Carcinogenesis bioassay of trichloroethylene.
Trichloroethylene (TCE), a halogenated chemical, has been tested for carcinogenicity in the National Cancer Institute's Carcinogenesis Bioassay Program. Trichloroethylene has been used primarily as a solvent in industrial degreasing operations. Other uses have been as a solvent in dry cleaning and food processing, as an ingredient in printing inks, paints. etc., and as a general anesthetic or analgesic. Industrial grade (>99% pure) trichloroethylene was tested using 50 animals per group at 2 doses and with both sexes of Osborne-Mendel rats and B6C3F1 mice. Twenty of each sex and species were maintained as matched controls, in addition to colony and positive carcinogen controls. Animals were exposed to the compound by oral gavage 5 times per week for 78 weeks. At the end of treatment, animals were observed until terminal sacrifice at 110 weeks for rats and 90 weeks for mice. A complete necropsy and microscopic evaluation of all animals (except 7 of the original 480) was conducted. Two doses were used with animals started on test at approximately 6 weeks of age. The initial doses used in this test were the estimated maximum tolerated dose (MTD) and 1/2 MTD, as predicted from data obtained in a 6-week toxicity study. For rats, the initial doses were 1,300 and 650 mg/kg body weight. These were changed, based upon survival and body weight data, so that the "time-weighted average" doses were 549 and 1,097 mg/kg for both male and female rats. For mice, the initial doses were 1,000 and 2,000 mg/kg for males and 700 and 1,400 mg/kg for females. The doses were increased so that the "time-weighted average" doses were 1,169 and 2,339 mg/kg for male mice and 869 and 1,739 mg/kg for female mice. Clinical signs of toxicity, including reduction in weight, were evident in treated rats. These, along with an increased mortality rate necessitated a reduction in doses during the test. In contrast, very little evidence of toxicity was seen in mice, so doses were increased slightly during the study. The increased mortality in treated male mice appears related to the presence of liver tumors. A variety of neoplastic lesions were observed in rats with no significant difference between trichloroethylene-treated and control animals. The only lesion that might be attributed to the treatment was a chronic nephropathy found in both sexes and at both dose levels. With both male and female mice, primary malignant tumors of the liver, i.e., hepatocellular carcinoma, were observed in high numbers. For males, 26/50 low dose and 31/48 high dose animals had hepatocellular carcinomas as compared with 1/20 matched controls and 5/77 colony controls. The differences between treated and matched control males at both doses were highly significant (P<0.01). For females, hepatocellular carcinomas were observed in 4/50 low dose and 11/47 high dose animals as compared with 0/20 matched controls and 1/80 colony controls. While the difference between the high dose female mice and matched controls was also highly significant (P<0.01), the difference at the low dose was less (P=0.09). For both male and female mice, age-adjusted tests for linear trend (dose response) were highly significant for hepatocellular carcinoma (P<0.001 for males and P=0.002 for females). In male mice at the high doses, hepatocellular carcinomas were observed early in the study. The first was seen at 27 weeks; 9 others were found in male mice dying by the 78th week. The tumor was not observed so early in low dose male or female mice. The diagnosis of hepatocellular carcinoma was based on size, histologic appearance, and presence of metastasis, especially to the lung. No other lesion was significantly elevated (P<0.05) in treated mice. The incidence of hepatocellular carcinomas in the trichloroethylene-matched controls was typical of that observed in colony controls. Carbon tetrachloride (CCl4) was used as a positive control for the series of chlorinated chemicals which included trichloroethylene. While virtually all male and female mice developed hepatocellular carcinomas following carbon tetrachloride treatment, the response in the Osborne-Mendel rats was considerably less. Only about 5% developed hepatocellular carcinomas. Thus, there appears to be a marked difference in sensitivity to induction of carcinomas by chlorinated compounds between the B6C3F1 mouse and the Osborne-Mendel rat. The results of this carcinogenesis test of trichloroethylene clearly indicate that trichloroethylene induced a hepatocellular carcinoma response in mice. While the absence of a similar effect in rats appears most likely attributable to a difference in sensitivity between the Osborne-Mendel rat and the B6C3F1 mouse, the early mortality of rats due to toxicity must also be considered.


Circadian Control of cab Gene Transcription and mRNA Accumulation in Arabidopsis.
An intriguing property of many organisms is their ability to exhibit rhythmic cellular events that continue independently of environmental stimuli. These rhythmic processes are generated by an endogenous mechanism known as the biological clock. We wished to determine whether Arabidopsis thaliana will serve as a model plant system for a molecular genetic dissection of the circadian clock. To this end, we investigated the expression of Arabidopsis chlorophyll a/b-binding protein (cab) genes throughout the circadian cycle. Steady-state mRNA levels of the cab2 and cab3 genes showed a dramatic circadian cycling in plants shifted from light/dark cycles to constant darkness, whereas the cab1 mRNA level exhibited little or no cycling under the same conditions. Analysis of cab promoter fusions in transgenic tobacco revealed that both the cab1 and cab2 5[prime] upstream regions confer circadian-regulated expression on a chloramphenicol acetyltransferase (cat) reporter gene. In vitro nuclear run-on transcription assays also indicated that the transcription of the cab1 and cab2 genes is circadian regulated in Arabidopsis. Taken together, these data suggest that a post-transcriptional mechanism influences cab1 mRNA levels in Arabidopsis. The identification of circadian-regulated cis-acting elements in the cab1 and cab2 upstream regions will provide powerful tools for both molecular and genetic analysis of the higher plant circadian clock.


Assessing wastewater dilution in small rivers with high resolution conductivity probes.
Installation and maintenance of flow gauging stations to assess wastewater dilution in small creeks is expensive. The method outlined in this paper provides a flexible and cheap alternative for situations with dilution factors smaller than 10 and unambiguous flow direction. It is shown that conductivity profiles from three sampling locations enable accurate identification of the dilution factor with an uncertainty of +/-10-30%. Furthermore, much insight can be gained on the temporal behavior of both, the receiving water and the wastewater treatment system by combined analysis of conductivity and temperature data. In this case study the data also enabled identification and tracking down of illegal polluters.


Effectiveness and Safety of High- vs Low-Dose Swallowed Topical Steroids for Maintenance Treatment of Eosinophilic Esophagitis: A Multicenter Observational Study.
Data evaluating efficacy of different doses of swallowed topical corticosteroids (STC) in the long-term management of eosinophilic esophagitis (EoE) are lacking. We assessed long-term effectiveness and safety of different STC doses for adults with EoE after achievement of histological remission. We performed a retrospective multicenter study at five EoE referral centers (US and Switzerland). We analyzed data on 82 patients with EoE in histological remission and ongoing STC treatment with therapeutic adherence of ≥75% (58 males; mean age at diagnosis, 37.2±14.4 years). Patients were followed for a median of 2.2 years (interquartile range [IQR], 1.0-3.8 years). We collected data from 217 follow-up endoscopy visits. The primary endpoint was time to histological relapse. Histological relapse occurred in 67% of patients. Relapse rates were comparable in patients taking low dose (≤0.5 mg per day, n = 58) and high dose STC (>0.5 mg per day, n = 24) with 72 vs 54% (ns). However, histological relapse occurred significantly earlier with low dose STC (1.0 vs 1.8 years, P = .030). There was no difference regarding rates of and time to stricture formation for low vs high dose STC. Esophageal candidiasis was observed in 6% of patients (5% for low dose, 8% for high dose, ns). No dysplasia or mucosal atrophy was detected. Histological relapse frequently occurs in EoE despite ongoing STC treatment regardless of STC doses. However, relapse develops later in patients on high dose STC without an increase in side-effects. Doses higher than 0.5 mg/day may be considered for EoE maintenance treatment, but advantage over lower doses appears to be small.


Meat quality characteristics of springbok (Antidorcas marsupialis). 3: Fatty acid composition as influenced by age, gender and production region.
The effects of age, gender and production region on the fatty acid composition of springbok Musculus longissimus dorsi (LD) were investigated. The major fatty acid of the LD muscle was stearic acid (C18:0), which contributed 23.92-27.02%. Oleic acid (C18:1) represented the largest component (16.33-20.45%) of the mono-unsaturated fatty acids (MUFA). The major n-6 polyunsaturated fatty acid (PUFA) was C18:2n-6, which formed 18.77-21.62%, whereas C18:3n-3 (3.33-4.00%) was the most abundant n-3 PUFA. The n-6:n-3 ratio of the meat varied from 3.02 to 3.35, with an average ratio of 3.2. Polyunsaturated to saturated (P:S) ratios varied between 0.96 and 1.18 and averaged at 1.06. Total MUFA was found to be higher (P<0.05) in males (20.99%) than females (16.67%). The cholesterol content of the meat varied from 54.45 to 59.34mg/100g muscle. Linear correlations between the fatty acid and the intramuscular fat (IMF) content indicated a significant increase in certain saturated and mono-unsaturated fatty acids with increasing IMF content of the meat.


A systematic overview of the medicinal importance of sanguivorous leeches.
Leeches are a class of segmented invertebrates, known for their blood-feeding habits and used in phlebotomy to treat various ailments since antiquity. In Europe, medicinal leeches have recently been rediscovered and are used by maxillofacial and other microsurgeons to aid salvage of compromised venous engorged tissue and amputations, such as digits, ears, and nasal tips. Because of their important salivary components, blood-sucking (sanguivorous) leeches, such as Hirudo medicinalis and related species, have engendered great interest from pharmaceutical companies searching for anticoagulants to prevent blood clotting during microsurgeries. Scientific research reveals that the beneficial effects of leeching, in addition to decongestion, include injection of a cocktail of several medicinally useful bioactive molecules present in their saliva. Owing to its therapeutic potential, the research is continuing as many new salivary compounds are being isolated and synthesized.


Higher Fluorosis Severity Makes Enamel Less Resistant to Demineralization.
Fluorotic teeth could either be more resistant or more susceptible to the caries process than sound ones due to their higher enamel fluoride concentration and higher porosity (subsurface hypomineralization), respectively; however, there is no consensus on this subject. In this study, a total of 49 human unerupted third molars presenting Thylstrup and Fejerskov (TF) fluorosis scores 0-4 were used. Two enamel slabs were obtained from each tooth. The rest of the tooth crown was powdered, and the enamel was separated from the dentine. In purified powdered enamel, the calcium (Ca), inorganic phosphate (Pi), and fluoride (F) concentrations were determined. The F concentration gradient throughout the enamel and in the enamel volume was determined in one slab. The other enamel slab was isolated with acid-resistant varnish, subjecting the exposed enamel surface half to a pH-cycling model to evaluate its demineralization resistance and to calculate the demineralization area. The nonexposed surface was used to determine the natural hypomineralization area found in fluorotic enamel and normalize the demineralization data. The hypomineralization and demineralization areas were assessed by cross-sectional microhardness. For statistical analyses, the data for TF1 and 2, and for TF3 and 4 were pooled. Concentrations of powered enamel Ca and Pi were not significantly different (p > 0.05) among groups TF0, TF1-2 and TF3-4, but a higher F concentration was found in fluorotic enamel (p < 0.05). Highly fluorotic teeth (TF3-4) presented a greater hypomineralization subsurface area and demonstrated lower demineralization resistance than sound enamel (p < 0.05). The findings suggest that a higher severity of fluorosis makes enamel less resistant to the caries process due to its greater subsurface mineral area exposed to demineralization and deeper acid diffusion through the enamel.


The mitotic kinase Aurora--a promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα(+) breast cancer cells.
In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα(+) breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα(+) breast cancer patients resistant to conventional endocrine therapy.


RNA leaching of transcription factors disrupts transcription in myotonic dystrophy.
Myotonic dystrophy type 1 (DM1) is caused by a CUGn expansion (n approximately 50 to 5000) in the 3' untranslated region of the mRNA of the DM protein kinase gene. We show that mutant RNA binds and sequesters transcription factors (TFs), with up to 90% depletion of selected TFs from active chromatin. Diverse genes are consequently reduced in expression, including the ion transporter CIC-1, which has been implicated in myotonia. When TF specificity protein 1 (Sp1) was overexpressed in DM1-affected cells, low levels of messenger RNA for CIC-1 were restored to normal. Transcription factor leaching from chromatin by mutant RNA provides a potentially unifying pathomechanistic explanation for this disease.


An orbital-overlap model for minimal work functions of cesiated metal surfaces.
We introduce a model for the effect of cesium adsorbates on the work function of transition metal surfaces. The model builds on the classical point-dipole equation by adding exponential terms that characterize the degree of orbital overlap between the 6s states of neighboring cesium adsorbates and its effect on the strength and orientation of electric dipoles along the adsorbate-substrate interface. The new model improves upon earlier models in terms of agreement with the work function-coverage curves obtained via first-principles calculations based on density functional theory. All the cesiated metal surfaces have optimal coverages between 0.6 and 0.8 monolayers, in accordance with experimental data. Of all the cesiated metal surfaces that we have considered, tungsten has the lowest minimum work function, also in accordance with experiments.


Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand.
The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.


Changes in the Use of Fresh-Frozen Plasma Transfusions in Preterm Neonates: A Single Center Experience.
The aim of this study was to evaluate changes in the use of fresh-frozen plasma (FFP) transfusions and the use of clotting tests in preterm neonates in our center over the past two decades. In this retrospective cohort analysis, we included all consecutive neonates with a gestational age at birth between 24 + 0 and 31 + 6 weeks admitted to our neonatal intensive care unit (NICU) between 2004 and 2019. We divided all included neonates into three consecutive time epochs according to date of birth: January 2004 to April 2009, May 2009 to August 2014 and September 2014 to December 2019. The main outcomes were the use of FFP transfusion, coagulation testing and the indications for FFP transfusion. The percentage of preterm neonates receiving FFP transfusion decreased from 5.7% (47/824) to 3.7% (30/901) to 2.0% (17/852) from the first epoch to the last epoch (p < 0.001). Additionally, the rate of neonates undergoing coagulation testing decreased from 24.3% (200/824) to 14.5% (131/901) to 8% (68/852) over the epochs (p < 0.001). Most FFP transfusions were prescribed prophylactically based on prolongation of activated partial thromboplastin time (aPTT) or prothrombin time (PT) (56%). In conclusion, both the use of FFP transfusions and the use of coagulation tests decreased significantly over the years. The majority of the FFP transfusions were administrated prophylactically for abnormal coagulation tests.


Adolescents with intellectual disability have reduced postural balance and muscle performance in trunk and lower limbs compared to peers without intellectual disability.
For adolescent people with ID, falls are more common compared to peers without ID. However, postural balance among this group is not thoroughly investigated. The aim of this study was to compare balance and muscle performance among adolescents aged between 16 and 20 years with a mild to moderate intellectual disability (ID) to age-matched adolescents without ID. A secondary purpose was to investigate the influence of vision, strength, height and Body Mass Index (BMI) on balance. A group of 100 adolescents with ID and a control group of 155 adolescents without ID were investigated with five balance tests and three strength tests: timed up and go test, one leg stance, dynamic one leg stance, modified functional reach test, force platform test, counter movement jump, sit-ups, and Biering-Sørensen trunk extensor endurance test. The results showed that adolescents with an ID in general had significantly lower scores in the balance and muscle performance tests. The group with ID did not have a more visually dominated postural control compared to the group without ID. Height, BMI or muscle performance had no strong correlations with balance performance. It appears as if measures to improve balance and strength are required already at a young age for people with an ID.


Swollen digits due to pachydermodactyly resembling inflammatory arthritis.
Pachydermodactyly is a rare digital fibromatosis, characterized by painless, fusiform swelling in proximal interphalangeal joints. Since it may be misdiagnosed as inflammatory arthritis, it is important to call physicians' attention to this rarely seen condition to avoid unnecessary immunosuppressive treatment. Hereby, we present a case of a pachydermodactyly that had been previously followed-up and treated as an inflammatory arthritis for 4 years. We describe the clinical, radiological, and dermoscopic features of the case.


The assessment of antimalarial drug efficacy in vivo.
Currently recommended methods of assessing the efficacy of uncomplicated falciparum malaria treatment work less well in high-transmission than in low-transmission settings. There is also uncertainty how to assess intermittent preventive therapies and seasonal malaria chemoprevention (SMC), and Plasmodium vivax radical cure. A pharmacometric antimalarial resistance monitoring (PARM) approach is proposed specifically for evaluating slowly eliminated antimalarial drugs in areas of high transmission. In PARM antimalarial drug concentrations at recurrent parasitaemia are measured to identify outliers (i.e., recurrent parasitaemias in the presence of normally suppressive drug concentrations) and to evaluate changes over time. PARM requires characterization of pharmacometric profiles but should be simpler and more sensitive than current molecular genotyping-based methodologies. PARM does not require parasite genotyping and can be applied to the assessment of both prevention and treatment.


Assessment of the genotoxic potential of nitric oxide-induced guanine lesions by in vitro reactions with Escherichia coli DNA polymerase I.
It has been suggested that carcinogenesis associated with chronic inflammation involves DNA damage by nitric oxide (NO) and other reactive species secreted from macrophages and neutrophils. The guanine moiety of DNA reacts with NO, yielding two major deamination products: xanthine (Xan) and oxanine (Oxa). Oxa reacts further with polyamines and DNA binding proteins to form cross-link adducts. In the present study, we characterized the structure of the cross-link adducts of Oxa with spermine (Oxa-Sp). Spectrometric analysis of Oxa-Sp adducts showed that they are ring-opened adducts of Oxa covalently bonded to the terminal amino (major product) and internal imino (minor product) groups of spermine. To assess genotoxic potential, Xan, Oxa, Oxa-Sp and an abasic (AP) site were site specifically incorporated into oligonucleotide templates. These lesions differentially blocked in vitro DNA synthesis catalyzed by DNA polymerase I Klenow fragment (Pol I Kf). The relative efficiency of translesion synthesis was G (1) > Oxa (0.19) > Xan (0.12) > AP (0.088) > Oxa-Sp (0.035). Primer extension assays with a single nucleotide and Pol I Kf revealed that non-mutagenic dCMP was inserted most efficiently opposite Xan and Oxa, with the extent of primer elongation being 65% for Xan and 68% for Oxa. However, mutagenic nucleotides were also inserted. The extent of primer elongation for Xan was 16% with dTMP and 14% with dGMP, whereas that for Oxa was 49% with dTMP. For Oxa-Sp, mutagenic dAMP (13%) was preferentially inserted. Accordingly, when generated in vivo, Xan and Oxa would constitute moderate blocks to DNA synthesis and primarily elicit G:C to A:T transitions when bypassed, whereas Oxa-Sp would strongly block DNA synthesis and elicit G:C to T:A transversions.


Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination.
We have developed and tested a wide-field coherent anti-Stokes Raman scattering (CARS) microscopy technique, which provides the simultaneous imaging of an extended illuminated area without scanning. This method is based on the non-phase-matching illumination of a sample and imaging of a CARS signal with a CCD camera using conventional microscope optics. We have identified a set of conditions on the illumination and imaging optics, as well as on sample preparation. Imaging of test objects proved high spatial resolution and chemical selectivity of this technique.


Studies of Beauty Suppression via Nonprompt D^{0} Mesons in Pb-Pb Collisions at sqrt[s_{NN}]=5.02 TeV.
The transverse momentum spectra of D^{0} mesons from b hadron decays are measured at midrapidity (|y|<1) in pp and Pb-Pb collisions at a nucleon-nucleon center of mass energy of 5.02 TeV with the CMS detector at the LHC. The D^{0} mesons from b hadron decays are distinguished from prompt D^{0} mesons by their decay topologies. In Pb-Pb collisions, the B→D^{0} yield is found to be suppressed in the measured p_{T} range from 2 to 100 GeV/c as compared to pp collisions. The suppression is weaker than that of prompt D^{0} mesons and charged hadrons for p_{T} around 10 GeV/c. While theoretical calculations incorporating partonic energy loss in the quark-gluon plasma can successfully describe the measured B→D^{0} suppression at higher p_{T}, the data show an indication of larger suppression than the model predictions in the range of 2<p_{T}<5 GeV/c.


Screening of Cyanobacterial Peptide Toxin, Microcystins in Hyperscum Water Samples from an Inland Sub Saharan Drinking Freshwater Reservoir.
A study which probed the occurrence and quantitative variations hepatotoxic microcystin in a Sub Saharan drinking freshwater reservoir was carried out between November 2014 and March 2015. Results reveal the presence of MCYST-YR, MCYST-LR, MCYST-RR, MCYST-LA and MCYST-LF variants either in cells collected directly from bloom or toxic isolates cultured under laboratory conditions. Two minor microcystin congeners (MCYST-(H4)YR) and (D-Asp3, Dha7) MCYST-RR) were identified, but not quantified. Variants dominance were in the order MCYST-LR > MCYST-RR > MCYST-YR > MCYST-LA > MCYST-LF across sampling sites. Maximum and minimum concentrations of quantified MCYSTs congeners were (489.25, 50.95 µg toxin/g DW), (98.92, 9.11 µg toxin/g DW), (140.25, 12.07 µg toxin/g DW), (56.99, 6.20 µg toxin/g DW) and (50.46, 3.65 µg toxin/g DW) for MCYST-LR, MCYST-YR, MCYST-RR, MCYST-LA and MCYST-LF, respectively. Analysis of variance (ANOVA) revealed there was a high significant difference between mean microcystin concentrations across sampling sites (p < 0.05).


Non-viraemic transmission of Thogoto virus: vector efficiency of Rhipicephalus appendiculatus and Amblyomma variegatum.
Previous studies have demonstrated that Thogoto virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. Furthermore, tick to tick transmission is potentiated by factors associated with the salivary glands of ticks (saliva activated transmission). The vector efficiency of 2 ixodid tick species, Rhipicephalus appendiculatus and Amblyomma variegatum, for Thogoto virus was assessed using this model. The number of uninfected recipient ticks that acquired Thogoto virus when co-feeding with virus-infected ticks (donors) on uninfected guinea-pigs was determined. When nymphs of either tick species were employed as donors, there was no significant difference in the number of infected recipient nymphs. In contrast, a significant difference in the vector efficiency of adults ticks was observed: 77.0% of recipient ticks which co-fed with R. appendiculatus donor adults acquired Thogoto virus compared to 44.7% of recipient ticks which co-fed with A. variegatum donors. No significant difference in susceptibility to Thogoto virus infection was observed between recipient ticks of the 2 species. Thus, adults of R. appendiculatus are more efficient than A. variegatum in mediating non-viraemic transmission.


Inotropic contractile reserve can risk-stratify patients with HIV cardiomyopathy: a dobutamine stress echocardiography study.
The purpose of this study was to assess whether inotropic contractile reserve (ICR) during dobutamine stress echocardiography (DSE) could risk-stratify patients with human immunodeficiency virus (HIV) cardiomyopathy and predict improvement of left ventricular ejection fraction (LVEF). HIV cardiomyopathy is an important cause of heart failure and death. ICR is associated with better survival and improvement of LVEF in patients with ischemic and nonischemic cardiomyopathies. However, the prognostic value of ICR in patients with HIV cardiomyopathy is unknown. Patients with HIV cardiomyopathy and a LVEF <45% who were referred for DSE were enrolled. ICR was evaluated by the delta wall motion score index (ΔWMSI), calculated as the difference between rest and peak WMSI. Patients were followed for cardiac death and change in LVEF on follow-up. Sixty patients (75% men; age, 54 ± 9 years) with HIV cardiomyopathy (mean LVEF, 28 ± 11%) formed the study group. After 2.4 ± 2.1 years, 11 cardiac deaths occurred (event rate of 7.6%/year). A receiver-operating characteristic curve identified a ΔWMSI of 0.38 as an optimal cut point for the presence of ICR, with a specificity of 88% and a sensitivity of 73% for the prediction of cardiac death. On univariable analysis, the absence of ICR (hazard ratio: 6.6; 95% confidence interval: 1.93 to 22.62; p = 0.003) and New York Heart Association functional class IV (hazard ratio: 7.2; 95% confidence interval: 2.20 to 23.65; p = 0.001) were the only predictors of cardiac death. After 2.1 ± 1.8 years, 41 patients had a follow-up echocardiogram. LVEF improvement from baseline occurred in 23 patients (56%), more so in patients with ICR than without ICR. A ΔWMSI of 0.59 predicted improvement in the LVEF with a specificity of 78% and a sensitivity of 74%. The presence of ICR during DSE can risk-stratify and predict subsequent improvement in LVEF in patients with HIV cardiomyopathy.


Diffractive imaging of a rotational wavepacket in nitrogen molecules with femtosecond megaelectronvolt electron pulses.
Imaging changes in molecular geometries on their natural femtosecond timescale with sub-Angström spatial precision is one of the critical challenges in the chemical sciences, as the nuclear geometry changes determine the molecular reactivity. For photoexcited molecules, the nuclear dynamics determine the photoenergy conversion path and efficiency. Here we report a gas-phase electron diffraction experiment using megaelectronvolt (MeV) electrons, where we captured the rotational wavepacket dynamics of nonadiabatically laser-aligned nitrogen molecules. We achieved a combination of 100 fs root-mean-squared temporal resolution and sub-Angstrom (0.76 Å) spatial resolution that makes it possible to resolve the position of the nuclei within the molecule. In addition, the diffraction patterns reveal the angular distribution of the molecules, which changes from prolate (aligned) to oblate (anti-aligned) in 300 fs. Our results demonstrate a significant and promising step towards making atomically resolved movies of molecular reactions.


The Indigenous Faculty Forum: A Longitudinal Professional Development Program to Promote the Advancement of Indigenous Faculty in Academic Medicine.
American Indian, Alaska Native (AIAN) and Native Hawaiian and other Pacific Islander (NHPI) faculty, are substantially under-represented (<1%) at US medical schools. The Oregon Health & Science University's Northwest Native American Center of Excellence and The University of Hawai'i Native Hawaiian Center of Excellence have created an Indigenous Faculty Forum (IFF), a one-day structured course with flanking social activities, specifically designed to meet the unique needs of AIAN and NHPI academic faculty. It provided: (1) Indigenous space, (2) skill building, (3) networking, and (4) ongoing mentorship, each of which were included to specifically mitigate isolation and tokenism that negatively affects promotion and advancement. Two Forums have been conducted, first in Portland, OR in 2017 and the second in Hilo, Hawai'i in 2018. Nine of eighteen AIAN faculty in the three-state region (CA, OR, WA) attended IFF Session #1, representing 50% of known AIAN faculty in this region. Thirty-four Indigenous faculty from around the world attended IFF Session #2, with twenty-nine completing program evaluations. Respondents were predominantly female (81.6%), under age 44 (52.7%), and either instructors or assistant professors (52.6%). In terms of career choice, both sessions included primary care physicians as the most represented group (55.6% at Session #1 and 62.1% at Session #2). Increasing Indigenous faculty representation in US medical schools, while simultaneously fostering their career advancement and meaning in work, is vitally important. We have begun the work needed to address this problem and look forward to conducting more efforts, including longitudinal evaluation designs to study effectiveness.


Moderate and vigorous exercise programs in children with asthma: safety, parental satisfaction, and asthma outcomes.
Previous research suggests that physical activity programs may improve fitness and reduce symptoms in children with asthma, but few studies have included severe asthmatics and focused on safety and parental satisfaction with the programs. To examine safety, parental satisfaction, and pre- to post-intervention changes in symptoms and quality of life (QOL) in a pilot study of the impact of vigorous physical activity (swimming) and moderate-intensity activity (golf) on inner-city children with asthma. Children with asthma (7-14 years old) residing in Milwaukee's highest asthma prevalence zip codes were randomized to a 9-week swimming or golf program. Pre- and post-intervention data were obtained on safety, parental satisfaction, asthma symptoms, quality of life, and urgent asthma physician visits. Twenty-eight children in the swimming group and 17 in the golf group completed the program. Combined group analysis (N = 45) revealed that only six symptom exacerbations occurred during 1,125 person-sessions of swimming and golf (all resolved with bronchodilator therapy), 92% of parents were very or extremely satisfied with the program, and post-exercise decreases were observed in asthma symptom severity scores (9.3-7.3, P < 0.001), improved parental QOL (4.9-5.4, P < 0.001), and reduced urgent physician visits for asthma (1.3-0.2 visits per person, P = 0.04). The study lacked sufficient power to perform intergroup comparisons. Findings from this pilot study indicate that vigorous (swimming) and moderate-intensity (golf) physical activity programs are well-tolerated, safe, and achieve high parental satisfaction. Participants and parents reported reduced childhood asthma symptoms and physician office visits and improved parental QOL. These findings suggest a potentially beneficial role for moderate to vigorous physical activity in childhood asthma.


The metabolomic signatures of alcohol consumption in young adults.
Metabolomic analysis may help us to understand the association between alcohol consumption and cardio-metabolic health. We aimed to: (i) replicate a previous study of alcohol consumption and metabolic profiles, (ii) examine associations between types of alcoholic beverages and metabolites and (iii) include potential confounders not examined in previous studies. Cross-sectional data of 1785 participants (age 26-36 years, 52% women) from the 2004-2006 Childhood Determinants of Adult Health study were used. Consumption of beer, wine and spirits was assessed by questionnaires. Metabolites were measured by a high-throughput nuclear magnetic resonance platform and multivariable linear regression examined their association with alcohol consumption (combined total and types) adjusted for covariates including socio-demographics, health behaviours and mental health. Alcohol consumption was associated with 23 out of 37 lipids, 12 out of 16 fatty acids and six out of 20 low-molecular-weight metabolites independent of confounders with similar associations for combined total alcohol consumption and different types of alcohol. Many metabolites (lipoprotein lipids in high-density lipoprotein (HDL) subclasses, HDL cholesterol, apolipoprotein A-1, phosphotriglycerides, total fatty acids, monounsaturated fatty acids, omega-3 fatty acids) had positive linear associations with alcohol consumption but some showed negative linear (low-density lipoprotein particle size, omega-6 fatty acids ratio to total fatty acids, citrate) or U-shaped (lipoprotein lipids in very-low-density lipoprotein (VLDL) subclasses, VLDL triglycerides) associations. Our results were similar to those of the only previous study. Associations with metabolites were similar for total and types of alcohol. Alcohol consumption in young adults is related to a diverse range of metabolomic signatures associated with benefits and harms to health.


Facial dysmorphism in Leigh syndrome with SURF-1 mutation and COX deficiency.
Leigh syndrome is an inherited, progressive neurodegenerative disorder of infancy and childhood. Mutations in the nuclear SURF-1 gene are specifically associated with cytochrome C oxidase-deficient Leigh syndrome. This report describes two patients with similar facial features. One of them was a 2(1/2)-year-old male, and the other was a 3-year-old male with a mutation in SURF-1 gene and facial dysmorphism including frontal bossing, brachycephaly, hypertrichosis, lateral displacement of inner canthi, esotropia, maxillary hypoplasia, hypertrophic gums, irregularly placed teeth, upturned nostril, low-set big ears, and retrognathi. The first patient's magnetic resonance imaging at 15 months of age indicated mild symmetric T2 prolongation involving the subthalamic nuclei. His second magnetic resonance imaging at 2 years old revealed a symmetric T2 prolongation involving the subthalamic nuclei, substantia nigra, and medulla lesions. In the second child, at the age of 2 the first magnetic resonance imaging documented heavy brainstem and subthalamic nuclei involvement. A second magnetic resonance imaging, performed when he was 3 years old, revealed diffuse involvement of the substantia nigra and hyperintense lesions of the central tegmental tract in addition to previous lesions. Facial dysmorphism and magnetic resonance imaging findings, observed in these cases, can be specific findings in Leigh syndrome patients with cytochrome C oxidase deficiency. SURF-1 gene mutations must be particularly reviewed in such patients.


Robustness of gut microbiota of healthy adults in response to probiotic intervention revealed by high-throughput pyrosequencing.
Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.


Results of a feasibility randomised controlled trial of osteopathy on neck-shoulder pain in computer users.
Computer use is a well-known source of chronic pain, leading to absenteeism and reduced productivity and well-being. This study evaluated the feasibility of conducting a full-scale randomised controlled trial. Several methodological variables defined trial feasibility. Thirty adults, daily computer users reporting pain, were recruited. Data collection took place at LABIOMEP. Participants were randomised into 1 of 3 parallel groups and received either osteopathic, sham or no treatment. Only the volunteers were blind to group assignments. The primary objective was to study the feasibility and acceptability of the protocol. Of 77 participants interested, 30 were included and randomised into three groups of ten. All participants concluded the study, and all the data was analysed. The feasibility outcomes were deemed appropriate. No adverse events or severe side effects were reported or identified. Studying the efficacy of osteopathic consultation on computer users by conducting an RCT is feasible and safe. With adjustments, a full-scale study can be designed. ClinicalTrials.gov with the identifier: NCT04501575. Date registered August 06, 2020.


Allergic rhinitis and associated comorbidities: prevalence in oman with knowledge gaps in literature.
Allergic rhinitis (AR) is a global health problem and its impact on health related quality of life for patients is substantial, and the economic impact often underestimated. The prevalence of allergic rhinitis in Oman is unknown. This study aimed to estimate the prevalence of AR and associated co-morbidities among adults in Oman. Its secondary objective was to identify knowledge gaps in the literature with the aim of directing future research. A prospective, cross-sectional study of patients who presented to the outpatient otolaryngology clinic at Sultan Qaboos University Hospital with nasal symptoms between June 2010 and June 2011 was conducted. A total of 887 patients were seen with nasal complaints. Among them 127 patients were diagnosed with non-infective rhinitis, the mean age of presentation was 27 years. AR was noted in 48% of patients, and non-allergic rhinitis in 52%. The prevalence of AR was 7%, with females being more affected than males, and age ranging from 18 to 51 years. Prevalence of perennial AR was 84% compared to seasonal AR which was 16%. The most common perennial antigens were house dust mites (80%) followed by cockroaches (67%). All patients diagnosed with seasonal AR were found to be sensitive to Russian thistle. The prevalence of chronic rhinosinusitis in patients with AR was 34%. The prevalence of AR in the adult population presenting with nasal symptoms was found to be 7%, with associated chronic rhinosinusitis present in a third of these patients. However, there appears to be substantial knowledge gaps regarding the association of other comorbidities, like otitis media, bronchitis and bronchial asthma, the long-term outcomes of medical management, and indication of surgical intervention in patients with AR. Future research in AR among Omani patients should aim to address these issues.


Testing evolutionary models of senescence in a natural population: age and inbreeding effects on fitness components in song sparrows.
Mutation accumulation (MA) and antagonistic pleiotropy (AP) have each been hypothesized to explain the evolution of 'senescence' or deteriorating fitness in old age. These hypotheses make contrasting predictions concerning age dependence in inbreeding depression in traits that show senescence. Inbreeding depression is predicted to increase with age under MA but not under AP, suggesting one empirical means by which the two can be distinguished. We use pedigree and life-history data from free-living song sparrows (Melospiza melodia) to test for additive and interactive effects of age and individual inbreeding coefficient (f) on fitness components, and thereby assess the evidence for MA. Annual reproductive success (ARS) and survival (and therefore reproductive value) declined in old age in both sexes, indicating senescence in this short-lived bird. ARS declined with f in both sexes and survival declined with f in males, indicating inbreeding depression in fitness. We observed a significant agexf interaction for male ARS (reflecting increased inbreeding depression as males aged), but not for female ARS or survival in either sex. These analyses therefore provide mixed support for MA. We discuss the strengths and limitations of such analyses and therefore the value of natural pedigreed populations in testing evolutionary models of senescence.


Investigating outbreaks of initially unknown aetiology in complex settings: findings and recommendations from 10 case studies.
Outbreaks of unknown aetiology in complex settings pose challenges and there is little information about investigation methods. We reviewed investigations into such outbreaks to identify methods favouring or impeding identification of the cause. We used two approaches: reviewing scientific literature and soliciting key informants. Case studies were developed through interviews with people involved and triangulated with documents available from the time of the investigation. Ten outbreaks in African or Asian countries within the period 2007-2017 were selected. The cause was identified in seven, of which two had an unclear mode of transmission, and in three, neither origin nor transmission mode was identified. Four events were caused by infectious agents and three by chemical poisoning. Despite differences in the outbreaks, similar obstacles were noted: incomplete or delayed description of patients, comorbidities confounding clinical pictures and case definitions wrongly attributed. Repeated rounds of data collection and laboratory investigations were common and there was limited capacity to ship samples. It was not possible to define activities that led to prompt identification of the cause in the case studies selected. Based on the observations, we conclude that basing case definitions on precise medical observations, implementing initial comprehensive data collection, including environmental, social and behavioural information; and involving local informants could save precious time and hasten implementation of control measures.


Crystal or jelly? Effect of color on the perception of translucent materials with photographs of real-world objects.
Translucent materials are ubiquitous in nature (e.g. teeth, food, and wax), but our understanding of translucency perception is limited. Previous work in translucency perception has mainly used monochromatic rendered images as stimuli, which are restricted by their diversity and realism. Here, we measure translucency perception with photographs of real-world objects. Specifically, we use three behavior tasks: binary classification of "translucent" versus "opaque," semantic attribute rating of perceptual qualities (see-throughness, glossiness, softness, glow, and density), and material categorization. Two different groups of observers finish the three tasks with color or grayscale images. We find that observers' agreements depend on the physical material properties of the objects such that translucent materials generate more interobserver disagreements. Further, there are more disagreements among observers in the grayscale condition in comparison to that in the color condition. We also discover that converting images to grayscale substantially affects the distributions of attribute ratings for some images. Furthermore, ratings of see-throughness, glossiness, and glow could predict individual observers' binary classification of images in both grayscale and color conditions. Last, converting images to grayscale alters the perceived material categories for some images such that observers tend to misjudge images of food as non-food and vice versa. Our result demonstrates that color is informative about material property estimation and recognition. Meanwhile, our analysis shows that mid-level semantic estimation of material attributes might be closely related to high-level material recognition. We also discuss individual differences in our results and highlight the importance of such consideration in material perception.


Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation.
In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.


Adsorption of methylene blue onto silylated silica surfaces, studied using visible attenuated total reflection spectroscopy with a slab optical waveguide.
The behavior of methylene blue (MB) molecules at silica/water and silylated silica/water interfaces was examined using visible attenuated total reflection spectroscopy with a slab optical waveguide (SOWG). An alkyl silane coating changed the adsorbed form of MB on the surface from a dimer (lambda max = 600 nm, bare silica surface) to a monomer (lambda max = 670 nm), and the carbon number of the silylation reagent influenced the amount of adsorption and the orientation of the molecule. Moreover, the addition of an anionic surfactant, dodecylbenzenesulfonate (DBS), caused the deposition of MB/DBS ion pairs, which gave an identical attenuated total reflection (ATR) spectrum to that of the dimer. Linear dichroism measurements revealed that the ion pairs were adsorbed onto the silylated silica surface randomly in terms of the orientation angle of MB, while the MB monomer was strongly oriented, i.e., the direction of the transition moment of MB roughly parallels the surface plane. This difference in the orientation angles of the adsorbed species can be utilized for their selective detection using polarization SOWG measurements.


A resonant chain of four transiting, sub-Neptune planets.
Surveys have revealed many multi-planet systems containing super-Earths and Neptunes in orbits of a few days to a few months. There is debate whether in situ assembly or inward migration is the dominant mechanism of the formation of such planetary systems. Simulations suggest that migration creates tightly packed systems with planets whose orbital periods may be expressed as ratios of small integers (resonances), often in a many-planet series (chain). In the hundreds of multi-planet systems of sub-Neptunes, more planet pairs are observed near resonances than would generally be expected, but no individual system has hitherto been identified that must have been formed by migration. Proximity to resonance enables the detection of planets perturbing each other. Here we report transit timing variations of the four planets in the Kepler-223 system, model these variations as resonant-angle librations, and compute the long-term stability of the resonant chain. The architecture of Kepler-223 is too finely tuned to have been formed by scattering, and our numerical simulations demonstrate that its properties are natural outcomes of the migration hypothesis. Similar systems could be destabilized by any of several mechanisms, contributing to the observed orbital-period distribution, where many planets are not in resonances. Planetesimal interactions in particular are thought to be responsible for establishing the current orbits of the four giant planets in the Solar System by disrupting a theoretical initial resonant chain similar to that observed in Kepler-223.


The sensitivity of endpoint forces produced by the extrinsic muscles of the thumb to posture.
This study utilizes a biomechanical model of the thumb to estimate the force produced at the thumb-tip by each of the four extrinsic muscles. We used the principle of virtual work to relate joint torques produced by a given muscle force to the resulting endpoint force and compared the results to two separate cadaveric studies. When we calculated thumb-tip forces using the muscle forces and thumb postures described in the experimental studies, we observed large errors. When relatively small deviations from experimentally reported thumb joint angles were allowed, errors in force direction decreased substantially. For example, when thumb posture was constrained to fall within +/-15 degrees of reported joint angles, simulated force directions fell within experimental variability in the proximal-palmar plane for all four muscles. Increasing the solution space from +/-1 degrees to an unbounded space produced a sigmoidal decrease in error in force direction. Changes in thumb posture remained consistent with a lateral pinch posture, and were generally consistent with each muscle's function. Altering thumb posture alters both the components of the Jacobian and muscle moment arms in a nonlinear fashion, yielding a nonlinear change in thumb-tip force relative to muscle force. These results explain experimental data that suggest endpoint force is a nonlinear function of muscle force for the thumb, support the continued use of methods that implement linear transformations between muscle force and thumb-tip force for a specific posture, and suggest the feasibility of accurate prediction of lateral pinch force in situations where joint angles can be measured accurately.


Marking and quantifying IL-17A-producing cells in vivo.
Interleukin (IL)-17A plays an important role in host defense against a variety of pathogens and may also contribute to the pathogenesis of autoimmune diseases. However, precise identification and quantification of the cells that produce this cytokine in vivo have not been performed. We generated novel IL-17A reporter mice to investigate expression of IL-17A during Klebsiella pneumoniae infection and during experimental autoimmune encephalomyelitis, conditions previously demonstrated to potently induce IL-17A production. In both settings, the majority of IL-17A was produced by non-CD4(+) T cells, particularly γδ T cells, but also invariant NKT cells and other CD4(-)CD3ε(+) cells. As measured in dual-reporter mice, IFN-γ-producing Th1 cells greatly outnumbered IL-17A-producing Th17 cells throughout both challenges. Production of IL-17A by cells from unchallenged mice or by non-T cells under any condition was not evident. Administration of IL-1β and/or IL-23 elicited rapid production of IL-17A by γδ T cells, invariant NKT cells and other CD4(-)CD3ε(+) cells in vivo, demonstrating that these cells are poised for rapid cytokine production and likely comprise the major sources of this cytokine during acute immunologic challenges.


Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates.
Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.


Seroadaptive Strategies of Gay & Bisexual Men (GBM) with the Highest Quartile Number of Sexual Partners in Vancouver, Canada.
Despite continued research among men with more sexual partners, little information exists on their seroadaptive behavior. Therefore, we examined seroadaptive anal sex strategies among 719 Vancouver gay and bisexual men (GBM) recruited using respondent-driven sampling. We provide descriptive, bivariable, and multivariable adjusted statistics, stratified by HIV status, for the covariates of having ≥7 male anal sex partners in the past 6 months (Population fourth quartile versus <7). Sensitivity Analysis were also performed to assess the robustness of this cut-off. Results suggest that GBM with more sexual partners are more likely to employ seroadaptive strategies than men with fewer partners. These strategies may be used in hopes of offsetting risk, assessing needs for subsequent HIV testing, and balancing personal health with sexual intimacy. Further research is needed to determine the efficacy of these strategies, assess how GBM perceive their efficacy, and understand the social and health impacts of their widespread uptake.


Influence of astigmatism on the fabrication of diffractive structures by use of focused ion-beam milling.
Astigmatism exists in a focused-ion-beam (FIB) system and causes the shape of a beam spot to change from a normal circle to an ellipse. This variation influences the fabrication of diffractive structures ?by use of programmable controlled milling of a FIB. It is analyzed combined with the fabrication of blazed gratings and Fresnel diffractive lenses. Fabrication errors caused by a beam spot with astigmatism is discussed in detail for four cases of the long axis of an ellipse (a) in accordance with the X axis, (b) in accordance with the Y axis, (c) at 45 masculine with the X axis, and (d) at -45 masculine with the X axis. Finally, a method is given for correction of the astigmatism and how to determine the circularity of the beam spot qualitatively.


Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad antibody responses in humans, a RAYS-based analysis.
Antibody responses to tumor antigens play an important role in initiating a cellular antitumor response with respect to antigen cross-presentation and T cell cross-priming. Successful vaccination strategies rely on an optimally timed activation of the humoral and cellular immune system by using appropriate adjuvant stimulation. The LUD99-008 trial used the cancer testis antigen NY-ESO-1 formulated with ISCOMATRIX adjuvant injected into patients intramuscularly. It was shown that this vaccination strategy is a safe and highly potent activator of the humoral and cellular immune system. Using the RAYS technology, we analyzed in detail the humoral immune response in these patients before and after vaccination: during the course of repeated vaccinations with the adjuvant, antibody titers against NY-ESO-1 and cross-reactivity to LAGE 1A and B increased as an indicator of an enhanced immune response, whereas no antibody response could be detected after vaccination without the adjuvant. Analysis of single fragments of the NY-ESO-1 protein revealed that the humoral response was almost exclusively directed against the N-terminal fragments and the number of fragments and their length being recognized by the NY-ESO-1-specific antibodies increased during the course of vaccination. The humoral immune response mainly consisted of antibodies of the IgG1 and IgG3 subclass. We rarely found IgG2 and IgG4 subclass antibodies. Our results support the implication that target-specific antibodies raised after vaccination contribute to the stimulation of an effective T cell response against the target antigen.


Short and long-term effectiveness of couple counselling: a study protocol.
Healthy couple relationships are fundamental to a healthy society, whereas relationship breakdown and discord are linked to a wide range of negative health and wellbeing outcomes. Two types of relationship services (couple counselling and relationship education) have demonstrated efficacy in many controlled studies but evidence of the effectiveness of community-based relationship services has lagged behind. This study protocol describes an effectiveness evaluation of the two types of community-based relationship services. The aims of the Evaluation of Couple Counselling study are to: map the profiles of clients seeking agency-based couple counselling and relationship enhancement programs in terms of socio-demographic, relationship, health, and health service use indicators; to determine 3 and 12-month outcomes for relationship satisfaction, commitment, and depression; and determine relative contributions of client and therapy factors to outcomes. A quasi-experimental pre-post-post evaluation design is used to assess outcomes for couples presenting for the two types of community-based relationship services. The longitudinal design involves a pre-treatment survey and two follow-up surveys at 3- and 12-months post-intervention. The study is set in eight Relationships Australia Victoria centres, across metropolitan, outer suburbs, and regional/rural sites. Relationships Australia, a non-government organisation, is the largest provider of couple counselling and relationship services in Australia. The key outcomes are couple satisfaction, relationship commitment, and depression measured by the CESD-10. Multi-level modelling will be used to account for the dyadic nature of couple data. The study protocol describes the first large scale investigation of the effectiveness of two types of relationship services to be conducted in Australia. Its significance lies in providing more detailed profiles of couples who seek relationship services, in evaluating both 3 and 12-month relationship and health outcomes, and in determining factors that best predict improvements. It builds on prior research by using a naturalistic sample, an effectiveness research design, a more robust measure of relationship satisfaction, robust health indicators, a 12-month follow-up period, and a more rigorous statistical procedure suitable for dyadic data. Findings will provide a more precise description of those seeking relationship services and factors associated with improved relationship and health outcomes.


A tetrad of bicuspid aortic valve association: A single-stage repair.
We report a 27 years old male who presented with a combination of both congenital and acquired cardiac defects. This syndrome complex includes congenital bicuspid aortic valve, Seller's grade II aortic regurgitation, juxta- subclavian coarctation, stenosis of ostium of left subclavian artery and ruptured sinus of Valsalva aneurysm without any evidence of infective endocarditis. This type of constellation is extremely rare. Neither coarctation of aorta with left subclavian artery stenosis nor the rupture of sinus Valsalva had a favorable pathology for percutaneus intervention. Taking account into morbidity associated with repeated surgery and anesthesia patient underwent a single stage surgical repair of both the defects by two surgical incisions. The approaches include median sternotomy for rupture of sinus of Valsalva and lateral thoracotomy for coarctation with left subclavian artery stenosis. The surgery was uneventful. After three months follow up echocardiography showed mild residual gradient across the repaired coarctation segment, mild aortic regurgitation and no residual left to right shunt. This patient is under follow up. This is an extremely rare case of single stage successful repair of coarctation and rupture of sinus of Valsalva associated with congenital bicuspid aortic valve.


'Mainland-island' population structure of a terrestrial salamander in a forest-bocage landscape with little evidence for in situ ecological speciation.
Adaptation to different ecological environments can, through divergent selection, generate phenotypic and genetic differences between populations, and eventually give rise to new species. The fire salamander (Salamandra salamandra) has been proposed to represent an early stage of ecological speciation, driven by differential habitat adaptation through the deposition and development of larvae in streams versus ponds in the Kottenforst near Bonn (Germany). We set out to test this hypothesis of ecological speciation in an area different from the one where it was raised and we took the opportunity to explore for drivers of genetic differentiation at a landscape scale. A survey over 640 localities demonstrated the species' presence in ponds and streams across forests, hilly terrain and areas with hedgerows ('bocage'). Genetic variation at 14 microsatellite loci across 41 localities in and around two small deciduous forests showed that salamander effective population sizes were higher in forests than in the bocage, with panmixia in the forests (Fst < 0.010) versus genetic drift or founder effects in several of the small and more or less isolated bocage populations (Fst > 0.025). The system fits the 'mainland-island' metapopulation model rather than indicating adaptive genetic divergence in pond versus stream larval habitats. A reanalysis of the Kottenforst data indicated that microsatellite genetic variation fitted a geographical rather than an environmental axis, with a sharp transition from a western pond-breeding to an eastern, more frequently stream-breeding group of populations. A parallel changeover in mitochondrial DNA exists but remains to be well documented. The data support the existence of a hybrid zone following secondary contact of differentiated lineages, more so than speciation in situ.


The stability of antimycobacterial drugs in media used for drug susceptibility testing.
The emergence of drug-resistant tuberculosis and disease caused by nontuberculous mycobacteria has increased the need for accurate drug susceptibility testing of mycobacteria. The stability of the tested drugs in relevant test media have been understudied. We assessed the stability of isoniazid, rifampicin, clarithromycin, linezolid and amikacin in Middlebrook 7H9 medium and that of clarithromycin, amikacin and cefoxitin in the cation-adjusted Mueller Hinton broth. We used ultra-performance liquid chromatography (UPLC) methods for rifampicin and isoniazid and a microbiological assay for rifampicin, clarithromycin, amikacin, cefoxitin and linezolid. Rifampicin and isoniazid concentrations in Middlebrook 7H9 medium had decreased by 92% and 54% after 7 days. The microbiological assay revealed decreases in drug concentration of ≥75% (rifampicin, clarithromycin, cefoxitin) and 60% (linezolid) after 14 days. With the exception of amikacin, all antimycobacterial drugs were unstable during 14 days of incubation in the preferred media for DST. Drug stability may influence minimum inhibitory concentration measurements.


Low hygiene and exposure to infections may be associated with increased risk for ulcerative colitis in a North Indian population.
Previous studies have attempted to link hygiene hypothesis with IBD. However most of these studies come from developed countries where the level of hygiene is high and universal. Very little data is available from developing countries. The present study explores the truth of hygiene hypotheses and other risk factors for ulcerative colitis (UC) in a North Indian population where the prevalence of UC has been increasing. A total of 518 patients diagnosed with UC and 188 age-matched controls were included in the study. A structured questionnaire concerning socio-demographics and level of hygiene was completed by all participants. Logistic regression analysis was used to study the association between hygiene-related factors and the risk for UC. Odds ratios and 95% confidence intervals were estimated. There was a higher proportion of females (P<0.001), and a higher educational status (P=0.01) in UC patients compared with controls. A family history of IBD was present in 7.2% of cases and non-existent in controls. On multivariate analysis, after accounting for potential confounders, having a private bed (P<0.001), and having better toilet facilities [(RCA versus none, P=0.01; Flush toilet versus none, P=0.01), (RCA LATRINE as a toilet technology used in rural areas where no flush facility exists. It was developed under RCA project)] were inversely associated with risk for UC whereas owning a pet (P=0.01) and stressful events like a death in the family (P=0.01) were associated with greater risks for UC. Our study does not provide definitive evidence to support hygiene hypothesis and rather suggests that the rising incidence of UC in North India may be attributable to inadequate sanitary measures or other as yet unidentified factors.


Bright monomeric red fluorescent protein with an extended fluorescence lifetime.
Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.


Combination of a proteomics approach and reengineering of meso scale network models for prediction of mode-of-action for tyrosine kinase inhibitors.
In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.


The Streptococcus agalactiae cell wall-anchored protein PbsP mediates adhesion to and invasion of epithelial cells by exploiting the host vitronectin/αv integrin axis.
Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin αv subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface and host integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS.


Molecular survey and microscopic examination of Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) in lacertid lizards from the western Mediterranean.
The genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) is composed of intracellular haemogregarine parasites that are widely distributed among all tetrapod groups. The present study combines microscopic and molecular data on haemogregarine parasites from lizards in the western Mediterranean. We screened tissue samples and examined blood smears for the presence of species of Hepatozoon from four lizards, namely Algyroides marchi Valverde, endemic to Southeast Spain, Podarcis bocagei Seoane from Spain and Portugal, P hispanica Steindachner from Spain, and P lilfordi Günther from Cabrera, Balearic Islands (Spain). Our results show that prevalence and intensity of Hepatozoon parasites vary between and within lizard species from different regions. Algyroides marchi and P bocagei from Spain had the lowest values, whereas P hispanica had the highest. Phylogeny based on 18S rRNA gene sequences indicates that most of the new Hepatozoon sequences are part of a clade exclusive from North African and Iberian lizards, except for a single P bocagei isolate that is found related to another clade including isolates from other reptile host species and rodents. Interestingly, isolates from Algyroides form a distinct monophyletic subgroup, which could be a signal of strict host-specificity within this host genus.


Assessment of need for limiting family after two children: A cross-sectional study from a Northern State of India.
Success of a programme of free-choice participation, like Family Planning Programme, can be envisaged only when focus is on the needs of the people. Although India was the first country to introduce Family Planning Programme, total fertility rate of 2.1 has not yet been achieved, highlighting that the need of limiting family after two children is not yet felt by the people of India. Therefore, the study was undertaken to assess need for limiting family and permanent methods, and factors influencing it among couples with two or more living children. A community-based cross-sectional study was conducted in rural and urban areas of Haldwani Tehsil, Uttarakhand. Multistage sampling technique was used for selection of study sample. Data were collected using a pretested questionnaire. Statistical analysis was done using Epi Info version 184.108.40.206. Out of 221 women interviewed, only one-third had adopted permanent methods of contraception for limiting family, one-third were using temporary methods, and remaining were not using any contraception. Merely one-third participants were willing to adopt permanent contraception in future. Unmet need for permanent methods was higher among women of younger age, literate woman, and/or husband and joint families. It was lower in women with three or more living children in comparison with women with two children. Unfelt need for limiting the family was 8.6%. Acceptance of permanent methods of contraception was low with temporary methods being equally preferred for limiting family. Unfelt need for limiting still remains in the community despite promotion of small family norm.


New multilocus sequence typing of MRSA in São Paulo, Brazil.
An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE) profile and molecular sequence typing (MLST) genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5), showing a new yqiL allele gene, resulting in a new sequence typing (ST) (1176). Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.


Overexpression of phospho-Akt correlates with phosphorylation of EGF receptor, FKHR and BAD in nasopharyngeal carcinoma.
The Akt pathway is one of the most common molecular alterations in various human malignancies. However, its involvement in nasopharyngeal carcinoma (NPC) tumorigenesis has not been well established. In this study, the status of Akt activation and expression of its upstream and downstream molecules was investigated in 64 NPC and 38 non-malignant nasopharyngeal tissues by immunohistochemistry. The hotspot mutations of PIK3CA, encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), were also determined in 25 of these NPC tissues. No hotspot mutations were found in any of the samples tested. Akt was activated in 27 (42.2%) and 23 (35.9%) NPCs, as indicated by p-Akt (Thr308) and p-Akt (Ser473) immunoreactivity, respectively. PTEN loss did not correlate statistically with activated Akt. However, a positive correlation was observed between activated Akt and phospho-epidermal growth factor receptor (p-EGFR), suggesting that the EGFR signaling might be one of the upstream regulators of the Akt pathway. The phosphorylation of forkhead (FKHR) and Bcl-2 associated death domain (BAD), but not mammalian target of rapamycin and glycogen synthase kinase-3beta, was significantly correlated with Akt activation. This implies that Akt promotes cell proliferation (as estimated by Ki-67) and survival, at least, through the inactivation of FKHR and BAD in NPC. Our data revealed that the EGFR/PI3K/Akt signaling pathway is important in NPC pathogenesis and that PIK3CA hotspot mutations are rare in NPC.


Flexible and stretchable thin-film transistors based on molybdenum disulphide.
The outstanding physical and chemical properties of two-dimensional materials, which include graphene and transition metal dichalcogenides, have allowed significant applications in next generation electronics. In particular, atomically thin molybdenum disulphide (MoS2) is attracting widespread attention because of its large bandgap, effective carrier mobility, and mechanical strength. In addition, recent developments in large-area high-quality sample preparation methods via chemical vapour deposition have enabled the use of MoS2 in novel functional applications, such as flexible and stretchable electronic devices. In this perspective, we focus on the current progress in generating MoS2-based flexible and stretchable thin-film transistors. The reported virtues and novelties of MoS2 provide significant advantages for future flexible and stretchable electronics.


Nurse anaesthetists' experiences with pre-operative anxiety.
To explore nurse anaesthetists' experiences and actions when administering and caring for children requiring anaesthesia. A qualitative design employing critical incident technique was used. Interviews were carried out with a purposeful sample of nurse anaesthetists (n=32). The nurse anaesthetists' experiences were grouped into two main areas: organisation focused and interrelational focused. Actions were grouped into two main areas: optimising the situation and creating interpersonal interaction. The categories and subcategories of the nurses' experiences appeared to influence the outcome for the child. The nurse anaesthetists' first priority was to create an optimal environment and increase sensitivity in their interactions with the child. Sensitivity to the child and flexibility in altering actions are key strategies to avoid physical restraint.


Effects of prostaglandin E2 (PGE2) and drugs affecting PGE2 degradation on acute rejection of rat cardiac allografts.
Systemic administration of prostaglandin E2 (PgE2) has been reported to prolong graft survival of heart transplants. We investigated the influence of systemic injection of two compounds which inhibit the endogenous degradation of PgE2 (CL42A and CL68A) and of local infusion of PgE2 into the transplant on the survival time of rat cardiac allografts. Both CL42A and CL68A gave increased graft survival time in two rat strain combinations, though this was not predictable in individual rats. Locally infused PgE2 gave slight, but not significant prolongation of graft survival in some recipients. Combined PgE2 and cyclosporin A, however, gave significant prolongation of graft survival time compared with cyclosporin A treatment alone. When local PgE2 treatment was begun 5 days after transplantation, graft survival time was prolonged in almost all the rats. Manipulation of the local PgE2 concentration thus seemed to have a positive effect on graft survival, possibly due to down-regulation of certain cells of the immune system by PgE2.


Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase.
R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O2, but prefers manganese in the presence of O2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.


Different Exosomal microRNA Profile in Aquaporin-4 Antibody Positive Neuromyelitis Optica Spectrum Disorders.
Neuromyelitis optica spectrum disorders (NMOSD) and multiple sclerosis (MS) are inflammatory demyelinating diseases of the central nervous system. Exosomal microRNAs (miRNAs) are emerging biomarkers for demyelinating diseases. In this study, 52 aquaporin-4 antibody serum-positive NMOSD patients, 18 relapsing-remitting multiple sclerosis (RRMS) patients and 17 healthy controls (HCs) were included for the next-generation sequencing (NGS). To validate the NGS results, the valuable miRNAs were selected for validation by real-time quantitative polymerase chain reaction in another cohort of patients, comprising 31 NMOSD patients and 14 HCs. In addition, these miRNAs were also validated in a longitudinal study. NGS data revealed the exosomal miRNAs profile in NMOSD patients was different from HCs. Among those potential exosomal miRNAs which can distinguish NMOSD status, hsa-miR-122-3p and hsa-miR-200a-5p were the most abundant miRNAs. In addition, hsa-miR-122-3p and hsa-miR-200a-5p were significantly upregulated in the serum exosome of relapsing NMOSD compared with that in remitting NMOSD. Hsa-miR-122-3p and hsa-miR-200a-5p had positive correlations with disease severity in NMOSD patients. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the MAPK, Wnt and Ras signaling pathways were enriched. Further biological function analysis demonstrated that these two miRNAs might be involved in the immunoregulation of NMOSD pathogenesis. Our results indicated that miRNAs delivered by exosomes could be applied as potential biomarkers for NMOSD.


The expression patterns and correlations of claudin-6, methy-CpG binding protein 2, DNA methyltransferase 1, histone deacetylase 1, acetyl-histone H3 and acetyl-histone H4 and their clinicopathological significance in breast invasive ductal carcinomas.
Claudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferase 1 (DNMT1)) and histone modification associated proteins (histone deacetylase 1 (HDAC1), acetyl-histone H3 (H3Ac) and acetyl- histone H4 (H4Ac)). We have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry. Claudin-6 protein expression was reduced in breast invasive ductal carcinomas (P < 0.001). In contrast, expression of MeCP2 (P < 0.001), DNMT1 (P = 0.001), HDAC1 (P < 0.001) and H3Ac (P = 0.004) expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021). Increased expression of HDAC1 was correlated with histological grade (P < 0.001), age (P = 0.004), clinical stage (P = 0.007) and lymph node metastasis (P = 0.001). H3Ac expression was associated with tumor size (P = 0.044) and clinical stage of cancers (P = 0.034). MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P > 0.05). We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression. Our results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1) or histone modification associated proteins (HDAC1, H3Ac, H4Ac). Interestingly, the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac protein expression was positively correlated with the expression of H4Ac in breast invasive ductal carcinoma The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452.


cGMP-dependent protein kinase I is crucial for angiogenesis and postnatal vasculogenesis.
Endothelium-derived nitric oxide plays an important role for the bone marrow microenvironment. Since several important effects of nitric oxide are mediated by cGMP-dependent pathways, we investigated the role of the cGMP downstream effector cGMP-dependent protein kinase I (cGKI) on postnatal neovascularization. In a disc neovascularization model, cGKI(-/-) mice showed an impaired neovascularization as compared to their wild-type (WT) littermates. Infusion of WT, but not cGKI(-/-) bone marrow progenitors rescued the impaired ingrowth of new vessels in cGKI-deficient mice. Bone marrow progenitors from cGKI(-/-) mice showed reduced proliferation and survival rates. In addition, we used cGKIalpha leucine zipper mutant (LZM) mice as model for cGKI deficiency. LZM mice harbor a mutation in the cGKIalpha leucine zipper that prevents interaction with downstream signaling molecules. Consistently, LZM mice exhibited reduced numbers of vasculogenic progenitors and impaired neovascularization following hindlimb ischemia compared to WT mice. Our findings demonstrate that the cGMP-cGKI pathway is critical for postnatal neovascularization and establish a new role for cGKI in vasculogenesis, which is mediated by bone marrow-derived progenitors.


Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis.
Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine- or cytosine deamination in DNA by its ability to release thymine or uracil from G.T and G.U mismatches. Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions. Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG. Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal. Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity. These results establish that the structure function model postulated for the E. coli enzyme is largely applicable to the human TDG. We further provide evidence for G.U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.


The 'caring experience': Testing the psychometric properties of the Caring Efficacy Scale.
The purpose of the study was to undertake rigorous psychometric testing of the Caring Efficacy Scale in a sample of Registered Nurses. A cross-sectional survey of 2000 registered nurses was undertaken. The Caring Efficacy Scale was utilized to inform the psychometric properties of the selected items of the Caring Efficacy Scale. Cronbach's Alpha identified reliability of the data. Exploratory Factor Analysis and Confirmatory Factor Analysis were undertaken to validate the factors. Confirmatory factor analysis confirmed the development of two factors; Confidence to Care and Doubts and Concerns. The Caring Efficacy Scale has undergone rigorous psychometric testing, affording evidence of internal consistency and goodness-of-fit indices within satisfactory ranges. The Caring Efficacy Scale is valid for use in an Australian population of registered nurses. The scale can be used as a subscale or total score reflective of self-efficacy in nursing. This scale may assist nursing educators to predict levels of caring efficacy.


Nerve signaling regulates basal keratinocyte proliferation in the blastema apical epithelial cap in the axolotl (Ambystoma mexicanum).
The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration.


A prospective study of shoulder pain in primary care: prevalence of imaged pathology and response to guided diagnostic blocks.
The prevalence of imaged pathology in primary care has received little attention and the relevance of identified pathology to symptoms remains unclear. This paper reports the prevalence of imaged pathology and the association between pathology and response to diagnostic blocks into the subacromial bursa (SAB), acromioclavicular joint (ACJ) and glenohumeral joint (GHJ). Consecutive patients with shoulder pain recruited from primary care underwent standardised x-ray, diagnostic ultrasound scan and diagnostic injections of local anaesthetic into the SAB and ACJ. Subjects who reported less than 80% reduction in pain following either of these injections were referred for a magnetic resonance arthrogram (MRA) and GHJ diagnostic block. Differences in proportions of positive and negative imaging findings in the anaesthetic response groups were assessed using Fishers test and odds ratios were calculated a for positive anaesthetic response (PAR) to diagnostic blocks. In the 208 subjects recruited, the rotator cuff and SAB displayed the highest prevalence of pathology on both ultrasound (50% and 31% respectively) and MRA (65% and 76% respectively). The prevalence of PAR following SAB injection was 34% and ACJ injection 14%. Of the 59% reporting a negative anaesthetic response (NAR) for both of these injections, 16% demonstrated a PAR to GHJ injection. A full thickness tear of supraspinatus on ultrasound was associated with PAR to SAB injection (OR 5.02; p < 0.05). Ultrasound evidence of a biceps tendon sheath effusion (OR 8.0; p < 0.01) and an intact rotator cuff (OR 1.3; p < 0.05) were associated with PAR to GHJ injection. No imaging findings were strongly associated with PAR to ACJ injection (p ≤ 0.05). Rotator cuff and SAB pathology were the most common findings on ultrasound and MRA. Evidence of a full thickness supraspinatus tear was associated with symptoms arising from the subacromial region, and a biceps tendon sheath effusion and an intact rotator cuff were associated with an intra-articular GHJ pain source. When combined with clinical information, these results may help guide diagnostic decision making in primary care.


Effect of crystalloid and colloid preloading on uteroplacental and maternal haemodynamic state during spinal anaesthesia for caesarean section.
We have studied the effects of crystalloid 1 litre (lactated Ringer's) or colloid 0.5 litre (hydroxyethyl starch) preloading in 26 healthy parturients undergoing elective Caesarean section under spinal anaesthesia. Maternal placental uterine artery circulation was measured using a pulsed colour Doppler technique with simultaneous measurement of maternal haemodynamics. A high incidence of maternal hypotension was observed during spinal anaesthesia in the crystalloid group (62%) but the incidence was lower in the colloid group (38%). Central venous pressure was increased significantly in both groups after preload but decreased shortly after induction of spinal anaesthesia to baseline values. The mean pulsatility index (PI) in the uterine arteries did not change during preload or spinal block. A surprising finding was the widespread variation and some high values for the uterine artery PI after spinal anaesthesia. These individual increases in PI were transient and always returned to baseline values within 2 min. These results suggest that preloading with either solution is ineffective in preventing maternal hypotension and that changes in maternal heart rate, systolic arterial pressure and central venous pressure during spinal anaesthesia were not associated with rapid individual increases in uteroplacental vascular resistance. These changes seemed not to have any major effect, however, on the clinical condition of the newborn, as assessed by Apgar scores and umbilical artery pH values.