Habitual cold-water swimming and upper respiratory tract infection.

It has long been claimed that non-wetsuit cold water swimming (CWS) benefits health (1), and anecdotally cold-water swimmers claimed to suffer fewer and milder infections, though this was not directly measured. A boost to immunity is biologically plausible: stress hormones are released during cold-water immersion (2), and short-term stress may ready the immune system for injury or infection (3). However, very few studies have investigated immune system markers and/or actual illness in habitual cold-water swimmers.

Substitution of starch for palm oil during gestation: impact on offspring survival and hepatic gene expression in the pig.

Piglet neonatal mortality rates are high (~20%), so nutritional strategies to reduce this are highly desirable. Maternal fat substitution (FS) may promote the preweaning survival of piglets by improving their energy status. Therefore, the aim of the present study was to investigate the effects of FS throughout pregnancy on offspring viability, together with the gene expression of stress-related markers in the liver. Sixteen pregnant sows were randomly allocated to one of two isocaloric diets, control (C) or FS in the form of palm oil, fed from 0 to 110 days gestation. Glucose tolerance was examined on Day 108. Median and low birthweight offspring were allocated to tissue sampling at either 7 days or 6 months postnatal age. In response to a glucose tolerance test, FS sows exhibited a raised glucose area under the curve with no change in basal glucose. Average piglet mortality (up to Day 28) was increased fourfold in the FS group, with surviving median-sized piglets exhibiting significantly lower fatty acid binding protein 1 (FABP1) expression at 7 days. There were no effects on the abundance of any other stress- or metabolic-related genes examined. Thus, this study demonstrates that maternal FS throughout gestation causes maternal glucose intolerance that may be linked to the observed increase in piglet mortality. However, the surviving offspring do not exhibit any detectable differences in postnatal growth or hepatic gene profile in later life.

An electromyographic evaluation of dual role breathing and upper body muscles in response to front crawl swimming.

The upper body trunk musculature is key in supporting breathing, propulsion, and stabilization during front crawl swimming. The aim of this study was to determine if the latissimus dorsi, pectoralis major, and serratus anterior contributed to the development of inspiratory muscle fatigue observed following front crawl swimming. Fourteen trained swimmers completed a 200-m front crawl swim at 90% of race pace. Maximal inspiratory and expiratory mouth pressures (PImax and PEmax) were assessed before (baseline) and after each swim, and electromyography was recorded from the three muscles. Post-swim PImax fell by 11% (P < 0.001, d = 0.57) and the median frequency (MDF: a measure of fatigue) of the latissimus dorsi, pectoralis major, and serratus anterior fell to 90% (P = 0.001, d = 1.57), 87% (P = 0.001, r = -0.60) and 89% (P = 0.018, d = 1.04) of baseline, respectively. The fall in serratus anterior MDF was correlated with breathing frequency (r = 0.675, P = 0.008) and stroke rate (r = 0.639, P = 0.014). The results suggest that the occurrence of inspiratory muscle fatigue was partly caused by fatigue of these muscles, and that breathing frequency and stroke rate particularly affect the serratus anterior.

What is the prevalence of the experience of death of a patient by suicide among medical students and residents? A systematic review.

OBJECTIVE: The purposes of this study were to examine the literature on the proportion of medical students and residents who experience the death of a patient by suicide and to identify curricula with data on outcomes that assist medical students or residents in preparing for or managing the psychological stress in dealing with those suicides. METHODS: The authors searched PubMed, Scopus, Google Scholar, and ScienceDirect databases using search terms patient suicide, trainee, medical student, and resident. They conducted a separate search to identify relevant curricula using the same terms in combination with coping, teaching, programs, and education. RESULTS: Eight studies met inclusion criteria, all of which concerned psychiatry residents alone. We found no studies that determined the prevalence of the experience of death of a patient by suicide among medical students or residents in specialties other than psychiatry. The prevalences were 31, 33, 43, 47, 54, 61, 68, and 69 %. All studies were cross-sectional, and none collected data prospectively. Limitations of these data included single-site studies, lack of clarity of the specific question asked, low response rates, and uncertain reporting periods. The authors found two curricula with outcome data that assisted medical trainees in managing the psychologically distressing consequences of the death of a patient. CONCLUSION: Although the data are limited, psychiatry residents commonly experience the death of a patient by suicide. There is a paucity of data on this topic concerning the experiences of medical students and of residents in other specialties.

Risk of infection and sepsis in severely injured patients related to single nucleotide polymorphisms in the lectin pathway.

BACKGROUND: Infectious complications remain a serious threat to patients with multiple trauma. Susceptibility and response to infection is, in part, heritable. The lectin pathway plays a major role in innate immunity. The aim of this study was to assess whether single nucleotide polymorphisms (SNPs) in three key genes within the lectin pathway affect susceptibility to infectious complications in severely injured patients. METHODS: A prospective cohort of severely injured patients admitted to a level I trauma centre between January 2008 and April 2011 were genotyped for SNPs in MBL2 (mannose-binding lectin 2), MASP2 (MBL-associated serine protease 2) and FCN2 (ficolin 2). Association of genotype with prevalence of positive culture findings and infection was tested by χ(2) and logistic regression analysis. RESULTS: A total of 219 patients were included, of whom 112 (51·1 per cent) developed a positive culture from sputum, wounds, blood or urine. A systemic inflammatory response syndrome (SIRS) developed in 139 patients (63·5 per cent), sepsis in 79 (36·1 per cent) and septic shock in 37 (16·9 per cent). Patients with a MBL2 exon 1 variant allele were more prone to positive wound cultures (odds ratio (OR) 2·51, 95 per cent confidence interval 1·12 to 5·62; P = 0·025). A MASP2 Y371D DD genotype predisposed to SIRS (OR 4·78, 1·06 to 21·59; P = 0·042) and septic shock (OR 2·53, 1·12 to 4·33; P = 0·003). A FCN2 A258S AS genotype predisposed to positive wound cultures (OR 3·37, 1·45 to 7·85; P = 0·005) and septic shock (OR 2·18, 1·30 to 4·78; P = 0·011). CONCLUSION: Severely injured patients with SNPs in MBL2, MASP2 Y371D and FCN2 A258S of the lectin pathway of complement activation are significantly more susceptible to positive culture findings, and to infectious complications, SIRS and septic shock than patients with a wildtype genotype.

Construction of a doxycycline inducible adipogenic lentiviral expression system.

To provide a tool for research on regulating adipocyte differentiation, tetracycline inducible (Tet on) lentiviral expression vectors under the control of an adipose-specific promoter were constructed. The lowest basal expression in the absence of doxycycline and most efficient dose-dependent, doxycycline-induced transient overexpression was observed using vectors constructed with a combination of Tetracycline Responsive Element (TRE) and reverse tetracycline-controlled TransActivator advanced (rtTAadv), transfected in white (3T3-L1) and brown (HIB-1B) preadipocytes cell lines. The results demonstrate that doxycycline adipogenic inducible expression can be achieved using a pLenti TRE / rtTA adv under the control of the truncated aP2 promoter in HIB-1B preadipocytes.

Cognitive performance of military men and women during prolonged load carriage.

BACKGROUND: This study evaluated cognitive workload in soldiers undertaking a long duration march wearing different loads. METHODS: Military participants (n=12 men and n=10 women) performed four 3-hour loaded marches (12.25 km at 4.9 km/hour) wearing either 21 kg, 26 kg, 33 kg or 43 kg. During the march, accuracy and response time were measured using the verbal working memory n-back test (0, 1, 2 and 3) and two bespoke Go/No Go tests (visual/auditory) to assess inhibition of a pre-potent response. RESULTS: The physical demands of the march increased with load and march duration but remained at moderate intensity. N-back test accuracy ranged from 74% to 98% in men and 62% to 98% in women. Reduced accuracy was observed as load and time increased. Accuracy during the visual Go/No Go test also reduced with load, accuracy ranged from 69% to 89% in men and 65% to 90% in women. No differences due to load or time were observed during completion of the auditory Go/No Go task; accuracy ranged from 93% to 97% in men and 77% to 95% in women. A number of participants were unable to complete the march due to discomfort. Reports of discomfort were more frequent in women, which may have contributed to the greater reductions in accuracy observed. CONCLUSION: These data provide further evidence that cognitive performance of military personnel can be affected during long duration loaded marching. Women reported discomfort from equipment more frequently than men, which may make them more susceptible to declines in cognitive performance. These findings highlight important considerations for equipment procurement.

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue.

AIMS/HYPOTHESIS: Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues. METHODS: Methylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding. RESULTS: We show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter-reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment. CONCLUSIONS/INTERPRETATION: The results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails.

Cloned ribosomal RNA genes from chloroplasts of Euglena gracilis.

Fragments of Euglena chloroplast DNA generated by endonuclease R-Eco RI were separated by agarose-gel electrophoresis into 24 distinct bands. At least five fragments contain sequences complementary to chloroplast ribosomal RNA, Most of the Eco RI fragments have been cloned in a plasmid of Escherichia coli. Three of the cloned fragments were shown to contain chloroplast ribosomal RNA sequences by DNA-RNA hybridization.

Influence of prior activity (warm-up) and inspiratory muscle training upon between- and within-day reliability of maximal inspiratory pressure measurement.

BACKGROUND: A specific inspiratory muscle 'warm-up' (IWU) prior to assessment of maximal inspiratory mouth pressure (PI(max)) may reduce the number of measurements required to obtain reproducible, representative estimates of PI(max). The influence of inspiratory muscle training (IMT) upon this phenomenon is unknown. OBJECTIVE: Compare the impact of an IWU on the between- and within-day reliability of PI(max) before and after IMT. METHOD: Eight participants were assessed on 4 separate occasions: 2 trials preceded IMT and 2 followed it. At each assessment, the highest of 3 initial efforts was recorded as the pre-IWU value (PI). The highest of 9 subsequent efforts that followed 2 sets of 30 breaths at 40% PI was recorded as PI(max). Following 4 weeks of IMT, the trials were repeated. RESULTS: IWU increased PI by 11-17% (p < or = 0.01), irrespective of IMT status. After IWU, 5-6 efforts were required to determine PI(max), irrespective of IMT status. PI(max) was similar between the 2 trials before IMT and the 2 trials after IMT (p > or = 0.05), and was 21% higher after IMT (p < or = 0.01). The coefficient of variation was excellent before and after IWU, both before (1.9 and 0.6%, respectively) and after IMT (1.1 and 0.3%, respectively). Limits of agreement and sample sizes for effect sizes < or =10% were substantially smaller after IWU in all trials. CONCLUSIONS: (1) IWU enhances the between-day reliability of PI(max) measurement, and this is unaffected by IMT, and (2) judgements regarding acceptability in relation to PI(max) reliability should be made in relation to analytical goals and we present data to facilitate this.

Tissue-specific genes for respiratory proteins.

All but one of the mitochondrial respiratory complexes are composed of products of both the mitochondrial and the nuclear genomes. The recent isolation of cDNAs for several nuclear-encoded respiratory proteins reveals that some of them are present in at least two forms. Although some of these forms are traditional in differing somewhat in amino acid sequence, a new class, termed silent isoforms, differs in the presequence but contains identical processed proteins. What are the roles of tissue isoforms in oxidative metabolism?

Gene expression of inflammatory markers in adipose tissue between obese women with polycystic ovary and normal obese women.

OBJECTIVE: The pathogenesis of polycystic ovary syndrome (PCOS), a common endocrine disease and metabolic disturbance, is still unknown. The aim of the study was to investigate whether patients with PCOS display increased expression of inflammatory markers in adipose tissue. PATIENTS AND METHODS: Two groups of women were investigated, those diagnosed with PCOS (n = 8) and age and BMI-matched normal women (n = 12). Their age was between 20-45 years and all subjects were apparently healthy and did not take any medications. Adipose tissue levels of mRNA of inflammatory markers were determined by use of real-time PCR. RESULTS: There were no differences between obese patients and obese PCOS in levels of adipocytokines. CONCLUSIONS: There were no effects of PCOS on the expression of any of the adipocytokines genes measured in subcutaneous adipose tissue.

Beta 4 integrin transfection of UM-UC-2 (human bladder carcinoma) cells: stable expression of a spontaneous cytoplasmic truncation mutant with rapid loss of clones expressing intact beta 4.

The alpha 6 beta 4 integrin is a component of the hemidesmosome, the anchoring structure in the basal membrane of epithelial cells. alpha 6 beta 4 expression is frequently altered in neoplastic cells. It is sometimes lost and sometimes overexpressed, which suggests that disruption of normal function is involved in neoplastic transformation. To examine the effect of this integrin on the growth and behavior of malignant cells that have lost beta 4, we transfected a full-length beta 4 cDNA into the UM-UC-2 cell line that expresses alpha 6 but not beta 4. Although large numbers of clones were obtained when a control vector was used in the transfection, only 12 clones could be isolated that expressed beta 4. Of these, only two beta 4-positive clones, clones 8 and 11, persisted long enough for further study. Clone 8 cells initially expressed beta 4, but within 2 weeks, all positive cells were lost from the culture. Clone 11 persisted in culture and retained strong surface expression of alpha 6 beta 4. Biochemical analysis and Western blotting revealed that this clone contained a truncated form of beta 4 that had lost the distal cytoplasmic domain. We conclude that expression of wild-type beta 4 in UM-UC-2 inhibits cell growth, presumably by an integrin-mediated signaling pathway. Clone 11 escaped from normal signaling because the cytoplasmic domain, a region essential for basal polar localization, was lost. The alpha 6 beta 4 integrin appears to have tumor suppressor activity in epithelial tumors.

Polyoma virus disrupts ARF signaling to p53.

Polyoma virus (Py) differs from other small DNA tumor viruses in not encoding a protein that inactivates p53. The complete Py early region encoding the large T-antigen (PyLT), middle T-antigen (PyMT) and small T-antigen (PyST) will transform primary rodent cells and REF52 cells, but PyMT, the main Py oncogene, by itself will only transform these cells when p53 or ARF is inactivated. We have related Py oncogene cooperation with the effects of the Py T-antigens on the ARF-p53 signaling pathway. PyMT activates an ARF-induced p53-mediated block to cell division explaining the inability of PyMT alone to generate dividing transformed cells. In contrast, in REF52 cells transformed by the whole Py early region (PyREF52), ARF is upregulated but p53 is not activated. Thus PyLT and/or PyST negates the PyMT-induced ARF-mediated block to cell division by disrupting the signaling pathway from ARF to p53. Although there is no detectable interaction or co-localization of endogenous ARF (nucleoli) and MDM2 (nucleoplasm) in PyREF52 cells, expression of transfected ectopic ARF results in an MDM2/ARF interaction and sequestration of MDM2 into the nucleoli. Sequestration of MDM2 by ARF in the nucleoli is not essential for a p53 response in REF52 cells as activation of Raf in REF52Raf-ER cells results in an ARF-induced p53-mediated cell cycle block in the absence of a detectable ARF-MDM2 interaction. Py may provide new insights into the cellular ARF-p53 signaling pathway.

Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family.

Previous investigations of a Li - Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was confirmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.

Characterization of p53 oligomerization domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family members.

p53 is a tumour suppressor gene which functions as a transcription factor to upregulate genes for growth arrest and apoptosis following DNA damage. p53 mutations are associated with Li-Fraumeni and Li-Fraumeni like syndromes. Recently mutations of the oligomerization domain have been isolated from an LFS and an LFL family affecting respectively codon 344 (Leu to Pro) and 337 (Arg to Cys). The present study was designed to determine the affect of these mutations on the function of p53 protein. p53 344 Leu to Pro existed only in a monomeric form and could not bind to DNA. It was inactive at inducing apoptosis, transactivating luciferase from a bax promoter and inhibiting cell growth. In contrast, p53 337 Arg to Cys could form tetramers and could bind to DNA. However, p53 337 Arg to Cys was not fully active and could only induce apoptosis, transactivate luciferase from a bax promoter and inhibit cell growth with approximately 60% of the ability of wild-type p53. Both mutant proteins had reduced ability to bind to MDM2, p53 337 Arg to Cys being more reduced than p53 344 Leu to Pro. These results indicate that point mutations in the oligomerization domain can disrupt p53 function. In addition, the value of LFS and LFL families for the further understanding of the biological and biochemical properties of p53 is demonstrated.

Structural organization of the bovine gene for the heart/muscle isoform of cytochrome c oxidase subunit VIa.

The bovine gene for the nuclear-encoded heart/muscle isoform of cytochrome c oxidase subunit VIa (COX6A1) was isolated from a library of bovine genomic DNA in lambda EMBL3 and sequenced. The gene spans 760 bp and comprises three exons and two small introns. Exon 1 encodes a 193 bp 5' untranslated region, a 12 amino acid presequence, and the first 12 amino acids of the mature COX VIa protein. Exon 2 encodes amino acids 13 to 58, and exon 3 amino acids 59 to 85 plus the 35 bp 3' untranslated region. Exons 2 and 3 are separated by a small intron of only 96 bp. All exon-intron boundaries matched the consensus splice junction sequences. COX6A1 transcripts are present in RNA from bovine heart but not brain. Primer extension and ribonuclease protection assays were used to map the 5' ends of COX6A1 transcripts in heart; both methods identified several clusters of transcription initiation sites, indicating that COX6A1 mRNA is heterogeneous at the 5' end. The proximal 5' flanking region is AT-rich and contains potential basal promoter elements, such as TATA and CCAAT boxes, associated with tissue-specific genes. A single consensus binding site for the muscle-specific transcription factor, MyoD1, was also located within this AT-rich region. The distal promoter region contained a perfect AP4 site plus potential binding sites for enhancer elements (NRF-1, Mt1, Mt3, and Mt4) proposed to regulate expression of genes for mitochondrial proteins.

Sequence of the cDNA for the liver/non-muscle isoform of mouse cytochrome-c oxidase subunit VIII.

We have isolated and sequenced the cDNA for the liver (L) or non-muscle isoform of mouse cytochrome-c oxidase subunit VIII (COX VIII-L). Comparison of deduced COX VIII-L protein sequences from three mammalian species indicated that the human gene has sustained more amino acid replacement substitutions than either the mouse or the cow. The most highly conserved regions of this subunit are the N-terminal presequence and the C-terminal domain of the mature protein.

The chicken cDNA for ornithine decarboxylase antizyme.

Two full length avian cDNAs for ornithine decarboxylase antizyme were isolated from a chicken cochlear cDNA library and differed in length through use of alternative poly(A) addition signals. The chick antizyme protein sequence predicted by translational frameshifting of the mRNA is 216 amino acids long and is more similar to Xenopus antizyme than to the mammalian protein.

The cDNA for the heart/muscle isoform of bovine cytochrome c oxidase subunit VIa encodes a presequence.

We have used mixed oligonucleotide probes to isolate a cDNA for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa (COX VIa-H) from a bovine heart cDNA library in lambda gt10. This cDNA, and a second one isolated upon rescreening, predict a 97 amino acid COX VIa precursor protein comprised of a 12 amino acid, basic presequence plus an 85 residue mature VIa protein. The presence of a presequence contrasts with the rat heart COX VIa cDNA.