Extracting DNA from 'jaws': high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material.

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield.

Effects of continuous positive airway pressure on systemic inflammation in patients with moderate to severe obstructive sleep apnoea: a randomised controlled trial.

BACKGROUND: Obstructive sleep apnoea syndrome (OSAS) has been associated with cardiovascular disease in epidemiological and observational studies. Continuous positive airway pressure (CPAP) is the treatment of choice for OSAS, but the impact of this intervention on systemic inflammation involved in the atherosclerotic process remains unclear. METHODS: 100 men with moderate-severe OSAS were randomised to therapeutic (n = 51) or subtherapeutic (n = 49) CPAP treatment for 4 weeks to investigate the effects of active treatment on inflammatory markers such as highly sensitive C reactive protein (hsCRP), interleukin (IL)6, interferon gamma (IFNgamma) and anti-inflammatory adiponectin. RESULTS: 4 weeks of therapeutic CPAP did not significantly change blood levels of hsCRP compared with the subtherapeutic control group (difference between median changes -0.24 mg/l (95% CI -0.88 to +0.24); p = 0.30). Plasma levels of IL6 and IFNgamma did not change significantly following therapeutic compared with subtherapeutic CPAP (difference between median changes +0.52 and -0.07 pg/ml (95% CI -0.72 to +1.94 and -0.81 to +0.44); p = 0.45 and p = 0.82, respectively). Furthermore, 4 weeks of therapeutic CPAP did not significantly change levels of adiponectin in plasma compared with the subtherapeutic control group (difference between median changes +0.05 pg/ml (95% CI -0.36 to +0.47); p = 0.84). If patients with hsCRP values above 8 mg/l at baseline were excluded, differences between the changes in hsCRP, IL6, IFNgamma and adiponectin after 4 weeks of CPAP were smaller, and again not statistically different between groups. CONCLUSIONS: 4 weeks of CPAP treatment has no beneficial effect on blood markers of inflammation and adiponectin in patients with moderate-severe obstructive sleep apnoea.

CPAP and measures of cardiovascular risk in males with OSAS.

Obstructive sleep apnoea syndrome (OSAS) has been associated with hypertension, stroke and myocardial ischaemia in epidemiological and observational studies. Continuous positive airway pressure (CPAP) is the treatment of choice for OSAS, but the impact of this intervention on established risk factors for cardiovascular disease remains incompletely understood. A total of 102 males with moderate-to-severe OSAS were randomised to therapeutic (n = 51) or subtherapeutic (n = 51) CPAP treatment for 4 weeks to investigate the effects of active treatment on 24-h urinary catecholamine excretion, baroreflex sensitivity (BRS), arterial stiffness (augmentation index) and 24-h ambulatory blood pressure (ABP). After 4 weeks of therapeutic CPAP, significant reductions were seen in urine normetanephrine excretion (from mean+/-sd 179.7+/-80.1 to 132.7+/-46.5 micromol x mol(-1) creatinine) and augmentation index (from 14.5+/-11.3 to 9.1+/-13.8%) compared with the subtherapeutic control group. Furthermore, therapeutic CPAP significantly improved BRS (from 7.1+/-3.3 to 8.8+/-4.2 ms x mmHg(-1)) and reduced mean arterial ABP by 2.6+/-5.4 mmHg. In conclusion, treatment of obstructive sleep apnoea with continuous positive airway pressure may lower cardiovascular risk by reducing sympathetic nerve activity, ambulatory blood pressure and arterial stiffness and by increasing sensitivity of the arterial baroreflex.

Pheasant rearer's lung.

A 47-year-old gamekeeper presented with an 8 month history of variable breathlessness, cough and clinical features of severe interstitial lung disease. Open lung biopsy showed an extrinsic allergic alveolitis, which we believe related to his work rearing pheasants. Initially he was resistant, despite advice, to changing his occupation but subsequently, although ceasing exposure to pheasants and beginning treatment with corticosteroids, his disease progressed to the point where he developed respiratory failure and was referred for lung transplantation. Sadly, he died of progressive respiratory failure and cor pulmonale complicated by bronchopneumonia before this could be achieved.

Circulating cardiovascular risk factors in obstructive sleep apnoea: data from randomised controlled trials.

BACKGROUND: Obstructive sleep apnoea (OSA) is associated with high cardiovascular morbidity and mortality and is an independent risk factor for hypertension. Novel circulating cardiovascular risk markers enabling a more accurate prediction of cardiovascular risk have been identified. Examination of these markers may clarify the increased risk in OSA and contribute to an analysis of the benefits of treatment. METHODS: Plasma levels of total cholesterol and triglyceride and activated coagulation factors XIIa and VIIa, factors VII, VIII, XII, fibrinogen, thrombin-antithrombin (TAT), von Willebrand factor antigen (vWFAg), soluble P-selectin (sP-sel), and homocysteine were measured before and after treatment for 1 month with therapeutic or subtherapeutic (control) continuous positive airways pressure (CPAP) in 220 patients with OSA. RESULTS: Levels of activated coagulation factors XIIa, VIIa, TAT and sP-sel were higher in OSA patients at baseline than in unmatched controls, but did not fall with 1 month of therapeutic CPAP treatment. The raised sP-sel levels correlated only with body mass index (p = 0.002). There was a trend towards a significant fall in total cholesterol with therapeutic CPAP (p = 0.06) compared with the control group. In the therapeutic group there was a clinically significant mean fall in total cholesterol of 0.28 mmol/l (95% confidence interval 0.11 to 0.45, p = 0.001) which may reduce cardiovascular risk by about 15%. CONCLUSION: A number of activated coagulation factors are increased in untreated OSA patients, potentially contributing to vascular risk, but they do not fall with 1 month of CPAP treatment. Nasal CPAP may produce a clinically relevant fall in total cholesterol level, potentially reducing cardiovascular risk, but this needs to be verified in a larger prospective study.

Caffeine levels following treatment of obstructive sleep apnoea.

BACKGROUND: Randomised trials show that treatment of obstructive sleep apnoea (OSA) with nasal continuous positive airway pressure (CPAP) greatly improves sleepiness and also lowers diurnal systemic blood pressures (BP). Such patients consume more coffee than controls (presumably to combat their sleepiness) and might reduce their consumption following effective treatment, thus lowering BP by this mechanism rather than via a direct effect of alleviating OSA. METHODS: Plasma caffeine levels before and after treatment with either therapeutic (n=52) or subtherapeutic (control, n=49) CPAP were measured in stored blood samples from a previous randomised controlled trial of CPAP for 4 weeks in patients with OSA. RESULTS: There was a small significant rise in caffeine levels when the two groups were analysed as a whole (p=0.02), but not individually. Despite the fall in sleepiness measured objectively in the therapeutic CPAP group, there was no difference in absolute (or change in) caffeine levels between the two groups (mean (SE) micro mol/l; therapeutic CPAP 9.2 (1.2), 10.2 (1.0), subtherapeutic 6.7 (0.9), 8.6 (0.9) before and after treatment, respectively). CONCLUSION: Reduced coffee consumption is unlikely to be the explanation for the falls in BP following treatment of OSA.

Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study.

BACKGROUND: The opportunistic fungus Pneumocystis jiroveci is a common cause of respiratory infection in immunocompromised patients. By contrast, pneumocystis pneumonia (PCP) occurs only rarely in immunocompetent individuals. Asymptomatic colonisation with P jiroveci has recently been described in patients who are either minimally immunosuppressed or who have underlying lung disorders such as bronchiectasis. We sought to determine the prevalence of asymptomatic colonisation by P jiroveci in a cohort of adult patients undergoing diagnostic bronchoscopy. METHODS: A prospective observational cohort study was performed in patients who required bronchoscopy and bronchoalveolar lavage (BAL) as part of their routine clinical assessment. All the samples underwent standard microbiological analysis and a Grocott methenamine silver stain was performed where clinically indicated to detect the presence of P jiroveci. Polymerase chain reaction for detection of P jiroveci specific DNA was also performed. RESULTS: Ninety three consecutive BAL fluid samples were analysed, 17 (18%) of which contained P jiroveci DNA. Of the potential predictors examined, only glucocorticoid use was significantly associated with detectable P jiroveci DNA. Eighteen patients were receiving oral glucocorticoids (equivalent to >20 mg/day prednisolone) at the time of bronchoscopy, of whom eight (44%) had detectable P jiroveci DNA. In contrast, P jiroveci was detected in only nine of 75 patients (12%) who were not receiving glucocorticoids (difference between proportions 32%, 95% CI 8 to 57; p=0.004, two tailed Fisher's exact test). CONCLUSIONS: P jiroveci colonisation, as determined by detection of P jiroveci DNA in BAL fluid, is common in HIV negative patients with primary respiratory disorders undergoing bronchoscopy and BAL. The higher prevalence in patients receiving corticosteroids suggests that oral glucocorticoid therapy is an independent risk factor for colonisation. In contrast, underlying lung cancer or COPD did not appear to be risk factors.

Sleep studies for sleep apnoea.

Sleep studies have grown to encompass a broad range of technologies employed to study and diagnose a variety of sleep disorders. From their inception in neurophysiology laboratories interested in investigating primary disorders of sleep architecture from psychiatric illness, their remit has widened such that their most common role is currently to diagnose secondary sleep disruption from respiratory, cardiovascular or other systemic causes. This review outlines the pathophysiology of obstructive sleep apnoea in particular and how sleep studies have improved our understanding of the complex dynamic changes in blood gas tensions, cardiovascular control and cerebral arousal that occur with these repetitive events. We review the historical development of standard laboratory-based sleep studies and discuss their limitations in staging sleep, reflecting the episodes of increased upper airway resistance that underlie these disorders and their ability to predict individuals' symptoms or response to medical or surgical therapies. We then describe some alternative signals that have been employed to monitor the physiological changes in upper airway resistance and arousal with a discussion of some of the evidence that these 'limited' studies may provide diagnostic information that can guide clinical decision making and may predict the outcome without the need, in some cases, for more complex and costly laboratory-based studies.

Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova.

Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i.

Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore.

OBJECTIVE: To develop an acceptable model system to study calcium activation of human oocytes. DESIGN: Study of oocyte development and intracellular calcium [Ca]i dynamics of activated oocytes. SETTING: Research center affiliated with infertility service. MAIN OUTCOME MEASURE: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca]i levels, oocytes were incubated with fura-2 (2 microM) for 30 minutes and [Ca]i was determined by rationing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. RESULT(S): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca]i, followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. CONCLUSION(S): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca]i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes.

The ovarian renin-angiotensin system. A paracrine-intracrine regulator of ovarian function.

We present a review that considers the regulation of ovarian function by the ovarian renin-angiotensin system. Evidence is presented that shows the ovarian renin-angiotensin system can regulate normal ovarian function by paracrine and intracrine mechanisms. In addition, the complexity that is inherent in the renin-angiotensin system conferred by multiple biological angiotensin peptides and multiple receptors is discussed. Evidence that a dysfunctional OVRAS induces ovarian pathology such as polycystic ovarian disease is also presented.

The basis and evidence of a role for the ovarian renin-angiotensin system in health and disease.

OBJECTIVE: We reviewed the evidence for an intrinsic ovarian renin-angiotensin system (OVRAS), highlighting potential diverse signaling in this system through different bioactive angiotensin peptides, their specific receptors, and second messengers. In addition, sites of action for OVRAS in the regulation of ovarian function in health and disease were reviewed. DATA SOURCES: We used published journals and abstracts from national scientific meetings. Current developments in the renin-angiotensin field are historically set. STUDY SELECTION: One hundred referenced articles provided studies on renin-angiotensin systems in mammalian species, including humans. DATA ABSTRACTION: Interpretation of the reviewed publication was in line with the original authors' conclusions and statistical analysis. DATA SYNTHESIS: Techniques in molecular biology, biochemistry, and immunohistochemistry have identified an OVRAS in mammalian species. Ovarian tissues contain all the elements for the production of angiotensin, including prorenin/renin, angiotensinogen, and angiotensin-converting enzyme. In addition, angiotensin II is present in ovarian compartments, and receptors for angiotensin II are demonstrated on specific ovarian cells. Angiotensin II is implicated to play a role in ovulation, steroidogenesis, follicular atresia, and hyperandrogenic syndromes. CONCLUSIONS: The newly identified OVRAS may have important actions in the ovary that range from regulation of ovulation to ovarian dysfunction, such as hyperandrogenic syndromes in women. In this respect, the OVRAS is a putative paracrine/autocrine regulator in the ovary, and pharmacologic regulation of the OVRAS may provide new methods for the management of fertility and reproduction.

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells.

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.

The in vitro perifused rat ovary: III. Interrelationship of the follicular and stromal compartments on steroid release.

The preovulatory ovary is composed of two primary tissue components, stroma and follicles. To assess the role of these tissue compartments in ovarian steroidogenesis, stromal tissue, follicular tissue, and a mixture of both tissues from pregnant mare serum gonadotropin (PMSG)-treated, prepubertal rats were perifused simultaneously for 8 h. The basal level of estradiol secretion by stromal tissue was lower (24 +/- 3 pg/mg/30 min), than that secreted by follicles (64 +/- 5.6 pg/mg/30 min, n = 6; p < 0.05). On the other hand, the mean basal levels of progesterone and testosterone secreted by stromal tissue (252 +/- 14 pg/mg/30 min and 162 +/- 18 pg/mg/30 min, respectively) were greater than those secreted by follicular tissue (84 +/- 3 pg/mg/30 min and 81 +/- 4 pg/mg/30 min, respectively). When stromal and follicular tissue were combined the secretion of progesterone, testosterone and estradiol was intermediate to that of the separate tissues. Under gonadotropin stimulation (human menopausal gonadotropins plus follicle stimulating hormone), the follicular tissue secreted greater amounts of steroids than did the stromal tissue, or stromal plus follicular tissue. When stromal tissue and follicular tissue were combined, the levels of basal progesterone and testosterone secreted by both tissues were significantly lower than those of stromal tissue alone. However, the reduction in follicular estradiol secretion induced by stromal tissue under basal conditions, was in large part overcome during gonadotropin perifusion. These observations suggest that locally produced factors may play an inhibitory, paracrine role in the regulation of ovarian steroidogenesis.

The in vitro perifused rat ovary: V. The significance of the follicle stimulating hormone and luteinizing hormone ratio on steroid release.

In these studies, an in vitro perifusion model was used to compare stimulation of ovarian tissue with either human menopausal gonadotropin (hMG), which is an equal mixture of luteinizing hormone (LH) and follicle stimulating hormone (FSH), or with hMG plus added human FSH. Eight-hour perifusion studies were conducted on either whole, or dissected clusters of follicles from pregnant mare serum gonadotropin (PMSG)-treated rats. In the two groups, similar stimulatory protocols were used, consisting of a ramp stimulation over 60 min with either hMG (0-8 mIU/ml) or hMG plus FSH (0-8 mIU/ml hMG + 0-8 mIU/ml FSH), followed by hourly pulse stimulation with hMG (8-18 mIU/ml) or hMG plus FSH (8-18 mIU/ml hMG + 8-18 mIU/ml FSH), respectively. In the whole ovaries, no differences were detected in progesterone, testosterone, or estradiol secretion. However, in the cluster of follicles, an elevated hFSH/hMG ratio resulted in a significantly higher secretion of progesterone, testosterone and estradiol (n = 8; p < 0.05) than the steroids secreted by follicles perifused with hMG alone. In conclusion, an elevated FSH: LH ratio led to greater steroidogenic responses by the perifused cluster of follicles, but not by the whole ovary.