD (Leaflet = +1.0% p.a., Osteoporosis self-management course = + 1.3% p.a., p = 0.4). Femoral neck BMD change was only significantly associated with starting calcium supplements (1.3 % p.a, 95%CI +0.49, +2.17) and persistent self-reported change in physical activity levels (0.7% p.a., 95%CI +0.22, +1.22). CONCLUSION: Individualized BMD feedback combined with a minimal educational intervention is effective at increasing hip but not spine bone density in premenopausal women. The changes in behavior through which this was mediated are potentially important in the prevention of other diseases, thus measuring BMD at a young age may have substantial public health benefits, particularly if these changes are sustained

DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes

Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. Methodology/Principal Findings: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38uC and 42uC. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation. Conclusion/Significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart

Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252Cf neutron and X-rays

<p>Abstract</p> <p>Background</p> <p>The aim of the study was to obtain stable radioresistant sub-lines from the human cervical cancer cell line HeLa by prolonged exposure to ²⁵²Cf neutron and X-rays. Radioresistance mechanisms were investigated in the resulting cells using microarray analysis of DNA damage repair genes.</p> <p>Methods</p> <p>HeLa cells were treated with fractionated ²⁵²Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HeLaNR and HeLaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay. Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by Western blotting and real-time PCR.</p> <p>Results</p> <p>The radioresistant sub-lines HeLaNR and HeLaXR were more radioresisitant to ²⁵²Cf neutron and X-rays than parental HeLa cells by detecting their radioresistant characteristics, respectively. Compared to HeLa cells, the expression of 24 genes was significantly altered by at least 2-fold in HeLaNR cells. Of these, 19 genes were up-regulated and 5 down-regulated. In HeLaXR cells, 41 genes were significantly altered by at least 2-fold; 38 genes were up-regulated and 3 down-regulated.</p> <p>Conclusions</p> <p>Chronic exposure of cells to ionizing radiation induces adaptive responses that enhance tolerance of ionizing radiation and allow investigations of cellular radioresistance mechanisms. The insights gained into the molecular mechanisms activated by these "radioresistance" genes will lead to new therapeutic targets for cervical cancer.</p

Targeting insulin-like growth factor pathways

Some cancer cells depend on the function of specific molecules for their growth, survival, and metastatic potential. Targeting of these critical molecules has arguably been the best therapy for cancer as demonstrated by the success of tamoxifen and trastuzumab in breast cancer. This review will evaluate the type I IGF receptor (IGF-IR) as a potential target for cancer therapy. As new drugs come forward targeting this receptor system, several issues will need to be addressed in the early clinical trials using these agents

A Novel Multi-Antigen Virally Vectored Vaccine against Mycobacterium avium Subspecies paratuberculosis

BACKGROUND: Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. METHODS: We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. PRINCIPAL FINDINGS: Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. CONCLUSIONS/SIGNIFICANCE: A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly immunogenic without adverse effect in mice and both attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection and conferred protection against subsequent challenge. Further studies of the present vaccine in naturally infected animals and humans are indicated

SNi from SN2: a front-face mechanism ‘synthase’ engineered from a retaining hydrolase

SNi or SNi-like mechanisms, in which leaving group departure and nucleophile approach occur on the same ‘front’ face, have been observed previously experimentally and computationally in both the chemical and enzymatic (glycosyltransferase) substitution reactions of α-glycosyl electrophiles. Given the availability of often energetically comparable competing pathways for substitution (SNi vs SN1 vs SN2) the precise modulation of this archetypal reaction type should be feasible. Here, we show that the drastic engineering of a protein that catalyzes substitution, a retaining β-glycosidase (from Sulfolobus solfataricus SSβG), apparently changes the mode of reaction from “SN2” to “SNi”. Destruction of the nucleophilic Glu387 of SSβG-WT through Glu387Tyr mutation (E387Y) created a catalyst (SSβG-E387Y) with lowered but clear transglycosylation substitution activity with activated substrates, altered substrate and reaction preferences and hence useful synthetic (‘synthase’) utility by virtue of its low hydrolytic activity with unactivated substrates. Strikingly, the catalyst still displayed retaining β stereoselectivity, despite lacking a suitable nucleophile; pH-activity profile, mechanism-based inactivators and mutational analyses suggest that SSβG-E387Y operates without either the use of nucleophile or general acid/base residues, consistent with a SNi or SNi-like mechanism. An x-ray structure of SSβG-E387Y and subsequent metadynamics simulation suggest recruitment of substrates aided by a π-sugar interaction with the introduced Tyr387 and reveal a QM/MM free energy landscape for the substitution reaction catalyzed by this unnatural enzyme similar to those of known natural, SNi-like glycosyltransferase (GT) enzymes. Proton flight from the putative hydroxyl nucleophile to the developing p-nitrophenoxide leaving group of the substituted molecule in the reactant complex creates a hydrogen bond that appears to crucially facilitate the mechanism, mimicking the natural mechanism of SNi-GTs. An oxocarbenium ion-pair minimum along the reaction pathway suggests a step-wise SNi-like DN*ANss rather than a concerted SNi DNAN mechanism. This first observation of a front face mechanism in a β-retaining glycosyl transfer enzyme highlights, not only that unusual SNi reaction pathways may be accessed through direct engineering of catalysts with suitable environments, but also suggests that ‘β-SNi’ reactions are also feasible for glycosyl transfer enzymes and the more widespread existence of SNi or SNi-like mechanism in nature