WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Comparison of four-year toxicities and local control of ultra-hypofractionated vs moderate-hypofractionated image guided prostate radiation with HDR brachytherapy boost: A phase I-II single institution trial.

Purpose/Objectives: To analyze the long term efficacy and safety of an ultra-hypofractionated (UHF) radiation therapy prostate treatment regimen with HDR brachytherapy boost (BB) and compare it to moderate-hypofractionated regimens (MHF). Materials/Methods: In this single arm, prospective monocentric study, 28 patients with intermediate risk prostate cancer were recruited in an experimental treatment arm of 25 Gy in 5 fractions plus a 15 Gy HDR BB. They were then compared to two historical control groups, treated with either 36 Gy in 12 fractions or 37.5 Gy in 15 fractions with a similar HDR BB. The control groups included 151 and 311 patients respectively. Patient outcomes were reported using the International Prostate Symptom Score (IPSS) and Expanded Prostate Index Composite (EPIC-26) questionnaires at baseline and at each follow-up visit. Results: Median follow-up for the experimental arm was 48.5 months compared to 47 months and 60 months compared to the 36/12 and 37,5/15 groups respectively. The IPSS and EPIC scores did not demonstrate any significant differences in the gastrointestinal or genitourinary domains between the three groups over time. No biochemical recurrence occurred in the UHF arm as defined by the Phoenix criterion. Conclusion: The UHF treatment scheme with HDR BB seems equivalent to standard treatment arms in terms of toxicities and local control. Randomized control trials with larger cohorts are ongoing and needed to further confirm our findings.

Effect of hypoxia exposure on the recovery of skeletal muscle phenotype during regeneration.

Hypoxia impairs the muscle fibre-type shift from fast-to-slow during post-natal development; however, this adaptation could be a consequence of the reduced voluntary physical activity associated with hypoxia exposure rather than the result of hypoxia per se. Moreover, muscle oxidative capacity could be reduced in hypoxia, particularly when hypoxia is combined with additional stress. Here, we used a model of muscle regeneration to mimic the fast-to-slow fibre-type conversion observed during post-natal development. We hypothesised that hypoxia would impair the recovery of the myosin heavy chain (MHC) profile and oxidative capacity during muscle regeneration. To test this hypothesis, the soleus muscle of female rats was injured by notexin and allowed to recover for 3, 7, 14 and 28 days under normoxia or hypobaric hypoxia (5,500 m altitude) conditions. Ambient hypoxia did not impair the recovery of the slow MHC profile during muscle regeneration. However, hypoxia moderately decreased the oxidative capacity (assessed from the activity of citrate synthase) of intact muscle and delayed its recovery in regenerated muscle. Hypoxia transiently increased in both regenerated and intact muscles the content of phosphorylated AMPK and Pgc-1α mRNA, two regulators involved in mitochondrial biogenesis, while it transiently increased in intact muscle the mRNA level of the mitophagic factor BNIP3. In conclusion, hypoxia does not act to impair the fast-to-slow MHC isoform transition during regeneration. Hypoxia alters the oxidative capacity of intact muscle and delays its recovery in regenerated muscle; however, this adaptation to hypoxia was independent of the studied regulators of mitochondrial turn-over.

Ambient hypoxia enhances the loss of muscle mass after extensive injury.

Hypoxia induces a loss of skeletal muscle mass and alters myogenesis in vitro, but whether it affects muscle regeneration in vivo following injury remains to be elucidated. We hypothesized that hypoxia would impair the recovery of muscle mass during regeneration. To test this hypothesis, the soleus muscle of female rats was injured by notexin and allowed to recover for 3, 7, 14, and 28 days under normoxia or hypobaric hypoxia (5,500 m) conditions. Hypoxia impaired the formation and growth of new myofibers and enhanced the loss of muscle mass during the first 7 days of regeneration, but did not affect the final recovery of muscle mass at 28 days. The impaired regeneration under hypoxic conditions was associated with a blunted activation of mechanical target of rapamycin (mTOR) signaling as assessed by p70(S6K) and 4E-BP1 phosphorylation that was independent of Akt activation. The decrease in mTOR activity with hypoxia was consistent with the increase in AMP-activated protein kinase activity, but not related to the change in regulated in development and DNA response 1 protein content. Hypoxia increased the mRNA levels of the atrogene muscle ring finger-1 after 7 days of regeneration, though muscle atrophy F box transcript levels remained unchanged. The increase in MyoD and myogenin mRNA expression with regeneration was attenuated at 7 days with hypoxia. In conclusion, our results support the notion that the enhanced loss of muscle mass observed after 1 week of regeneration under hypoxic conditions could mainly result from the impaired formation and growth of new fibers resulting from a reduction in protein synthesis and satellite cell activity.

Effect of hypoxia exposure on the phenotypic adaptation in remodelling skeletal muscle submitted to functional overload.

AIM: To determine whether hypoxia influences the phenotypic adaptation of skeletal muscle induced by mechanical overload. METHODS: Plantaris muscles of female rats were submitted to mechanical overload following synergist ablation. After 3 days of overload, rats were exposed to either hypobaric hypoxia (equivalent to 5500 m) or normoxia. Muscles were collected after 5, 12 and 56 days of overload (i.e. after 3, 9 and 53 days of hypoxia). We determined the myosin heavy chain (MHC) distribution, mRNA levels of myocyte-enriched calcineurin-integrating protein 1 (MCIP1) to indirectly assess calcineurin activity, the changes in oxidative capacity from the activities of citrate synthase (CS) and cytochrome c oxidase (COX), and the expression of regulators involved in mitochondrial biogenesis (Pgc-1α, NRF1 and Tfam) and degradation (BNIP-3). RESULTS: Hypoxia did not alter the fast-to-slow MHC shift and the increase in calcineurin activity induced by overload; it only transiently slowed down the overload-induced transition in MHC isoforms. Hypoxia similarly decreased CS and COX activities in overloaded and control muscles. Nuclear respiratory factor 1 (NRF1) and transcription factor A (Tfam) mRNA and BNIP-3 protein were not influenced by hypoxia in overloaded muscles, whereas Pgc-1α mRNA and protein contents did not correlate with changes in oxidative capacity. CONCLUSION: Hypoxia is not a critical stimulus to modulate the fast-to-slow MHC transition associated with overload. Thus, the impairment of the fast-to-slow fibre shift often observed during post-natal development in hypoxia could be explained by the lower voluntary locomotor activity associated with hypoxia. Hypoxia alters mitochondrial oxidative capacity, but this adaptive response is similar in overloaded and control muscles.

Pitfalls in target mRNA quantification for real-time quantitative RT-PCR in overload-induced skeletal muscle hypertrophy.

Quantifying target mRNA using real-time quantitative reverse transcription-polymerase chain reaction requires an accurate normalization method. Determination of normalization factors (NFs) based on validated reference genes according to their relative stability is currently the best standard method in most usual situations. This method controls for technical errors, but its physiological relevance requires constant NF values for a fixed weight of tissue. In the functional overload model, the increase in the total RNA concentration must be considered in determining the NF values. Here, we pointed out a limitation of the classical geNorm-derived normalization. geNorm software selected reference genes despite that the NF values extensively varied under experiment. Only the NF values calculated from four intentionally selected genes were constant between groups. However, a normalization based on these genes is questionable. Indeed, three out of four genes belong to the same functional class (negative regulator of muscle mass), and their use is physiological nonsense in a hypertrophic model. Thus, we proposed guidelines for optimizing target mRNA normalization and quantification, useful in models of muscle mass modulation. In our study, the normalization method by multiple reference genes was not appropriate to compare target mRNA levels between overloaded and control muscles. A solution should be to use an absolute quantification of target mRNAs per unit weight of tissue, without any internal normalization. Even if the technical variations will stay present as a part of the intergroup variations, leading to less statistical power, we consider this method acceptable because it will not generate misleading results.

Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo.

OBJECTIVES: The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts. MATERIALS AND METHODS: Rat L6 and primary human myoblasts were grown in 21% or 1% O(2) in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT-PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique. RESULTS: After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O(2) conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts. CONCLUSIONS: The data show that exposure to hypoxic conditions (1% O(2)) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.

Nutritional status and dietary adequacy in rural communities of a protected area in Gabon.

OBJECTIVE: As part of a larger study designed to understand how to protect the food and nutrition security of individuals living in a protected area of Gabon, we assessed their nutritional status and its relationship to dietary adequacy and health status. DESIGN: A 7 d food consumption survey was conducted during each of the two major seasons using a weighing method. Data were also collected on weight, height and health of individuals as well as on sociodemographic characteristics and potential determinants of the nutrition situation. SETTING: Four rural communities were intentionally selected to represent both inland and coastal settings and access to food markets. SUBJECTS: Approximately 500 individuals representing over 90% of the population of these communities participated in the survey during each season. RESULTS: Undernutrition was present in the area, particularly among children <5 years of age and the elderly. Health was generally good and under-fives were most frequently ill. Energy, Fe and vitamin A requirements of individuals were generally not satisfied; the opposite was true for protein. The estimated prevalence of inadequate intakes of energy and vitamin A was very high in most age groups. Global nutrient adequacy was associated with nutritional outcome. CONCLUSIONS: Individuals do not eat enough and breast-feeding practices are poor. Many suffer from undernutrition, particularly young children and the elderly. The results confirm the need to investigate the determinants of this poor nutrition situation to ensure that protection of natural resources will not be associated with harm to the well-being of the population.

A polysomnographic study of daytime sleepiness in myotonic dystrophy type 1.

OBJECTIVES: To assess contributors to excessive daytime sleepiness (EDS) in myotonic dystrophy type 1 (DM1), to characterise subjects with sleep-onset REM periods (SOREMPs), and to verify whether self-reported instruments and respiratory function tests can predict multiple sleep latency test (MSLT) and sleep-disordered breathing. METHODS: A sample of 43 DM1 patients without selection bias underwent polysomnography (PSG) for two consecutive nights and MSLT, completed a sleep diary and Epworth Sleepiness Scale (ESS), and were assessed for respiratory function and narcolepsy symptoms. RESULTS: ESS scores (ES) > or =11 and MSLT mean sleep latency (MSL) < or =8 min were found in 21 (50.0%) and 19 (44.2%) subjects, and either in 30 (69.8%) subjects. ES did not relate to MSL. Subjects with subjective sleepiness (ES> or =11) reported more cataplexy-like and sleep paralysis symptoms, longer habitual sleep times, and higher sleep efficiency and REM sleep per cent than those without. Subjects with objective sleepiness (MSL< or =8 min) had a higher stage 4 sleep per cent. Subjects with > or =2 SOREMPs (25.6%) showed higher muscular impairment, lower MSL, higher ES, and more cataplexy-like symptoms than those with < or =1 SOREMP. Apnoea-hypopnoea index (AHI) > or =5, predominantly obstructive, was found in 37 (86.0%) subjects, and AHI >30 in 12 (27.9%). Neither subjective nor objective sleepiness could be explained by AHI, nor satisfactorily predicted by daytime respiratory abnormalities. CONCLUSIONS: DM1 entails frequent EDS but with different phenotypes and distinct mechanisms involved. The high prevalence of daytime sleepiness and severe sleep apnoeas found in this study supports the routine use of clinical sleep interviews, PSG and MSLT in DM1, and emphasises the need for more randomised trials of psychostimulants.

Determinants of undernutrition in rural communities of a protected area in Gabon.

OBJECTIVE: To understand how access to natural resources may contribute to nutrition. DESIGN: In each of the two major seasons, data were collected during a 7 d period using observations, semi-structured interviews, anthropometric measures and a weighed food consumption survey. SETTING: Four rural communities selected to represent inland and coastal areas of the Gamba Complex in Gabon. SUBJECTS: In each community, all individuals from groups vulnerable to malnutrition, i.e. children aged 0-23 months (n 41) and 24-59 months (n 63) and the elderly (n 101), as well as women caregivers (n 96). RESULTS: In most groups, household access to natural resources was associated with household access to food but not with individual nutritional status. In children aged 0-23 months, access to care and to health services and a healthy environment were the best predictors of length-for-age (adjusted R2: 14%). Health status was the only predictor of weight-for-height in children aged 24-59 months (adjusted R2: 14%). In women caregivers, household food security was negatively associated with nutritional status, as was being younger than 20 years (adjusted R2: 16%). Among the elderly, only nutrient adequacy predicted nutritional status (adjusted R2: 5%). CONCLUSION: Improving access to care and health for young children would help reverse the process of undernutrition. Reaching a better understanding of how the access of individuals to both food and other resources relate to household access could further our appreciation of the constraints to good nutrition. This is particularly relevant in women to ensure that their possibly important contribution to the household is not at their own expense.

Brain stem NO modulates ventilatory acclimatization to hypoxia in mice.

The objective of our study was to assess the role of neuronal nitric oxide synthase (nNOS) in the ventilatory acclimatization to hypoxia. We measured the ventilation in acclimatized Bl6/CBA mice breathing 21% and 8% oxygen, used a nNOS inhibitor, and assessed the expression of N-methyl-d-aspartate (NMDA) glutamate receptor and nNOS (mRNA and protein). Two groups of Bl6/CBA mice (n = 60) were exposed during 2 wk either to hypoxia [barometric pressure (PB) = 420 mmHg] or normoxia (PB = 760 mmHg). At the end of exposure the medulla was removed to measure the concentration of nitric oxide (NO) metabolites, the expression of NMDA-NR1 receptor, and nNOS by real-time RT-PCR and Western blot. We also measured the ventilatory response [fraction of inspired O(2) (Fi(O(2))) = 0.21 and 0.08] before and after S-methyl-l-thiocitrulline treatment (SMTC, nNOS inhibitor, 10 mg/kg ip). Chronic hypoxia caused an increase in ventilation that was reduced after SMTC treatment mainly through a decrease in tidal volume (Vt) in normoxia and in acute hypoxia. However, the difference observed in the magnitude of acute hypoxic ventilatory response [minute ventilation (Ve) 8% - Ve 21%] in acclimatized mice was not different. Acclimatization to hypoxia induced a rise in NMDA receptor as well as in nNOS and NO production. In conclusion, our study provides evidence that activation of nNOS is involved in the ventilatory acclimatization to hypoxia in mice but not in the hypoxic ventilatory response (HVR) while the increased expression of NMDA receptor expression in the medulla of chronically hypoxic mice plays a role in acute HVR. These results are therefore consistent with central nervous system plasticity, partially involved in ventilatory acclimatization to hypoxia through nNOS.

Role of maximal heart rate and arterial O2 saturation on the decrement of VO2max in moderate acute hypoxia in trained and untrained men.

We aimed to evaluate 1) the altitude where maximal heart rate (HR (max)) decreases significantly in both trained and untrained subjects in moderate acute hypoxia, and 2) if the HR (max) decrease could partly explain the drop of V.O (2max). Seventeen healthy males, nine trained endurance athletes (TS) and eight untrained individuals (US) were studied. Subjects performed incremental exercise tests at sea level and at 5 simulated altitudes (1000, 1500, 2500, 3500, 4500 meters). Power output (PO), heart rate (HR), arterial oxygen saturation (SaO (2)), oxygen uptake (V.O (2)), arterialized blood pH and lactate were measured. Both groups showed a progressive reduction in V.O (2max). The decrement in HR (max) (DeltaHR (max)) was significant from 1000 m for TS and 2500 m for US and more important in TS than US (at 1500 m and 3500 m). At maximal exercise, TS had a greater reduction in SaO (2) (DeltaSaO (2)) at each altitude. DeltaHR (max) observed in TS was correlated with DeltaSaO (2). When the two groups were pooled, simple regressions showed that DeltaV.O (2max) was correlated with both DeltaSaO (2) and DeltaHR (max). However, a multiple regression analysis demonstrated that DeltaSaO (2) alone may account for DeltaV.O (2max). Furthermore, in spite of a greater reduction in SaO (2) and HR (max) in TS, no difference was evidenced in relative DeltaV.O (2max) between groups. Thus, in moderate acute hypoxia, the reduction in SaO (2) is the primary factor to explain the drop of V.O (2max) in trained and untrained subjects.

The faculty portfolio: documenting the scholarship of teaching.

The scholarship of teaching is considered an essential component of scholarship within academic settings. To promote the transfer of knowledge specific to the discipline of nursing, this category of scholarship must include inquiry into the practice of teaching, program development, and professional role modeling, in addition to excellence in teaching itself. Conveying what constitutes teaching scholarship in nursing may present special challenges for nurse faculty in the university setting. A faculty teaching portfolio is one mechanism for explicating, communicating, and enhancing the scholarship of teaching. The methodology for creating, improving, and maintaining a teaching portfolio includes analyzing the mission of the university, articulating a philosophy of teaching, deciding on goals and objectives, designing evaluative mechanisms, processing data, conducting a self-evaluation, applying new approaches, and revisiting and reflecting on the outcomes. Faculty teaching portfolios serve to display, communicate, and document the scholarship of teaching. The creative endeavors surrounding portfolio development are ongoing and recursive, necessitating self-reflection and new approaches. J Prof Nurs 17:180-186, 2001.

Relations between the stage of cell maturation and lactate transporter activities in rat neonatal muscle cells in culture.

Lactate transport was investigated in newborn rat muscle cells in culture. The aim was to study the lactate transport function at two stages of cell differentiation in culture: (i) during the proliferative phase characterized by myoblasts and myotubes (MyB/MyT2) obtained after 2-3 seedings, (ii) when myotubes (MyT1) grow old in culture after 8-9 seedings. In both developmental stages MyB/MyT2, lactate was carried following a saturable and sigmoidal velocity curve: the Hill and the Scatchard plot analyses confirmed an allosteric or multisite mechanism of lactate transport with two classes of carriers: one of low and one of high affinity i.e., 8.6 and 0.95 mm, respectively, which are associated with high and low transport capacities (V(m)) i.e., 9.1 and 0.67 nm/min/mg, respectively. With MyT1, the velocity curve of lactate transport presented a hyperbolic profile, and the Hill plot analysis gave a Hill number near one suggesting that for cell aging in culture the decrease in cooperativity shows that lactate transport essentially occurs through the low affinity transport system. Inhibitor effects also contributed to evidence for at least two systems of transport. Results obtained from primary cells give evidence for the early activity of lactate transport system at the Myb/MyT2 stage and its evolution during cell aging in culture (MyT1). Sarcolemmal lactate transport in primary cultures of myocytes is accomplished by multiple carriers, neither of which are MCT1 or MCT2 as confirmed by immunoblots.

Food insecurity: consequences for the household and broader social implications.

A conceptual framework showing the household and social implications of food insecurity was elicited from a qualitative and quantitative study of 98 households from a heterogeneous low income population of Quebec city and rural surroundings; the study was designed to increase understanding of the experience of food insecurity in order to contribute to its prevention. According to the respondents' description, the experience of food insecurity is characterized by two categories of manifestations, i.e., the core characteristics of the phenomenon and a related set of actions and reactions by the household. This second category of manifestations is considered here as a first level of consequences of food insecurity. These consequences at the household level often interact with the larger environment to which the household belongs. On a chronic basis, the resulting interactions have certain implications that are tentatively labeled "social implications" in this paper. Their examination suggests that important aspects of human development depend on food security. It also raises questions concerning the nature of socially acceptable practices of food acquisition and food management, and how such acceptability can be assessed. Guidelines to that effect are proposed. Findings underline the relevance and urgency of working toward the realization of the right to food.

Dietary patterns of adults in Québec and their nutritional adequacy.

The purpose of this study was to identify dietary patterns among adults in Québec and to determine their relationship to nutritional adequacy of the diet. We used 24-hour food recall data on 2,104 adults from the Québec nutrition survey (1990). Nutritional adequacy was assessed based on the 1990 Nutrition Recommendations for Canadians; dietary patterns were assessed via a factor analysis of the 30 food groups consumed. The three major patterns identified ('high-energy density', 'traditional' and 'health-conscious') explained 18% of the variation in food intake. Only the 'health-conscious' pattern correlated positively with the four chosen indicators of nutritional adequacy. Generally, men scored positively on the 'high-energy density' and the 'traditional' pattern whereas women scored positively on the 'health-conscious' pattern. Aside from sex, scoring was most related to age and education. The use of these patterns to define and target nutrition interventions should be tested in the aim of improving the effectiveness of health promotion.