Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans.
The extensive oxygen gradient between the air we breathe (Po2 ~21 kPa) and its ultimate distribution within mitochondria (as low as ~0.5-1 kPa) is testament to the efforts expended in limiting its inherent toxicity. It has long been recognized that cell culture undertaken under room air conditions falls short of replicating this protection in vitro. Despite this, difficulty in accurately determining the appropriate O2 levels in which to culture cells, coupled with a lack of the technology to replicate and maintain a physiological O2 environment in vitro, has hindered addressing this issue thus far. In this review, we aim to address the current understanding of tissue Po2 distribution in vivo and summarize the attempts made to replicate these conditions in vitro. The state-of-the-art techniques employed to accurately determine O2 levels, as well as the issues associated with reproducing physiological O2 levels in vitro, are also critically reviewed. We aim to provide the framework for researchers to undertake cell culture under O2 levels relevant to specific tissues and organs. We envisage that this review will facilitate a paradigm shift, enabling translation of findings under physiological conditions in vitro to disease pathology and the design of novel therapeutics.


The impact of kidney function at highly active antiretroviral therapy initiation on mortality in HIV-infected women.
In the early highly active antiretroviral therapy (HAART) era, kidney dysfunction was strongly associated with death among HIV-infected individuals. We re-examined this association in the later HAART period to determine whether chronic kidney disease remains a predictor of death after HAART initiation. To evaluate the effect of kidney function at the time of HAART initiation on time to all-cause mortality, we evaluated 1415 HIV-infected women initiating HAART in the Women's Interagency HIV Study. Multivariable proportional hazards models with survival times calculated from HAART initiation to death were constructed; participants were censored at the time of the last available visit or December 31, 2006. Chronic kidney disease (estimated glomerular filtration rate less than 60 mL/min/1.73 m) at HAART initiation was associated with higher mortality risk adjusting for age, race, hepatitis C serostatus, AIDS history, and CD4 cell count (hazard ratio 2.23, 95% confidence interval: 1.45-3.43). Adjustment for hypertension and diabetes history attenuated this association (hazard ratio = 1.89, confidence interval: 0.94-3.80). Lower kidney function at HAART initiation was weakly associated with increased mortality risk in women with prior AIDS (hazard ratio = 1.09, confidence interval: 1.00-1.19, per 20% decrease in estimated glomerular filtration rate). Kidney function at HAART initiation remains an independent predictor of death in HIV-infected individuals, especially in those with a history of AIDS. Our study emphasizes the necessity of monitoring kidney function in this population. Additional studies are needed to determine mechanisms underlying the increased mortality risk associated with chronic kidney disease in HIV-infected persons.


Structural color in Myxomycetes.
In this paper we report evidence of structural color in Myxomycetes, a group of eukaryotic microorganisms with an uncertain taxonomic position. We investigated the Diachea leucopoda, which belongs to the Physarales order, Myxomycetes class, and found that its peridium -protective layer that encloses the mass of spores- is basically a corrugated layer of a transparent material, which produces a multicolored pointillistic effect, characteristic of this species. Scanning (SEM) and transmission (TEM) electron microscopy techniques have been employed to characterize the samples. A simple optical model of a planar slab is proposed to calculate the reflectance. The chromaticity coordinates are obtained, and the results confirm that the color observed is a result of an interference effect.


Maternal vitamin D supplementation during pregnancy and lactation to promote infant growth in Dhaka, Bangladesh (MDIG trial): study protocol for a randomized controlled trial.
Vitamin D regulates bone mineral metabolism and skeletal development. Some observational studies have suggested that prenatal vitamin D deficiency increases the risk of adverse pregnancy and/or birth outcomes; however, there is scant evidence from controlled trials, leading the World Health Organization to advise against routine vitamin D supplementation in pregnancy. Importantly, little is known about the effect of maternal vitamin D status on infant linear growth in communities in South Asia where stunting is highly prevalent and maternal-infant vitamin D status is commonly suboptimal. The Maternal Vitamin D for Infant Growth study is a randomized, placebo-controlled, dose-ranging trial of maternal vitamin D supplementation during pregnancy and lactation in Dhaka, Bangladesh. The primary aims are to estimate (1) the effect of maternal prenatal oral vitamin D3 supplementation (4200 IU/wk, 16,800 IU/wk, or 28,000 IU/wk, administered as weekly doses) versus placebo on infant length at 1 year of age and (2) the effect of maternal postpartum oral vitamin D3 supplementation (28,000 IU/wk) versus placebo on length at 1 year of age among infants born to women who received vitamin D 28,000 IU/wk during pregnancy. Generally healthy pregnant women (n = 1300) in the second trimester (17-24 weeks of gestation) are randomized to one of five parallel arms: placebo 4200 IU/wk, 16,800 IU/wk, or 28,000 IU/wk in the prenatal period and placebo in the postpartum period or 28,000 IU/wk in the prenatal period and 28,000 IU/wk in the postpartum period. Household- and clinic-based follow-up of mother-infant pairs is conducted weekly by trained personnel until 26 weeks postpartum and every 3 months thereafter. The primary trial outcome measure is length for age z-score at 1 year of age. Anthropometric measurements, clinical information, and biological specimens collected at scheduled intervals will enable the assessment of a range of maternal, perinatal, and infant outcomes. The role of vitamin D in maternal and infant health remains unresolved. This trial is expected to contribute unique insights into the effects of improving maternal-infant vitamin D status in a low-income setting where stunting and adverse perinatal outcomes represent significant public health burdens. ClinicalTrials.gov identifier: NCT01924013. Registered on 13 August 2013.


Object preference by walking fruit flies, Drosophila melanogaster, is mediated by vision and graviperception.
Walking fruit flies, Drosophila melanogaster, use visual information to orient towards salient objects in their environment, presumably as a search strategy for finding food, shelter or other resources. Less is known, however, about the role of vision or other sensory modalities such as mechanoreception in the evaluation of objects once they have been reached. To study the role of vision and mechanoreception in exploration behavior, we developed a large arena in which we could track individual fruit flies as they walked through either simple or more topologically complex landscapes. When exploring a simple, flat environment lacking three-dimensional objects, flies used visual cues from the distant background to stabilize their walking trajectories. When exploring an arena containing an array of cones, differing in geometry, flies actively oriented towards, climbed onto, and explored the objects, spending most of their time on the tallest, steepest object. A fly's behavioral response to the geometry of an object depended upon the intrinsic properties of each object and not a relative assessment to other nearby objects. Furthermore, the preference was not due to a greater attraction towards tall, steep objects, but rather a change in locomotor behavior once a fly reached and explored the surface. Specifically, flies are much more likely to stop walking for long periods when they are perched on tall, steep objects. Both the vision system and the antennal chordotonal organs (Johnston's organs) provide sufficient information about the geometry of an object to elicit the observed change in locomotor behavior. Only when both these sensory systems were impaired did flies not show the behavioral preference for the tall, steep objects.


Mouse hypoxia-inducible factor-1alpha is encoded by two different mRNA isoforms: expression from a tissue-specific and a housekeeping-type promoter.
Hypoxic induction of erythropoietin (Epo) and other oxygen-dependent genes is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transactivator consisting of an alpha and a beta subunit. We previously found that the mouse gene encoding HIF-1alpha harbors two alternative first exons (I.1 and I.2), giving rise to two different HIF-1alpha mRNA isoforms. Here, we show by RNase protection analysis that the exon I.1-derived mRNA isoform is differentially expressed in mouse tissues, being highest in kidney, tongue, stomach, and testis, but undetectable in liver, whereas the exon I.2 mRNA isoform is ubiquitously expressed. Sequence and methylation analysis showed that, in contrast to exon I.1, exon I.2 resides within a region showing typical features of a CpG island, known to be associated with the 5' end of housekeeping genes. We identified a 232-bp minimal exon I.2 promoter that strongly induced reporter gene expression in mouse L929 fibroblasts and Hepa1 hepatoma cells. In contrast to L929 cells, the exon I.1 promoter was inactive in Hepa1 cells and hypoxic exposure (1% O2) markedly reduced exon I.2 promoter activity in Hepa1 cells. Prolonged exposure of mice to hypoxia (7.5% O2 for up to 72 hours) also caused a decrease in liver HIF-1alpha mRNA, whereas aldolase mRNA levels increased. These findings might be related to the relatively low Epo levels in the adult liver.


Neonatal sepsis in Egypt associated with bacterial contamination of glucose-containing intravenous fluids.
Rates of sepsis exceeding 50% in a neonatal intensive care unit (NICU) in Cairo, Egypt, were not controlled by routine antimicrobial therapy. We investigated these conditions in September 2001. Case series and retrospective cohort studies were conducted on 2 groups of NICU infants admitted to an academic medical center between February 12 and July 31, 2001. Observation of clinical practices led us to culture in-use intravenous (i.v.) fluids and medications. We monitored rates of i.v. fluid contamination, clinical sepsis and mortality after interventions to establish new procedures for handling and disposal of i.v. fluids, infection control training and improved clinical laboratory capacity. Among infants in the retrospective cohort group, 88 (77%) of 115 had clinical sepsis, and 59 (51%) died. In the case series group, we documented the time of initial positive blood culture; 21 (64%) of 33 were septic <24 hours after birth. Klebsiella pneumoniae accounted for 24 (73%) of 33 isolates; 14 (58%) of 24 were extended spectrum beta-lactamase-producing and aminoglycoside-resistant. On admission, all neonates received glucose-containing i.v. fluids; i.v. bottles (500 mL) were divided among multiple infants. The i.v. fluids were prepared at the bedside; poor hand hygiene and poor adherence to aseptic techniques were observed. K. pneumoniae was isolated from 13 (65%) of 20 in-use glucose-containing i.v. fluids. Fluid contamination, sepsis and mortality rates declined significantly after intervention. Extrinsically contaminated i.v. fluids resulted in sepsis and deaths. Standard infection control precautions significantly improve mortality and sepsis rates and are prerequisites for safe NICU care.


Gastroschisis in a developing country: poor resuscitation is a more significant predictor of mortality than postnasal transfer time.
The time from birth to the first paediatric surgical consultation of neonates with gastroschisis is a predictor of mortality in dveloping countries. This is contrary to findings in the developed world. We set out to document this relationship within our population. Neonates with gastroschisis who were transferred to Steve Biko Academic Hospital within the study period were included. The association between mortality and demographic, clinical and biochemical variables was assessed. Significant variables after univariate analysis were subjected to multivariates regression. Sixty patients were included. The mortality rate was 65%. Mean transfer time and distance were 14.9 hours and 225km. Forty-eight per cent of the neonates were either dehydrated or in hypovolaemic shock clinically on arrival. Eight neonates arrived hypothermic. It was shown through univariate analysis that female sex, appropriate weight for gestational age, hydration status, gestation, transfer time, serum urea, base deficit and serum bicarbonate (HCO3) were significant predictors of mortality. Only female sex, appropriate weight for gestational age and serum HC03 were shown to be significant using ultivariate analysis. Our high mortality rate was not due to lengthy transfer times. The poor clinical condition of the patients on arrival at our hospital, which relates to deficiencies in the neonatal transfer system, had a direct impact on the survival of neonates with gastroschisis.


Natural course of scoliosis and lifetime risk of scoliosis surgery in spinal muscular atrophy.
To investigate the natural course of scoliosis and to estimate lifetime probability of scoliosis surgery in spinal muscular atrophy (SMA). We analyzed cross-sectional data from 283 patients from our population-based cohort study. Additional longitudinal data on scoliosis progression and spinal surgery were collected from 36 consecutive patients who received scoliosis surgery at our center. The lifetime probability of receiving scoliosis surgery was ≈80% in SMA types 1c and 2. Patients with type 2 who only learned to sit (type 2a) were significantly younger at time of surgery than those who learned to sit and stand (type 2b). The lifetime risk of surgery was lower in type 3a (40%) and strongly associated with age at loss of ambulation: 71% in patients losing ambulation before 10 years of age vs 22% losing ambulation after the age of 10 years (p = 0.005). In type 3a, preserving the ability to walk 1 year longer corresponded to a 15% decrease in lifetime risk of scoliosis surgery (hazard ratio 0.852, p = 0.017). Scoliosis development was characterized by initial slow progression, followed by acceleration in the 1.5- to 2-year period before surgery. The lifetime probability of scoliosis surgery is high in SMA types 1c and 2 and depends on age at loss of ambulation in type 3. Motor milestones such as standing that are not part of the standard classification system are of additional predictive value. Our data may act as a reference to assess long-term effects of new SMA-specific therapies.


Phosphatidylcholine synthesis through cholinephosphate cytidylyltransferase is dispensable in Leishmania major.
Phosphatidylcholine (PC) is a major cell membrane constituent and precursor of important second messengers. In Leishmania parasites, PC synthesis can occur via the choline branch of the Kennedy pathway, the N-methylation of phosphatidylethanolamine (PE), or the remodeling of exogenous phospholipids. To investigate the role of de novo PC synthesis in Leishmania major, we focused on the cholinephosphate cytidylyltransferase (CPCT) which catalyzes the formation of CDP-choline, a key intermediate in the choline branch of the Kennedy pathway. Without CPCT, L. major parasites cannot incorporate choline into PC, yet the CPCT-null mutants contain similar levels of PC and PE as wild type parasites. Loss of CPCT does not affect the growth of parasites in complete medium or their virulence in mice. These results suggest that other mechanisms of PC synthesis can compensate the loss of CPCT. Importantly, CPCT-null parasites exhibited severe growth defects when ethanolamine and exogenous lipids became limited or when they were co-cultured with certain bacteria that are known to be members of sandfly midgut microbiota. These findings suggest that Leishmania employ multiple PC synthesis pathways to utilize a diverse pool of nutrients, which may be crucial for their survival and development in the sandfly.


Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2.
P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na(+),K(+)-ATPase and Ca(2+)-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.


Electrospun fiber constructs for vocal fold tissue engineering: effects of alignment and elastomeric polypeptide coating.
Vocal fold lamina propria extracellular matrix (ECM) is highly aligned and when injured, becomes disorganized with loss of the tissue's critical biomechanical properties. This study examines the effects of electrospun fiber scaffold architecture and elastin-like polypeptide (ELP4) coating on human vocal fold fibroblast (HVFF) behavior for applications toward tissue engineering the vocal fold lamina propria. Electrospun Tecoflex™ scaffolds were made with aligned and unaligned fibers, and were characterized using scanning electron microscopy and uniaxial tensile testing. ELP4 was successfully adsorbed onto the scaffolds; HVFFs were seeded and their viability, proliferation, morphology and gene expression were characterized. Aligned and unaligned scaffolds had initial elastic moduli of ∼14 MPa, ∼5 MPa and ∼0.3 MPa, ∼0.6 MPa in the preferred and cross-preferred directions, respectively. Scaffold topography had an effect on the orientation of the cells, with HVFFs seeded on aligned scaffolds having a significantly different (p<0.001) angle of orientation than HVFFs cultured on unaligned scaffolds. This same effect and significant difference (p<0.001) was seen on aligned and unaligned scaffolds coated with ELP4. Scaffold alignment and ELP4 coating impacted ECM gene expression. ELP4 coating, and aligned scaffolds upregulated elastin synthesis when tested on day 7 without a concomitant upregulation of collagen III synthesis. Collectively, results indicate that aligned electrospun scaffolds and ELP4 coating are promising candidates in the development of biodegradeable vocal fold lamina propria constructs.


Continuing professional development: investment or expectation?
The aim of this study was to explore the reason for nurses' participation in postregistration education. The study was located in one third level institution in Ireland and prospective candidates who applied to undertake a programme of studies were invited to complete the postal questionnaire and return it to the college anonymously in advance of commencing their studies. A descriptive survey research design was adopted with the use of a questionnaire for data collection. The respondents had an opportunity to make additional comments in a questionnaire, which generated some qualitative data. A total of 243 questionnaires were returned which represented a 46.7% response rate. The major reasons for participating in postregistration education were 'to obtain promotion to a higher grade/position' (99%) and 'to enable me extend my clinical role' (98%). Investment in nursing education should take into account the reasons for participation in continuing education and professional development as identified in this study and in other studies so as to focus efforts that improve planning for long-term continuing education and professional development. The adoption of such a strategic approach by employers will ensure more precise targeting of scarce continuing education and professional development resources. Equally, expectation without adequate investment is not realistic if the profession wants to move forward in this era of rapid change in the delivery of health care.


Ultrastructural changes, increased oxidative stress, inflammation, and altered cardiac hypertrophic gene expressions in heart tissues of rats exposed to incense smoke.
Incense smoke exposure has recently been linked to cardiovascular disease risk, heart rate variability, and endothelial dysfunction. To test the possible underlying mechanisms, oxidative stress, and inflammatory markers, gene expressions of cardiac hypertrophic and xenobiotic-metabolizing enzymes and ultrastructural changes were measured, respectively, using standard, ELISA-based, real-time PCR, and transmission electron microscope procedures in heart tissues of Wistar rats after chronically exposing to Arabian incense. Malondialdehyde, tumor necrosis alpha (TNF)-α, and IL-4 levels were significantly increased, while catalase and glutathione levels were significantly declined in incense smoke-exposed rats. Incense smoke exposure also resulted in a significant increase in atrial natriuretic peptide, brain natriuretic peptide, β-myosin heavy chain, CYP1A1 and CYP1A2 messenger RNAs (mRNAs). Rats exposed to incense smoke displayed marked ultrastructural changes in heart muscle with distinct cardiac hypertrophy, which correlated with the augmented hypertrophic gene expression as well as markers of cardiac damage including creatine kinase-myocardial bound (CK-MB) and lactate dehydrogenase (LDH). Increased oxidative stress, inflammation, altered cardiac hypertrophic gene expression, tissue damage, and architectural changes in the heart may collectively contribute to increased cardiovascular disease risk in individuals exposed to incense smoke. Increased gene expressions of CYP1A1 and CYP1A2 may be instrumental in the incense smoke-induced oxidative stress and inflammation. Thus, incense smoke can be considered as a potential environmental pollutant and its long-term exposure may negatively impact human health.


Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene.
The microenvironment of rapidly growing tumors is associated with increased energy demand and diminished vascular supply, resulting in focal areas of prominent hypoxia. A number of hypoxia-responsive genes have been associated with growing tumors, and here we demonstrate that the multidrug resistance (MDR1) gene product P-glycoprotein, a Mr approximately 170,000 transmembrane protein associated with tumor resistance to chemotherapeutics, is induced by ambient hypoxia. Initial studies using quantitative microarray analysis of RNA revealed an approximately 7-fold increase in MDR in epithelial cells exposed to hypoxia (pO(2) 20 torr, 18 h). These findings were further confirmed at the mRNA and protein level. P-Glycoprotein function was studied by analysis of verapamil-inhibitable efflux of digoxin and rhodamine 123 in intact T84 cells and revealed that hypoxia enhances P-glycoprotein function by as much as 7 +/- 0.4-fold over normoxia. Subsequent studies confirmed hypoxia-elicited MDR1 gene induction and increased P-glycoprotein expression in nontransformed, primary cultures of human microvascular endothelial cells, and analysis of multicellular spheroids subjected to hypoxia revealed increased resistance to doxorubicin. Examination of the MDR1 gene identified a binding site for hypoxia inducible factor-1 (HIF-1), and inhibition of HIF-1 expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible MDR1 expression and a nearly complete loss of basal MDR1 expression. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subjected to hypoxia, and such hypoxia inducibility was lost in truncated constructs lacking the HIF-1 site and in HIF-1 binding site mutants. Extensions of these studies also identified a role for Sp1 in this hypoxia response. Taken together, these data indicate that the MDR1 gene is hypoxia responsive, and such results may identify hypoxia-elicited P-glycoprotein expression as a pathway for resistance of some tumors to chemotherapeutics.


Calorie restriction increases telomerase activity, enhances autophagy, and improves diastolic dysfunction in diabetic rat hearts.
The aims of this study were to investigate the impact of caloric restriction (CR) on cardiac telomere biology in an animal model of diabetes and to examine the signal transduction involved in cell senescence as well as cardiac function. Male 8-week-old Otsuka Long-Evans Tokushima fatty (OLETF) diabetic rats were divided into two groups: a group fed ad libitum (OLETF-AL) and a group fed with CR (OLETF-CR: 30% energy reduction). Long-Evans Tokushima Otsuka (LETO) non-diabetic rats were used as controls. LETO rats were also divided into two groups: a CR (LETO-CR) group and a group fed AL (LETO-AL). At 40 weeks of age, the body weight was decreased by 9.7% and the insulin resistance was less in OLETF-CR rats. Telomerase activity in OLETF-CR rats was significantly increased, and telomerase reverse transcriptase was more highly expressed in those rats. However, the telomere length (TL) was not different between AL- and CR-treated rats of each strain. The protein expressions for FoxO1 and FoxO3 were increased in OLETF-AL rats, but the levels of phosphorylated (p)-Akt were decreased compared to those in OLETF-CR rats. Autophagic LC3II signals revealed significant increases in OLETF-CR rats. Echocardiography showed that OLETF-CR improved the left ventricular diastolic dysfunction without changes in the left ventricular dimension. This study revealed that CR increases cardiac telomerase activity without TL attrition, and significantly ameliorates diastolic dysfunction. These findings suggest that cardiac telomerase activity may play an important role in the maintenance of normal cardiac function.


Symptoms of obstructive defecation syndrome: functional outcome after stapled haemorrhoidectomy.
Patients with haemorrhoids often present obstructive defecation symptoms. Preoperative they can be classified in three groups: slight, severe and mild symptoms. Aim of this study was to postoperatively evaluate functional outcome in patients who underwent stapled haemorrhoidectomy in mild obstructive defecation syndrome. We enrolled 98 patients with mild obstructive defecation symptoms using Wexner score preoperatively (range 8-16) from the 332 consecutive patients treated with stapled haemorrhoidectomy between January 2009 and December 2011. All patients were clinically examined 1 month after surgical treatment and every 6 months with a mean followup of 30 months (range 24-42 months). The 98 patients enrolled included 58 men and 40 women. There were no intraoperative complications. Postoperative complications included four patients. A reduction of Wexner score from a mean preoperative score of 12 to a mean postoperative score of 6 just 6 months after surgery was observed. This difference was statistically significant (p<0.001: Mann-Whitney U test). Careful clinical evaluation of patients with haemorrhoids and obstructive defecation symptom is required to establish the most appropriate treatment. The results of this study indicated that stapled haemorrhoidectomy can improves the functional symptoms for patients with constipation symptoms of moderate degree. Functional outcome, Obstructive defecation symptoms, Stapled haemorrhoidectomy.


Psychological and behavioural interventions in bipolar disorder that target sleep and circadian rhythms: A systematic review of randomised controlled trials.
Sleep and circadian disruptions are prominent symptoms of bipolar disorder (BD) and potential targets for adjunctive interventions. The aim of this review was to appraise the effectiveness of psychological and behavioural interventions in BD that target sleep and circadian rhythms, as reported by randomised controlled trials. Nineteen studies met the inclusion/exclusion criteria. They were summarised via narrative synthesis and meta-analysis wherever appropriate. Six studies delivered bright light therapy, five interpersonal and social rhythm therapy, two blue-light blocking glasses, one cognitive behavioural therapy for insomnia, one total sleep deprivation, and four combination treatments. More than half of the studies (N = 10, 52 %) did not measure sleep or circadian rhythms despite being the principal target of the intervention. Overall, the evidence base for the effectiveness of these interventions was limited. There was a small number of studies for each intervention, and a lack of consistency in protocols and outcomes. Meta-analysis was possible for the effect of bright light therapy on depression, revealing a medium-to-large post-treatment effect (Nc = 6; g=-0.74 [95 % CI=-1.05 to -0.42], p < 0.001).


Aneuploidy in the human blastocyst.
Studies of human cleavage stage embryos, 3 days after fertilization of the oocyte, have revealed remarkably high levels of chromosome abnormality. In addition to meiotic errors derived from the gametes, principally the oocyte, mitotic errors occurring after fertilization are also common, leading to widespread chromosomal mosaicism. The prevalence of chromosome anomalies in embryos may explain the relatively poor fertility and fecundity in humans and the low success rates of assisted reproductive treatments (e.g., IVF). While much is known concerning the incidence of aneuploidy during the first 3 days following fertilization, it is only in the last couple of years that large numbers of embryos at the final stage of preimplantation development, the blastocyst stage, 5 days after fertilization, have been subjected to detailed analysis. Here we discuss the latest data from the comprehensive cytogenetic analysis of blastocysts. These findings indicate that the majority of selection against chromosome abnormalities does not occur until the time of implantation or shortly after, with aneuploidy typically affecting more than 50% of blastocysts. Additionally, clinical results presented suggest that screening of blastocyst stage embryos for chromosome abnormality, with preferential transfer to the uterus of those found to be euploid, may help to improve the success rates of assisted reproductive treatments.


Micronutrients in pregnancy in low- and middle-income countries.
Pregnancy is one of the more important periods in life when increased micronutrients, and macronutrients are most needed by the body; both for the health and well-being of the mother and for the growing foetus and newborn child. This brief review aims to identify the micronutrients (vitamins and minerals) likely to be deficient in women of reproductive age in Low- and Middle-Income Countries (LMIC), especially during pregnancy, and the impact of such deficiencies. A global prevalence of some two billion people at risk of micronutrient deficiencies, and multiple micronutrient deficiencies of many pregnant women in LMIC underline the urgency to establishing the optimal recommendations, including for delivery. It has long been recognized that adequate iron is important for best reproductive outcomes, including gestational cognitive development. Similarly, iodine and calcium have been recognized for their roles in development of the foetus/neonate. Less clear effects of deficiencies of zinc, copper, magnesium and selenium have been reported. Folate sufficiency periconceptionally is recognized both by the practice of providing folic acid in antenatal iron/folic acid supplementation and by increasing numbers of countries fortifying flours with folic acid. Other vitamins likely to be important include vitamins B12, D and A with the water-soluble vitamins generally less likely to be a problem. Epigenetic influences and the likely influence of micronutrient deficiencies on foetal origins of adult chronic diseases are currently being clarified. Micronutrients may have other more subtle, unrecognized effects. The necessity for improved diets and health and sanitation are consistently recommended, although these are not always available to many of the world's pregnant women. Consequently, supplementation programmes, fortification of staples and condiments, and nutrition and health support need to be scaled-up, supported by social and cultural measures. Because of the life-long influences on reproductive outcomes, including inter-generational ones, both clinical and public health measures need to ensure adequate micronutrient intakes during pregnancy, but also during adolescence, the first few years of life, and during lactation. Many antenatal programmes are not currently achieving this. We aim to address the need for micronutrients during pregnancy, the importance of micronutrient deficiencies during gestation and before, and propose the scaling-up of clinical and public health approaches that achieve healthier pregnancies and improved pregnancy outcomes.


FACT Sets a Barrier for Cell Fate Reprogramming in Caenorhabditis elegans and Human Cells.
The chromatin regulator FACT (facilitates chromatin transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen using Caenorhabditis elegans, we identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACT's role as a barrier to cell fate conversion is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts. Such activity is unexpected because FACT is known as a positive regulator of gene expression, and previously described reprogramming barriers typically repress gene expression. While FACT depletion in human fibroblasts results in decreased expression of many genes, a number of FACT-occupied genes, including reprogramming-promoting factors, show increased expression upon FACT depletion, suggesting a repressive function of FACT. Our findings identify FACT as a cellular reprogramming barrier in C. elegans and humans, revealing an evolutionarily conserved mechanism for cell fate protection.


Thrombosis in Inherited Fibrinogen Disorders.
Although inherited fibrinogen disorders (IFD) are primarily considered to be bleeding disorders, they are associated with a higher thrombotic complication risk than defects in other clotting factors. Managing IFD patients with thrombosis is challenging as anticoagulant treatment may exacerbate the underlying bleeding risk which can be life-threatening. Due to the low prevalence of IFD, there is little information on pathophysiology or optimal treatment of thrombosis in these patients. We searched the literature for cases of thrombosis among IFD patients and identified a total of 128 patient reports. In approximately half of the cases, thromboses were spontaneous, while in the others trauma, surgery, and parturition contributed to the risk. The true mechanism(s) of thrombosis in IFD patients remain to be elucidated. A variety of anticoagulant treatments have been used in the treatment or prevention of thrombosis, sometimes with concurrent fibrinogen replacement therapy. There is no definite evidence that fibrinogen supplementation increases the risk of thrombosis, and it may potentially be effective in the treatment and prevention of both thrombosis and hemorrhage in IFD patients.


Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development.
During blood-stage infection by Plasmodium falciparum, merozoites invade RBCs. Currently there is limited knowledge of cellular and molecular invasion events, and no established assays are available to readily measure and quantify invasion-inhibitory antibodies or compounds for vaccine and drug studies. We report the isolation of viable merozoites that retain their invasive capacity, at high purity and yield, purified by filtration of highly synchronous populations of schizonts. We show that the half-life of merozoite invasive capacity after rupture is 5 min at 37 degrees C, and 15 min at room temperature. Studying the kinetics of invasion revealed that 80% of invasion events occur within 10 min of mixing merozoites and RBCs. Invasion efficiency was maximum at low merozoite-to-RBC ratios and occurred efficiently in the absence of serum and with high concentrations of dialyzed nonimmune serum. We developed and optimized an invasion assay by using purified merozoites that enabled invasion-inhibitory activity of antibodies and compounds to be measured separately from other mechanisms of growth inhibition; the assay was more sensitive for detecting inhibitory activity than established growth-inhibition assays. Furthermore, with the use of purified merozoites it was possible to capture and fix merozoites at different stages of invasion for visualization by immunofluorescence microscopy and EM. We thereby demonstrate that processing of the major merozoite antigen merozoite surface protein-1 occurs at the time of RBC invasion. These findings have important implications for defining invasion events and molecular interactions, understanding immune interactions, and identifying and evaluating inhibitors to advance vaccine and drug development.


Collecting Validity Evidence: A Hands-on Workshop for Medical Education Assessment Instruments.
There is an increasing call for developing validity evidence in medical education assessment. The literature lacks a practical resource regarding an actual development process. Our workshop teaches how to apply principles of validity evidence to existing assessment instruments and how to develop new instruments that will yield valid data. The literature, consensus findings of curricula and content experts, and principles of adult learning guided the content and methodology of the workshop. The workshop underwent stringent peer review prior to presentation at one international and three national academic conferences. In the interactive workshop, selected domains of validity evidence were taught with sequential cycles of didactics, demonstration, and deliberate practice with facilitated feedback. An exercise guide steered participants through a stepwise approach. Using Likert-scale items and open-response questions, an evaluation form rated the workshop's effectiveness, captured details of how learners reached the objectives, and determined participants' plans for future work. The workshop demonstrated generalizability with successful implementation in diverse settings. Sixty-five learners, the majority being clinician-educators, completed evaluations. Learners rated the workshop favorably for each prompt. Qualitative comments corroborated the workshop's effectiveness. The active application and facilitated feedback components allowed learners to reflect in real time as to how they were meeting a particular objective. This feasible and practical educational intervention fills a literature gap by showing the medical educator how to apply validity evidence to both existing and in-development assessment instruments. Thus, it holds the potential to significantly impact learner and, subsequently, patient outcomes.


Determinants of coronavirus disease 2019 infection by artificial intelligence technology: A study of 28 countries.
The coronavirus disease 2019 pandemic has affected countries around the world since 2020, and an increasing number of people are being infected. The purpose of this research was to use big data and artificial intelligence technology to find key factors associated with the coronavirus disease 2019 infection. The results can be used as a reference for disease prevention in practice. This study obtained data from the "Imperial College London YouGov Covid-19 Behaviour Tracker Open Data Hub", covering a total of 291,780 questionnaire results from 28 countries (April 1~August 31, 2020). Data included basic characteristics, lifestyle habits, disease history, and symptoms of each subject. Four types of machine learning classification models were used, including logistic regression, random forest, support vector machine, and artificial neural network, to build prediction modules. The performance of each module is presented as the area under the receiver operating characteristics curve. Then, this study further processed important factors selected by each module to obtain an overall ranking of determinants. This study found that the area under the receiver operating characteristics curve of the prediction modules established by the four machine learning methods were all >0.95, and the RF had the highest performance (area under the receiver operating characteristics curve is 0.988). Top ten factors associated with the coronavirus disease 2019 infection were identified in order of importance: whether the family had been tested, having no symptoms, loss of smell, loss of taste, a history of epilepsy, acquired immune deficiency syndrome, cystic fibrosis, sleeping alone, country, and the number of times leaving home in a day. This study used big data from 28 countries and artificial intelligence methods to determine the predictors of the coronavirus disease 2019 infection. The findings provide important insights for the coronavirus disease 2019 infection prevention strategies.


[Biological versus mechanical valves in the mitral position].
The long-term late results of isolated mitral valve replacement before April 1981 were retrospectively evaluated in 54 patients receiving a Hancock valve (HX) with a total follow-up of 424 patient-years (py) and 37 patients receiving a Björk-Shiley valve (BS) with a total follow-up of 366 py. There was no significant difference in regard to preoperative age, cardiothoracic ratio, electrocardiogram, or New York Heart Association functional class between the two groups. All of the BS group and 43 of the HX patients received long-term anticoagulant therapy. No significant difference was notable between the two groups in hospital mortality. Actuarial survival rate excluding hospital deaths was 75.5 +/- 6.7% for the HX group, and 80.8 +/- 7.1% for the BS group at ten years (not significant). There was no significant difference between the two groups in overall incidence of thromboembolism (HX 1.4% py, BS 1.9% py), anticoagulant related bleeding (HX 0.5% py, BS 0.8% py), or endocarditis (HX 0.5% py, BS 0.3% py). Actuarial freedom from reoperation at ten years was 69.6 +/- 8.1% for the HX group, and 93.5 +/- 4.4% for the BS group (p less than 0.01). Event free survival excluding hospital deaths at ten years was 49.2 +/- 8.1% for the HX group, 70.6 +/- 8.3% for the BS group (p less than 0.05). We prefer a mechanical prosthesis except in patients over 65 years old, who have a short life expectancy, in whom anticoagulation is thought to be difficult, and who hope a biological valve.


FOXP3 recognizes microsatellites and bridges DNA through multimerization.
FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity1-5. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain-a DNA-binding domain that is commonly thought to function as a monomer or dimer-to form a higher-order multimer after binding to TnG repeat microsatellites. The cryo-electron microscopy structure of FOXP3 in a complex with T3G repeats reveals a ladder-like architecture, whereby two double-stranded DNA molecules form the two 'side rails' bridged by five pairs of FOXP3 molecules, with each pair forming a 'rung'. Each FOXP3 subunit occupies TGTTTGT within the repeats in a manner that is indistinguishable from that of FOXP3 bound to the forkhead consensus motif (TGTTTAC). Mutations in the intra-rung interface impair TnG repeat recognition, DNA bridging and the cellular functions of FOXP3, all without affecting binding to the forkhead consensus motif. FOXP3 can tolerate variable inter-rung spacings, explaining its broad specificity for TnG-repeat-like sequences in vivo and in vitro. Both FOXP3 orthologues and paralogues show similar TnG repeat recognition and DNA bridging. These findings therefore reveal a mode of DNA recognition that involves transcription factor homomultimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.


Discovering protein-protein interaction stabilisers by native mass spectrometry.
Protein-protein interactions (PPIs) are key therapeutic targets. Most PPI-targeting drugs in the clinic inhibit these important interactions; however, stabilising PPIs is an attractive alternative in cases where a PPI is disrupted in a disease state. The discovery of novel PPI stabilisers has been hindered due to the lack of tools available to monitor PPI stabilisation. Moreover, for PPI stabilisation to be detected, both the stoichiometry of binding and the shift this has on the binding equilibria need to be monitored simultaneously. Here, we show the power of native mass spectrometry (MS) in the rapid search for PPI stabilisers. To demonstrate its capability, we focussed on three PPIs between the eukaryotic regulatory protein 14-3-3σ and its binding partners estrogen receptor ERα, the tumour suppressor p53, and the kinase LRRK2, whose interactions upon the addition of a small molecule, fusicoccin A, are differentially stabilised. Within a single measurement the stoichiometry and binding equilibria between 14-3-3 and each of its binding partners was evident. Upon addition of the fusicoccin A stabiliser, a dramatic shift in binding equilibria was observed with the 14-3-3:ERα complex compared with the 14-3-3:p53 and 14-3-3:LRRK2 complexes. Our results highlight how native MS can not only distinguish the ability of stabilisers to modulate PPIs, but also give important insights into the dynamics of ternary complex formation. Finally, we show how native MS can be used as a screening tool to search for PPI stabilisers, highlighting its potential role as a primary screening technology in the hunt for novel therapeutic PPI stabilisers.


Cross-talk between language processes and overt motor behavior in the first 200 msec of processing.
A recently emerging view sees language understanding as closely linked to sensory and motor processes. The present study investigates this issue by examining the influence of processing action verbs and concrete nouns on the execution of a reaching movement. Fine-grained analyses of movement kinematics revealed that relative to nouns, processing action verbs significantly affects overt motor performance. Within 200 msec after onset, processing action verbs interferes with a concurrent reaching movement. By contrast, the same words assist reaching movement when processed before movement onset. The cross-talk between language processes and overt motor behavior provides unambiguous evidence that action words and motor action share common cortical representations and could thus suggest that cortical motor regions are indeed involved in action word retrieval.


Geographical distribution and prevalence of podoconiosis in Rwanda: a cross-sectional country-wide survey.
Podoconiosis is a type of tropical lymphoedema that causes massive swelling of the lower limbs. The disease is associated with both economic insecurity, due to long-term morbidity-related loss of productivity, and intense social stigma. Reliable and detailed data on the prevalence and distribution of podoconiosis are scarce. We aimed to fill this data gap by doing a nationwide community-based study to estimate the number of cases throughout Rwanda. We did a population-based cross-sectional survey to determine the national prevalence of podoconiosis. A podoconiosis case was defined as a person with bilateral, asymmetrical lymphoedema of the lower limb present for more than 1 year, who tested negative for Wuchereria bancrofti antigen (determined by Filariasis Test Strip) and specific IgG4 (determined by Wb123 test), and had a history of any of the associated clinical signs and symptoms. All adults (aged ≥15 years) who resided in any of the 30 districts of Rwanda for 10 or more years were invited at the household level to participate. Participants were interviewed and given a physical examination before Filariasis Test Strip and Wb123 testing. We fitted a binomial mixed model combining the site-level podoconiosis prevalence with continuous environmental covariates to estimate prevalence at unsampled locations. We report estimates of cases by district combining our mean predicted prevalence and a contemporary gridded map of estimated population density. Between June 12, and July 28, 2017, 1 360 612 individuals-719 730 (53%) women and 640 882 (47%) men-were screened from 80 clusters in 30 districts across Rwanda. 1143 individuals with lymphoedema were identified, of whom 914 (80%) had confirmed podoconiosis, based on the standardised diagnostic algorithm. The overall prevalence of podoconiosis was 68·5 per 100 000 people (95% CI 41·0-109·7). Podoconiosis was found to be widespread in Rwanda. District-level prevalence ranged from 28·3 per 100 000 people (16·8-45·5, Nyarugenge, Kigali province) to 119·2 per 100 000 people (59·9-216·2, Nyamasheke, West province). Prevalence was highest in districts in the North and West provinces: Nyamasheke, Rusizi, Musanze, Nyabihu, Nyaruguru, Burera, and Rubavu. We estimate that 6429 (95% CI 3938-10 088) people live with podoconiosis across Rwanda. Despite relatively low prevalence, podoconiosis is widely distributed geographically throughout Rwanda. Many patients are likely to be undiagnosed and morbidity management is scarce. Targeted interventions through a well coordinated health system response are needed to manage those affected. Our findings should inform national level planning, monitoring, and implementation of interventions. Wellcome Trust.


Service delivery characteristics associated with contraceptive use among youth clients in integrated voluntary counseling and HIV testing clinics in Kenya.
Youth attending voluntary counseling and HIV testing (VCT) services often have unmet need for contraception. Integrated family planning (FP) and HIV services can address dual HIV and unintended pregnancy prevention needs. However, little is known about which VCT service characteristics, such as quality of care, strength of FP-HIV service integration, and youth-friendliness, affect contraceptive behavior. This study explores the facility- and provider-level characteristics that may be associated with same day uptake or intention to use contraception after a VCT visit, and contraceptive use three months later among youth clients, controlling for client characteristics. This was a descriptive study conducted in 20 integrated VCT clinics across Kenya. Data collection included 20 structured clinic observations, 349 baseline interviews with male and female clients aged 15-24 after their VCT visit plus 277 follow-up interviews with clients three months later, and interviews with 46 providers who served the clients at the study clinics. Client, provider, and structured observation data were linked and multi-level logistic path models were used for analyses. Results revealed little evidence of specific service characteristics being associated with contraceptive behavior. However, VCT providers were not routinely screening for risk of unintended pregnancy or counseling on contraception. Results are likely a reflection of suboptimal integrated services. The clinics were serving youth with unmet contraceptive need and therefore integrated services should be strengthened overall to ensure no missed opportunities.


Cystathionine gamma-lyase: Clinical, metabolic, genetic, and structural studies.
We report studies of six individuals with marked elevations of cystathionine in plasma and/or urine. Studies of CTH, the gene that encodes cystathionine gamma-lyase, revealed the presence among these individuals of either homozygous or compound heterozygous forms of a novel large deletion, p.Gly57_Gln196del, two novel missense mutations, c.589C>T (p.Arg197Cys) and c.932C>T (p.Thr311Ile), and one previously reported alteration, c.200C>T (p.Thr67Ile). Another novel missense mutation, c.185G>T (p.Arg62His), was found in heterozygous form in three mildly hypercystathioninemic members of a Taiwanese family. In one severely hypercystathioninemic individual no CTH mutation was found. Brief clinical histories of the cystathioninemic/cystathioninuric patients are presented. Most of the novel mutations were expressed and the CTH activities of the mutant proteins determined. The crystal structure of the human enzyme, hCTH, and the evidence available as to the effects of the mutations in question, as well as those of the previously reported p.Gln240Glu, on protein structure, enzymatic activity, and responsiveness to vitamin B(6) administration are discussed. Among healthy Czech controls, 9.3% were homozygous for CTH c.1208G>T (p.Ser403Ile), previously found homozygously in 7.5% of Canadians for whom plasma total homocysteine (tHcy) had been measured. Compared to wild-type homozygotes, among the 55 Czech c.1208G>T (p.Ser403Ile) homozygotes a greater level of plasma cystathionine was found only after methionine loading. Three of the four individuals homozygous or compound heterozygous for inactivating CTH mutations had mild plasma tHcy elevations, perhaps indicating a cause-and-effect relationship. The experience with the present patients provides no evidence that severe loss of CTH activity is accompanied by adverse clinical effects.


A Cross-Sectional Study on the Prevalence, Risk Factors, and Ill Effects of Internet Addiction Among Medical Students in Northeastern India.
To evaluate Internet addiction among medical students in northeastern India and gain detailed knowledge about the prevalence, risk factors, and ill effects commonly associated with the disorder. The cross-sectional study sample comprised 188 medical students from Silchar Medical College and Hospital (Silchar, Assam, India). Students completed a sociodemographic form and an Internet use questionnaire, both created for this study, and the Young's 20-Item Internet Addiction Test after they received brief instructions. Data were collected during a10-day period in June 2015. Of the 188 medical students, 46.8% were at increased risk of Internet addiction. Those who were found to be at increased risk had longer years of Internet exposure (P = .046) and always online status (P = .033). Also, among this group, the men were more prone to develop an online relationship. Excessive Internet usage also led to poor performance in college (P < .0001) and feeling moody, anxious, and depressed (P < .0001). The ill effects of Internet addiction include withdrawal from real-life relationships, deterioration in academic activities, and a depressed and nervous mood. Internet use for nonacademic purposes is increasing among students, thus there is an immediate need for strict supervision and monitoring at the institutional level. The possibility of becoming addicted to the Internet should be emphasized to students and their parents through awareness campaigns so that interventions and restrictions can be implemented at the individual and family levels.


Combined kidney and pancreas transplantation. A 3-year experience.
Between May 1988 and September 1991, we performed 26 simultaneous kidney and pancreas transplants and one pancreas transplant after a kidney transplant. All transplants consisted of bladder drainage via a duodenal segment. Actuarial patient, kidney, and pancreas graft survival rates at 12 months were 96%, 88%, and 85%, respectively, and at 24 months were 96%, 88%, and 81%, respectively, and were not significantly different from those of diabetic recipients of cadaver kidney transplants alone. Excellent long-term glycemic control was obtained as monitored by fasting blood glucose and glycosylated hemoglobin levels and by oral glucose tolerance tests. The mean period of hospitalization and number of hospital admissions in the first year posttransplant were significantly greater for patients who received combined kidney and pancreas transplants than for those who received cadaver kidney transplants alone. Combined kidney and pancreas transplants can be performed with patient and graft survival comparable to those of kidney transplants alone, with excellent long-term glycemic control, but result in increased morbidity in the first postoperative year.


Identification of a locus for type I punctate palmoplantar keratoderma on chromosome 15q22-q24.
The identification of the molecular basis of disorders of keratinisation has significantly advanced our understanding of skin biology, revealing new information on key structures in the skin, such as the intermediate filaments, desmosomes, and gap junctions. Among these disorders, there is an extraordinarily heterogeneous group known as palmoplantar keratodermas (PPK), for which only a few molecular defects have been described. A particular form of PPK, known as punctate PPK, has been described in a few large autosomal dominant pedigrees, but its genetic basis has yet to be identified. Identification of the gene for punctate PPK. Clinical examination and linkage analysis in three families with punctate PPK. A genomewide scan was performed on an extended autosomal dominant pedigree, and linkage to chromosome 15q22-q24 was identified. With the addition of two new families with the same phenotype, we confirmed the mapping of the locus for punctate PPK to a 9.98 cM interval, flanked by markers D15S534 and D15S818 (maximum two point lod score of 4.93 at theta = 0 for marker D15S988). We report the clinical and genetic findings in three pedigrees with the punctate form of PPK. We have mapped a genetic locus for this phenotype to chromosome 15q22-q24, which indicates the identification of a new gene involved in skin integrity.


Smoke invigoration versus inhibition of clouds over the Amazon.
The effect of anthropogenic aerosols on clouds is one of the most important and least understood aspects of human-induced climate change. Small changes in the amount of cloud coverage can produce a climate forcing equivalent in magnitude and opposite in sign to that caused by anthropogenic greenhouse gases, and changes in cloud height can shift the effect of clouds from cooling to warming. Focusing on the Amazon, we show a smooth transition between two opposing effects of aerosols on clouds: the microphysical and the radiative. We show how a feedback between the optical properties of aerosols and the cloud fraction can modify the aerosol forcing, changing the total radiative energy and redistributing it over the atmospheric column.


Androgen therapy does not prevent bone loss and arterial calcifications in male rats with chronic kidney disease.
Patients suffering from chronic kidney disease (CKD) often experience bone loss and arterial calcifications. It is unclear if hypogonadism contributes to the development of these complications and whether androgen therapy might prevent them. Male adult rats were randomized into four groups. The first group received standard chow (control), while three other groups were fed a 0.25% adenine/low vitamin K diet (CKD). Two CKD groups were treated with testosterone or dihydrotestosterone (DHT), whereas the control group and one CKD group received vehicle (VEH). CKD animals had 10-fold higher serum creatinine and more than 15-fold higher parathyroid hormone levels compared to controls. Serum testosterone levels were more than two-fold lower in the CKDVEH group compared to control + VEH and CKD + testosterone groups. Seminal vesicle weight was reduced by 50% in CKDVEH animals and restored by testosterone and DHT. CKD animals showed a low bone mass phenotype with decreased trabecular bone volume fraction and increased cortical porosity, which was not rescued by androgen treatment. Aortic calcification was much more prominent in CKD animals and not unequivocally prevented by androgens. Messenger RNA expression of the androgen receptor-responsive genes Acta1 and Col1a1 was reduced by CKD and stimulated by androgen treatment in levator ani muscle but not in the bone or aortic tissue. We conclude that adenine-induced CKD results in the development of hypogonadism in male rats. Androgen therapy is effective in restoring serum testosterone levels and androgen-sensitive organ weights but does not prevent bone loss or arterial calcifications, at least not in the presence of severe hyperparathyroidism.


Application of near-infrared spectroscopy for monitoring and control of cell culture and fermentation.
Near-infrared (NIR) spectroscopy can potentially provide on-line information on substrate, biomass, product, and metabolite concentrations in fermentation processes, which could be useful for improved monitoring or control. However, several factors can negatively influence the quality of chemometric models built for interpretation of the spectra, thus impairing the analyte concentration predictions. The aim of this review was to provide an overview of necessary conditions and challenges that one has to face when developing a NIR application for monitoring of cell culture or fermentation processes. Important practical aspects are introduced, such as sampling, modeling of biomass concentration, influence of microorganism morphology on the spectra, effects of the hydrodynamic conditions in the fermenter, temperature influence, instrument settings, and signal optimization. Several examples from the literature are provided, which will hopefully guide the reader interested in the topic. Furthermore, the general procedure used for the development of calibration models is presented, and the influence of microorganism metabolism-potential source of correlation between analytes-is commented. Other important issues such as wavelength selection and evaluation of robustness are shortly introduced. Finally, some examples of potential applications of NIR monitoring are provided, including the implementation of control strategies, the combination with other monitoring tools (the so-called sensor fusion), and the description of process trajectories. On the basis of the review, we conclude that acceptance of NIR spectroscopy as a standard monitoring tool by the fermentation industry will necessitate considerably more on-line studies using industrially relevant-and highly challenging-fermentation conditions (high aeration intensity, high biomass concentration and viscosity, and filamentous production strain).


Melatonin receptor expression in the zebra finch brain and peripheral tissues.
The circadian endocrine hormone melatonin plays a significant role in many physiological processes, such as modulating the sleep/wake cycle and oxidative stress. Melatonin is synthesized and secreted during the night by the pineal gland and released into the circulatory system. It binds to numerous membrane, cytosolic, and nuclear receptors in the brain and peripheral organs. Three G-protein-linked membrane receptors (Mel1A, Mel1B, and Mel1C) have been identified in numerous species. Considering the importance of this hormone and its receptors, this study looks at the location and rhythmicity of these three avian melatonin receptors using reverse transcriptase-polymerase chain reaction (RT-PCR) mRNA analysis techniques. This study shows successful partial cloning of the three receptors, and gene expression analysis reveals significant rhythms of the Mel1A receptor in the cerebellum, diencephalon, tectum opticum, telencephalon, and retina. Significant rhythms were found in the diencephalon, pineal gland, retina, tectum opticum, and cerebellum of the Mel1B receptor, whereas Mel1C appeared not to be rhythmically expressed in brain tissues studied. Mel1A, Mel1B, and Mel1C receptor mRNAs were also present in peripheral tissues, showing tissue-specific expression patterns.


Lead Contamination in Opium, Opium Tincture, and Methadone Oral Solution, in Iran.
The present study is conducted with the aim to assess the lead contamination in opium tincture, methadone oral solution, and opium. 10 samples from each of the matters of opium tinctures, methadone oral solutions, and opium (provided by the Law Enforcement Force of the Islamic Republic of Iran) were collected. Then, an atomic absorption spectrophotometer (AAS) was used to measure lead concentration in each of the samples. Data were analyzed using the SPSS software. A P value of 0.05 or less was considered to be statistically significant (Two-tailed). In this study, the amount of lead measured in all samples was equal or less than 5 parts per million (ppm) and the only exception was the lead level of 5.6 ppm in one of the opium tincture samples, which was slightly higher than the standard lead level. The results of the current study showed that lead was present in opium tincture, methadone oral solution, and opium, but it was not in toxic levels. It is reasonable for opium derived medicinal products, but the low levels of lead in opium may need to be addressed at different times in different regions of the country.


Metabolic Consequences of LDHA inhibition by Epigallocatechin Gallate and Oxamate in MIA PaCa-2 Pancreatic Cancer Cells.
Lactate dehydrogenase A (LDHA) is the enzyme that converts pyruvate to lactate and oxidizes the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD+. Several human cancers including the pancreas display elevated expression of LDHA. Because of its essential role in cancer metabolism, LDHA has been considered to be a potential target for cancer therapy. Recently, we have shown that a green tea extract significantly down-regulated LDHA in HPAF-II pancreatic cancer cells using global proteomics profiling. The present study is to investigate how EGCG, a major biological active constituent of green tea, targets the metabolism of human pancreatic adenocarcinoma MIA PaCa-2 cells. We compared the effect of EGCG to that of oxamate, an inhibitor of LDHA, on the multiple metabolic pathways as measured by extracellular lactate production, glucose consumption, as well as intracellular aspartate and glutamate production, fatty acid synthesis, acetyl-CoA, RNA ribose and deoxyribose. Specific metabolic pathways were studied using [1, 2-13C2]-d-glucose as the single precursor metabolic tracer. Isotope incorporations in metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS) and stable isotope-based dynamic metabolic profiling (SiDMAP). We found that the EGCG treatment of MIA PaCa-2 cells significantly reduced lactate production, anaerobic glycolysis, glucose consumption and glycolytic rate that are comparable to the inhibition of LDHA by oxamate treatment. Significant changes in intracellular glucose carbon re-distribution among major glucose-utilizing macromolecule biosynthesis pathways in response to EGCG and oxamate treatment were observed. The inhibition of LDHA by EGCG or oxamate impacts on various pathways of the cellular metabolic network and significantly modifies the cancer metabolic phenotype. These results suggest that phytochemical EGCG and LDHA inhibitor oxamate confer their anti-cancer activities by disrupting the balance of flux throughout the cellular metabolic network.


The role of parkinsonism and antiparkinsonian therapy in the subsequent development of tardive dyskinesia.
Tardive dyskinesia (TD) is a side effect of prolonged neuroleptic treatment presenting as abnormal involuntary movements. This troublesome disorder occurs in only 15-30% of patients taking neuroleptics, suggesting that these individuals may be physiologically distinct so as to be predisposed. This study analyzed possible factors contributing to TD development. Fifty patients on depot neuroleptics for more than 7.1 years were prospectively examined for TD and drug-induced parkinsonism (DIP) using the Smith-Trims rating scale for an average of 5 years. The patients were assessed for the severity of the movement and if the movement increased or decreased with respect to neuroleptic dosage, anticholinergic dosage, parkinsonism, and other related factors. Both TD and DIP increased over time. In the patients whose dose of neuroleptic decreased, the increase in TD ratings was not significant. Using a forward stepwise regression DIP was found to increase as TD worsened but did not appear to predict subsequent TD development. Anticholinergic treatment showed a less significant correlation with the change in TD. These results have implications for the management of combined TD and DIP presentation.


Melatonin and agomelatine for preventing seasonal affective disorder.
Seasonal affective disorder (SAD) is a seasonal pattern of recurrent major depressive episodes that most commonly occurs during autumn or winter and remits in spring. The prevalence of SAD in the United States ranges from 1.5% to 9%, depending on latitude. The predictable seasonal aspect of SAD provides a promising opportunity for prevention. This is one of four reviews on the efficacy and safety of interventions to prevent SAD; we focus on agomelatine and melatonin as preventive interventions. To assess the efficacy and safety of agomelatine and melatonin (in comparison with each other, placebo, second-generation antidepressants, light therapy, psychological therapy or lifestyle interventions) in preventing SAD and improving patient-centred outcomes among adults with a history of SAD. We conducted a search of the Specialised Register of the Cochrane Depression, Anxiety and Neurosis Review Group (CCDANCTR) to 11 August 2015. The CCDANCTR contains reports of relevant randomised controlled trials from EMBASE (1974 to date), MEDLINE (1950 to date), PsycINFO (1967 to date) and the Cochrane Central Register of Controlled Trials (CENTRAL). Furthermore, we searched the Cumulative Index to Nursing and Allied Health Literature (CINAHL), Web of Knowledge, The Cochrane Library and the Allied and Complementary Medicine Database (AMED) (to 26 May 2014). We conducted a grey literature search (e.g. in clinical trial registries) and handsearched the reference lists of all included studies and pertinent review articles. To examine efficacy, we planned to include randomised controlled trials (RCTs) on adults with a history of winter-type SAD who were free of symptoms at the beginning of the study. To examine adverse events, we intended to include non-randomised studies. We planned to include studies that compared agomelatine versus melatonin, or agomelatine or melatonin versus placebo, any second-generation antidepressant (SGA), light therapy, psychological therapies or lifestyle changes. We also intended to compare melatonin or agomelatine in combination with any of the comparator interventions listed above versus the same comparator intervention as monotherapy. Two review authors screened abstracts and full-text publications against the inclusion criteria. Two review authors planned to independently extract data and assess risk of bias of included studies. We planned to pool data for meta-analysis when participant groups were similar and when studies assessed the same treatments by using the same comparator and presented similar definitions of outcome measures over a similar duration of treatment; however, we identified no studies for inclusion. We identified 2986 citations through electronic searches and reviews of reference lists after de-duplication of search results. We excluded 2895 records during title and abstract review and assessed 91 articles at full-text level for eligibility. We identified no controlled studies on use of melatonin and agomelatine to prevent SAD and to improve patient-centred outcomes among adults with a history of SAD. No available methodologically sound evidence indicates that melatonin or agomelatine is or is not an effective intervention for prevention of SAD and improvement of patient-centred outcomes among adults with a history of SAD. Lack of evidence clearly shows the need for well-conducted, controlled studies on this topic. A well-conducted RCT of melatonin or agomelatine for prevention of SAD would assess the comparative benefits and risks of these interventions against others currently used to treat the disorder.


Estimating Allee dynamics before they can be observed: polar bears as a case study.
Allee effects are an important component in the population dynamics of numerous species. Accounting for these Allee effects in population viability analyses generally requires estimates of low-density population growth rates, but such data are unavailable for most species and particularly difficult to obtain for large mammals. Here, we present a mechanistic modeling framework that allows estimating the expected low-density growth rates under a mate-finding Allee effect before the Allee effect occurs or can be observed. The approach relies on representing the mechanisms causing the Allee effect in a process-based model, which can be parameterized and validated from data on the mechanisms rather than data on population growth. We illustrate the approach using polar bears (Ursus maritimus), and estimate their expected low-density growth by linking a mating dynamics model to a matrix projection model. The Allee threshold, defined as the population density below which growth becomes negative, is shown to depend on age-structure, sex ratio, and the life history parameters determining reproduction and survival. The Allee threshold is thus both density- and frequency-dependent. Sensitivity analyses of the Allee threshold show that different combinations of the parameters determining reproduction and survival can lead to differing Allee thresholds, even if these differing combinations imply the same stable-stage population growth rate. The approach further shows how mate-limitation can induce long transient dynamics, even in populations that eventually grow to carrying capacity. Applying the models to the overharvested low-density polar bear population of Viscount Melville Sound, Canada, shows that a mate-finding Allee effect is a plausible mechanism for slow recovery of this population. Our approach is generalizable to any mating system and life cycle, and could aid proactive management and conservation strategies, for example, by providing a priori estimates of minimum conservation targets for rare species or minimum eradication targets for pests and invasive species.


Serial magnetic resonance imaging in experimental primate stroke: validation of MRI for pre-clinical cerebroprotective trials.
Precise assessment of stroke outcome is critical for pre-clinical testing of cerebroprotective strategies. Differences in stroke volume measured by various magnetic resonance imaging (MRI) techniques are documented in humans, but not well described in experimental primate stroke. This study characterizes changes in stroke volume using serial MRI scans in a baboon model of reperfused cerebral ischemia. The location/area of hyperintensity on MRI corresponded with the TTC-stained infarct region. T2-weighted fast spin echo (T2W FSE), fluid attenuated inversion recovery (FLAIR), and diffusion weighted imaging (DWI) showed a decrease in infarct volume between 72 h and nine days post-ischemia (p = ns, p = 0.029, and p = 0.006). T2W FSE and FLAIR demonstrated an increase in infarct volume from 24 h to nine days post-ischemia, while DWI displayed a decrease over the same period. While early T2W FSE, FLAIR, and DWI all correlated with late infarct volume (p < 0.001), 72 h T2W FSE was the best direct measure (2.39% +/- 1.40% unity deviation). Serial MRI in a nonhuman primate model of focal cerebral ischemia recapitulates findings in clinical stroke. MRI at 72 h accurately predicts late infarct volume.


Differences in the glucose and fructose consumption profiles in diverse Saccharomyces wine species and their hybrids during grape juice fermentation.
Yeasts with a high fructose consumption capability are very important for winemakers to solve problems associated with sluggish or stuck fermentations causing undesirable sweetness in wines. In the present study, we analyze the kinetics of glucose and fructose consumption during wine fermentations performed at low (12 degrees C) and high (28 degrees C) temperatures by twelve different yeast strains belonging to the species Saccharomyces cerevisiae, S. bayanus var. uvarum, S. kudriavzevii as well as interspecific Saccharomyces hybrids. Different mathematical equations (sigmoid, exponential and linear decay functions) were used to fit, by means of linear and nonlinear regressions, the sugar degradation along the fermentative process. Temperature had an important influence on glucose and fructose consumption, and clearly different degradation profiles were observed at 12 and 28 degrees C. From the obtained equations, times to consume half and total of the initial glucose and fructose concentrations present in the must were calculated, allowing a quantitative comparison among yeasts in order to select the fastest fermentative yeast according to the fermentation temperature. In general, all yeasts assayed showed a slightly higher preference for glucose than fructose at both temperatures, confirming the glucophilic character of Saccharomyces wine yeasts. However, at low temperatures, some Saccharomyces yeasts showed a fructophilic character at the beginning of fermentation. This kind of studies can be very useful for the wine industry to select yeast strains with different glucose/fructose preferences.


HIV-1 induced activation of CD4+ T cells creates new targets for HIV-1 infection in human lymphoid tissue ex vivo.
We demonstrate mechanisms by which HIV-1 appears to facilitate its own infection in ex vivo-infected human lymphoid tissue. In this system, HIV-1 readily infects various CD4+ T cells, but productive viral infection was supported predominantly by activated T cells expressing either CD25 or HLA-DR or both (CD25/HLA-DR) but not other activation markers: There was a strong positive correlation (r=0.64, P=.001) between virus production and the number of CD25+/HLA-DR+ T cells. HIV-1 infection of lymphoid tissue was associated with activation of both HIV-1-infected and uninfected (bystanders) T cells. In these tissues, apoptosis was selectively increased in T cells expressing CD25/HLA-DR and p24gag but not in cells expressing either of these markers alone. In the course of HIV-1 infection, there was a significant increase in the number of activated (CD25+/HLA-DR+) T cells both infected and uninfected (bystander). By inducing T cells to express particular markers of activation that create new targets for infection, HIV-1 generates in ex vivo lymphoid tissues a vicious destructive circle of activation and infection. In vivo, such self-perpetuating cycle could contribute to HIV-1 disease.


Estimates of the contribution of single neurons to perception depend on timescale and noise correlation.
The sensitivity of a population of neurons, and therefore the amount of sensory information available to an animal, is limited by the sensitivity of single neurons in the population and by noise correlation between neurons. For decades, therefore, neurophysiologists have devised increasingly clever and rigorous ways to measure these critical variables (Parker and Newsome, 1998). Previous studies examining the relationship between the responses of single middle temporal (MT) neurons and direction-discrimination performance uncovered an apparent paradox. Sensitivity measurements from single neurons suggested that small numbers of neurons may account for a monkey's psychophysical performance (Britten et al., 1992), but trial-to-trial variability in activity of single MT neurons are only weakly correlated with the monkey's behavior, suggesting that the monkey's decision must be based on the responses of many neurons (Britten et al., 1996). We suggest that the resolution to this paradox lies (1) in the long stimulus duration used in the original studies, which led to an overestimate of neural sensitivity relative to psychophysical sensitivity, and (2) mistaken assumptions (because no data were available) about the level of noise correlation in MT columns with opposite preferred directions. We therefore made new physiological and psychophysical measurements in a reaction time version of the direction-discrimination task that matches neural measurements to the actual decision time of the animals. These new data, considered together with our recent data on noise correlation in MT (Cohen and Newsome, 2008), provide a substantially improved account of psychometric performance in the direction-discrimination task.


Effect of light emitting diodes in the photodynamic therapy of rheumatoid arthritis.
Complex and painful surgical removal of synovium was replaced by arthroscopic synovectomy as an early treatment of rheumatoid arthritis (RA), which being limited to bigger joints, was replaced by laser synovectomy. Having been more time consuming, laser photodynamic therapy (PDT) replaced this method. Due to thermal side effects of laser PDT, an alternative source of light has been sought. Therefore, to make RA treatment cheaper, less hazardous and suitable according to anatomical geometry, light emitting diodes (LEDs) were used in this study as a potential source of light. Red, white, yellow and infra-red (IR) LEDs were tested to measure the optical penetration for soft tissue and their scattering. In vitro study of the cellular response of normal and inflamed lymphocytes from healthy and RA patients was conducted respectively. Methotrexate was injected as photosensitizer to achieve cell-specific precision. IR LEDs showed the maximum penetration and least scattering of all LEDs used. Specimen with drug administration and with subsequent exposure to IR LEDs exhibited massive suppression of inflamed activated lymphocytes in comparison to other controls. The properly selected wavelength and intensity of light beam were incident with great precision so that they would not affect unwanted cells, but inflamed activated cells were suppressed due to intense light energy following Methotrexate injection. Without invasion, IR LED PDT showed an effective and cheaper treatment solution for RA.


Prediction of mortality in adult patients with severe acute lung failure receiving veno-venous extracorporeal membrane oxygenation: a prospective observational study.
Veno-venous extracorporeal membrane oxygenation (vvECMO) can be a life-saving therapy in patients with severe acute lung failure refractory to conventional therapy. Nevertheless, vvECMO is a procedure associated with high costs and resource utilization. The aim of this study was to assess published models for prediction of mortality following vvECMO and optimize an alternative model. Established mortality risk scores were validated to assess their usefulness in 304 adult patients undergoing vvECMO for refractory lung failure at the University Medical Center Regensburg from 2008 to 2013. A parsimonious prediction model was developed based on variables available before ECMO initiation using logistic regression modelling. We then assessed whether addition of variables available one day after ECMO implementation enhanced mortality prediction. Models were internally validated and calibrated by bootstrapping (400 runs). Predictive ability, goodness-of-fit and model discrimination were compared across the different models. In the present study population, existing mortality prediction tools for vvECMO patients showed suboptimal performance. Evaluated before vvECMO initiation, a logistic prediction model comprising age, immunocompromised state, artificial minute ventilation, pre-ECMO serum lactate and hemoglobin concentrations showed best mortality prediction in our patients (area under curve, AUC: 0.75). Additional information about norepinephrine dosage, fraction of inspired oxygen, C-reactive protein and fibrinogen concentrations the first day following ECMO initiation further improved discrimination (AUC: 0.79, P = 0.03) and predictive ability (likelihood ratio test, P < 0.001). When classifying patients as lower (<40%) or higher (>80%) risk based on their predicted mortality, the pre-ECMO and day1-on-ECMO models had negative/positive predictive values of 76%/82% and 82%/81%, respectively. While pre-ECMO mortality prediction remains a challenge due to large patient heterogeneity, evaluation one day after ECMO initiation may improve the ability to separate lower- and higher-risk patients. Our findings support the clinical perception that chronic health condition, high comorbidity and reduced functional reserves are strongly related to survival during and following ECMO support. Renewed evaluation the first day after ECMO initiation may provide enhanced guidance for further handling of ECMO patients. Despite the usefulness of prediction models, thorough clinical evaluation should always represent the cornerstone in decision for ECMO.


Cross talk between cytokine and hyperthermia-induced pathways: identification of different subsets of NF-κB-dependent genes regulated by TNFα and heat shock.
Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of NF-κB-dependent genes is limited. Using genomic approaches, i.e., expression microarrays and ChIP-Seq, we aimed to establish a global picture for heat shock-mediated impact on the expression of genes regulated by TNFα cytokine. We found that 193 genes changed expression in human U-2 osteosarcoma cells stimulated with cytokine (including 77 genes with the κB motif in the proximal promoters). A large overlap between sets of genes modulated by cytokine or by heat shock was revealed (86 genes were similarly affected by both stimuli). Binding sites for heat shock-induced HSF1 were detected in regulatory regions of 1/3 of these genes. Furthermore, pre-treatment with heat shock affected the expression of 2/3 of cytokine-modulated genes. In the largest subset of co-affected genes, heat shock suppressed the cytokine-mediated activation (antagonistic effect, 83 genes), which genes were associated with the canonical functions of NF-κB signaling. However, subsets of co-activated and co-repressed genes were also revealed. Importantly, pre-treatment with heat shock resulted in the suppression of NF-κB binding in the promoters of the cytokine-upregulated genes, either antagonized or co-activated by both stimuli. In conclusion, we confirmed that heat shock inhibited activation of genes involved in the classical cytokine-mediated functions of NF-κB. On the other hand, genes involved in transcription regulation were over-represented in the subset of genes upregulated by both stimuli. This suggests the replacement of NF-κB-mediated regulation by heat shock-mediated regulation in the latter subset of genes, which may contribute to the robust response of cells to both stress conditions.


Neuregulin 1 and schizophrenia: genetics, gene expression, and neurobiology.
Neuregulin 1 (NRG1) is a leading schizophrenia susceptibility gene. The NRG1 locus on chromosome 8p shows linkage to the disorder, and genetic association has been found between schizophrenia and various non-coding polymorphisms and haplotypes, especially at the 5' end of the NRG1 gene, in many but not all case-control and family studies. NRG1 is a pleiotropic growth factor, important in nervous system development and functioning; roles include the modulation of neuronal migration, synaptogenesis, gliogenesis, neuron-glia communication, myelination, and neurotransmission. Understanding the neurobiology of NRG1 and its involvement in schizophrenia is challenged by the complexity of the gene, which gives rise to multiple functionally distinct isoforms, including six "types" of NRG1 defined by 5' exon usage. Type IV and type I NRG1 may be particularly relevant to schizophrenia, with initial data showing altered expression of these isoforms in the disorder or in association with NRG1 risk alleles. We review the structure and functions of NRG1, consider the evidence for and against it being a schizophrenia susceptibility gene, and discuss mechanisms that might underlie the contribution of NRG1 to disease pathophysiology.


Effects of bidi smoking on all-cause mortality and cardiorespiratory outcomes in men from south Asia: an observational community-based substudy of the Prospective Urban Rural Epidemiology Study (PURE).
Bidis are minimally regulated, inexpensive, hand-rolled tobacco products smoked in south Asia. We examined the effects of bidi smoking on baseline respiratory impairment, and prospectively collected data for all-cause mortality and cardiorespiratory events in men from this region. This substudy of the international, community-based Prospective Urban Rural Epidemiology (PURE) study was done in seven centres in India, Pakistan, and Bangladesh. Men aged 35-70 years completed spirometry testing and standardised questionnaires at baseline and were followed up yearly. We used multilevel regression to compare cross-sectional baseline cardiorespiratory symptoms, spirometry measurements, and follow-up events (all-cause mortality, cardiovascular events, respiratory events) adjusted for socioeconomic status and baseline risk factors between non-smokers, light smokers of bidis or cigarettes (≤10 pack-years), heavy smokers of cigarettes only (>10 pack-years), and heavy smokers of bidis (>10 pack-years). 14 919 men from 158 communities were included in this substudy (8438 non-smokers, 3321 light smokers, 959 heavy cigarette smokers, and 2201 heavy bidi smokers). Mean duration of follow-up was 5·6 years (range 1-13). The adjusted prevalence of self-reported chronic wheeze, cough or sputum, dyspnoea, and chest pain at baseline increased across the categories of non-smokers, light smokers, heavy cigarette smokers, and heavy bidi smokers (p<0·0001 for association). Adjusted cross-sectional age-related changes in forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) ratio were larger for heavy bidi smokers than for the other smoking categories. Hazard ratios (relative to non-smokers) showed increasing hazards for all-cause mortality (light smokers 1·28 [95% CI 1·02-1·62], heavy cigarette smokers 1·59 [1·13-2·24], heavy bidi smokers 1·56 [1·22-1·98]), cardiovascular events (1·45 [1·13-1·84], 1·47 [1·05-2·06], 1·55 [1·17-2·06], respectively) and respiratory events (1·30 [0·91-1·85], 1·21 [0·70-2·07], 1·73 [1·23-2·45], respectively) across the smoking categories. Bidi smoking is associated with severe baseline respiratory impairment, all-cause mortality, and cardiorespiratory outcomes. Stricter controls and regulation of bidis are needed to reduce the tobacco-related disease burden in south Asia. Population Health Research Institute, Canadian Institutes of Health Research, and Heart and Stroke Foundation of Ontario.


Ketamine and ketamine metabolites as novel estrogen receptor ligands: Induction of cytochrome P450 and AMPA glutamate receptor gene expression.
Major depressive disorder (MDD) is the most common psychiatric illness worldwide, and it displays a striking sex-dependent difference in incidence, with two thirds of MDD patients being women. Ketamine treatment can produce rapid antidepressant effects in MDD patients, effects that are mediated-at least partially-through glutamatergic neurotransmission. Two active metabolites of ketamine, (2R,6R)-hydroxynorketamine (HNK) and (2S,6S)-HNK, also appear to play a key role in ketamine's rapid antidepressant effects through the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors. In the present study, we demonstrated that estrogen plus ketamine or estrogen plus active ketamine metabolites displayed additive effects on the induction of the expression of AMPA receptor subunits. In parallel, the expression of estrogen receptor alpha (ERα) was also significantly upregulated. Even more striking, radioligand binding assays demonstrated that [3H]-ketamine can directly bind to ERα (KD: 344.5 ± 13 nM). Furthermore, ketamine and its (2R,6R)-HNK and (2S,6S)-HNK metabolites displayed similar affinity for ERα (IC50: 2.31 ± 0.1, 3.40 ± 0.2, and 3.53 ± 0.2 µM, respectively) as determined by [3H]-ketamine displacement assays. Finally, induction of AMPA receptors by either estrogens or ketamine and its metabolites was lost when ERα was knocked down or silenced pharmacologically. These results suggest a positive feedback loop by which estrogens can augment the effects of ketamine and its (2R,6R)-HNK and (2S,6S)-HNK metabolites on the ERα-induced transcription of CYP2A6 and CYP2B6, estrogen inducible enzymes that catalyze ketamine's biotransformation to form the two active metabolites. These observations provide novel insight into ketamine's molecular mechanism(s) of action and have potential implications for the treatment of MDD.


Comparison of the effects of increased myocardial oxygen consumption and adenosine on the coronary microvascular resistance.
The purposes of this study were to determine if coronary dilation secondary to an increase in myocardial oxygen consumption (MVO2) affects the microcirculation in a homogeneous or heterogeneous manner and to determine if comparable degrees of coronary dilation produced by increasing MVO2 or exogenous (intravenous adenosine) or endogenous (intravenous dipyridamole) adenosine have similar effects in the coronary microcirculation. The epimyocardial coronary microcirculation was observed through an intravital microscope by stroboscopic epi-illumination in anesthetized open-chest dogs. Aortic pressure and heart rate were controlled by an aortic snare and atrioventricular sequential pacing, respectively, during experimental procedures. In group 1 (n = 15), coronary arterial microvessel diameters were measured under control condition and during rapid pacing at 300 beats/min, which doubled MVO2. Increases in MVO2 caused heterogeneous vasodilation in coronary arterial microvessels (40-380 microns). There was an inverse relation between control diameter and percent increase in diameter. In group 2 (n = 15) or group 3 (n = 10), adenosine or dipyridamole was infused intravenously to increase myocardial perfusion to the same level as that obtained with rapid pacing. Adenosine and dipyridamole did not change MVO2. Adenosine and dipyridamole also caused heterogeneous vasodilation, but the effects of adenosine and dipyridamole were restricted to arterial microvessels smaller than 150 microns. From these results, we conclude that increases in MVO2 produce widespread but heterogeneous vasodilation, that is, greater dilation in smaller arterial microvessels. Comparable increases in coronary flow produced by increasing MVO2 or endogenous and exogenous adenosine do not produce identical changes in the distribution of coronary microvascular resistance.


Treatment patterns among elderly patients with stage IV breast cancer treated with HER-2-targeted therapy.
To evaluate treatment patterns among elderly, newly diagnosed stage IV breast cancer patients receiving HER-2-targeted therapy. Women aged 65+ with an incident diagnosis of stage IV breast cancer (index) and no history of other cancer were identified from 2006 to 2010 linked Surveillance, Epidemiology and End RESULTS and Medicare data. Continuous enrollment from 1 year preindex (baseline) through disenrollment, death or the end of the data (follow-up) was required. Patients were required to receive HER-2-targeted therapy (trastuzumab or lapatinib) during follow-up. Treatment therapies during follow-up were evaluated, as was the distribution of treatment combinations. Initial treatment regimens were evaluated based on the treatment(s) received after index. A 42-day gap in therapy or the addition of a biologic therapy was used as a marker for a subsequent regimen. A total of 173 patients were identified (mean [standard deviation] age: 73.9 [6.7] years). The majority received trastuzumab (>93%) during follow-up (mean [standard deviation] duration: 24.3 [11.3] months), with 9.8% receiving lapatinib. Most received chemotherapy (83.2%), approximately half received surgery (55.5%), over 40% received hormonal therapy and a third received radiation (35.3%). Trastuzumab + chemotherapy was the most common initial treatment regimen (43.9%); less common therapies include trastuzumab alone (17.3%), and trastuzumab + chemotherapy + hormonal (13.3%). Among patients receiving chemotherapy, the majority received a taxane-based chemotherapy. The average treatment duration for any treatment regimen was just less than a year (44.9-52.5 weeks). Among this population, the majority received taxane-based combination chemotherapy, consistent with National Comprehensive Cancer Network guidelines.