Mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury.
Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV. Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and 2003, and infected patients developed an atypical pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) leading to pulmonary fibrosis and death. We identified sets of differentially expressed genes that contribute to ALI and ARDS using lethal and sublethal SARS-CoV infection models. Mathematical prioritization of our gene sets identified the urokinase and extracellular matrix remodeling pathways as the most enriched pathways. By infecting Serpine1-knockout mice, we showed that the urokinase pathway had a significant effect on both lung pathology and overall SARS-CoV pathogenesis. These results demonstrate the effective use of unbiased modeling techniques for identification of high-priority host targets that regulate disease outcomes. Similar transcriptional signatures were noted in 1918 and 2009 H1N1 influenza virus-infected mice, suggesting a common, potentially treatable mechanism in development of virus-induced ALI.


Luminance and edge information in grouping: a study using visual search.
Preattentive grouping is supported by 2 systems, a brightness system that is contrast polarity sensitive and an edge system that is relatively insensitive to contrast polarity. Search was spatially parallel for pairs of same contrast polarity vertically aligned circles, among horizontal pairs, and serial for pairs of circles that had the opposite contrast polarity (Experiments 1-3). By replacing the circles with squares, the authors investigated the effect of adding collinear edge information. When collinear edges were present, the polarity difference between paired items did not disrupt grouping (Experiments 4-6). These results support models of grouping in which brightness and edge information are processed separately (e.g., S. Grossberg & E. Mingolla, 1985) and models of visual search in which complex relations between stimuli can be computed in parallel across the display.


Auricular acupuncture for pain relief after ambulatory knee surgery: a randomized trial.
Auricular acupuncture is a promising method for postoperative pain relief. However, there is no evidence for its use after ambulatory surgery. Our aim was to test whether auricular acupuncture is better than invasive needle control for complementary analgesia after ambulatory knee surgery. One hundred and twenty patients undergoing ambulatory arthroscopic knee surgery under standardized general anesthesia were randomly assigned to receive auricular acupuncture or a control procedure. Fixed indwelling acupuncture needles were inserted before surgery and retained in situ until the following morning. Postoperative rescue analgesia was directed to achieve pain intensity less than 40 mm on a 100-mm visual analogue scale. The primary outcome measure was the postoperative requirement for ibuprofen between surgery and examination the following morning. Intention-to-treat analysis showed that patients from the control group (n = 59) required more ibuprofen than patients from the auricular acupuncture group (n = 61): median (interquartile range) 600 (200-800) v. 200 (0-600) mg (p = 0.012). Pain intensity on a visual analogue scale was similar in both groups at all time points registered. The majority of patients in both groups believed that they had received true acupuncture and wanted to repeat it in future. Auricular acupuncture reduced the requirement for ibuprofen after ambulatory knee surgery relative to an invasive needle control procedure.


Establishment of tolerance to commensal bacteria requires a complex microbiota and is accompanied by decreased intestinal chemokine expression.
Intricate regulation of tolerance to the intestinal commensal microbiota acquired at birth is critical. We hypothesized that epithelial cell tolerance toward early gram-positive and gram-negative colonizing bacteria is established immediately after birth, as has previously been shown for endotoxin. Gene expression in the intestine of mouse pups born to dams that were either colonized with a conventional microbiota or monocolonized (Lactobacillus acidophilus or Eschericia coli) or germ free was examined on day 1 and day 6 after birth. Intestinal epithelial cells from all groups of pups were stimulated ex vivo with L. acidophilus and E. coli to assess tolerance establishment. Intestine from pups exposed to a conventional microbiota displayed lower expression of Ccl2, Ccl3, Cxcl1, Cxcl2, and Tslp than germ-free mice, whereas genes encoding proteins in Toll-like receptor signaling pathways and cytokines were upregulated. When comparing pups on day 1 and day 6 after birth, a specific change in gene expression pattern was evident in all groups of mice. Tolerance to ex vivo stimulation with E. coli was only established in conventional animals. Colonization of the intestine was reflected in the spleen displaying downregulation of Cxcl2 compared with germ-free animals on day 1 after birth. Colonization reduced the expression of genes involved in antigen presentation in the intestine-draining mesenteric lymph nodes, but not in the popliteal lymph nodes, as evidenced by gene expression on day 23 after birth. We propose that microbial detection systems in the intestine are upregulated by colonization with a diverse microbiota, whereas expression of proinflammatory chemokines is reduced to avoid excess recruitment of immune cells to the maturing intestine.


Increased resting metabolic rates in obese subjects with non-insulin-dependent diabetes mellitus and the effect of sulfonylurea therapy.
Obese subjects with non-insulin-dependent diabetes mellitus (NIDDM) lose weight soon after diagnosis and tend to gain weight during hypoglycemic therapy. One explanation for these weight shifts is the change in caloric loss from glycosuria. We compared 24 obese Pima Indians with NIDDM to 24 Pima Indians with normal glucose tolerance to determine whether resting metabolic rate changes may be an additional factor influencing the weight shifts. The diabetic and nondiabetic subjects were equally obese, body fat 38 +/- 1% versus 37 +/- 1% (mean +/- SEM), respectively, as determined by densitometry. In the morning after an overnight fast, resting metabolic rate (RMR) was measured by indirect calorimetry. The mean RMR of the diabetic subjects, 32.9 +/- 0.5 kcal/day X kg fat-free mass (FFM), was 5% higher than that of the nondiabetic subjects, 31.4 +/- 0.5 kcal/day X kg FFM (P less than 0.05). In nine of the diabetic subjects, 6 wk of tolazamide therapy was associated with reductions in mean FPG, 253 +/- 16 to 144 +/- 14 mg/dl (P less than 0.01), mean daily urine glucose loss, 128 +/- 26 to 11 +/- 4 g (P less than 0.01), and mean RMR, 31.9 +/- 0.8 to 30.2 +/- 0.6 kcal/day X kg FFM (P less than 0.04). Weight of the subjects was maintained constant from beginning to end of therapy (106.5 +/- 9.6 versus 108.1 +/- 9.9 kg) by decreasing daily calorie intake from 3070 +/- 103 to 2784 +/- 163 kcal (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Emerging roles of cadmium and heme oxygenase in type-2 diabetes and cancer susceptibility.
Many decades after an outbreak of severe cadmium poisoning, known as Itai-itai disease, cadmium continues to pose a significant threat to human health worldwide. This review provides an update on the effects of this environmental toxicant cadmium, observed in numerous populations despite modest exposure levels. In addition, it describes the current knowledge on the link between heme catabolism and glycolysis. It examines novel functions of heme oxygenase-2 (HO-2) that protect against type 2-diabetes and obesity, which have emerged from diabetic/obese phenotypes of the HO-2 knockout mouse model. Increased cancer susceptibility in type-2 diabetes has been noted in several large cohorts. This is a cause for concern, given the high prevalence of type-2 diabetes worldwide. A lifetime exposure to cadmium is associated with pre-diabetes, diabetes, and overall cancer mortality with sex-related differences in specific types of cancer. Liver and kidney are target organs for the toxic effects of cadmium. These two organs are central to the maintenance of blood glucose levels. Further, inhibition of gluconeogenesis is a known effect of heme, while cadmium has the propensity to alter heme catabolism. This raises the possibility that cadmium may mimic certain HO-2 deficiency conditions, resulting in diabetic symptoms. Intriguingly, evidence has emerged from a recent study to suggest the potential interaction and co-regulation of HO-2 with the key regulator of glycolysis: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4). HO-2 could thus be critical to a metabolic switch to cancer-prone cells because the enzyme PFKFB and glycolysis are metabolic requirements for cell proliferation and resistance to apoptosis.


Time Explains Regional Richness Patterns within Clades More Often than Diversification Rates or Area.
Most groups of organisms occur in multiple regions and have different numbers of species in different regions. These richness patterns are directly explained by speciation, extinction, and dispersal. Thus, regional richness patterns may be explained by differences in when regions were colonized (more time for speciation in regions colonized earlier), differences in how often they were colonized, or differences in diversification rates (speciation minus extinction) among regions (with diversification rates potentially influenced by area, climate, and/or many other variables). Few studies have tested all three factors, and most that did examined them only in individual clades. Here, we analyze a diverse set of 15 clades of plants and animals to test the causes of regional species richness patterns within clades. We find that time was the sole variable significantly explaining richness patterns in the best-fitting models for most clades (10/15), whereas time combined with other factors explained richness in all others. Time was the most important factor explaining richness in 13 of 15 clades, and it explained 72% of the variance in species richness among regions across all 15 clades (on average). Surprisingly, time was increasingly important in older and larger clades. In contrast, the area of the regions was relatively unimportant for explaining these regional richness patterns. A systematic review yielded 15 other relevant studies, which also overwhelmingly supported time over diversification rates (13 to 1, with one study supporting both diversification rates and time). Overall, our results suggest that colonization time is a major factor explaining regional-scale richness patterns within clades (e.g., families).


Blue moons and Martian sunsets.
The familiar yellow or orange disks of the moon and sun, especially when they are low in the sky, and brilliant red sunsets are a result of the selective extinction (scattering plus absorption) of blue light by atmospheric gas molecules and small aerosols, a phenomenon explainable using the Rayleigh scattering approximation. On rare occasions, dust or smoke aerosols can cause the extinction of red light to exceed that for blue, resulting in the disks of the sun and moon to appear as blue. Unlike Earth, the atmosphere of Mars is dominated by micron-size dust aerosols, and the sky during sunset takes on a bluish glow. Here we investigate the role of dust aerosols in the blue Martian sunsets and the occasional blue moons and suns on Earth. We use the Mie theory and the Debye series to calculate the wavelength-dependent optical properties of dust aerosols most commonly found on Mars. Our findings show that while wavelength selective extinction can cause the sun's disk to appear blue, the color of the glow surrounding the sun as observed from Mars is due to the dominance of near-forward scattering of blue light by dust particles and cannot be explained by a simple, Rayleigh-like selective extinction explanation.


Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.
CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.


Computerized adaptive testing--ready for ambulatory monitoring?
Computerized adaptive tests (CATs) have abundant theoretical advantages over established static instruments, which could improve ambulatory monitoring of patient-reported outcomes (PROs). However, an empirical demonstration of their practical benefits is warranted. We reviewed the literature and evaluated existing data to discuss the potential of CATs for use in ambulatory monitoring outside clinical facilities. Computerized adaptive tests are not being used for ambulatory monitoring, but initial results from their use in health care research allow for discussion of some issues relevant to ambulatory care. Evidence shows that CATs can capture the most relevant health outcomes as well as established static tools, with substantially decreased respondent burden. They can be more precise than static tools of similar length and can reduce floor and ceiling effects. Computerized adaptive tests can reliably measure a construct over time with different items, which yields the potential of introducing item exposure control in ambulatory monitoring. Studies have shown that CATs can be at least as valid as well-designed static tools in group comparisons, but further investigation is needed to determine whether psychometric advantages lead to increased responsiveness of CATs. Ambulatory monitoring of PROs demands short, yet very precise measurements, which can be repeated up to many times a day. Computerized adaptive tests may address several present shortcomings in ambulatory monitoring of PROs efficiently. However, most CAT developments have primarily focused on psychometric improvements. To use the full potential of CATs for ambulatory monitoring purposes, content must also be carefully considered.


Measuring COVID-19-related fear and threat in Australian, Indian, and Nepali university students.
The present study describes the adaption and validation of a brief measure of contagion-related fear and threat in Australian, Indian, and Nepali university students in Australia at the height of the first wave of the COVID-19 pandemic. Adapted from Ho, Kwong-Lo, Mak, and Wong's (2005) SARS-related fear scale, the Contagion Fear and Threat Scale (CFTS) was rapidly adapted to capture the experience of COVID-19 pandemic-related fear. The factor structure and validity of the 6-item scale were established among Australian (n = 154), Indian (n = 111), and Nepali (n = 149) university students studying in Australia in May-June 2020. Factor analysis revealed two 3-item factors in the Australian student sample: Fear of Infection and Existential Threat. These factors were confirmed in the Indian and Nepali student samples and mirror those found by the Ho et al. (2005) in their original instrument. The convergent and discriminant validity of the full CFTS, Fear of Infection, and Existential Threat scales are indicated via correlations with established measures of depression, anxiety, stress, subjective wellbeing, and religiosity. Differences in the performance of the Fear of Infection and Existential Threat scales are considered in terms of the respective objective and subjective nature of the constructs.


Working IT out in medical practice: IT systems design and development as co-realisation.
The paper explores possibilities for situating IT design and development work within the context of use so as to support the co-realisation of technology and 'design in use'. The aim is to build a new understanding between IT professionals and users which is grounded upon what happens as the latter grapple with the problems of applying IT, appropriating its functionalities and affordances into their work practices and relations. Following a discussion of participatory design and ethnomethodology, a novel method called co-realisation, which aims to provide a synthesis of the preceding methods, is suggested as an alternative. Through a discussion of findings from a case study of IT systems design and development in healthcare we show how the co-realisation approach might provide work-affording systems and how user-designer relations might be reformulated. We suggest that work-affording systems can be developed through the deployment of an engaged facilitator who works with the users to unpack the work site-specific potentialities of technology. The case study shows how risk of non-adoption might be minimised through the development of partnerships, and how the presence of the facilitator in the workplace capitalises on the mundane work undertaken therein and how the facilitator might work with the users to develop artefacts that support this work as opposed to reconfiguring it. The case study illustrates co-realisation in action and how it might be seen to reconfigure relations between users and designers in a way that appears productive. Co-realisation can help address the widely observed problem of IT systems failures in healthcare.


The use of household cleaning sprays and adult asthma: an international longitudinal study.
Cleaning work and professional use of certain cleaning products have been associated with asthma, but respiratory effects of nonprofessional home cleaning have rarely been studied. To investigate the risk of new-onset asthma in relation to the use of common household cleaners. Within the follow-up of the European Community Respiratory Health Survey in 10 countries, we identified 3,503 persons doing the cleaning in their homes and who were free of asthma at baseline. Frequency of use of 15 types of cleaning products was obtained in a face-to-face interview at follow-up. We studied the incidence of asthma defined as physician diagnosis and as symptoms or medication usage at follow-up. Associations between asthma and the use of cleaning products were evaluated using multivariable Cox proportional hazards or log-binomial regression analysis. The use of cleaning sprays at least weekly (42% of participants) was associated with the incidence of asthma symptoms or medication (relative risk [RR], 1.49; 95% confidence interval [CI], 1.12-1.99) and wheeze (RR, 1.39; 95% CI, 1.06-1.80). The incidence of physician-diagnosed asthma was higher among those using sprays at least 4 days per week (RR, 2.11; 95% CI, 1.15-3.89). These associations were consistent for subgroups and not modified by atopy. Dose-response relationships (P < 0.05) were apparent for the frequency of use and the number of different sprays. Risks were predominantly found for the commonly used glass-cleaning, furniture, and air-refreshing sprays. Cleaning products not applied in spray form were not associated with asthma. Frequent use of common household cleaning sprays may be an important risk factor for adult asthma.


Feasibility of training practice nurses to deliver a psychosocial intervention within a collaborative care framework for people with depression and long-term conditions.
Practice nurses (PNs) deliver much of the chronic disease management in primary care and have been highlighted as appropriately placed within the service to manage patients with long-term physical conditions (LTCs) and co-morbid depression. This nested qualitative evaluation within a service development pilot provided the opportunity to examine the acceptability of a Brief Behavioural Activation (BBA) intervention within a collaborative care framework. Barriers and facilitators to engaging with the intervention from the patient and clinician perspective will be used to guide future service development and research. The study was conducted across 8 practices in one Primary Care Trust 1 in England. Through purposive sampling professionals (n = 10) taking part in the intervention (nurses, GPs and a mental health gateway worker) and patients (n = 4) receiving the intervention participated in semi-structured qualitative interviews. Analysis utilised the four Normalisation Process Theory (NPT) concepts of coherence, cognitive participation, collective action and reflexive monitoring to explore the how this intervention could be implemented in practice. Awareness of depression and the stigma associated with the label of depression meant that, from a patient perspective a PN being available to 'listen' was perceived as valuable. Competing practice priorities, perceived lack of time and resources, and lack of engagement by the whole practice team were considered the greatest barriers to the implementation of this intervention in routine primary care. Lack of understanding of, participation in, and support from the whole practice team in the collaborative care model exacerbated the pressures perceived by PNs. The need for formal supervision of PNs to enable them to undertake the role of case manager for patients with depression and long-term conditions is emphasised.


Short- and long-term benefits of lenalidomide treatment in patients with lower-risk del(5q) myelodysplastic syndromes.
The treatment of patients with myelodysplastic syndromes (MDS) begins with assessment of karyotype and risk. Lenalidomide is approved for the treatment of patients who have transfusion-dependent anemia due to lower-risk MDS with chromosome 5q deletion (del(5q)) with or without additional cytogenetic abnormalities, and isolated del(5q) only in the European Union. Mounting evidence suggests that lenalidomide is effective not only in reducing red blood cell (RBC) transfusion burden, but also in modifying the disease natural history by suppressing the malignant clone. Data discussed here from the pivotal phase 2 (MDS-003) and phase 3 (MDS-004) studies of lenalidomide demonstrate that lenalidomide treatment was associated with both short- and long-term benefits. These clinical benefits included high rates of RBC-transfusion independence (TI) with prolonged durations of response, high rates of cytogenetic response (CyR) associated with achievement of durable RBC-TI, no significant difference in rates of progression to acute myeloid leukemia (AML), and improvements in health-related quality of life (HRQOL). Achievement of RBC-TI and CyR with lenalidomide treatment was associated with extended survival and time to AML progression. Achievement of RBC-TI and hemoglobin response was additionally associated with HRQOL benefits. Recent data describing the impact of TP53 mutations and p53 expression on the prognosis of patients with del(5q) and the impact on response to lenalidomide are also discussed. The authors provide practical recommendations for the use of lenalidomide in patients with lower-risk del(5q) MDS.


CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum.
Engineering the small subunit of the key CO2-fixing enzyme Rubisco (SSU, encoded by rbcS) in plants currently poses a significant challenge, as many plants have polyploid genomes and SSUs are encoded by large multigene families. Here, we used CRISPR-Cas9-mediated genome editing approach to simultaneously knock-out multiple rbcS homologs in the model tetraploid crop tobacco (Nicotiana tabacum cv. Petit Havana). The three rbcS homologs rbcS_S1a, rbcS_S1b and rbcS_T1 account for at least 80% of total rbcS expression in tobacco. In this study, two multiplexing guide RNAs (gRNAs) were designed to target homologous regions in these three genes. We generated tobacco mutant lines with indel mutations in all three genes, including one line with a 670 bp deletion in rbcS-T1. The Rubisco content of three selected mutant lines in the T1 generation was reduced by ca. 93% and mutant plants accumulated only 10% of the total biomass of wild-type plants. As a second goal, we developed a proof-of-principle approach to simultaneously introduce a non-native rbcS gene while generating the triple SSU knockout by co-transformation into a wild-type tobacco background. Our results show that CRISPR-Cas9 is a viable tool for the targeted mutagenesis of rbcS families in polyploid species and will contribute to efforts aimed at improving photosynthetic efficiency through expression of superior non-native Rubisco enzymes in plants.


Expression of bone morphogenetic proteins during mandibular distraction osteogenesis in rabbits.
We examined the expression pattern of bone morphogenetic proteins (BMPs) during mandibular distraction osteogenesis in rabbits and also investigated the mechanism of membranous bone distraction. Twenty-three rabbits underwent mandibular distraction (protocol; no latency period, a 1-week distraction at 0.5 mm/d, and a 2-week consolidation period). Samples were collected at 3, 5, and 7 days of distraction and at 1-week and 2-week consolidation. We prepared undecalcified fresh-frozen sections and immunohistochemically evaluated the expression of BMPs 2 through 8. Both endochondral ossification and intramembranous ossification were observed. The expression of BMPs 2, 4, 5, and 6 was observed continuously from the beginning of distraction. BMP-7 was expressed weakly. The expression of BMP-3 was not observed conspicuously during distraction but was strongly expressed at 1- and 2-week consolidation. The expression pattern of BMPs during membranous bone distraction was similar to that during long bone distraction, but it differed from the expression pattern of long bone distraction in that the expression of BMPs was maintained for 2 weeks after the completion of distraction.


Epidemiological analysis of retinopathy of prematurity in a referral centre in Turkey.
To collect data towards the establishment of a guideline to predict the population under risk for the development of retinopathy of prematurity (ROP) in Turkey. Medical reports of 2950 preterm infants who underwent screening examinations for ROP between 1996 and 2010 at Istanbul Faculty of Medicine, Department of Ophthalmology were reviewed. The study population was classified as inpatient infants and referred infants. A risk factor analysis was performed limited to the 788 inpatient infants. The demographical variables regarding birth weight (BW), gestational age (GA) and postnatal age at the time of treatment were compared independently between 403 referred and 64 inpatient infants who were treated for severe ROP. The mean GA of the infants with severe ROP was 31.2±2.7 weeks, mean BW was 1543.4±508.3 g. The risk factor analysis of inpatient infants revealed BW<1500 g, GA<32 weeks; intraventricular haemorrhage, respiratory distress syndrome, sepsis, apnoea and phototherapy were associated with a higher incidence of ROP. In addition, sepsis, male gender, multiple gestations, BW<1500 g were associated with a higher incidence of progression to severe ROP, whereas maternal pre-eclampsia was associated with a lower incidence. On the other hand, comparison of demographic features revealed that the referred infants with ROP were born at a significantly higher BW and needed significantly earlier treatment than inpatient infants with ROP. The high BW and GA of infants with severe ROP indicate wider screening criteria should be used in our country. BW might be a more important criterion than GA for ROP screening.


Hepatocellular damage from non-steroidal anti-inflammatory drugs.
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the management of rheumatological disorders, and as analgesics and antipyretics. Hepatotoxicity is an uncommon, but potentially lethal complication, which usually occurs within 12 weeks of starting therapy. It can occur with all NSAIDs, but appears to be more common with diclofenac and particularly sulindac. Female patients aged >50 years, with autoimmune disease, and those on other potentially hepatotoxic drugs, appear to be particularly susceptible. Liver function test abnormalities generally settle within 4-6 weeks of stopping the causative drug. However, some patients may develop acute liver failure and successful orthotopic liver transplantation may be undertaken in such patients. Recent in vitro animal studies have shown that the mechanism of diclofenac toxicity relates both to impairment of ATP synthesis by mitochondria, and to production of active metabolites, particularly n,5-dihydroxydiclofenac, which causes direct cytotoxicity. Mitochondrial permeability transition (MPT) has also been shown to be important in diclofenac-induced liver injury, resulting in generation of reactive oxygen species, mitochondrial swelling and oxidation of NADP and protein thiols. Physicians and hepatologists must be vigilant to the hepatotoxic potential of any NSAID, as increased awareness, surveillance and reporting of these events will lead to a better understanding of the risk factors and the pathophysiology of NSAID-related hepatotoxicity.


Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.
Atrial fibrillation is the most common clinical cardiac arrhythmia. It is often initiated by ectopic beats arising from the pulmonary veins and atrium, but the source and mechanism of these beats remains unclear. The melanin synthesis enzyme dopachrome tautomerase (DCT) is involved in intracellular calcium and reactive species regulation in melanocytes. Given that dysregulation of intracellular calcium and reactive species has been described in patients with atrial fibrillation, we investigated the role of DCT in this process. Here, we characterize a unique DCT-expressing cell population within murine and human hearts that populated the pulmonary veins, atria, and atrioventricular canal. Expression profiling demonstrated that this population expressed adrenergic and muscarinic receptors and displayed transcriptional profiles distinct from dermal melanocytes. Adult mice lacking DCT displayed normal cardiac development but an increased susceptibility to atrial arrhythmias. Cultured primary cardiac melanocyte-like cells were excitable, and those lacking DCT displayed prolonged repolarization with early afterdepolarizations. Furthermore, mice with mutations in the tyrosine kinase receptor Kit lacked cardiac melanocyte-like cells and did not develop atrial arrhythmias in the absence of DCT. These data suggest that dysfunction of melanocyte-like cells in the atrium and pulmonary veins may contribute to atrial arrhythmias.


Off- Versus On-Pump Coronary Surgery and the Effect of Follow-Up Length and Surgeons' Experience: A Meta-Analysis.
Background The debate on the relative benefits of off-pump and on-pump coronary artery bypass surgery ( OPCABG and ONCABG ) is still open. We aimed to provide an updated and complete summary of the evidence on the differences between OPCABG and ONCABG and to explore whether the length of the follow-up and the surgeons' experience in OPCABG modify the comparative results. Methods and Results All randomized clinical trials comparing OPCABG and ONCABG were included. Primary outcome was follow-up mortality. Secondary outcomes were operative mortality, perioperative stroke, perioperative myocardial infarction, and late repeated revascularization. Subgroup analyses were performed based on the length of the follow-up and the percentage of crossover from the OPCABG group (used as a surrogate of surgeon experience with OPCABG ). One hundred four trials were included (20 627 patients, OPCABG : 10 288; ONCABG : 10 339). Weighted mean follow-up time was 3.7 years (range 1-7.5 years). OPCABG was associated with a higher risk of follow-up mortality (incidence rate ratio 1.11, 95% confidence interval 1.00-1.23, P=0.05). The difference was significant only for trials with mean follow-up of ≥3 years and for studies with a crossover rate of ≥10%. There was a trend toward lower risk of perioperative stroke and higher need for late repeated revascularization in the OPCABG arm. Conclusions OPCABG is associated with a higher incidence of incomplete revascularization, an increased need for repeated revascularization, and decreased midterm survival compared with ONCABG . Surgeon inexperience in OPCABG is associated with late mortality.


Community-based screening for cardiovascular disease and diabetes using HbA1c.
Persons from inner-city immigrant and mixed-ethnic communities are known to be at high risk for cardiovascular disease and diabetes. Such communities may also be underserved for preventive medical care. The authors hypothesized that hemoglobin A1c (HbA1c) could be used as a screening test for a community-based program to detect new cases of diabetes and persons at risk for diabetes and cardiovascular disease. Screenings took place in churches, group homes, shelters, community centers, and street corners of the Bronx. Screening data included history of diabetes, age, ethnicity, body mass index, blood pressure, lipid panel, random glucose, and HbA1c. Data were analyzed for number of cases of new diabetes (HbA1c > or =7%), for patients at risk for diabetes (HbA1c 6%-6.99%), and for associations between HbA1c and other variables. The effect of location of screening and self-reported ethnicity on outcome variables was also analyzed. Seven hundred four persons were screened in 25 different sessions. HbA1c and lipid profile were obtained on 539 persons, which formed the cohort for analysis. Mean HbA1c for the cohort was 6.00%. Thirty-two percent of the cohort had HbA1c of more than 6%, and 11.4% had HbA1c of more than 7%. Excluding known diabetics (13% of cohort), 24% had HbA1c of more than 6%, and 3.4% had HbA1c of more than 7%. HbA1c was significantly correlated with total cholesterol, triglycerides, low-density lipoprotein, systolic blood pressure, body mass index, and age; in all cases, correlation coefficients were higher with HbA1c than with random glucose. In addition, significantly higher cardiovascular disease risk factors were found in persons with HbA1c of more than 6%; 6% may be a threshold value for the metabolic syndrome. Mean HbA1c was higher in persons from the South Bronx (which has a higher poverty rate) than the North Bronx (6.08% v 5.74%, p=0.013). There were no statistically significant differences between self-reported ethnic groupings. There was a high prevalence of undiagnosed diabetes, and of patients at risk for diabetes, in this community setting. Community-based screening can be used as a method for identifying high percentages of patients at risk for diabetes or with undiagnosed diabetes in an inner city, immigrant, mixed-ethnic population.


Speciation in Indo-Pacific swiftlets (Aves: Apodidae): integrating molecular and phenotypic data for a new provisional taxonomy of the Collocalia esculenta complex.
White-bellied swiftlets of the Collocalia esculenta complex constitute a radiation of colony-breeding swifts distributed throughout the tropical Indo-Pacific region. Resolution of their taxonomy is challenging due to their morphological uniformity. To analyze the evolutionary history of this complex, we combine new biometric measurements and results from plumage assessment of museum specimens with novel as well as previously published molecular data. Together, this body of information constitutes the largest systematic dataset for white-bellied swiftlets yet compiled, drawn from 809 individuals belonging to 32 taxa for which new molecular, biometric, and/or plumage data are presented. We propose changing the classification of white-bellied swiftlets, for which two species are currently recognized, to elevate eight regional forms to species level, and we also describe two new subspecies. The ten taxa we recommend recognizing at the species level are: Collocalia linchi (Java to Lombok, Sumatran hills), C. dodgei (montane Borneo), C. natalis (Christmas Island), C. affinis (Greater Sundas, including the Thai-Malay Peninsula and Andaman-Nicobar Islands), C. marginata (Philippines), C. isonota (Philippines), C. sumbawae (west Lesser Sundas), C. neglecta (east Lesser Sundas), C. esculenta (Sulawesi, Moluccas, New Guinea, Bismarck Archipelago, Solomon Islands), and C. uropygialis (Vanuatu, New Caledonia). Future molecular and morphological work is needed to resolve questions of speciation and population affinities in the Philippines, Christmas Island, Wallacea and central Melanesia, and to shed light on historic diversification and patterns of gene flow in the complex.


Abasic pivot substitution harnesses target specificity of RNA interference.
Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA-target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼ 80-100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications.


Neofunctionalization of Duplicated P450 Genes Drives the Evolution of Insecticide Resistance in the Brown Planthopper.
Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3-5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6-8]. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait.


Axial stretch enhances sarcoplasmic reticulum Ca2+ leak and cellular Ca2+ reuptake in guinea pig ventricular myocytes: experiments and models.
Cardiac cellular calcium (Ca2+) handling is the well-investigated mediator of excitation-contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse--effects of mechanical stimulation on cardiomyocyte Ca2+ handling--are much less well understood, in particular during the inter-beat period, called 'diastole'. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca2+ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca2+ content [Ca2+]SR, and the reloading of the SR after prior depletion of Ca2+ from the cell. Cells were loaded with Fura-2 AM (an indicator of the cytosolic 'free' Ca2+ concentration, [Ca2+]i), and pre-conditioned by field-stimulation (2 Hz) at 37 degrees C, while [Ca2+]i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10mM of caffeine (in Na+/Ca2+-free solution), which causes the release of Ca2+ from the SR (caffeine), but largely prevents extrusion of Ca2+ from the cytosol to the cell exterior (Na+/Ca2+-free solution). By comparing the [Ca2+]i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca2+]SR. To assess SR reloading after initial Ca2+ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10mM of caffeine in normal Tyrode solution (which causes Ca2+ to be released from the SR and extruded from the cell via the Na+/Ca2+ exchanger; NCX). Axial stretch enhanced the rate of both rest-decay and reloading of [Ca2+]SR. Application of 40 microM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe-Kohl-Noble model of single cardiac myocyte Ca2+ handling, suggesting that stretch increases both Ca2+ leak from the SR and Ca2+ influx via the sarcolemma. This may have important implications for the mobilisation of Ca2+ in stretched cells, and could contribute to the regional 'matching' of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue.


First evidence of the presence of S-cysteinylated and S-glutathionylated precursors in tannins.
Tannins are widely used in winemaking and food and beverage preparation for the many different contributions they can give to the overall characteristics of the product (e.g., colour stability, mouthfeel and aromatic composition). Varietal thiols and their precursors are one of the most interesting research areas in food science and a lot of effort has been put to further the current understanding on their formation and on the impact of different production strategies on their concentration in the final product. This paper reports the identification of two important thiol precursors (Cys-3MH and GSH-3MH) in commercial grape oenological tannins and, to the best of our knowledge, this information is reported here for the first time. This finding allows potential new perspectives in the winemaking and the food industry, offering the possibility of controlled additions of thiol precursors in pre-fermentative stages in order to tune the aroma profile of fermented products.


Home-Based Physical Activity Program Improves Depression and Anxiety in Older Adults.
Late-life depression and anxiety among older adults is an important public health concern. This study examined the effect of a DVD-delivered exercise intervention on the secondary outcomes of depression and anxiety in older adults and the extent to which physical self-worth mediated the relationship between leisure-time physical activity and depression and anxiety. Older adults (N = 307) were randomized to a 6-month flexibility, toning, and balance DVD (FlexToBa™, FTB) or healthy aging DVD control. Self-reported physical activity and questionnaires were administered at baseline and postintervention. Statistical analyses were conducted in the total sample and in a subsample of participants with elevated levels of depression or anxiety. FTB participants with elevated depression and anxiety symptoms at baseline had significantly greater reductions in depression and anxiety (d = 1.66 and 2.90) than the control condition (d = 0.77 and 0.73). The effect of physical activity on depression and anxiety was partially mediated by increases in physical self-worth in the total sample but not in those with elevated depression or anxiety. A home-based physical activity intervention may be a viable treatment for reducing depression and anxiety in older adults with elevated baseline scores.


Extracorporeal Membrane Oxygenation (ECMO) in Critically Ill Patients with Coronavirus Disease 2019 (COVID-19) Pneumonia and Acute Respiratory Distress Syndrome (ARDS).
Traced back to December 2019, an unexpected outbreak of a highly contagious new coronavirus pneumonia (COVID-19) has rapidly swept around China and the globe. There have now been an estimated 2 580 000 infections and more than 170 000 fatal cases around the world. The World Health Organization (WHO) estimated that approximately 14% of infections developed into severe disease, 5% were critically ill, and the mortality rate of critically ill patients is reported to be over 50%. The shortage of specific anti-viral treatment and vaccines remains a huge challenge. In COVID-19, refractory hypoxemia is common among the critically ill with acute respiratory distress syndrome (ARDS) despite invasive mechanical ventilation, and is further complicated by respiratory and circulatory failure. This difficult situation calls for the use of extracorporeal membrane oxygenation (ECMO) for assisting respiration and circulation if necessary. This article reviews the pertinent clinical literature, technical guidance, and expert recommendations on use of ECMO in critically ill cases of COVID-19. Here, we present basic knowledge and opinions about COVID-19 and ECMO, review the evidence on ECMO use in Middle East Respiratory Syndrome (MERS) and H1N1 influenza, share the technical guidance and recommendations on use of ECMO in COVID-19, summarize the current use of ECMO against COVID-19 in China, and discuss the issues in use of ECMO in COVID-19.


Group-velocity-matched optical parametric oscillator in tilted quasi-phase-matched gratings.
An achromatic phase-matching scheme is reported for an optical parametric oscillator in tilted quasi-phase-matched gratings. The spectral angular dispersion is introduced in interaction waves such that each wave component satisfies the two-dimensional (noncollinear) quasi-phase matching. This is equivalent to simultaneous quasi-phase matching and group-velocity matching for ultrashort pulses. The phase-matching bandwidth for 10 mm periodically poled KTP increases by a factor of 12 at lambdas = 1.7 microm compared with one-dimensional quasi-phase matching. The effective interaction length will increase as a result of the matching.


Carboplatin dosage: prospective evaluation of a simple formula based on renal function.
A dosage formula has been derived from a retrospective analysis of carboplatin pharmacokinetics in 18 patients with pretreatment glomerular filtration rates (GFR) in the range of 33 to 136 mL/min. Carboplatin plasma clearance was linearly related to GFR (r = 0.85, P less than .00001) and rearrangements of the equation describing the correlation gave the dosage formula dose (mg) = target area under the free carboplatin plasma concentration versus time curve (AUC) x (1.2 x GFR + 20). In a prospective clinical and pharmacokinetic study the formula was used to determine the dose required to treat 31 patients (GFR range, 33 to 135 mL/min) with 40 courses of carboplatin. The target AUC was escalated from 3 to 8 mg carboplatin/mL/min. Over this AUC range the formula accurately predicted the observed AUC (observed/predicted ratio 1.24 +/- 0.11, r = 0.886) and using these additional data, the formula was refined. Dose (mg) = target AUC x (GFR + 25) is now the recommended formula. AUC values of 4 to 6 and 6 to 8 mg/mL. min gave rise to manageable hematological toxicity in previously treated and untreated patients, respectively, and hence target AUC values of 5 and 7 mg/mL min are recommended for single-agent carboplatin in these patient groups. Pharmacokinetic modeling demonstrated that the formula was reasonably accurate regardless of whether a one- or two-compartment model most accurately described carboplatin pharmacokinetics, assuming that body size did not influence nonrenal clearance. The validity of this assumption was demonstrated in 13 patients where no correlation between surface area and nonrenal clearance was found (r = .31, P = .30). Therefore, the formula provides a simple and consistent method of determining carboplatin dose in adults. Since the measure of carboplatin exposure in the formula is AUC, and not toxicity, it will not be influenced by previous or concurrent myelosuppressive therapy or supportive measures. The formula is therefore applicable to combination and high-dose studies as well as conventional single-agent therapy, although the target AUC for carboplatin will need to be redefined for combination chemotherapy.


Clinically detectable copy number variations in a Canadian catchment population of schizophrenia.
Copy number variation (CNV) is a highly topical area of research in schizophrenia, but the clinical relevance is uncertain and the translation to clinical practice is under-studied. There is a paucity of research involving truly community-based samples of schizophrenia and widely available laboratory techniques. Our objective was to determine the prevalence of clinically detectable CNVs in a community sample of schizophrenia, while mimicking typical clinical practice conditions. We used a brief clinical screening protocol for developmental features in adults with schizophrenia for identifying individuals with 22q11.2 deletions and karyotypically detectable chromosomal anomalies in 204 consecutive patients with schizophrenia from a single Canadian catchment area. Twenty-seven (13.2%) subjects met clinical criteria for a possible syndrome, and 26 of these individuals received clinical genetic testing. Five of these, representing 2.5% of the total sample (95% CI: 0.3%-4.6%), including two of ten patients with mental retardation, had clinically detectable anomalies: two 22q11.2 deletions (1.0%), one 47, XYY, and two other novel CNVs--an 8p23.3-p23.1 deletion and a de novo 19p13.3-p13.2 duplication. The results support the utility of screening and genetic testing to identify genetic syndromes in adults with schizophrenia in clinical practice. Identifying large, rare CNVs (particularly 22q11.2 deletions) can lead to significant changes in management, follow-up, and genetic counselling that are helpful to the patient, family, and clinicians.


AI on a chip.
Artificial intelligence (AI) has dramatically changed the landscape of science, industry, defence, and medicine in the last several years. Supported by considerably enhanced computational power and cloud storage, the field of AI has shifted from mostly theoretical studies in the discipline of computer science to diverse real-life applications such as drug design, material discovery, speech recognition, self-driving cars, advertising, finance, medical imaging, and astronomical observation, where AI-produced outcomes have been proven to be comparable or even superior to the performance of human experts. In these applications, what is essentially important for the development of AI is the data needed for machine learning. Despite its prominent importance, the very first process of the AI development, namely data collection and data preparation, is typically the most laborious task and is often a limiting factor of constructing functional AI algorithms. Lab-on-a-chip technology, in particular microfluidics, is a powerful platform for both the construction and implementation of AI in a large-scale, cost-effective, high-throughput, automated, and multiplexed manner, thereby overcoming the above bottleneck. On this platform, high-throughput imaging is a critical tool as it can generate high-content information (e.g., size, shape, structure, composition, interaction) of objects on a large scale. High-throughput imaging can also be paired with sorting and DNA/RNA sequencing to conduct a massive survey of phenotype-genotype relations whose data is too complex to analyze with traditional computational tools, but is analyzable with the power of AI. In addition to its function as a data provider, lab-on-a-chip technology can also be employed to implement the developed AI for accurate identification, characterization, classification, and prediction of objects in mixed, heterogeneous, or unknown samples. In this review article, motivated by the excellent synergy between AI and lab-on-a-chip technology, we outline fundamental elements, recent advances, future challenges, and emerging opportunities of AI with lab-on-a-chip technology or "AI on a chip" for short.


Physiological, Perceptual and Performance Responses Associated With Self-Selected Versus Standardized Recovery Periods During a Repeated Sprint Protocol in Elite Youth Football Players: A Preliminary Study.
To examine the physiological and perceptual responses of youth footballers to a repeated sprint protocol employing standardized and self-selected recovery. Eleven male participants (13.7 ± 1.1 years) performed a repeated sprint assessment comprising 10 × 30 m efforts. Employing a randomized cross-over design, repeated sprints were performed using 30 s and self-selected recovery periods. Heart rate was monitored continuously with ratings of perceived exertion (RPE) and lower body muscle power measured 2 min after the final sprint. The concentration of blood lactate was measured at 2, 5 and 7 min post sprinting. Magnitude of effects were reported using effect size (ES) statistics ± 90% confidence interval and percentage differences. Differences between trials were examined using paired student t tests (p < .05). Self-selected recovery resulted in most likely shorter recovery times (57.7%; ES 1.55 ± 0.5; p < .01), a most likely increase in percentage decrement (65%; ES 0.36 ± 0.21; p = .12), very likely lower heart rate recovery (-58.9%; ES -1.10 ± 0.72; p = .05), and likely higher blood lactate concentration (p = .08-0.02). Differences in lower body power and RPE were unclear (p > .05). Self-selected recovery periods compromise repeated sprint performance.


Human post-mortem synapse proteome integrity screening for proteomic studies of postsynaptic complexes.
Synapses are fundamental components of brain circuits and are disrupted in over 100 neurological and psychiatric diseases. The synapse proteome is physically organized into multiprotein complexes and polygenic mutations converge on postsynaptic complexes in schizophrenia, autism and intellectual disability. Directly characterising human synapses and their multiprotein complexes from post-mortem tissue is essential to understanding disease mechanisms. However, multiprotein complexes have not been directly isolated from human synapses and the feasibility of their isolation from post-mortem tissue is unknown. Here we establish a screening assay and criteria to identify post-mortem brain samples containing well-preserved synapse proteomes, revealing that neocortex samples are best preserved. We also develop a rapid method for the isolation of synapse proteomes from human brain, allowing large numbers of post-mortem samples to be processed in a short time frame. We perform the first purification and proteomic mass spectrometry analysis of MAGUK Associated Signalling Complexes (MASC) from neurosurgical and post-mortem tissue and find genetic evidence for their involvement in over seventy human brain diseases. We have demonstrated that synaptic proteome integrity can be rapidly assessed from human post-mortem brain samples prior to its analysis with sophisticated proteomic methods. We have also shown that proteomics of synapse multiprotein complexes from well preserved post-mortem tissue is possible, obtaining structures highly similar to those isolated from biopsy tissue. Finally we have shown that MASC from human synapses are involved with over seventy brain disorders. These findings should have wide application in understanding the synaptic basis of psychiatric and other mental disorders.


Delay differential analysis of time series.
Nonlinear dynamical system analysis based on embedding theory has been used for modeling and prediction, but it also has applications to signal detection and classification of time series. An embedding creates a multidimensional geometrical object from a single time series. Traditionally either delay or derivative embeddings have been used. The delay embedding is composed of delayed versions of the signal, and the derivative embedding is composed of successive derivatives of the signal. The delay embedding has been extended to nonuniform embeddings to take multiple timescales into account. Both embeddings provide information on the underlying dynamical system without having direct access to all the system variables. Delay differential analysis is based on functional embeddings, a combination of the derivative embedding with nonuniform delay embeddings. Small delay differential equation (DDE) models that best represent relevant dynamic features of time series data are selected from a pool of candidate models for detection or classification. We show that the properties of DDEs support spectral analysis in the time domain where nonlinear correlation functions are used to detect frequencies, frequency and phase couplings, and bispectra. These can be efficiently computed with short time windows and are robust to noise. For frequency analysis, this framework is a multivariate extension of discrete Fourier transform (DFT), and for higher-order spectra, it is a linear and multivariate alternative to multidimensional fast Fourier transform of multidimensional correlations. This method can be applied to short or sparse time series and can be extended to cross-trial and cross-channel spectra if multiple short data segments of the same experiment are available. Together, this time-domain toolbox provides higher temporal resolution, increased frequency and phase coupling information, and it allows an easy and straightforward implementation of higher-order spectra across time compared with frequency-based methods such as the DFT and cross-spectral analysis.


Superpixel-based spatial amplitude and phase modulation using a digital micromirror device.
We present a superpixel method for full spatial phase and amplitude control of a light beam using a digital micromirror device (DMD) combined with a spatial filter. We combine square regions of nearby micromirrors into superpixels by low pass filtering in a Fourier plane of the DMD. At each superpixel we are able to independently modulate the phase and the amplitude of light, while retaining a high resolution and the very high speed of a DMD. The method achieves a measured fidelity F = 0.98 for a target field with fully independent phase and amplitude at a resolution of 8 × 8 pixels per diffraction limited spot. For the LG10 orbital angular momentum mode the calculated fidelity is F = 0.99993, using 768 × 768 DMD pixels. The superpixel method reduces the errors when compared to the state of the art Lee holography method for these test fields by 50% and 18%, with a comparable light efficiency of around 5%. Our control software is publicly available.


The effect of scissor-type versus non-scissor-type knives on the technical outcomes in endoscopic submucosal dissection for superficial esophageal cancer: a multi-center retrospective study.
The Clutch Cutter was invented as a scissor-type knife for endoscopic submucosal dissection (ESD) of gastrointestinal neoplasms. ESD with the scissor-type knife (ESD-S) may be considered a technically easier procedure than ESD with non-scissor-type knives (ESD-NS). Therefore, this study aimed to compare the technical outcomes of ESD-S with those of ESD-NS for superficial esophageal cancer. This was a multicenter retrospective study. Patients with superficial esophageal cancer treated with ESD between October 2015 and March 2018 at three hospitals were retrospectively reviewed. The ESD-S group had 48 patients and the ESD-NS group had 114 patients. A propensity score matching analysis was performed to compensate for the confounding bias between both groups. Multivariate analyses and propensity score matching were used to adjust for age, sex, the tumor size, tumor location, tumor depth, degree of tumor circumference, operator level, usage of the traction method, and the sedation method. The primary outcome was the procedure time of the ESD. Secondary outcomes were the rate of en-bloc/complete resection and the rate of complications including perforation, delayed bleeding, and stricture. Propensity score matching analysis provided 36 matched pairs. Median procedure time in the ESD-S group was significantly shorter than that in the ESD-NS group (44.0 min vs. 66.5 min, P = 0.020). In addition, the treatment outcomes were similar in both groups (en-bloc resection: 100% vs. 97.2%, P = 1; complete resection: 88.9% vs. 86.1%, P = 1; curative resection: 80.6% vs. 77.8%, P = 1; perforation: 0% vs. 5.6%, P = 0.49; delayed bleeding: 0% in both groups; stricture: 2.8% vs. 8.3%, P = 0.61). ESD-S was associated with a shorter procedure time than ESD-NS, without an increase in the incidence of complications. Therefore, the scissor-type knife should be considered as an endo-knife for ESD of superficial esophageal cancers.


Transcriptome profiling of mice testes following low dose irradiation.
Radiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis. Transcriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi). We identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster. The five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.


[Respiratory virus infections in adult patients hospitalized in an internal medicine unit].
Respiratory viral infections (RVi) can be associated with a wide range of clinical manifestations. To investigate the frequency and clinical manifestations of RVi among adult patients during winter hospitalizations. All patients admitted to the hospital with flu like disease and those with fever or exacerbation of any underlying disease during hospitalization without an evident cause, were prospectively enrolled. A direct immunofluorescence (DIF) of nasopharyngeal aspirate for influenza A (IA) and B, parainfluenza 1, 2 and 3, adenovirus, respiratory syncytial virus (RSV) and metapneumovirus, was performed. Epidemiological and clinical data were recorded. Between May and September 2012, 975 adults were admitted to the Internal Medicine Unit of Puerto Montt Hospital and in 128 (13%) patients, DIF was carried out. DIF was positive in 44 patients (34%) aged 65 ± 20 years, 68.2% females, corresponding to 4.5% of total hospitalizations. Eighty six percent of the latter had at least one co-morbidity, mainly asthma and chronic respiratory diseases in 34.1%, diabetes in 29.5%, cardiac problems in 25% and congestive heart failure in 20.5%. The most common RVi were RSV (n = 21, 48%) and IA (n = 17, 39%). Six patients had a nosocomial RVi. Patients infected with IA had a significantly higher frequency of fever and bronchial hyper reactivity than those infected with RSV. RVi were associated with exacerbation of underlying disease in 62% of cases and pneumonia in 27%. Two patients had a viral pericarditis. RVi are an important cause of adult morbidity and their detection should be routine in adult patients hospitalized during winter.


Economic Evidence for US Asthma Self-Management Education and Home-Based Interventions.
The health and economic burden of asthma in the United States is substantial. Asthma self-management education (AS-ME) and home-based interventions for asthma can improve asthma control and prevent asthma exacerbations, and interest in health care-public health collaboration regarding asthma is increasing. However, outpatient AS-ME and home-based asthma intervention programs are not widely available; economic sustainability is a common concern. Thus, we conducted a narrative review of existing literature regarding economic outcomes of outpatient AS-ME and home-based intervention programs for asthma in the United States. We identified 9 outpatient AS-ME programs and 17 home-based intervention programs with return on investment (ROI) data. Most programs were associated with a positive ROI; a few programs observed positive ROIs only among selected populations (eg, higher health care utilization). Interpretation of existing data is limited by heterogeneous ROI calculations. Nevertheless, the literature suggests promise for sustainable opportunities to expand access to outpatient AS-ME and home-based asthma intervention programs in the United States. More definitive knowledge about how to maximize program benefit and sustainability could be gained through more controlled studies of specific populations and increased uniformity in economic assessments.


Intranet-based quality improvement documentation at the Veterans Affairs Maryland Health Care System.
The Pathology and Laboratory Medicine Service of the Veterans Affairs Maryland Health Care System is inspected biannually by the College of American Pathologists (CAP). As of the year 2000, all documentation in the Anatomic Pathology Section is available to all staff through the VA Intranet. Signed, supporting paper documents are on file in the office of the department chair. For the year 2000 CAP inspection, inspectors conducted their document review by use of these Web-based documents, in which each CAP question had a hyperlink to the corresponding section of the procedure manual. Thus inspectors were able to locate the documents relevant to each question quickly and efficiently. The procedure manuals consist of 87 procedures for surgical pathology, 52 procedures for cytopathology, and 25 procedures for autopsy pathology. Each CAP question requiring documentation had from one to three hyperlinks to the corresponding section of the procedure manual. Intranet documentation allows for easier sharing among decentralized institutions and for centralized updates of the laboratory documentation. These documents can be upgraded to allow for multimedia presentations, including text search for key words, hyperlinks to other documents, and images, audio, and video. Use of Web-based documents can improve the efficiency of the inspection process.


Comparative effectiveness of wide antral versus ostial pulmonary vein isolation: a systematic review and meta-analysis.
For the past decade, electric pulmonary vein isolation (PVI) has become a procedure implemented worldwide for the treatment of atrial fibrillation. Currently, 2 main approaches are used for PVI: ostial isolation of the PVs and wide antral PVI. The aims of this systematic review are to evaluate the relative merits of each technique with a pooled comparative analysis of efficacy and complications. Studies were identified by searching electronic databases for studies on ostial versus antral PVI. Information was extracted from each included trial. Odds ratio was the primary measure of treatment effect or side effects. The proportion of patients with recurrences of atrial fibrillation or other atrial tachyarrhythmias was evaluated at the end of the follow-up periods in 12 trials, including 1183 patients. The recurrence rate of total supraventricular arrhythmias was significantly lower in wide antral than in segmental PVI group (odds ratio, 0.42; 95% confidence interval, 0.32-0.56; P<0.00001). Atrial fibrillation recurrence was significantly lower in the wide antral group (odds ratio, 0.33; 95% confidence interval, 0.24-0.46; P<0,00001). A trend toward a higher incidence of left atrial tachycardia occurrence in the wide antral circumferential ablation group was detected, which did not reach statistical significance (odds ratio, 1.53; 95% confidence interval, 0.88-2.69; P=0.13). Our primary finding is that PVI performed with a wide antral approach is more effective than ostial PVI in achieving freedom from total atrial tachyarrhythmia recurrence at long-term follow-up.


A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes.
This study identifies a dendritic cell (DC) subset that constitutively transports apoptotic intestinal epithelial cell remnants to T cell areas of mesenteric lymph nodes in vivo. Rat intestinal lymph contains two DC populations. Both populations have typical DC morphology, are major histocompatibility complex class II(hi), and express OX62, CD11c, and B7. CD4(+)/OX41(+) DCs are strong antigen-presenting cells (APCs). CD4(-)/OX41(-) DCs are weak APCs and contain cytoplasmic apoptotic DNA, epithelial cell-restricted cytokeratins, and nonspecific esterase (NSE)(+) inclusions, not seen in OX41(+) DCs. Identical patterns of NSE electrophoretic variants exist in CD4(-)/OX41(-) DCs, intestinal epithelial cells, and mesenteric node DCs but not in other DC populations, macrophages, or tissues. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive DCs and strongly NSE(+) DCs are present in intestinal lamina propria. Peyer's patches and mesenteric but not other lymph nodes contain many strongly NSE(+) DCs in interfollicular and T cell areas. Similar DCs are seen in the ileum and in T cell areas of mesenteric nodes in gnotobiotic rats. These results show that a distinct DC subset constitutively endocytoses and transports apoptotic cells to T cell areas and suggest a role for these DCs in inducing and maintaining peripheral self-tolerance.


A method for sensitivity analysis to assess the effects of measurement error in multiple exposure variables using external validation data.
Measurement error in self-reported dietary intakes is known to bias the association between dietary intake and a health outcome of interest such as risk of a disease. The association can be distorted further by mismeasured confounders, leading to invalid results and conclusions. It is, however, difficult to adjust for the bias in the association when there is no internal validation data. We proposed a method to adjust for the bias in the diet-disease association (hereafter, association), due to measurement error in dietary intake and a mismeasured confounder, when there is no internal validation data. The method combines prior information on the validity of the self-report instrument with the observed data to adjust for the bias in the association. We compared the proposed method with the method that ignores the confounder effect, and with the method that ignores measurement errors completely. We assessed the sensitivity of the estimates to various magnitudes of measurement error, error correlations and uncertainty in the literature-reported validation data. We applied the methods to fruits and vegetables (FV) intakes, cigarette smoking (confounder) and all-cause mortality data from the European Prospective Investigation into Cancer and Nutrition study. Using the proposed method resulted in about four times increase in the strength of association between FV intake and mortality. For weakly correlated errors, measurement error in the confounder minimally affected the hazard ratio estimate for FV intake. The effect was more pronounced for strong error correlations. The proposed method permits sensitivity analysis on measurement error structures and accounts for uncertainties in the reported validity coefficients. The method is useful in assessing the direction and quantifying the magnitude of bias in the association due to measurement errors in the confounders.


Safety and efficacy of essential oil, oleoresin and tincture from Zingiber officinale Roscoe when used as sensory additives in feed for all animal species.
Following a request from the European Commission, the EFSAPanel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of essential oil, oleoresin and tincture from Zingiber officinale Roscoe when used as sensory additives in feed for all animal species. The FEEDAPPanel concludes that the additives under consideration are safe for the target species at the following use levels: (i) ginger essential oil up to the maximum proposed use level of 80 mg/kg for veal calves (milk replacer) and 20 mg/kg complete feed (or 20 mg/L water for drinking) for all other species; (ii) ginger oleoresin at the maximum proposed concentrations of 20 mg/kg complete feed for fish, sheep, goats and horses and of 1 mg/kg for pets. For the remaining species, the calculated maximum safe concentration of ginger oleoresin in feed is less than that proposed use level and ranges from 5 mg/kg complete feed for chickens for fattening to 21 mg/kg for veal calves; (iii) ginger tincture at the maximum proposed concentrations of 1.6 mL/kg complete feed for horses and 0.26 mL/kg for dogs. For poultry species, the calculated maximum safe dose ranges between 0.2 and 0.3 mg/L water for drinking. No concerns for consumers were identified following the use of the additives up to the highest safe level in animal nutrition. The additives should be considered as irritants to skin and eyes and the respiratory tract and as a skin sensitisers. The use of the additives in feed is not expected to pose a risk for the environment. Since ginger and its preparations are recognised to flavour food and their function in feed would be essentially the same as that in food, no further demonstration of efficacy is considered necessary.


Identification of up-regulated Ras-like GTPase, Rap1b, by suppression subtractive hybridization.
Diabetic nephropathy accounts for over 30% of the end-stage renal disease (ESRD). A number of defined mechanisms and molecules that are involved in its pathogenesis are known, while others remain to be identified. Suppression subtraction hybridization (SSH)-polymerase chain reaction (PCR) was employed to search for new genes that may be relevant to the pathogenesis of diabetic nephropathy during embryonic development, the time when the kidney is most susceptible to various forms of stress. A diabetic state was induced in pregnant mice at day-13 of gestation by administration of streptozotocin. The kidneys of newborn mice with blood glucose level> 200 mg/dL were harvested, mRNA isolated and subjected to SSH-PCR. Several differentially expressed cDNA fragments with up-regulated expression were isolated. One of the cDNA fragments had homology with human Ras-like guanine 5'-triphosphate (GTPase), Rap1b gene. By utilizing the lambdaZAP II mouse cDNA library and SMART RACE amplification, a full-length Rap1b cDNA was isolated. A recombinant protein was generated in pET15b bacterial expression system. An anti-Rap1b antibody was raised in rabbits by immunizing them with the fusion protein, and its specificity was confirmed by Western blot analysis. Rap1b cDNA had an open reading frame of 552 bp with a predicted putative protein size of approximately 21 kD. In vitro translation verified the authentication of the Rap1b cDNA clone. Northern blot analyses revealed a single approximately 2.3 kb Rap1b mRNA transcript. Its expression was up-regulated in several tissues, including the kidney of newborn diabetic mice. The degree of up-regulation of Rap1b mRNA expression was proportional to the blood glucose levels. Western blot analyses confirmed the hyperglycemia-induced up-regulation of the Rap1b expression. In situ hybridization and immunofluorescence studies revealed that Rap1b was expressed in the inner medullary collecting tubules. During hyperglycemia, its expression was accentuated and extended into the outer medullary and cortical collecting tubules. Similar up-regulation of Rap1b was observed when embryonic kidneys, harvested at day-13 of gestation, were exposed to high glucose ambience. The data suggest that Rap1b, a GTP-binding protein that plays a critical role in various signaling intracellular events, is another molecule that may be relevant to the pathobiology of diabetic nephropathy.


Assessment of Blood Brain Barrier Leakage with Gadolinium-Enhanced MRI.
The integrity of the blood-brain barrier (BBB) can be noninvasively monitored by magnetic resonance imaging (MRI). Conventional MR contrast agents (CAs) containing gadolinium are used in association with MRI in routine clinical practice to detect and quantify BBB leakage. Under normal circumstances CAs do not cross the intact BBB. However due to their small size they extravasate from the blood into the brain tissue even when the BBB is partially compromised. Here we describe an MR method based on T1-weighted images taken prior to and after CA injection. This MR method is useful for investigating BBB permeability in in vivo mouse models and can be easily applied in a number of experimental disease conditions including neuroinflammation disorders, or to assess (un)wanted drug effects.


TRPV4 mutations and cytotoxic hypercalcemia in axonal Charcot-Marie-Tooth neuropathies.
To improve understanding of TRPV4-associated axonal Charcot-Marie-Tooth (CMT) neuropathy phenotypes and their debated pathologic mechanism. A total of 17 CMT2C phenotypic families with vocal cord and diaphragmatic involvement and 36 clinically undifferentiated CMT2 subjects underwent sequencing analysis of the coding region of TRPV4. Functional studies of mutant proteins were performed using transiently transfected cells for TRPV4 subcellular localization, basal and stimulated Ca(2+) channel analysis, and cell viability assay with or without channel blockade. Two TRPV4 mutations R232C and R316H from 17 CMT2C families were identified in the ankyrin repeat domains. The R316H is a novel de novo mutation found in a patient with CMT2C phenotype. The family with R232C mutation had individuals with and without vocal cord and diaphragm involvement. Both mutant TRPV4 proteins had normal subcellular localization in HEK293 and HeLa cells. Cells transfected with R232C and R316H displayed increased intracellular Ca(2+) levels and reversible cell death by the TRPV channel antagonist, ruthenium red. TRPV4 ankyrin domain alterations including a novel de novo mutation cause axonal CMT2. Individuals with the same mutation may have nondistinct CMT2 or have phenotypic CMT2C with vocal cord paresis. Reversible hypercalcemic gain-of-function of mutant TRPV4 instead of loss-of-function appears to be pathologically important. The reversibility of cell death by channel blockade provides an attractive area of investigation in consideration of treatable axonal degeneration.


Translocation of solutes and proteins across the glycosomal membrane of trypanosomes; possibilities and limitations for targeting with trypanocidal drugs.
Glycosomes are specialized peroxisomes found in all kinetoplastid organisms. The organelles are unique in harbouring most enzymes of the glycolytic pathway. Matrix proteins, synthesized in the cytosol, cofactors and metabolites have to be transported across the membrane. Recent research on Trypanosoma brucei has provided insight into how these translocations across the membrane occur, although many details remain to be elucidated. Proteins are imported by a cascade of reactions performed by specialized proteins, called peroxins, in which a cytosolic receptor with bound matrix protein inserts itself in the membrane to deliver its cargo into the organelle and is subsequently retrieved from the glycosome to perform further rounds of import. Bulky solutes, such as cofactors and acyl-CoAs, seem to be translocated by specific transporter molecules, whereas smaller solutes such as glycolytic intermediates probably cross the membrane through pore-forming channels. The presence of such channels is in apparent contradiction with previous results that suggested a low permeability of the glycosomal membrane. We propose 3 possible, not mutually exclusive, solutions for this paradox. Glycosomal glycolytic enzymes have been validated as drug targets against trypanosomatid-borne diseases. We discuss the possible implications of the new data for the design of drugs to be delivered into glycosomes.


Towards the classification of subpopulations of layer V pyramidal projection neurons.
The nature of cerebral cortical circuitry has been increasingly clarified by markers for the identification of precise cell types with specific morphology, connectivity and distinct physiological properties. Molecular markers are not only helpful in dissecting cortical circuitry, but also give insight into the mechanisms of cortical neuronal specification and differentiation. The two principal neuronal types of the cerebral cortex are the pyramidal and GABAergic cells. Pyramidal cells are excitatory and project to distant targets, while GABAergic neurons are mostly inhibitory non-pyramidal interneurons. Reliable markers for specific subtypes of interneurons are available and have been employed in the classification and functional analysis of cortical circuitry. Until recently, cortical pyramidal neurons have been considered a homogeneous class of cells. This concept is now changing as the powerful tools of molecular biology and genetics identify molecular tags for subtypes of pyramidal cells such as: Otx-1 [Frantz, G.D., Bohner, A.P., Akers, R.M., McConnell, S.K., 1994. Regulation of the POU domain gene SCIP during cerebral cortical development. J. Neurosci. 14, 472-485; Weimann, J.M., Zhang, Y.A., Levin, M.E., Devine, W.P., Brulet, P., McConnell, S.K., 1999. Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 24, 819-831]; SMI-32, N200 and FNP-7 [Voelker, C.C., Garin, N., Taylor, J.S., Gahwiler, B.H., Hornung, J.P., Molnár, Z., 2004. Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of layer V pyramidal neurons in vivo and in vitro. Cereb. Cortex 14, 1276-1286]; ER81 [Hevner, R.F., Daza, R.A., Rubenstein, J.L., Stunnenberg, H., Olavarria, J.F., Englund, C., 2003. Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons. Dev. Neurosci. 25 (2-4), 139-151; Yoneshima, H., Yamasaki, S., Voelker, C., Molnár, Z., Christophe, E., Audinat, E., Takemoto, M., Tsuji, S., Fujita, I., Yamamoto, N., 2006. ER81 is expressed in a subpopulation of layer 5 projection neurons in rodent cerebral cortices. Neuroscience, 137, 401-412]; Lmo4 [Bulchand, S., Subramanian, L., Tole, S., 2003. Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex. Dev. Dyn. 226, 460-469; Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207-221]; CTIP2 [Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207-221]; Fez1 [Molyneaux, B.J., Arlotta, P., Hirata, T., Hibi, M., Macklis, J.D., 2005. Fez1 is required for the birth and specification of corticospinal motor neurons. Neuron 47 (6), 817-831; Chen, B., Schaevitz, L.R., McConnell, S.K., 2005. Fez1 regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc. Natl. Acad. Sci. U.S.A. 102 (47), 17184-17189]. These genes outline the numerous subtypes of pyramidal cells and are increasingly refining our previous classifications. They also indicate specific developmental programs operate in cell fate decisions. This review will describe the progress made on the correlation of these markers to each other within a specific subtype of layer V neurons with identified, stereotypic projections. Further work is needed to link these data with observations on somatodendritic morphology and physiological properties. The integrated molecular, anatomical and physiological characterisation of pyramidal neurons will lead to a much better appreciation of functional cortical circuits.


Rapid surface water intervention performance comparison for urban planning.
Surface water flooding can be a significant source of damage and disruption in urban areas. The complexity of urban surfaces, the need for spatially disaggregated approaches and the multiplicity of interventions makes management challenging from a number of perspectives. This research responds to the challenge of selecting appropriate surface water management interventions by applying a fast assessment framework to generate evidence for comparing strategies at low resource cost during initial design. This is demonstrated by simulating flood dynamics and comparing damage costs in 144 flood scenarios. The main finding of this work is that a high-level quantitative assessment of large numbers of scenarios is capable of providing evidence to identify performance trends and consider resilience to extreme events at an early stage of planning.


Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.
Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.


Lhx2-dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons.
Inactivation of the LIM-homeodomain 2 gene (Lhx2) results in a severe defect in specification of olfactory sensory neurons (OSNs). However, the ramifications of lack of Lhx2-dependent OSN specification for formation of the primary olfactory pathway have not been addressed, since mutant mice die in utero. We have analyzed prenatal and postnatal consequences of conditionally inactivating Lhx2 selectively in OSNs. A cell-autonomous effect is that OSN axons cannot innervate their target, the olfactory bulb. Moreover, the lack of Lhx2 in OSNs causes unpredicted, non-cell-autonomous phenotypes. First, the olfactory bulb shows pronounced hypoplasia in adults, and the data suggest that innervation by correctly specified OSNs is necessary for adult bulb size and organization. Second, absence of an olfactory nerve in the conditional mutant reveals that the vomeronasal nerve is dependent on olfactory nerve formation. Third, the lack of a proper vomeronasal nerve prevents migration of gonadotropin-releasing hormone (GnRH) cells the whole distance to their final positions in the hypothalamus during embryo development. As adults, the conditional mutants do not pass puberty, and these findings support the view of an exclusive nasal origin of GnRH neurons in the mouse. Thus, Lhx2 in OSNs is required for functional development of three separate systems.


Hypopituitarism caused by intrasellar aneurysms.
To determine the prevalence, clinical presentation, and outcome of hypopituitarism due to an intrasellar aneurysm. We performed a retrospective review of Mayo Clinic, Rochester, Minn, medical records from 1950 through 1995. We calculated the prevalence of hypopituitarism, characterized the clinical presentation, and evaluated postoperative outcomes. Of 4087 patients with a diagnosis of hypopituitarism, 7 had hypopituitarism due to an intrasellar aneurysm, accounting for a prevalence of 0.17%. Adrenal, thyroid, and gonadal deficiencies were observed in 7, 6, and 5 patients, respectively. The prolactin level was increased in the 2 patients in whom it was measured. No patient had diabetes insipidus. All had visual impairment. In 5 patients an intrasellar aneurysm of the internal carotid artery was diagnosed preoperatively based on 1 or more imaging procedures. In the other 2 patients (1 with an internal carotid artery aneurysm and 1 with an anterior cerebral artery aneurysm), the diagnosis was made at surgical exploration. Four patients underwent a surgical procedure: 2 had direct packing of the aneurysm, and 2 had a bypass with proximal carotid occlusion or aneurysm trapping. Pituitary function remained unchanged postoperatively. An intrasellar aneurysm is a rare cause of hypopituitarism (0.17% of cases). Preoperative diagnosis is essential for successful surgical outcomes. Hypopituitarism is usually permanent.


Psychosocial characteristics and outcomes in patients with left ventricular assist device implanted as destination therapy.
Psychosocial factors impact survival in patients undergoing cardiac transplantation, but it is unclear whether they affect outcomes in patients undergoing left ventricular assist device (LVAD) implantation as destination therapy (DT). Patients undergoing DT LVAD at the Mayo Clinic in Rochester, MN, from February 2007 to December 2013 were included. Psychosocial characteristics at the time of LVAD implantation were abstracted from the medical record. Andersen-Gill and Cox models were used to examine the association between psychosocial characteristics and all-cause readmission and death, respectively. Patients were censored at death or last follow-up through September 2014. Among 136 patients (mean age. 64 years; 17% female), most were married/living with a partner (82%), half (55%) had post-high school education, and a history of depression was common (32%). Although most patients were former tobacco users (60%) only a small proportion were current tobacco users (10%) and had a history of alcohol abuse (16%) or illegal drug use (7%). After a mean follow-up of 2.2 ± 1.8 years, 78% of patients had been readmitted (range, 0-14 per person) and 49% had died. There were no statistically significant differences in the risk of death according to psychosocial characteristics. However, current tobacco users had lower risk of readmission (adjusted HR, 0.57; 95% CI, 0.38-0.88), while illegal drug use (HR, 1.55; 95% CI, 1.01-2.35) and depression (HR, 1.77; 95% CI, 1.40-2.22) were associated with higher readmission risk. Psychosocial characteristics are not significant predictors of death but are associated with readmission risk after DT LVAD.


Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death.
To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell-growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, resulting in part from the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. Here, we circumvent this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1. We find that overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild-type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. We find that cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, we demonstrate that Atg1-induced autophagy strongly inhibits cell growth and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, we show that Atg1 expression results in negative feedback on the activity of TOR itself. Our results reveal a central role for Atg1 in mounting a coordinated autophagic response and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth.


Myeloperoxidase expression as a potential determinant of parthenolide-induced apoptosis in leukemia bulk and leukemia stem cells.
Given that parthenolide (PTL) is an effective antileukemic agent, identifying molecular markers that predict response to PTL is important. We evaluated the role of myeloperoxidase (MPO) in determining the sensitivity of leukemia cells to PTL-induced apoptosis. In this study, the level of PTL-induced generation of reactive oxygen species (ROS) and apoptosis was significantly higher in the MPO-high leukemia cell lines compared with the MPO-low leukemia cell lines. Pretreatment of MPO-high leukemia cells with a MPO-specific inhibitor, 4-aminobenzoic acid hydrazide, or a MPO-specific small interfering RNA (siRNA) abrogated the PTL-induced ROS generation and apoptosis, indicating that MPO plays a crucial role in PTL-induced apoptosis in leukemia cells. PTL-induced apoptosis was accompanied by down-regulation of nuclear factor-κB, Bcl-xL, Mcl-1, X-linked inhibitor of apoptosis protein, and survivin and selectively observed in primary acute myeloid leukemia (AML) cells expressing higher levels of MPO (≥50%) while sparing both AML cells with lower MPO and normal CD34-positive (CD34+) normal bone marrow cells. The extent of PTL-induced apoptosis of the CD34+CD38- cell fraction was significantly greater in the MPO-high AML cases, compared with the MPO-low AML (P < 0.01) and normal CD34+ marrow cells (P < 0.01). Nonobese diabetic/severe combined immunodeficient human leukemia mouse model also revealed that PTL preferentially targets the MPO-high AML cells. Our data suggest that MPO plays a crucial role in determining the susceptibility of leukemia cells to PTL-induced apoptosis. PTL can be considered a promising leukemic stem cell-targeted therapy for AML expressing high levels of MPO.