Bloodstream infections in pediatric ECLS: usefulness of daily blood culture monitoring and predictive value of biological markers. The British Columbia experience.
The incidence of bloodstream infection (BSI) in extracorporeal life support (ECLS) is reported between 0.9 and 19.5%. In January 2006, the Extracorporeal Life Support Organization (ELSO) reported an overall incidence of 8.78% distributed as follows: respiratory: 6.5% (neonatal), 20.8% (pediatric); cardiac: 8.2% (neonatal) and 12.6% (pediatric). At BC Children's Hospital (BCCH) daily surveillance blood cultures (BC) are performed and antibiotic prophylaxis is not routinely recommended. Positive BC (BC+) were reviewed, including resistance profiles, collection time of BC+, time to positivity and mortality. White blood cell count, absolute neutrophile count, immature/total ratio, platelet count, fibrinogen and lactate were analyzed 48, 24 and 0 h prior to BSI. A univariate linear regression analysis was performed. From 1999 to 2005, 89 patients underwent ECLS. After exclusion, 84 patients were reviewed. The attack rate was 22.6% (19 BSI) and 13.1% after exclusion of coagulase-negative staphylococci (n = 8). BSI patients were significantly longer on ECLS (157 h) compared to the no-BSI group (127 h, 95% CI: 106-148). Six BSI patients died on ECLS (35%; 4 congenital diaphragmatic hernias, 1 hypoplastic left heart syndrome and 1 after a tetralogy repair). BCCH survival on ECLS was 71 and 58% at discharge, which is comparable to previous reports. No patient died primarily because of BSI. No BSI predictor was identified, although lactate may show a decreasing trend before BSI (P = 0.102). Compared with ELSO, the studied BSI incidence was higher with a comparable mortality. We speculate that our BSI rate is explained by underreporting of "contaminants" in the literature, the use of broad-spectrum antibiotic prophylaxis and a higher yield with daily monitoring BC. We support daily surveillance blood cultures as an alternative to antibiotic prophylaxis in the management of patients on ECLS.


A genome wide linkage search for breast cancer susceptibility genes.
Mutations in known breast cancer susceptibility genes account for a minority of the familial aggregation of the disease. To search for further breast cancer susceptibility genes, we performed a combined analysis of four genome-wide linkage screens, which included a total of 149 multiple case breast cancer families. All families included at least three cases of breast cancer diagnosed below age 60 years, at least one of whom had been tested and found not to carry a BRCA1 or BRCA2 mutation. Evidence for linkage was assessed using parametric linkage analysis, assuming both a dominant and a recessive mode of inheritance, and using nonparametric methods. The highest LOD score obtained in any analysis of the combined data was 1.80 under the dominant model, in a region on chromosome 4 close to marker D4S392. Three further LOD scores over 1 were identified in the parametric analyses and two in the nonparametric analyses. A maximum LOD score of 2.40 was found on chromosome arm 2p in families with four or more cases of breast cancer diagnosed below age 50 years. The number of linkage peaks did not differ from the number expected by chance. These results suggest regions that may harbor novel breast cancer susceptibility genes. They also indicate that no single gene is likely to account for a large fraction of the familial aggregation of breast cancer that is not due to mutations in BRCA1 or BRCA2.


Apaf-1 deficiency and neural tube closure defects are found in fog mice.
The forebrain overgrowth mutation (fog) was originally described as a spontaneous autosomal recessive mutation mapping to mouse chromosome 10 that produces forebrain defects, facial defects, and spina bifida. Although the fog mutant has been characterized and available to investigators for several years, the underlying mutation causing the pathology has not been known. Because of its phenotypic resemblance to apoptotic protease activating factor-1 (Apaf-1) knockout mice, we have investigated the possibility that the fog mutation is in the Apaf-1 gene. Allelic complementation, Western blot analysis, and caspase activation assays indicate that fog mutant mice lack Apaf-1 activity. Northern blot and reverse transcription-PCR analysis show that Apaf-1 mRNA is aberrantly processed, resulting in greatly reduced expression levels of normal Apaf-1 mRNA. These findings are strongly suggestive of the fog mutation being a hypomorphic Apaf-1 defect and implicate neural progenitor cell death in the pathogenesis of spina bifida-a common human congenital malformation. Because a complete deficiency in Apaf-1 usually results in perinatal lethality and fog/fog mice more readily survive into adulthood, these mutants serve as a valuable model with which apoptotic cell death can be studied in vivo.


Production of bio-hydrogen by mesophilic anaerobic fermentation in an acid-phase sequencing batch reactor.
The pH and hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR) were varied to optimize the conversion of carbohydrate-rich synthetic wastewater into bio-hydrogen. A full factorial design using evolutionary operation (EVOP) was used to determine the effect of the factors and to find the optimum condition of each factor required for high hydrogen production rate. Experimental results from 20 runs indicate that a maximum hydrogen production rate of 4,460-5,540 mL/L/day under the volumetric organic loading rate (VOLR) of 75 g-COD/L/day obtained at an observed design point of HRT = 8 h and pH = 5.7. The hydrogen production rate was strongly dependent on the HRT, and the effect was statistically significant (P < 0.05). However, no significant effect (P > 0.05) was found for the pH on the hydrogen production rate. When the ASBR conditions were set for a maximum hydrogen production rate, the hydrogen production yield and specific hydrogen production rate were 60-74 mL/g-COD and 330-360 mL/g-VSS/day, respectively. The hydrogen composition was 43-51%, and no methanogenesis was observed. Acetate, propionate, butyrate, valerate, caproate, and ethanol were major liquid intermediate metabolites during runs of this ASBR. The dominant fermentative types were butyrate-acetate or ethanol-acetate, representing the typical anaerobic pathway of Clostridium species. This hydrogen-producing ASBR had a higher hydrogen production rate, compared with that produced using continuous-flow stirred tank reactors (CSTRs). This study suggests that the hydrogen-producing ASBR is a promising bio-system for prolonged and stable hydrogen production.


Alginate Microspheres Containing Temperature Sensitive Liposomes (TSL) for MR-Guided Embolization and Triggered Release of Doxorubicin.
The objective of this study was to develop and characterize alginate microspheres suitable for embolization with on-demand triggered doxorubicin (DOX) release and whereby the microspheres as well as the drug releasing process can be visualized in vivo using MRI. For this purpose, barium crosslinked alginate microspheres were loaded with temperature sensitive liposomes (TSL/TSL-Ba-ms), which release their payload upon mild hyperthermia. These TSL contained DOX and [Gd(HPDO3A)(H2O)], a T1 MRI contrast agent, for real time visualization of the release. Empty alginate microspheres crosslinked with holmium ions (T2* MRI contrast agent, Ho-ms) were mixed with TSL-Ba-ms to allow microsphere visualization. TSL-Ba-ms and Ho-ms were prepared with a homemade spray device and sized by sieving. Encapsulation of TSL in barium crosslinked microspheres changed the triggered release properties only slightly: 95% of the loaded DOX was released from free TSL vs. 86% release for TSL-Ba-ms within 30 seconds in 50% FBS at 42°C. TSL-Ba-ms (76 ± 41 μm) and Ho-ms (64 ± 29 μm) had a comparable size, which most likely will result in a similar in vivo tissue distribution after an i.v. co-injection and therefore Ho-ms can be used as tracer for the TSL-Ba-ms. MR imaging of a TSL-Ba-ms and Ho-ms mixture (ratio 95:5) before and after hyperthermia allowed in vitro and in vivo visualization of microsphere deposition (T2*-weighted images) as well as temperature-triggered release (T1-weighted images). The [Gd(HPDO3A)(H2O)] release and clusters of microspheres containing holmium ions were visualized in a VX2 tumor model in a rabbit using MRI. In conclusion, these TSL-Ba-ms and Ho-ms are promising systems for real-time, MR-guided embolization and triggered release of drugs in vivo.


The adaptor protein Shc integrates growth factor and ECM signaling during postnatal angiogenesis.
Angiogenesis requires integration of cues from growth factors, extracellular matrix (ECM) proteins, and their receptors in endothelial cells. In the present study, we show that the adaptor protein Shc is required for angiogenesis in zebrafish, mice, and cell-culture models. Shc knockdown zebrafish embryos show defects in intersegmental vessel sprouting in the trunk. Shc flox/flox; Tie2-Cre mice display reduced angiogenesis in the retinal neovascularization model and in response to VEGF in the Matrigel plug assay in vivo. Functional studies reveal a model in which Shc is required for integrin-mediated spreading and migration specifically on fibronectin, as well as endothelial cell survival in response to VEGF. Mechanistically, Shc is required for activation of the Akt pathway downstream of both integrin and VEGF signaling, as well as for integration of signals from these 2 receptors when cells are grown on fibronectin. Therefore, we have identified a unique mechanism in which signals from 2 critical angiogenic signaling axes, integrins and VEGFR-2, converge at Shc to regulate postnatal angiogenesis.


Efficacy of Moringa oleifera leaf powder as a hand-washing product: a crossover controlled study among healthy volunteers.
Moringa oleifera is a plant found in many tropical and subtropical countries. Many different uses and properties have been attributed to this plant, mainly as a nutritional supplement and as a water purifier. Its antibacterial activity against different pathogens has been described in different in vitro settings. However the potential effect of this plant leaf as a hand washing product has never been studied. The aim of this study is to test the efficacy of this product using an in vivo design with healthy volunteers. The hands of fifteen volunteers were artificially contaminated with Escherichia coli. Moringa oleifera leaf powder was tested as a hand washing product and was compared with reference non-medicated liquid soap using a cross over design following an adaptation of the European Committee for Standardization protocol (EN 1499). In a second part of tests, the efficacy of the established amount of Moringa oleifera leaf powder was compared with an inert powder using the same protocol. Application of 2 and 3 g of dried Moringa oleifera leaf powder (mean log10-reduction: 2.44 ± 0.41 and 2.58 ± 0.34, respectively) was significantly less effective than the reference soap (3.00 ± 0.27 and 2.99 ± 0.26, respectively; p < 0.001). Application of the same amounts of Moringa oleifera (2 and 3 g) but using a wet preparation, was also significantly less effective than reference soap (p < 0.003 and p < 0.02, respectively). However there was no significant difference when using 4 g of Moringa oleifera powder in dried or wet preparation (mean log10-reduction: 2.70 ± 0.27 and 2.91 ± 0.11, respectively) compared with reference soap (2.97 ± 0.28). Application of calcium sulphate inert powder was significantly less effective than the 4 g of Moringa oleifera powder (p < 0.01). Four grams of Moringa oleifera powder in dried and wet application had the same effect as non-medicated soap when used for hand washing. Efficacious and available hand washing products could be useful in developing countries in controlling pathogenic organisms that are transmitted through contaminated hands.


Dynamic tuft cell expansion during gastric metaplasia and dysplasia.
Tuft cells are chemosensory cells associated with luminal homeostasis, immune response, and tumorigenesis in the gastrointestinal tract. We aimed to elucidate alterations in tuft cell populations during gastric atrophy and tumorigenesis in humans with correlative comparison to relevant mouse models. Tuft cell distribution was determined in human stomachs from organ donors and in gastric pathologies including Ménétrier's disease, Helicobacter pylori gastritis, intestinal metaplasia (IM), and gastric tumors. Tuft cell populations were examined in Lrig1-KrasG12D , Mist1-KrasG12D , and MT-TGFα mice. Tuft cells were evenly distributed throughout the entire normal human stomach, primarily concentrated in the isthmal region in the fundus. Ménétrier's disease stomach showed increased tuft cells. Similarly, Lrig1-Kras mice and mice overexpressing TGFα showed marked foveolar hyperplasia and expanded tuft cell populations. Human stomach with IM or dysplasia also showed increased tuft cell numbers. Similarly, Mist1-Kras mice had increased numbers of tuft cells during metaplasia and dysplasia development. In human gastric cancers, tuft cells were rarely observed, but showed positive associations with well-differentiated lesions. In mouse gastric cancer xenografts, tuft cells were restricted to dysplastic well-differentiated mucinous cysts and were lost in less differentiated cancers. Taken together, tuft cell populations increased in atrophic human gastric pathologies, metaplasia, and dysplasia, but were decreased in gastric cancers. Similar findings were observed in mouse models, suggesting that, while tuft cells are associated with precancerous pathologies, their loss is most associated with the progression to invasive cancer.


The effect of low-dose oral vitamin K supplementation on INR stability in patients receiving warfarin. A randomised trial.
The anticoagulant effect of warfarin is influenced by variations in vitamin K intake. Concomitant use of daily low-dose oral vitamin K (LDVK) and warfarin may improve INR stability. We hypothesise that administration of LDVK improves INR control. To test this hypothesis we performed a multi-centre, placebo-controlled, randomised trial conducted at four university-affiliated hospitals in Canada. Patients on chronic warfarin therapy received oral vitamin K 150 mcg daily or a matching placebo for a total of six months after a one-month run in period. The primary outcome was a comparison of mean time in therapeutic range (TTR) in LDVK and placebo group during a six-month-period. The secondary outcome was number of INR excursions <1.5 or >4.5. There was no significant difference in the final TTR between the two groups (65.1 % vs 66 %, p =0.8). Mean TTR in both LDVK and placebo groups were statistically increased compared with prior to the study. The number of INR excursions were significantly decreased in the LDVK group (9.4 % and 5.4 %, absolute difference [pre- minus post-] = 4 %, 95 % CI, 2 to 6 %, p-value <0.001). We conclude that LDVK administration did not increase mean TTR, but did decrease the number of INR excursions. The observed improvement in mean TTR in both groups suggests that more attentive monitoring of warfarin therapy, rather than LDVK, was responsible for the improvement in TTR observed. The reduced excursions suggest that LDVK did reduce extreme INR variation. The study is registered at www.ClinicalTrial.gov# NCT00990158.


Gender differentials in the evolution of cigarette smoking habits in a general European adult population from 1993-2003.
Describe the recent evolution of cigarette smoking habits by gender in Geneva, where incidence rates of lung cancer have been declining in men but increasing in women. Continuous cross-sectional surveillance of the general adult (35-74 yrs) population of Geneva, Switzerland for 11 years (1993-2003) using a locally-validated smoking questionnaire, yielding a representative random sample of 12,271 individuals (6,164 men, 6,107 women). In both genders, prevalence of current cigarette smoking was stable over the 11-year period, at about one third of men and one quarter of women, even though smoking began at an earlier age in more recent years. Older men were more likely to be former smokers than older women. Younger men, but not women, tended to quit smoking at an earlier age. This continuous (1993-2003) risk factor surveillance system, unique in Europe, shows stable prevalence of smoking in both genders. However, sharp contrasts in age-specific prevalence of never and former smoking and of ages at smoking initiation indicate that smoking continues a long-term decline in men but has still not reached its peak in women.


I wept for four years and when I stopped I was blind.
The conversion phenomena of hysteria were the subject of intense study in the late nineteenth and early twentieth centuries, after which work on the subject went into decline. The patients are still with us, however, and I cite an epidemic of hysterical blindness among Cambodian refugees living in the U.S. as a poignant example. Since the advent of brain imaging technology, conversion hysteria has been receiving renewed attention. In this paper, I suggest that examining the ideas about hysteria from the past, especially those of Charcot and Janet are fertile areas of study, including the illness and its relation to hypnosis, shock, suggestion, and dissociation theory. I also address the role of the imaginary and the imagination in the illness and critique the implicit dualist model used in most brain imaging studies that distorts the integration of psyche and soma. I summon Merleau-Ponty's body-subject, infant research on intersubjectivity, and Vittorio Gallese's "embodied simulation" as possible windows onto the problem of hysterical conversion, and finally I suggest that along with imaging studies, more dynamic narrative strategies should be used if we hope to understand the metamorphoses, mimesis, and powerful emotions that all play a part in this mysterious disease.


Homo- and heterodinuclear helicates of lanthanide(III), zinc(II) and aluminium(III) based on 8-hydroxyquinoline ligands.
Homonuclear helicates with rare-earth-metal(III) ions or heteronuclear derivatives with rare-earth-metal and aluminium or zinc centres are obtained in alkali-metal-templated self-assembly processes from isobutenylidene-bridged homoditopic bis(2-carbamido-8-hydroxyquinoline)-derived ligands 1-H(2) and 2-H(2) or heteroditopic (8-hydroxyquinoline)(2-carbamido-8-hydroxyquinoline)-derived ligands 3-H(2) and 4-H(2). Diamagnetic coordination compounds possess a high stability in organic solvents such as CDCl(3), [D(4)]MeOH or [D(6)]DMSO and can be well characterised by (1)H NMR spectroscopy by using methylene protons and the protons of the vinylic units of the ligand as stereochemical or symmetry probes, respectively. Some of the homonuclear complexes could be crystallised and were characterised by using X-ray diffraction studies. The complexes adopt a triple-stranded helical structure with a central templating cation encapsulated in their interior. An unusual orientation of the double bond of one spacer towards this cation is observed. The homo- and heterodinuclear helicates with ytterbium(III), neodymium(III) or erbium(III) of ligands 2 and 4 were of special interest owing to their near-infrared (NIR) emitting properties, which were investigated depending on the lanthanide and on the encapsulated alkali-metal cation.


Randomized controlled trial to evaluate transdermal testosterone in female cancer survivors with decreased libido; North Central Cancer Treatment Group protocol N02C3.
Decreased libido is one of several changes in sexual function that are often experienced by female cancer patients. Transdermal testosterone therapy has been associated with increased libido among estrogen-replete women who report low libido. In a phase III randomized, placebo-controlled crossover clinical trial, we evaluated whether transdermal testosterone would increase sexual desire in female cancer survivors. Postmenopausal women with a history of cancer and no current evidence of disease were eligible if they reported a decrease in sexual desire and had a sexual partner. Eligible women were randomly assigned to receive 2% testosterone in Vanicream for a testosterone dose of 10 mg daily or placebo Vanicream for 4 weeks and were then crossed over to the opposite treatment for an additional 4 weeks. The primary endpoint was sexual desire or libido, as measured using the desire subscales of the Changes in Sexual Functioning Questionnaire, as assessed at baseline and at the end of 4 and 8 weeks of treatment. Serum levels of bioavailable testosterone were measured at the same times. All statistical tests were two-sided. We enrolled 150 women. Women who were on active testosterone cream had higher serum levels of bioavailable testosterone than women on placebo (mean change from baseline, testosterone versus placebo, week 4, 11.57% versus 0%, difference = 11.57%, 95% confidence interval [CI] = 8.49% to 14.65%; week 8, 10.21% versus 0.28%, difference = 9.92%, 95% CI = 5.42% to 14.42%; P<.001 for all). However, the average intrapatient libido change from baseline to weeks 4 and 8 was similar on both arms. Increased testosterone level did not translate into improved libido, possibly because women on this study were estrogen depleted.


Association between neurologic improvement with decline in blood pressure and recanalization in stroke.
Patients with stroke often have a decline in blood pressure after thrombolysis. Neurologic improvement could result from recanalization or better collateral flow despite persistent occlusion. We hypothesized that neurologic improvement with concurrent decline in blood pressure may be a clinical sign of recanalization after intravenous tissue plasminogen activator. Patients treated with intravenous tissue plasminogen activator at Suburban Hospital, Bethesda, Maryland, and MedStar Washington Hospital Center, Washington, DC, from 1999 to 2009 were included in the study if they had pretreatment and 24-hour magnetic resonance angiographic scans, National Institutes of Health Stroke Scale scores at those times, and proximal middle cerebral artery occlusion demonstrated prior to treatment. The recanalization status on 24-hour magnetic resonance angiography was classified as none, partial, or complete. Seventeen patients met study criteria. On 24-hour magnetic resonance angiography, 3 patients had no recanalization, 8 had partial recanalization, and 6 had complete recanalization. At 24 hours after thrombolysis, neurologic improvement with concurrent decline in systolic blood pressure of 20 mm Hg or greater was seen in 4 patients with partial recanalization, 4 patients with complete recanalization, and none of the patients with no recanalization. Neurologic improvement with concurrent decline in systolic blood pressure of 20 mm Hg or greater after intravenous tissue plasminogen activator may be a clinical sign of recanalization. This observation needs confirmation in a larger cohort.


De novo gain-of-function and loss-of-function mutations of SCN8A in patients with intellectual disabilities and epilepsy.
Mutations of SCN8A encoding the neuronal voltage-gated sodium channel NaV1.6 are associated with early-infantile epileptic encephalopathy type 13 (EIEE13) and intellectual disability. Using clinical exome sequencing, we have detected three novel de novo SCN8A mutations in patients with intellectual disabilities, and variable clinical features including seizures in two patients. To determine the causality of these SCN8A mutations in the disease of those three patients, we aimed to study the (dys)function of the mutant sodium channels. The functional consequences of the three SCN8A mutations were assessed using electrophysiological analyses in transfected cells. Genotype-phenotype correlations of these and other cases were related to the functional analyses. The first mutant displayed a 10 mV hyperpolarising shift in voltage dependence of activation (gain of function), the second did not form functional channels (loss of function), while the third mutation was functionally indistinguishable from the wildtype channel. Comparison of the clinical features of these patients with those in the literature suggests that gain-of-function mutations are associated with severe EIEE, while heterozygous loss-of-function mutations cause intellectual disability with or without seizures. These data demonstrate that functional analysis of missense mutations detected by clinical exome sequencing, both inherited and de novo, is valuable for clinical interpretation in the age of massive parallel sequencing.


Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study.
The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6-0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56-0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38-0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02-1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society.


Downregulation of vascular angiotensin II type 1 receptor by thyroid hormone.
Thyroid hormone has a broad effect on cardiovascular system. 3,3',5-triiodo-l-thyronine (T3), a biologically active form of thyroid hormone, increases cardiac contractility. T3 causes arterial relaxation and reduction of systemic vascular resistance, resulting in an increase in cardiac output. However, the molecular mechanisms of vascular relaxation by T3 are incompletely characterized. We studied the effect of T3 on the angiotensin (Ang) II type 1 receptor (AT1R) expression in vascular smooth muscle cells. T3 dose-dependently decreased expression levels of AT1R mRNA, with a peak at 6 hours of stimulation. Binding assay using [125I]Sar1-Ile8-Ang II revealed that AT1R number was decreased by stimulation with T3 without changing the affinity to Ang II. T3 reduced calcium response of vascular smooth muscle cells to Ang II by 26%. AT1R promoter activity measured by luciferase assay was reduced by 50% after 9 hours of T3 administration. mRNA stability was also decreased by T3. Real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis revealed that AT1R mRNA and protein were downregulated in the aorta of T3-treated rats. These results suggest that T3 downregulates AT1R expression both at transcriptional and posttranscriptional levels, and attenuates biological function of Ang II. Our results suggest that downregulation of AT1R gene expression may play an important role for T3-induced vascular relaxation.


Do children with grapheme-colour synaesthesia show cognitive benefits?
Grapheme-colour synaesthesia is characterized by conscious and consistent associations between letters and colours, or between numbers and colours (e.g., synaesthetes might see A as red, 7 as green). Our study explored the development of this condition in a group of randomly sampled child synaesthetes. Two previous studies (Simner & Bain, 2013, Frontiers in Human Neuroscience, 7, 603; Simner, Harrold, Creed, Monro, & Foulkes, 2009, Brain, 132, 57) had screened over 600 primary school children to find the first randomly sampled cohort of child synaesthetes. In this study, we evaluate this cohort to ask whether their synaesthesia is associated with a particular cognitive profile of strengths and/or weaknesses. We tested our child synaesthetes at age 10-11 years in a series of cognitive tests, in comparison with matched controls and baseline norms. One previous study (Green & Goswami, 2008, Cognition, 106, 463) had suggested that child synaesthetes might perform differently to non-synaesthetes in such tasks, although those participants may have been a special type of population independent of their synaesthesia. In our own study of randomly sampled child synaesthetes, we found no significant advantages or disadvantages in a receptive vocabulary test and a memory matrix task. However, we found that synaesthetes demonstrated above-average performance in a processing-speed task and a near-significant advantage in a letter-span task (i.e., memory/recall task of letters). Our findings point to advantages for synaesthetes that go beyond those expected from enhanced coding accounts and we present the first picture of the broader cognitive profile of a randomly sampled population of child synaesthetes.


The 13C method as a robust alternative to 14C-based measurements of primary productivity in the Mediterranean Sea.
Due to the increasing constraints on using the radioactive isotope 14C to measure primary productivity (14C-PP), we determined the surface carbon fixation rates in the Mediterranean Sea, using the alternative stable isotope 13C method (13C-PP). Rates obtained (13C-POCp) were compared with simultaneous 14C-POCp measurements in samples of different volumes (72 mL and 1.2 L). We also tested the variation of the percentage of dissolved primary production (PER), to the total productivity using organic and inorganic filters (14C method). 13C-POCp rates ranged from 0.4, in the Ionian basin, to 1.5 mgC m-3 h-1 in the Ligurian region. These results agreed with those found with the 14C-PP in 1.2-L samples (two sample t-test, t = 1.035, df = 22, P = 0.31). However, we found that 14C-POCp rates derived from 72-mL incubations were 46% lower than those measured with 13C-PP. The discrepancy between large and small volume incubations was likely due to differences in the number of large phytoplankton cells within the community. PER values measured with silver membrane filters yielded similar results than those obtained using polycarbonate filters. Our findings showed that when the sample size is carefully chosen, the 13C-PP provide comparable results to 14C-PP even in waters of low productivity in the Mediterranean Sea.


Factors influencing the composition of the intestinal microbiota in early infancy.
The aim of this study was to examine the contribution of a broad range of external influences to the gut microbiotic composition in early infancy. Fecal samples from 1032 infants at 1 month of age, who were recruited from the KOALA Birth Cohort Study in the Netherlands, were subjected to quantitative real-time polymerase chain reaction assays for the enumeration of bifidobacteria, Escherichia coli, Clostridium difficile, Bacteroides fragilis group, lactobacilli, and total bacterial counts. Information on potential determinants of the gut microbiotic composition was collected with repeated questionnaires. The associations between these factors and the selected gut bacteria were analyzed with univariate and multivariate analyses. Infants born through cesarean section had lower numbers of bifidobacteria and Bacteroides, whereas they were more often colonized with C difficile, compared with vaginally born infants. Exclusively formula-fed infants were more often colonized with E coli, C difficile, Bacteroides, and lactobacilli, compared with breastfed infants. Hospitalization and prematurity were associated with higher prevalence and counts of C difficile. Antibiotic use by the infant was associated with decreased numbers of bifidobacteria and Bacteroides. Infants with older siblings had slightly higher numbers of bifidobacteria, compared with infants without siblings. The most important determinants of the gut microbiotic composition in infants were the mode of delivery, type of infant feeding, gestational age, infant hospitalization, and antibiotic use by the infant. Term infants who were born vaginally at home and were breastfed exclusively seemed to have the most "beneficial" gut microbiota (highest numbers of bifidobacteria and lowest numbers of C difficile and E coli).


Interplay of autophagy and apoptosis during murine cytomegalovirus infection of RPE cells.
Previous studies have demonstrated that autophagy is involved in the pathogenesis of human cytomegalovirus (HCMV) infection. However, whether autophagy is regulated by murine cytomegalovirus (MCMV) infection has not yet been investigated. The purpose of these studies was to determine how autophagy is affected by MCMV infection of the retinal pigment epithelial (RPE) cells and whether there is a functional relationship between autophagy and apoptosis; and if so, how regulation of autophagy impacts apoptosis. RPE cells were isolated from C57BL/6 mice and infected with MCMV K181. The cells were cultured in medium containing rapamycin, chloroquine, or ammonium chloride. Green fluorescent protein-light chain 3 (GFP-LC3) plasmid was transfected to RPE cells, and the GFP-LC3 positive puncta were counted. Electron microscopic (EM) images were taken to visualize the structure of the autophagic vacuoles. Western blot was performed to detect the expression of related proteins. Trypan blue exclusion assay was used to measure the percentage of viable cells. Although the LC3B-II levels consistently increased during MCMV infection of RPE cells, administration of chloroquine or ammonium chloride increased LC3B-II expression only at the early stage of infection (6 h post-inoculation [p.i.] and 12 h p.i.), not at or after 24 h p.i. The punctate autophagic vacuoles in the GFP-LC3 transfected RPE cells were counted using light microscopy or by EM examination, the number of autophagic vacuoles was significantly increased in the MCMV-infected RPE cells compared to the uninfected controls. Compared to untreated MCMV-infected control cells, rapamycin treatment resulted in a significant decrease in the cleaved caspase 3 levels as well as a significant decrease in the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR and in the ratio of phosphorylated P70S6K to total P70S6K. In contrast, chloroquine treatment resulted in a significant increase in the cleaved caspase 3 levels in the MCMV-infected RPE cells. Autophagic vacuole accumulation was detected during MCMV infection of RPE cells. In contrast, autophagic flux was greatly decreased at or after 24 h p.i. The results suggest that MCMV might have a strategy for inhibiting or blocking autophagy activity by targeting a later autophagy process, such as the formation of autolysosomes or degradation of their content. Our data also suggest that there is a functional relationship between autophagy and apoptosis, which plays an important role during MCMV infection of the RPE.


[Towards the Swiss systemic lupus erythematosus cohort study (SSCS)].
Systemic lupus erythematosus (SLE) is a rare disease mainly affecting women of childbearing age. It is characterized by a very large spectrum of clinical manifestations accompanied by prototypic abnormalities of the immune system. While recent advances in therapeutic approaches have taken place, SLE still has a profound impact on the quality of life and life expectancy of affected persons. The Swiss cohort for longitudinally studying SLE named SSCS responds to the necessity of better understanding the history of the disease, the mechanisms involved in its pathogenesis, to identify and apply new therapeutic and prevention strategies, as well as to analyze the impact that SLE has at the social and personal levels in Switzerland. SSCS is a tool to be used by all researchers interested to provide answers to the many open questions in SLE.


The Novel Phages phiCD5763 and phiCD2955 Represent Two Groups of Big Plasmidial Siphoviridae Phages of Clostridium difficile.
Until recently, Clostridium difficile phages were limited to Myoviruses and Siphoviruses of medium genome length (32-57 kb). Here we report the finding of phiCD5763, a Siphovirus with a large extrachromosomal circular genome (132.5 kb, 172 ORFs) and a large capsid (205.6 ± 25.6 nm in diameter) infecting MLST Clade 1 strains of C. difficile. Two subgroups of big phage genomes similar to phiCD5763 were identified in 32 NAPCR1/RT012/ST-54 C. difficile isolates from Costa Rica and in whole genome sequences (WGS) of 41 C. difficile isolates of Clades 1, 2, 3, and 4 from Canada, USA, UK, Belgium, Iraq, and China. Through comparative genomics we discovered another putative big phage genome in a non-NAPCR1 isolate from Costa Rica, phiCD2955, which represents other big phage genomes found in 130 WGS of MLST Clade 1 and 2 isolates from Canada, USA, Hungary, France, Austria, and UK. phiCD2955 (131.6 kb, 172 ORFs) is related to a previously reported C. difficile phage genome, phiCD211/phiCDIF1296T. Detailed genome analyses of phiCD5763, phiCD2955, phiCD211/phiCDIF1296T, and seven other putative C. difficile big phage genome sequences of 131-136 kb reconstructed from publicly available WGS revealed a modular gene organization and high levels of sequence heterogeneity at several hotspots, suggesting that these genomes correspond to biological entities undergoing recombination. Compared to other C. difficile phages, these big phages have unique predicted terminase, capsid, portal, neck and tail proteins, receptor binding proteins (RBPs), recombinases, resolvases, primases, helicases, ligases, and hypothetical proteins. Moreover, their predicted gene load suggests a complex regulation of both phage and host functions. Overall, our results indicate that the prevalence of C. difficile big bacteriophages is more widespread than realized and open new avenues of research aiming to decipher how these viral elements influence the biology of this emerging pathogen.


A multicenter comparison study between the Endosafe PTS rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products.
Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.


Tracking and line integration of diffuse cellular subdomains by mesh advection.
Active molecular transport ensures a purposeful spatiotemporal distribution of cellular proteins and is therefore key to a wide range of processes such as morphogenesis, homeostasis or migration. However, redistributions of molecules in bulk are seldom quantified because the regions involved are too diffuse to be segmented consistently. To bridge this gap, we propose a Laplace-corrected Runge-Kutta advection that is based on mesh triangulation. Our framework can follow the movement and deformation of multiple parts of a diffuse region at once and offers a seamless combination with spatiotemporal line integration in Lagrangian coordinates. This allows the flexibility to taylor specific measures to the question at hand, e.g. mechanical work, bringing long-established physics concepts into biology grounds. We exemplify our approach by quantifying how the isotropy of intracellular protein distributions changes during cargo transport.


Cross-cultural validity of the demand-control questionnaire: Swedish and Brazilian workers.
OBJECTIVE To evaluate the cross-cultural validity of the Demand-Control Questionnaire, comparing the original Swedish questionnaire with the Brazilian version. METHODS We compared data from 362 Swedish and 399 Brazilian health workers. Confirmatory and exploratory factor analyses were performed to test structural validity, using the robust weighted least squares mean and variance-adjusted (WLSMV) estimator. Construct validity, using hypotheses testing, was evaluated through the inspection of the mean score distribution of the scale dimensions according to sociodemographic and social support at work variables. RESULTS The confirmatory and exploratory factor analyses supported the instrument in three dimensions (for Swedish and Brazilians): psychological demands, skill discretion and decision authority. The best-fit model was achieved by including an error correlation between work fast and work intensely (psychological demands) and removing the item repetitive work (skill discretion). Hypotheses testing showed that workers with university degree had higher scores on skill discretion and decision authority and those with high levels of Social Support at Work had lower scores on psychological demands and higher scores on decision authority. CONCLUSIONS The results supported the equivalent dimensional structures across the two culturally different work contexts. Skill discretion and decision authority formed two distinct dimensions and the item repetitive work should be removed.


Chronic oral administration of minocycline to sheep with ovine CLN6 neuronal ceroid lipofuscinosis maintains pharmacological concentrations in the brain but does not suppress neuroinflammation or disease progression.
The neuronal ceroid lipofuscinoses (NCLs; or Batten disease) are fatal inherited human neurodegenerative diseases affecting an estimated 1:12,500 live births worldwide. They are caused by mutations in at least 11 different genes. Currently, there are no effective treatments. Progress into understanding pathogenesis and possible therapies depends on studying animal models. The most studied animals are the CLN6 South Hampshire sheep, in which the course of neuropathology closely follows that in affected children. Neurodegeneration, a hallmark of the disease, has been linked to neuroinflammation and is consequent to it. Activation of astrocytes and microglia begins prenatally, starting from specific foci associated with the later development of progressive cortical atrophy and the development of clinical symptoms, including the occipital cortex and blindness. Both neurodegeneration and neuroinflammation generalize and become more severe with increasing age and increasing clinical severity. The purpose of this study was to determine if chronic administration of an anti-inflammatory drug, minocycline, from an early age would halt or reverse the development of disease. Minocycline, a tetracycline family antibiotic with activity against neuroinflammation, was tested by chronic oral administration of 25 mg minocycline/kg/day to presymptomatic lambs affected with CLN6 NCL at 3 months of age to 14 months of age, when clinical symptoms are obvious, to determine if this would suppress neuroinflammation or disease progression. Minocycline was absorbed without significant rumen biotransformation to maintain pharmacological concentrations of 1 μM in plasma and 400 nM in cerebrospinal fluid, but these did not result in inhibition of microglial activation or astrocytosis and did not change the neuronal loss or clinical course of the disease. Oral administration is an effective route for drug delivery to the central nervous system in large animals, and model studies in these animals should precede highly speculative procedures in humans. Minocycline does not inhibit a critical step in the neuroinflammatory cascade in this form of Batten disease. Identification of the critical steps in the neuroinflammatory cascade in neurodegenerative diseases, and targeting of specific drugs to them, will greatly increase the likelihood of success.


Pregnenolone sulfate and aging of cognitive functions: behavioral, neurochemical, and morphological investigations.
Neurosteroids are a subclass of steroids that can be synthesized in the central nervous system independently of peripheral sources. Several neurosteroids influence cognitive functions. Indeed, in senescent animals we have previously demonstrated a significant correlation between the cerebral concentration of pregnenolone sulfate (PREG-S) and cognitive performance. Indeed, rats with memory impairments exhibited low PREG-S concentrations compared to animals with correct memory performance. Furthermore, these memory deficits can be reversed by intracerebral infusions of PREG-S. Neurotransmitter systems modulated by this neurosteroid were unknown until our recent report of an enhancement of acetylcholine (ACh) release in basolateral amygdala, cortex, and hippocampus induced by central administrations of PREG-S. Central ACh neurotransmission is involved in the regulation of memory processes and is affected in normal aging and in human neurodegenerative pathologies like Alzheimer's disease. ACh neurotransmission is also involved in the modulation of sleep-wakefulness cycle and relationships between paradoxical sleep and memory are well documented in the literature. PREG-S infused at the level of ACh cell bodies induces a dramatic increase of paradoxical sleep in young animals. Cognitive dysfunctions, particularly those observed in Alzheimer's disease, have also been related to alterations of cerebral plasticity. Among these mechanisms, neurogenesis has been recently studied. Preliminary data suggest that PREG-S central infusions dramatically increase neurogenesis. Taken together these data suggest that PREG-S can influence cognitive processes, particularly in senescent subjects, through a modulation of ACh neurotransmission associated with paradoxical sleep modifications; furthermore our recent data suggest a role for neurosteroids in the modulation of hippocampal neurogenesis.


Characterization of a rice variety with high hydraulic conductance and identification of the chromosome region responsible using chromosome segment substitution lines.
The rate of photosynthesis in paddy rice often decreases at noon on sunny days because of water stress, even under submerged conditions. Maintenance of higher rates of photosynthesis during the day might improve both yield and dry matter production in paddy rice. A high-yielding indica variety, 'Habataki', maintains a high rate of leaf photosynthesis during the daytime because of the higher hydraulic conductance from roots to leaves than in the standard japonica variety 'Sasanishiki'. This research was conducted to characterize the trait responsible for the higher hydraulic conductance in 'Habataki' and identified a chromosome region for the high hydraulic conductance. Hydraulic conductance to passive water transport and to osmotic water transport was determined for plants under intense transpiration and for plants without transpiration, respectively. The varietal difference in hydraulic conductance was examined with respect to root surface area and hydraulic conductivity (hydraulic conductance per root surface area, L(p)). To identify the chromosome region responsible for higher hydraulic conductance, chromosome segment substitution lines (CSSLs) derived from a cross between 'Sasanishiki' and 'Habataki' were used. The significantly higher hydraulic conductance resulted from the larger root surface area not from L(p) in 'Habataki'. A chromosome region associated with the elevated hydraulic conductance was detected between RM3916 and RM2431 on the long arm of chromosome 4. The CSSL, in which this region was substituted with the 'Habataki' chromosome segment in the 'Sasanishiki' background, had a larger root mass than 'Sasanishiki'. The trait for increasing plant hydraulic conductance and, therefore, maintaining the higher rate of leaf photosynthesis under the conditions of intense transpiration in 'Habataki' was identified, and it was estimated that there is at least one chromosome region for the trait located on chromosome 4.


The Physiology, Pathology, and Potential Therapeutic Applications of the TREM2 Signaling Pathway.
Alzheimer's disease, obesity-related metabolic syndrome, and cancer are the leading causes of death and among the most costly medical conditions in the Western world. In all three cases, recent discoveries establish the TREM2 receptor as a major pathology-induced immune signaling hub that senses tissue damage and activates robust immune remodeling in response to it. In this review, we summarize and question what is known and remains to be discovered about TREM2 signaling pathway, track the consequences of its activation in physiological niches and pathological contexts, and highlight the promising potential of therapeutic manipulation of TREM2 signaling.


The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins.
The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.


Proceedings of the second international molecular pathological epidemiology (MPE) meeting.
Disease classification system increasingly incorporates information on pathogenic mechanisms to predict clinical outcomes and response to therapy and intervention. Technological advancements to interrogate omics (genomics, epigenomics, transcriptomics, proteomics, metabolomics, metagenomics, interactomics, etc.) provide widely open opportunities in population-based research. Molecular pathological epidemiology (MPE) represents integrative science of molecular pathology and epidemiology. This unified paradigm requires multidisciplinary collaboration between pathology, epidemiology, biostatistics, bioinformatics, and computational biology. Integration of these fields enables better understanding of etiologic heterogeneity, disease continuum, causal inference, and the impact of environment, diet, lifestyle, host factors (including genetics and immunity), and their interactions on disease evolution. Hence, the Second International MPE Meeting was held in Boston in December 2014, with aims to: (1) develop conceptual and practical frameworks; (2) cultivate and expand opportunities; (3) address challenges; and (4) initiate the effort of specifying guidelines for MPE. The meeting mainly consisted of presentations of method developments and recent data in various malignant neoplasms and tumors (breast, prostate, ovarian and colorectal cancers, renal cell carcinoma, lymphoma, and leukemia), followed by open discussion sessions on challenges and future plans. In particular, we recognized need for efforts to further develop statistical methodologies. This meeting provided an unprecedented opportunity for interdisciplinary collaboration, consistent with the purposes of the Big Data to Knowledge, Genetic Associations and Mechanisms in Oncology, and Precision Medicine Initiative of the US National Institute of Health. The MPE meeting series can help advance transdisciplinary population science and optimize training and education systems for twenty-first century medicine and public health.


Characterization and possible function of an enigmatic reflector in the eye of the shrimp Litopenaeus vannamei.
Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 μm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.


Fingerprints of a message: integrating positional information on the transcriptome.
The recent explosion of high-throughput sequencing methods applied to RNA molecules is allowing us to go beyond the description of sequence variants and their relative abundances, as measured by RNA-seq. We can now probe for RNA engagement in polysomes, for ribosomes, RNA binding proteins and microRNAs binding sites, for RNA secondary structure and for RNA methylation. These descriptors produce a steadily growing multidimensional array of positional information on RNA sequences, whose effective integration only would bring to decipher the regulatory interplay occurring between proteins, RNAs and their modifications on the transcriptome. This interplay ultimately dictates the degree of mRNA availability to translation, and thus the occurrence of cell phenotypes. However, several issues in data presentation are slowing down effective integration. A standardization effort for new dataset types produced should be urgently undertaken to solve these issues. Providing uniformed experimental details along with datasets processed to be directly usable and employing shared formats would greatly simplify integration efforts, strengthening hypotheses stemming from correlative observations and eventually bringing to mechanistic understanding.


[Inhibitors of RAS Might Be a Good Choice for the Therapy of COVID-19 Pneumonia].
The novel coronavirus 2019 (COVID-19) infected patients by binding human ACE2, leading to severe pneumonia and highly mortality rate in patients. At present, there is no definite and effective treatment for COVID-19. ACE2 plays an important role in the RAS, and the imbalance between ACE/Ang II/AT1R pathway and ACE2/Ang (1-7)/Mas receptor pathway in the RAS system will lead to multi-system inflammation. Increased ACE and Ang II are poor prognostic factors for severe pneumonia. Animal studies have shown that RAS inhibitors could effectively relieve symptoms of acute severe pneumonia and respiratory failure. The binding of COVID-19 and ACE2 resulted in the exhaustion of ACE2, and then ACE2/Ang (1-7)/Mas receptor pathway was inhibited. The balance of the RAS system was broken, and this would lead to the exacerbation of acute severe pneumonia. Therefore, we speculate that ACEI and AT1R inhibitors could be used in patients with COVID-19 pneumonia under the condition of controlling blood pressure, and might reduce the pulmonary inflammatory response and mortality.


Incidence of allergic reactions with fresh frozen plasma or cryo-supernatant plasma in the treatment of thrombotic thrombocytopenic purpura.
Plasma replacement for thrombotic thrombocytopenic purpura (TTP) is accomplished with various plasma products. This study sought to determine the incidence of allergic reactions with FFP or CPP as replacement in therapeutic plasma exchange (TPE). Forty-one TTP patients were identified retrospectively who received TPE replacement with either FFP (n=21) or CPP (n=20). Anti-histamine was administered prophylactically following the initial occurrence of an allergic reaction (urticaria, respiratory distress, or anaphylaxis with hypotension). Fifty-one allergic reactions occurred in 65.8% of patients. Urticaria comprised 49 of 51 (96%) of reactions and respiratory distress the remaining 4%. No anaphylaxis occurred. Nineteen urticarial reactions occurred in 50% of CPP recipients compared to 71% of FFP recipients (P=0.28). Anti-histamine breakthrough occurred in 36.3% of patients who experienced a previous allergic reaction with CPP and 37.5% with FFP (P=1.0). The overall risk of allergy per unit of plasma was 1.37% (1.23 % CPP, 1.48% FFP), comparable to estimates in non-TTP recipients. The median number of donor exposures preceding the first allergic reaction was 35 and 32, CPP and FFP, respectively (P=0.63). The mean volume of plasma transfused prior to reaction was 9,883 mL for CPP and 9,348 mL for FFP (P=0.85). Neither product was advantageous in preventing allergic complications. Because of the large volume, the number of donor exposures, and prolonged duration of therapy, allergic reactions to plasma are common (65.8%) in the treatment of TTP.


Decoupling of the PI3K Pathway via Mutation Necessitates Combinatorial Treatment in HER2+ Breast Cancer.
We report here on experimental and theoretical efforts to determine how best to combine drugs that inhibit HER2 and AKT in HER2(+) breast cancers. We accomplished this by measuring cellular and molecular responses to lapatinib and the AKT inhibitors (AKTi) GSK690693 and GSK2141795 in a panel of 22 HER2(+) breast cancer cell lines carrying wild type or mutant PIK3CA. We observed that combinations of lapatinib plus AKTi were synergistic in HER2(+)/PIK3CA(mut) cell lines but not in HER2(+)/PIK3CA(wt) cell lines. We measured changes in phospho-protein levels in 15 cell lines after treatment with lapatinib, AKTi or lapatinib + AKTi to shed light on the underlying signaling dynamics. This revealed that p-S6RP levels were less well attenuated by lapatinib in HER2(+)/PIK3CA(mut) cells compared to HER2(+)/PIK3CAwt cells and that lapatinib + AKTi reduced p-S6RP levels to those achieved in HER2(+)/PIK3CA(wt) cells with lapatinib alone. We also found that that compensatory up-regulation of p-HER3 and p-HER2 is blunted in PIK3CA(mut) cells following lapatinib + AKTi treatment. Responses of HER2(+) SKBR3 cells transfected with lentiviruses carrying control or PIK3CA(mut )sequences were similar to those observed in HER2(+)/PIK3CA(mut) cell lines but not in HER2(+)/PIK3CA(wt) cell lines. We used a nonlinear ordinary differential equation model to support the idea that PIK3CA mutations act as downstream activators of AKT that blunt lapatinib inhibition of downstream AKT signaling and that the effects of PIK3CA mutations can be countered by combining lapatinib with an AKTi. This combination does not confer substantial benefit beyond lapatinib in HER2+/PIK3CA(wt) cells.


Femoral neck response to exercise and subsequent deconditioning in young and adult rats.
Aged bones have been considered to have reduced capacity to respond to changes in incident loading. By subjecting young and adult rats to increased loading and subsequent deconditioning, we observed quantitatively similar adaptive responses of bone in these two groups, but young skeletons adapted primarily through geometric changes and adult bones through increased volumetric density. Loss of the exercise-induced bone benefits did not depend on age. Aging has been shown to decrease the sensitivity of the mechanosensory cells of bones to loading-induced stimuli, presumably resulting in not only reduced capacity but also different adaptive mechanism of the aged skeleton to altered loading, as well as poorer capacity to preserve exercise-induced bone benefits. Fifty young (5-week-old) and 50 adult (33-week-old) male rats were randomized into control and exercise (+deconditioning) groups. After a 14-week progressively intensified running program, one-half of the exercised rats (both young and adult) were killed, and the remaining rats underwent subsequent 14-week period of deconditioning (free cage activity). A comprehensive analysis of the femoral neck was performed using peripheral quantitative computed tomography and mechanical testing. In comparison with the controls, both young and adult exercised rats had significant increases in almost all measured parameters: +25% (p < 0.001) and +10% (not significant [NS]) in the cross-sectional area; +28% (p < 0.001) and +18% (p < 0.001) in bone mineral content; +11% (p < 0.05) and +23% (p < 0.001) in bone mineral density; and +30% (p < 0.01) and +28% (p < 0.01) in the breaking load, respectively. The skeletal responses were not statistically different between the young and adult rats. After the 14-week period of deconditioning, the corresponding exercised-to-controls differences were +17% (p < 0.05) and +10% (NS), +18% (p < 0.05) and +13% (p < 0.05), +2% (NS) and +2% (NS), and +11% (NS) and +6% (NS), respectively. Again, the response differences were not significant between the age groups. Quantitatively, the capacity of the young and adult skeleton to adapt to increased loading was similar, but the adaptive mechanisms appeared different: growing bones seemed to primarily display geometric changes (increase in bone size), whereas the adult skeleton responded mainly through an increase in density. Despite this apparent difference in the adaptive mechanism, aging did not modulate the ability of the skeleton to preserve the exercise-induced bone gain, because the bone loss was similar in the young and adult rats after cessation of training.


Validation of the Jackson Heart Study Physical Activity Survey in African Americans.
Physical inactivity has been consistently linked to cardiovascular disease, yet few instruments have been validated for assessment of physical activity in African Americans, a group particularly vulnerable to heart disease. The current study aimed to establish the psychometric properties of the activity survey used in the Jackson Heart Study (JHS) among African Americans, the JHS Physical Activity Cohort survey (JPAC). Test-retest reliability over 2 weeks was assessed using a convenience sample of 40 African Americans. Convergent validity with accelerometer and pedometer data were assessed in 2 samples from the JHS (N = 404 and 294, respectively). Test-retest reliability was excellent, with intraclass correlations = .99 for the JPAC total and index scores. Higher JPAC total scores were significantly associated with higher raw accelerometer and pedometer counts. Spearman correlations between JPAC total scores and accelerometer (rho = .24) and pedometer counts (rho = .32) were consistent with these results. Most subscales were significantly correlated with the objective measures. The JPAC total score was most strongly associated with objectively-measured activity. This study provides support for the reliability and validity of the JPAC as a tool for assessing physical activity among African Americans across a variety of domains.


Challenges for medical educators: results of a survey among members of the German Association for Medical Education.
Despite the increasing interest in medical education in the German-speaking countries, there is currently no information available on the challenges which medical educators face. To address this problem, we carried out a web-based survey among the members of the Association for Medical Education (Gesellschaft für medizinische Ausbildung, GMA). A comprehensive survey was carried out on the need for further qualifications, expertise and the general conditions of medical educators in Germany. As part of this study, the educators were asked to list the three main challenges which they faced and which required urgent improvement. The results were analysed by means of qualitative content analysis. The questionnaire was completed by 147 of the 373 members on the GMA mailing list (response rate: 39%). The educators named a total of 346 challenges and emphasised the following areas: limited academic recognition for engagement in teaching (53.5% of educators), insufficient institutional (31.5%) and financial support (28.4%), a curriculum in need of reform (22.8%), insufficient time for teaching assignments (18,9%), inadequate teacher competence in teaching methods (18.1%), restricted faculty development programmes (18.1%), limited networking within the institution (11.0%), lack of teaching staff (10.2%), varying preconditions of students (8.7%), insufficient recognition and promotion of medical educational research (5.5%), extensive assessment requirements (4.7%), and the lack of role models within medical education (3.2%). The medical educators found the biggest challenges which they faced to be limited academic recognition and insufficient institutional and financial support. Consequently, improvements should be implemented to address these issues.


Free-standing millimetre-long Bi2Te3 sub-micron belts catalyzed by TiO2 nanoparticles.
Physical vapour deposition (PVD) is used to grow millimetre-long Bi2Te3 sub-micron belts catalysed by TiO2 nanoparticles. The catalytic efficiency of TiO2 nanoparticles for the nanostructure growth is compared with the catalyst-free growth employing scanning electron microscopy. The catalyst-coated and catalyst-free substrates are arranged side-by-side, and overgrown at the same time, to assure identical growth conditions in the PVD furnace. It is found that the catalyst enhances the yield of the belts. Very long belts were achieved with a growth rate of 28 nm/min. A ∼1-mm-long belt with a rectangular cross section was obtained after 8 h of growth. The thickness and width were determined by atomic force microscopy, and their ratio is ∼1:10. The chemical composition was determined to be stoichiometric Bi2Te3 using energy-dispersive X-ray spectroscopy. Temperature-dependent conductivity measurements show a characteristic increase of the conductivity at low temperatures. The room temperature conductivity of 0.20 × 10(5) S m (-1) indicates an excellent sample quality.


mRNA modifications: Dynamic regulators of gene expression?
The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N(6)-methyladenosine (m(6)A), 5-methylcytosine (m(5)C), pseudouridine (Ψ) and N(1)-methyladenosine (m(1)A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression.


MLK3 regulates bone development downstream of the faciogenital dysplasia protein FGD1 in mice.
Mutations in human FYVE, RhoGEF, and PH domain-containing 1 (FGD1) cause faciogenital dysplasia (FGDY; also known as Aarskog syndrome), an X-linked disorder that affects multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase CDC42. However, the mechanisms by which mutations in FGD1 affect skeletal development are unknown. Here, we describe what we believe to be a novel signaling pathway in osteoblasts initiated by FGD1 that involves the MAP3K mixed-lineage kinase 3 (MLK3). We observed that MLK3 functions downstream of FGD1 to regulate ERK and p38 MAPK, which in turn phosphorylate and activate the master regulator of osteoblast differentiation, Runx2. Mutations in FGD1 found in individuals with FGDY ablated its ability to activate MLK3. Consistent with our description of this pathway and the phenotype of patients with FGD1 mutations, mice with a targeted deletion of Mlk3 displayed multiple skeletal defects, including dental abnormalities, deficient calvarial mineralization, and reduced bone mass. Furthermore, mice with knockin of a mutant Mlk3 allele that is resistant to activation by FGD1/CDC42 displayed similar skeletal defects, demonstrating that activation of MLK3 specifically by FGD1/CDC42 is important for skeletal mineralization. Thus, our results provide a putative biochemical mechanism for the skeletal defects in human FGDY and suggest that modulating MAPK signaling may benefit these patients.


Respiratory function is associated to marathon race time.
The purpose of this study was to analyze the relationship between marathon race time and expiratory pulmonary parameters in a heterogeneous group of amateur marathoners. A total of 110 marathon runners (age=41.9±9.4 yr, body mass=74.0±9.1 kg, height=175.0±8.0 cm) volunteered to participate in this study. First, they completed a questionnaire about running experience and best performance time in the 10-km, half-marathon and marathon competitions. Then, they performed a maximal spirometry test following guidelines for standardized spirometry. Measurement included peak expiratory flow (PEF), forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC). All these expiratory variables were normalized for the participant's body mass. Within 3 days of the spirometry test, participants competed in an official marathon and race time was measured by a chip-timing. After this, participants were grouped by their marathon race time as follows: <210 min, N.=33; between 210 and 240 min, N.=31; and >240 min, N.=46). Marathon race time correlated to the FVC·kg-1 (r=-0.41; P<0.001), to FEV1·kg-1 (r=-0.40; P<0.001), and PEF·kg-1 (r=-0.50; P=0.005). However, self-reported running experience did not show significant correlations to FVC·kg-1 and PEF·kg-1 (P>0.05). The group of faster marathoners (e.g., <210 min) had greater FEV1·kg-1 (<210 min group: 0.064±0.009; 210-240 min group: 0.058±0.008; >240 min group: 0.057±0.009; P<0.001) and higher FVC·kg-1 (<210 min group: 0.081±0.011; 210-240 min group: 0.075±0.012; >240 min group: 0.072±0.010; P<0.001) than the other two groups of slower runners. These results suggest a significant relationship between individual pulmonary function and marathon race time. Thus, a higher lung capacity per kg of body mass might be a key variable for marathon performance in amateur runners.


Benthic reef primary production in response to large amplitude internal waves at the Similan Islands (Andaman Sea, Thailand).
Coral reefs are facing rapidly changing environments, but implications for reef ecosystem functioning and important services, such as productivity, are difficult to predict. Comparative investigations on coral reefs that are naturally exposed to differing environmental settings can provide essential information in this context. One prevalent phenomenon regularly introducing alterations in water chemistry into coral reefs are internal waves. This study therefore investigates the effect of large amplitude internal waves (LAIW) on primary productivity in coral reefs at the Similan Islands (Andaman Sea, Thailand). The LAIW-exposed west sides of the islands are subjected to sudden drops in water temperature accompanied by enhanced inorganic nutrient concentrations compared to the sheltered east. At the central island, Ko Miang, east and west reefs are only few hundred meters apart, but feature pronounced differences. On the west lower live coral cover (-38 %) coincides with higher turf algae cover (+64 %) and growth (+54 %) compared to the east side. Turf algae and the reef sand-associated microphytobenthos displayed similar chlorophyll a contents on both island sides, but under LAIW exposure, turf algae exhibited higher net photosynthesis (+23 %), whereas the microphytobenthos displayed reduced net and gross photosynthesis (-19 % and -26 %, respectively) accompanied by lower respiration (-42 %). In contrast, the predominant coral Porites lutea showed higher chlorophyll a tissues contents (+42 %) on the LAIW-exposed west in response to lower light availability and higher inorganic nutrient concentrations, but net photosynthesis was comparable for both sides. Turf algae were the major primary producers on the west side, whereas microphytobenthos dominated on the east. The overall primary production rate (comprising all main benthic primary producers) was similar on both island sides, which indicates high primary production variability under different environmental conditions.


Reducing the inversion degree of MnFe2O4 nanoparticles through synthesis to enhance magnetization: evaluation of their (1)H NMR relaxation and heating efficiency.
Manganese ferrite (MnFe2O4) nanoparticles of identical size (9 nm) and with different inversion degrees were synthesized under solvothermal conditions as a candidate theranostic system. In this facile approach, a long-chain amine, oleylamine, was utilized as a reducing and surface-functionalizing agent. The synthesized nanoparticles were shown to have a cubic-spinel structure as characterized by TEM and XRD patterns. Control over their inversion degree was achieved by a simple change of manganese precursor from Mn(acac)2 to Mn(acac)3. The variation in the inversion degree is ascribed to the partial oxidation of Mn(2+) to Mn(3+), as was evidenced by X-ray absorption near edge structure spectroscopy and extended X-ray absorption fine structure spectroscopy at both the Fe and Mn K-edges. The reduction of the inversion degree from 0.42 to 0.22 is close to the corresponding bulk value of 0.20 and led to elevated magnetization (65.7 emu g(-1)), in contrast to the Néel temperature, which was decreased owing to the weaker superexchange interactions between the tetrahedral and octahedral sites within the spinel structure. In order to evaluate the performance of these nanoprobes as a possible bifunctional targeting system, the (1)H NMR relaxation of the samples was tested together with their specific loss power under an alternating magnetic field as a function of concentration. The hydrophobic as prepared MnFe2O4 nanoparticles converted to hydrophilic nanoparticles with cetyltrimethylammonium bromide (CTAB). The MnFe2O4 nanoparticles, well-dispersed in aqueous media, were shown to have r2 relaxivity of up to 345.5 mM(-1) s(-1) and heat release of up to 286 W g(-1), demonstrating their potential use for bioapplications.


SerbGO: searching for the best GO tool.
In recent years, the scientific community has provided many tools to assist with pathway analysis. Some of these programs can be used to manage functional annotation of gene products, others are oriented to exploring and analyzing data sets and many allow both possibilities. Potential users of these tools are faced with the necessity to decide which of the existing programs are the most appropriate for their needs. SerbGO is a user-friendly web tool created to facilitate this task. It can be used (i) to search for specific functionalities and determine which applications provide them and (ii) to compare several applications on the basis of different types of functionalities. Iterating and combining both functionalities can easily lead to selecting an appropriate tool. Data required by SerbGO is either the desired capabilities within a defined Standard Functionalities Set or the list of the tools to be compared. The analysis performed carries out a cross-classification that produces an easily readable output with the list of tools that implement the capabilities demanded or a table with the categorization of the GO tools that one wishes to compare. SerbGO is freely available and does not require a login. It can be accessed either directly at our server (http://estbioinfo.stat.ub.es/apli/serbgo) or at the GO Consortium website (http://www.geneontology.org/GO.tools.microarray.shtml#serbgo).


Evolution of senescence: late survival sacrificed for reproduction.
In so far as it is associated with declining fertility and increasing mortality, senescence is directly detrimental to reproductive success. Natural selection should therefore act in the direction of postponing or eliminating senescence from the life history. The widespread occurrence of senescence is explained by observing that (i) the force of natural selection is generally weaker at late ages than at early ages, and (ii) the acquisition of greater longevity usually involves some cost. Two convergent theories are the 'antagonistic pleiotropy' theory, based in population genetics, and the 'disposable soma' theory, based in physiological ecology. The antagonistic pleiotropy theory proposes that certain alleles that are favoured because of beneficial early effects also have deleterious later effects. The disposable soma theory suggests that because of the competing demands of reproduction less effort is invested in the maintenance of somatic tissues than is necessary for indefinite survival.


Provider cost analysis supports results-based contracting out of maternal and newborn health services: an evidence-based policy perspective.
There is dearth of evidence on provider cost of contracted out services particularly for Maternal and Newborn Health (MNH). The evidence base is weak for policy makers to estimate resources required for scaling up contracting. This paper ascertains provider unit costs and expenditure distribution at contracted out government primary health centers to inform the development of optimal resource envelopes for contracting out MNH services. This is a case study of provider costs of MNH services at two government Rural Health Centers (RHCs) contracted out to a non-governmental organization in Pakistan. It reports on four selected Basic Emergency Obstetrical and Newborn Care (BEmONC) services provided in one RHC and six Comprehensive Emergency Obstetrical and Newborn Care (CEmONC) services in the other. Data were collected using staff interviews and record review to compile resource inputs and service volumes, and analyzed using the CORE Plus tool. Unit costs are based on actual costs of MNH services and are calculated for actual volumes in 2011 and for volumes projected to meet need with optimal resource inputs. The unit costs per service for actual 2011 volumes at the BEmONC RHC were antenatal care (ANC) visit USD$ 18.78, normal delivery US$ 84.61, newborn care US$ 16.86 and a postnatal care (PNC) visit US$ 13.86; and at the CEmONC RHC were ANC visit US$ 45.50, Normal Delivery US$ 148.43, assisted delivery US$ 167.43, C-section US$ 183.34, Newborn Care US$ 41.07, and PNC visit US$ 27.34. The unit costs for the projected volumes needed were lower due to optimal utilization of resources. The percentage distribution of expenditures at both RHCs was largest for salaries of technical staff, followed by salaries of administrative staff, and then operating costs, medicines, medical and diagnostic supplies. The unit costs of MNH services at the two contracted out government rural facilities remain higher than is optimal, primarily due to underutilization. Provider cost analysis using standard treatment guideline (STG) based service costing frameworks should be applied across a number of health facilities to calculate the cost of services and guide development of evidence based resource envelopes and performance based contracting.


Metabotropic glutamate receptor agonist-induced hyperpolarizations in rat basolateral amygdala neurons: receptor characterization and ion channels.
1. Metabotropic glutamate receptor (mGluR)-agonist-induced hyperpolarizations and corresponding outward currents were analyzed in basolateral amygdala (BLA) neurons in rat brain slice preparations with current-clamp and single-electrode voltage-clamp recording to characterize the mGluR subtype(s) and the ion channel(s) mediating this response. 2. The mGluR agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) induced a membrane hyperpolarization or outward current in BLA neurons in a concentration-dependent manner (median effective concentration = 34 microM; range = 10-200 microM); the 1S,3R-ACPD hyperpolarizations are recorded in 89% of neurons that accommodate or cease firing in response to a 400-ms depolarizing current injection (0.5 nA). 3. mGluR agonists elicited hyperpolarizations or outward currents in a concentration-dependent manner in the following rank order of potency: (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) > 1S,3R-ACPD > (s)-4-carboxyphenylglycine = (RS)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) > L-aminophosphonobutyric acid > (1S,3S)-1-amino-cyclopentane-1,3-dicarboxylic acid. In contrast, the mGluR agonists quisqualate and ibotenate induced only depolarizations in the presence of D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione in BLA neurons. 4. The 1S,3R-ACPD-induced outward current is mediated through a large-conductance calcium-dependent potassium (BK) conductance. The BK channel blockers iberiotoxin and charybdotoxin blocked the response, as did the potassium channel blockers tetraethylammonium and 4-aminopyridine; the small-conductance calcium-activated potassium channel blocker apamin did not affect the response. 5. The mGluR-agonist-induced hyperpolarization is blocked in amygdala slices from animals pretreated with pertussis toxin (PTX). 1S,3R-ACPD hyperpolarizations were recorded in neurons contralateral but not ipsilateral to the site of PTX injection. 6. The antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) reduced significantly the 1S,3R-ACPD-induced hyperpolarization. 7. In conclusion, the relative potency of L-CCG-I and 4C3HPG in evoking only hyperpolarizations (outward currents) in accommodating neurons, and the observation that MCPG (500 microM) reduces the hyperpolarization, suggest that a group-II-like mGluR underlies the hyperpolarizing response. The mGluR-induced response is sensitive to iberiotoxin and to pretreatment with PTX, suggesting activation of BK channels through a group II mGluR linked to a PTX-sensitive G protein in BLA neurons.


Filling annotation gaps in yeast genomes using genome-wide contact maps.
De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional genomic features such as genes, non-coding RNAs or regulatory sequences, taking advantage of diverse datasets. These steps sometimes fail at detecting non-coding functional sequences: for example, origins of replication, centromeres and rDNA positions have proven difficult to annotate with high confidence. Here, we demonstrate an unconventional application of Chromosome Conformation Capture (3C) technique, which typically aims at deciphering the average 3D organization of genomes, by showing how functional information about the sequence can be extracted solely from the chromosome contact map. Specifically, we describe a combined experimental and bioinformatic procedure that determines the genomic positions of centromeres and ribosomal DNA clusters in yeasts, including species where classical computational approaches fail. For instance, we determined the centromere positions in Naumovozyma castellii, where these coordinates could not be obtained previously. Although computed centromere positions were characterized by conserved synteny with neighboring species, no consensus sequences could be found, suggesting that centromeric binding proteins or mechanisms have significantly diverged. We also used our approach to refine centromere positions in Kuraishia capsulata and to identify rDNA positions in Debaryomyces hansenii. Our study demonstrates how 3C data can be used to complete the functional annotation of eukaryotic genomes. The source code is provided in the Supplementary Material. This includes a zipped file with the Python code and a contact matrix of Saccharomyces cerevisiae. firstname.lastname@example.com Supplementary data are available at Bioinformatics online.


Horizontal and vertical targeting: a population-based comparison of public eldercare services in urban and rural areas of Sweden.
The concepts of target efficiency can be used to assess the extent to which service provision is in line with the needs of the population. Horizontal target efficiency denotes the extent to which those deemed to need a service receive it and vertical target efficiency is the corresponding extent to which those who receive services actually need them. The aim of this study was to assess the target efficiency of the Swedish eldercare system and to establish whether target efficiencies differ in different geographical areas such as large urban, midsize urban and rural areas. Vertical efficiency was measured by studying those people who received eldercare services and was expressed as a percentage of those who received services who were functionally dependent. To measure horizontal target efficiency, data collected at baseline in the longitudinal population study SNAC (Swedish National study on Aging and Care) during the years 2001-2004 were used. The horizontal efficiency was calculated as the percentage of functionally dependent persons who received services. Functional dependency was measured as having difficulty with instrumental activities of daily living (IADL) and/or personal activities of daily living (PADL). Services included long-term municipal eldercare services (LTC). Horizontal target efficiency for the public LTC system was reasonably high in all three geographical areas, when using dependency in PADL as the measure of need (70-90 %), but efficiency was lower when the less restrictive measure of IADL dependency was used (40-50 %). In both cases, the target efficiency was markedly higher in the large urban and the rural areas than in the midsize urban areas. Vertical target efficiency showed the same pattern-it was almost 100 % in all areas for IADL dependency, but only 50-60 % for PADL dependency. Household composition differed in the areas studied as did the way public long-term care was provided to people living alone as compared to those co-habiting.


Maintaining a wild phenotype in a conservation hatchery program for Chinook salmon: The effect of managed breeding on early male maturation.
In many salmonid species, age and size at maturation is plastic and influenced by the interaction between genetic and environmental factors. Hatchery reared salmon often mature at an earlier age and smaller size than wild fish. Modern salmon conservation efforts have focused on managing the level of gene flow between hatchery and natural origin fish to minimize potential genotypic and phenotypic change. In salmonids, maturation probability is dependent on exceeding a genetically set threshold in growth rate and energetic status (and by association, body size) referred to as the probabalisitic maturation reaction norm (PMRN). Over fourteen years, we monitored the frequency of age-2 precocious male maturation (common term: age-2 minijack rate) and the PMRN of natural founder (FNDR), integrated natural-hatchery (INT), and segregated hatchery (SEG) broodlines of spring Chinook salmon, Oncorhynchus tshawytscha. The average age-2 minijack rate (± SEM) of the FNDR, INT and SEG broodlines was 48.2 ± 5.2%, 41.9 ± 3.6% and 30.9 ± 4.7%, respectively. Additionally, the PMRN WP50 (predicted weight at 50% maturation) of the SEG broodline was significantly greater (20.5 g) than that of the FNDR/INT broodlines (18.2 g). We also conducted a common garden experiment exploring the effects of less than one [INT (0-1)], one [SEG (1)] or two [SEG (2)] generations of hatchery culture on the age-2 minijack rate and PMRN WP50. Growth was not significantly different among broodlines, but age-2 minijack rates were significantly lower following two consecutive generations of hatchery culture: [INT (0-1): 68.3 ± 1.7%], [SEG (1): 70.3 ± 1.8%] and [SEG (2): 58.6 ± 0.4%] and the PMRN WP50 was significantly higher by 6.1 g after two generations of SEG culture. These results indicate that managed gene flow reduces phenotypic divergence, but may serve to maintain potentially undesirably high age-2 minijack rates in salmon conservation hatchery programs.


Surveillance using serological and molecular methods for the detection of infectious agents in captive Brazilian neotropic and exotic felids.
The aim of the current study was to investigate the exposure of captive wild felids to various infectious pathogens using serological and molecular methods. One hundred and fifty-nine neotropic felids and 51 exotic felids from 28 captive settings in Brazil were tested. While antibodies against Feline parvovirus and Feline coronavirus (FCoV), Feline calicivirus and Bartonella spp. were frequently detected by serologic tests, antibodies against Felid herpesvirus 1 or infection with hemotropic mycoplasmas were less prevalent. Serologic evidence of exposure to Ehrlichia spp., Feline immunodeficiency virus, and Feline leukemia virus (FeLV) was detected rarely, and infections with FeLV, Ehrlichia spp., and Cytauxzoon spp. were found infrequently. The detected Bartonella sequence was molecularly similar to B. koehlerae and B. henselae; for Cytauxzoon, the sequence resembled those from domestic cats. No Anaplasma phagocytophilum and Theileria spp. infections were detected. The positive test results varied significantly among different facilities and species. Additionally, FCoV seropositivity was more prevalent in captivity than in free-ranging populations. Results suggest that testing is appropriate prior to relocation of felids.


Regulatable Lentiviral Hematopoietic Stem Cell Gene Therapy in a Mouse Model of Parkinson's Disease.
Glial cell line-derived neurotrophic factor (GDNF) exhibits potent neuroprotective properties in preclinical models of Parkinson's disease (PD), but challenges in GDNF delivery have been reported from clinical trials. To address this barrier, we developed a hematopoietic stem cell transplantation-based macrophage-mediated GDNF therapy platform. Here, we introduced a regulatable lentiviral vector (LV-MSP-Tet-Off-hGDNF) to allow the expression of human GDNF (hGDNF) to be adjusted or stopped by oral administration of doxycycline (Dox). C57BL/6J mice were lethally irradiated with head protection and then transplanted with syngeneic bone marrow cells transduced with either the hGDNF-expressing vector or a corresponding GFP-expressing vector, LV-MSP-Tet-Off-GFP. Suppression of vector gene expression was achieved through administration of Dox in drinking water. To create a toxin-induced Parkinsonian model, mice were injected in two cycles with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to yield nigral cell/striatal dopamine loss and behavioral deficits. During the presence of Dox in the drinking water, plasma GDNF was at a basal level, whereas during the absence of Dox, plasma GDNF was significantly elevated, indicating reliable regulation of therapeutic gene expression. Midbrain GDNF levels were altered in parallel, although these did not return completely to basal levels during the periods of Dox withdrawal. Motor activities of the MPTP-Tet-off-hGDNF group were comparable to those of the Tet-off-GFP (subject to no MPTP treatment) group, but substantially better than those of the MPTP-Tet-off-GFP group. Interestingly, the improvement in motor activities was sustained during the Dox-withdrawn periods in MPTP-Tet-off-hGDNF animals. Neuroprotection by therapeutic GDNF expression was further evidenced by significant amelioration of nigral tyrosine hydroxylase loss after both the first and second MPTP treatment cycles. These data suggest that neurotrophic factor expression can be upregulated to achieve efficacy or downregulated in case of off-target effects or adverse events, a feature that may eventually increase the acceptance of this potentially neuroprotective/disease-modifying PD therapy.


IGF1R signaling in Ewing sarcoma is shaped by clathrin-/caveolin-dependent endocytosis.
Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling.