Prediction of ligands to universally conserved binding sites of the influenza a virus nuclear export protein.
The nuclear export protein (NEP) of the influenza A virus exports viral ribonucleoproteins to the host cell cytoplasm following nuclear transcription. In this work conservation analysis of 3000 protein sequences and molecular modelling of full-length NEP identified ligand binding sites overlapping with high sequence conservation. Two binding hot spots were identified close to the first nuclear export signal and several hot spots overlapped with highly conserved amino acids such as Arg42, Asp43, Lys39, Ile80, Gln101 and Val109. Virtual screening with ~43,000 compounds against a binding site showed affinities of up to -8.95 kcal/mol, while ~1700 approved drugs showed affinities of up to -8.31 kcal/mol. A drug-like compounds predicted was ZINC01564229 that could be used as probe to investigate NEP function or as a new drug lead. The approved drugs Nandrolone phenylpropionate and Estropipate were predicted to bind with high affinity and may be investigated for repurposing as anti-influenza drugs. IMPORTANCE: The influenza A virus causes respiratory illness in humans and farm animals annually across the world. Antigenic shifts and drifts in the surface proteins lead to genome diversity and unpredictable pandemics and epidemics. The high evolution rate of the RNA genome can also limit the effectiveness of antivirals and is the cause of emerging resistance. From a human health perspective, it is important that compounds identified as potential influenza replication inhibitors remain effective long-term. This work presents results which are based on computational predictions that reveal interactions between available compounds and regions of the influenza A nuclear export protein which display high conservation. Due to a low probability of highly conserved regions undergoing genomic changes, these compounds may serve as ideal leads for new antivirals.


Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [(3)H]thymidine incorporation assay.
T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [(3)H]thymidine ([(3)H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [(3)H]TdR assay as a "gold standard." As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [(3)H]TdR incorporation in both study groups (HIV-1 positives, r = 0.96; HIV-1 negatives, r = 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [(3)H]TdR incorporation, both in HIV-1-positive (r = 0.96) and HIV-1-negative (r = 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.


The effect of progesterone on prolactin stimulation of fatty acid synthesis, glycerolipid synthesis and lipogenic-enzyme activities in mammary glands of pseudopregnant rabbits, after explant culture or intraductal injection.
The activities of lipogenic enzymes, such as acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase, and glycerolipid synthesis increased significantly in mammary explants of 11-day-pseudopregnant rabbits in response to prolactin, in the presence of near-physiological concentrations of insulin and corticosterone in culture. Increasing the concentration of progesterone in culture resulted in suppression of glycerolipid synthesis and activities of acetyl-CoA carboxylase and fatty acid synthetase, but not the pentose phosphate dehydrogenases. However, at near-physiological concentration of progesterone, only acetyl-CoA carboxylase activity was decreased. Injection of prolactin intraductally into 11-day-pseudopregnant rabbits stimulated glycerolipid synthesis, fatty acid synthesis and enzymes involved in fatty acid synthesis, after 3 days. Intraductal injection of progesterone separately or together with prolactin had no significant effect on basal or stimulated lipogenesis in mammary glands. Intramuscular injection of progesterone at 10 mg/day did not suppress fatty acid synthesis stimulated when prolactin was injected intraductally, but a significant inhibition was observed at a higher dose (80 mg/day).


Experimental Investigation of Thrust Force, Delamination and Surface Roughness in Drilling Hybrid Structural Composites.
Filled hybrid composites are widely used in various structural applications where machining is critical. Hence, it is essential to understand the performance of the fibre composites' machining behaviour. As such, a new hybrid structural composite was fabricated with redmud as filler and sisal fibre as reinforcement in polyester matrix. The composite was then tested for its drilling performance. A comprehensive drilling experiment was conducted using Taguchi L27 orthogonal array. The effect of the drill tool point angle, the cutting speed, the feed rate on thrust force, delamination, and burr formation were analysed for producing quality holes. The significance of each parameter was analysed, and the experimental outcomes revealed some important findings in the context of the drilling behaviour of sisal fibre/polyester composites with redmud as a filler. Spindle speed contributed 39% in affecting the thrust force, while the feed rate had the maximum influence of ca. 38% in affecting delamination.


Plant Natural compounds with antibacterial activity towards common pathogens of pond-cultured channel catfish (Ictalurus punctatus).
The bacteria Edwardsiella ictaluri and Flavobacterium columnare cause enteric septicemia and columnaris disease, respectively, in channel catfish (Ictalurus punctatus). Natural therapeutants may provide an alternative to current management approaches used by producers. In this study, a rapid bioassay identified plant compounds as potential therapeutants. Chelerythrine chloride and ellagic acid were the most toxic toward E. ictaluri, with 24-h IC50 of 7.3 mg/L and 15.1 mg/L, respectively, and MIC of 2.1 mg/L and 6.5 mg/L, respectively. Chelerythrine chloride, ellagic acid, β-glycyrrhetinic acid, sorgoleone, and wogonin were the most toxic towards two genomovars of F. columnare, and wogonin had the strongest antibacterial activity (MIC = 0.3 mg/L).


Distinct gene expression profiles characterize cellular responses to palmitate and oleate.
Obese individuals are both insulin resistant and have high levels of circulating free fatty acids (FFAs). In cell culture, saturated but not unsaturated fatty acids induce endoplasmic reticulum (ER) stress. We hypothesized that chronic exposure to low dose fatty acids would significantly attenuate the acute stress response to a saturated fatty acid challenge and that unsaturated fatty acids (oleate) would be more protective than saturated fatty acids (palmitate). The ER stress response to palmitate was reduced after low dose fatty acid exposure in human hepatoma cells. Palmitate and oleate gave distinctive transcript responses, both acutely and after chronic low dose exposure. Differentially regulated pathways included lipid, cholesterol, fatty acid, and triglyceride metabolism, and IkappaB kinase and nuclear factor kappaB kinase inflammatory cascades. Oleate reduced palmitate-induced changes significantly more than low dose palmitate and completely blocked palmitate-induced phosphoinositide 3 kinase inhibitor (PIK3IP1) as well as induction of GADD45A and B. These changes are predicted to alter the PI3 kinase pathway and the pro-apoptotic p38 MAPK pathway. We recapitulated the oleate response by small interfering RNA-mediated block of PIK3IP1 stimulation with palmitate and significantly protected cells from palmitate-mediated ER stress. We show that transcriptional responses to oleate and palmitate are distinct, broad, and often discordant. We identified several potential candidates that may direct the transcriptional networks and demonstrate that PIK3IP1 partially accounts for the protective effects of oleate.


Time-resolved molecular measurements reveal changes in astronauts during spaceflight.
From the early days of spaceflight to current missions, astronauts continue to be exposed to multiple hazards that affect human health, including low gravity, high radiation, isolation during long-duration missions, a closed environment and distance from Earth. Their effects can lead to adverse physiological changes and necessitate countermeasure development and/or longitudinal monitoring. A time-resolved analysis of biological signals can detect and better characterize potential adverse events during spaceflight, ideally preventing them and maintaining astronauts' wellness. Here we provide a time-resolved assessment of the impact of spaceflight on multiple astronauts (n = 27) by studying multiple biochemical and immune measurements before, during, and after long-duration orbital spaceflight. We reveal space-associated changes of astronauts' physiology on both the individual level and across astronauts, including associations with bone resorption and kidney function, as well as immune-system dysregulation.


Epstein-Barr Virus SM protein utilizes cellular splicing factor SRp20 to mediate alternative splicing.
Epstein-Barr virus (EBV) SM protein is an essential nuclear protein produced during the lytic cycle of EBV replication. SM is an RNA-binding protein with multiple mechanisms of action. SM enhances the expression of EBV genes by stabilizing mRNA and facilitating nuclear export. SM also influences splicing of both EBV and cellular pre-mRNAs. SM modulates splice site selection of the host cell STAT1 pre-mRNA, directing utilization of a novel 5' splice site that is used only in the presence of SM. SM activates splicing in the manner of SR proteins but does not contain the canonical RS domains typical of cellular splicing factors. Affinity purification and mass spectrometry of SM complexes from SM-transfected cells led to the identification of the cellular SR splicing factor SRp20 as an SM-interacting protein. The regions of SM and SRp20 required for interaction were mapped by in vitro and in vivo assays. The SRp20 interaction was shown to be important for the effects of SM on alternative splicing by the use of STAT1 splicing assays. Overexpression of SRp20 enhanced SM-mediated alternative splicing and knockdown of SRp20 inhibited the SM effect on splicing. These data suggest a model whereby SM, a viral protein, recruits and co-opts the function of cellular SRp20 in alternative splicing.


Three-dimensional upper-airway changes associated with various amounts of mandibular advancement in awake apnea patients.
Mandibular advancement appliances have been used for years in the treatment of obstructive sleep apnea (OSA). However, it has not been completely clarified how the upper airway changes after mandibular advancement. The purpose of this study was to examine the effects of mandibular advancement on anatomic structures of the upper airway in patients with OSA by magnetic resonance imaging. Eleven OSA patients (aged 48.0 +/- 5.86 years) with apnea and hypopnoea index values of 46.8 +/- 21.47 participated in this study. A series of magnetic resonance images was taken for each subject with a custom-made device in the mouth that could advance the mandible by 0, 2, 4, 6, and 8 mm. In addition, an image with the mandible in centric occlusion was taken. Changes in cross-sectional area, sagittal diameter, transverse diameter, and cross-sectional shape in the whole upper airway and the different regions were compared between the various amounts of mandibular advancement. The minimum and mean cross-sectional area of the velopharynx and the mean transverse diameter of the velopharynx increased significantly (P = 0.0001 and P = 0.0016, respectively) during mandibular advancement. However, no statistically significant changes (P > 0.05) in sagittal diameter were found between the various mandibular advancements. In cross-sectional shape, a significant increase (P = 0.0066) was found when the mandible was advanced to 8 mm from 0 mm or centric occlusion position. Mandibular advancement significantly increases the cross-sectional area of the velopharynx in awake OSA patients. This contributes to the increase in its transverse dimension rather than the sagittal, thus resulting in the change of its shape.


Correlation between serological and sequencing analyses of the PorB outer membrane protein in the Neisseria meningitidis serotyping system.
The current serological typing scheme for Neisseria meningitidis is not comprehensive; a proportion of isolates are not serotypeable. DNA sequence analysis and predicted amino acid sequences were used to characterize the structures of variable-region (VR) epitopes on N. meningitidis PorB proteins (PorB VR typing). Twenty-six porB gene sequences were obtained from GenBank and aligned with 41 new sequences. Primary amino acid structures predicted from those genes were grouped into 30 VR families of related variants that displayed at least 60% similarity. We correlated VR families with monoclonal antibody (MAb) reactivities, establishing a relationship between VR families and epitope locations for 15 serotype-defining MAbs. The current panel of serotype-defining MAbs underestimates by at least 50% the PorB VR variability because reagents for several major VR families are lacking or because a number of VR variants within some families are not recognized by serotype-defining MAbs. These difficulties, also reported for serosubtyping based on the PorA protein, are shown as inconsistent results between serological and sequence analyses, leading to inaccurate strain identification and incomplete epidemiological data. The information from this study enabled the expansion of the panel of MAbs currently available for serotyping, by including MAbs of previously undetermined specificities. Use of the expanded serotype panel enabled us to improve the sensitivity of serotyping by resolving a number of formerly nonserotypeable strains. In most cases, this information can be used to predict the VR family placement of unknown PorB proteins without sequencing the entire porB gene. PorB VR typing complements serotyping, and a combination of both techniques may be used for full characterization of meningococcal strains. The present work represents the most complete and integrated data set of PorB VR sequences and MAb reactivities of serogroup B and C meningococci produced to date.


Psychomotor functions at various weeks of chronic renal failure in rats.
In chronic renal failure there is a gradual retention of substances in the tissues and body fluids, called as uremic retention toxins, which can bring about a number of biochemical activities in the body. Chronic renal insufficiency also leads to progressive behavioural conflict. Uremic toxins can affect both the central and the peripheral nervous system. Uremic encephalopathy is also associated with problems in cognition and memory. To study the psychomotor functional disorders in rats with progressive chronic renal failure surgical nephrectomy was done by resection method. The animals were grouped into two control groups, Sham control (SC) and normal control (NC) and two uremic groups, moderate uremia (GM) and severe uremia (GS). Psychomotor analysis was done by passive avoidance and open field in these animals at 4, 8, 12, and 16 weeks. After the incubation period, the nephrectomised groups (GM and GS) showed significant changes in exploratory, locomotor and emotional behaviour when compared to the controls (NC and SC). Psychomotor changes involve poor cognition, reduced memory, reduced locomotor activity and decreased exploratory drive and emotional disturbance like increased fear during the initial stages. During the later stages a restless behaviour was noticed, associated with diminished fear.


[Risk assessment of health disorders and quality of life in employees of modern polyvinyl chloride production].
The authors analyse changes in exposure load and associated occupational risk, health state of workers engaged into contemporary polyvinyl chloride production. Consideration covered objective parameters--results of thorough medical examinations -and subjective ones--results of quantitative risk evaluation of general pathologic syndromes and life quality caused by health state. The latter were compared with general working population unexposed to the hazards. Findings are statistically significant increase of functional disorders risk connected with cardiovascular and nervous diseases. Correlation was found between cardiovascular diseases prevalence and exposure chemical load. Statistically significant differences were seen in all life quality components between the workers groups compared.


Arterial pulsatility improvement in a feedback-controlled continuous flow left ventricular assist device: an ex-vivo experimental study.
Continuous flow left ventricular assist devices (CF-LVADs) reduce arterial pulsatility, which may cause long-term complications in the cardiovascular system. The aim of this study is to improve the pulsatility by driving a CF-LVAD at a varying speed, synchronous with the cardiac cycle in an ex-vivo experiment. A Micromed DeBakey pump was used as CF-LVAD. The heart was paced at 140 bpm to obtain a constant cardiac cycle for each heartbeat. First, the CF-LVAD was operated at a constant speed. At varying-speed CF-LVAD assistance, the pump was driven such that the same mean pump output was generated. For synchronization purposes, an algorithm was developed to trigger the CF-LVAD each heartbeat. The pump flow rate was selected as the control variable and a reference model was used for regulating the CF-LVAD speed. Continuous and varying-speed CF-LVAD assistance provided the same mean arterial pressure and flow rate, while the index of pulsatility doubled in both arterial pressure and pump flow rate signals under pulsatile pump speed support. This study shows the possibility of improving the pulsatility in CF-LVAD support by regulating pump speed over a cardiac cycle without compromising the overall level of support.


A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America.
Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and animals comprising strains that cause clinical disease in people, dogs and horses (the pathogenic A. phagocytophilum "genospecies") and more distantly related strains. A rodent-adapted genospecies named DU1, found primarily in woodrats, is unable to infect horses. We developed a real-time PCR (RT-PCR) assay, which targets an 85 base pair region of the ank gene and is specific for the pathogenic genospecies of A. phagocytophilum from North America. Thirty DNA samples from A. phagocytophilum RT-PCR-positive rodents and ticks for which the ank gene had previously been sequenced and had been identified as either the pathogenic genospecies or DU1 were used for validation. All nine samples with the pathogenic genospecies tested positive using the new RT-PCR and all 21 samples with the DU1 genospecies tested negative. Two strains from which the whole genome has been sequenced and are known to be pathogenic (A. phagocytophilum, strain HZ isolated from a human from New York and strain MRK isolated from a horse from California) both tested positive using the new RT-PCR assay. We also tested blood from 20 horses and six dogs that were RT-PCR-positive using a previously validated RT-PCR protocol: all were PCR-positive using the new assay as well. The assay has high efficiency and reproducibility and there was no cross-reaction with related and tick-borne bacteria tested, although the assay cross-reacts with the ungulate-adapted genospecies Ap-Variant 1. This new RT-PCR assay will aid in future research on the ecology and epidemiology of A. phagocytophilum by allowing researchers to easily identify the pathogenic genospecies in reservoir hosts and vectors.


Intracellular lipid in clear cell renal cell carcinoma tumor thrombus and metastases detected by chemical shift (in and opposed phase) MRI: radiologic-pathologic correlation.
Clear cell renal cell carcinoma (RRC) characteristically contain intracellular lipid which is also detectable in tumor thrombus and metastases. To assess the incidence of intracellular lipid in clear cell RCC metastases and tumor thrombus using chemical shift MRI. With REB approval, 33 consecutive patients with clear cell RCC and tumor thrombus/metastatic disease underwent magnetic resonance imaging (MRI) over a 10-year period. Diagnosis was established by histopathology for tumor thrombi (n = 25) and metastases (n = 15) or growth for metastases (n = 14). Two blinded radiologists independently assessed for a signal intensity (SI) drop at chemical shift MRI (indicative of intracellular lipid) and a third radiologist established consensus. Chemical shift SI (CS-SI) index ([SItumorIP - SItumorOP]/SITumorIP x 100) was calculated. Inter-observer agreement was assessed using intra-class correlation (ICC) and tests of association were performed using the Chi-square test and Spearman correlation. Using CS-MRI, intracellular lipid was detected in 36.4% of clear cell RCC, with moderate agreement, (ICC = 0.5). Intracellular lipid was detected in 20% of tumor thrombi and 20% of metastases with strong agreement (ICC = 0.73). Intracellular lipid within tumor thrombi/metastases was not associated with lipid within the primary tumor (P = 0.09). There was a correlation in CS-SI index between primary tumor and thrombi/metastases when lipid was detected in both lesions (r = 0.91, P = 0.005); however, there was no correlation when lipid was not detected in both lesions (r = -0.09, P = 0.72). The presence of intracellular lipid in tumor thrombus and metastases from clear cell RCC is uncommon and, is not necessarily associated with lipid within the primary tumor at chemical shift MRI.


Combined Haploidentical Hematopoetic Stem Cell Transplantation and Liver Transplantation in a Pediatric Patient.
Solid organ transplantation from the same donor is an established procedure for end-stage organ failure that developed after a previous hematopoietic stem cell transplantation (HSCT); however, it is rarely done in patients transplanted with unmanipulated haplo-HSCT. There are no pediatric reports regarding the long-term performance of organ transplantation after haplo-HSCT with post-transplant cyclophosphamide (PTCY). A juvenile myelomonocytic leukemia patient, who underwent unmanipulated haplo-HSCT with PTCY from her mother at the age of 3 years, developed chronic liver graft versus host disease (GvHD) which was refractory to specific GvHD treatment. Liver transplantation (LT) from her mother (the donor of her haplo-HSCT) was decided as the next line of treatment. LT was performed on day 540 post-HSCT, and the donor's left lateral segment was appropriately removed and attached to the recipient. The symptoms of GvHD completely regressed in a month. The patient died on day 121 after LT, because of a possible hepato-pulmonary syndrome. Organ failure can develop after allo-HSCT secondary to GvHD and therefore performing HSCT from a haplo-donor may be superior to a matched unrelated donor in terms of subsequent organ transplantation for organ failure.


Validation of the Portuguese version of the Community Assessment of Psychic Experiences and characterization of psychotic experiences in a Brazilian sample.
We investigated: i) the reliability and validity of a Brazilian version of the Community Assessment of Psychic Experiences (CAPE), developed to detect and characterize psychotic experiences in the general population; and ii) the association between psychotic experiences, childhood adversity, and cannabis use in a population-based sample. We performed factorial analyses and generalized linear models with CAPE scores as the dependent variable in a sample composed of 217 first-episode psychosis patients, 104 unaffected biological siblings, and 319 non-psychotic population-based participants. After removing seven items from its positive dimension and two items from its negative dimension, a 33-item Brazilian version of the CAPE showed acceptable adjustment indices (confirmatory fit index = 0.895; goodness of fit index = 0.822; parsimony goodness of fit index = 0.761; root mean square error of approximation [RMSEA] = 0.055, p [RMSEA ≤ 0.05] = 0.04) and internal consistency in all its dimensions (> 0.70). Childhood adversity was associated with higher scores in all three dimensions, as well as with total score. Lifetime cannabis use was associated with higher scores only in the positive dimension. The proposed Brazilian version of the CAPE corroborates the tridimensional approach for assessing psychosis-proneness, and the frequency and severity of psychotic manifestations are distributed as a spectrum in the general population.


Effects of GLP-1-(7-36)NH2, GLP-1-(7-37), and GLP-1- (9-36)NH2 on intravenous glucose tolerance and glucose-induced insulin secretion in healthy humans.
Glucagon-like peptide 1 (GLP-1) is an insulin secretagogue synthesized in the intestine and released in response to meal ingestion. It is secreted primarily in two forms, GLP-1-(7-37) and GLP-1-(7-36)NH(2), both of which bind to a specific GLP-1 receptor (GLP-1r) on the pancreatic beta-cell and augment glucose-stimulated insulin secretion. Once secreted, GLP-1-(7-36)NH(2) is rapidly metabolized to GLP-1-(9-36)NH(2), which is the predominant form of GLP-1 in postprandial plasma because of its relatively slower clearance. Although no clear biological role for GLP-1-(9-36)NH(2) in humans has been identified, recent studies in animals suggest two potential effects: to antagonize the effects of intact GLP-1 and to promote glucose disappearance in peripheral tissues. In the studies reported here we compared the independent effects of GLP-1-(7-36)NH(2), GLP-1-(7-37), and GLP-1-(9-36)NH(2) on parameters of iv glucose tolerance and determined whether GLP-1-(9-36)NH(2) inhibits the insulinotropic actions of GLP-1. Ten healthy subjects underwent 4 separate frequently sampled iv glucose tolerance tests during infusions of GLP-1-(7-37), GLP-1-(7-36)NH(2), GLP-1-(9-36)NH(2), or saline. Results from the iv glucose tolerance test were used to obtain indexes of beta-cell function (acute insulin response to glucose) and iv glucose tolerance (glucose disappearance constant), and the minimal model of glucose kinetics was used to obtain indexes of glucose effectiveness and insulin sensitivity. Compared with control studies, both GLP-1-(7-36)NH(2) and GLP-1-(7-37) significantly increased acute insulin response to glucose, glucose disappearance constant, glucose effectiveness, and glucose effectiveness at zero insulin, but did not change the insulin sensitivity index. In contrast, none of the parameters of glucose tolerance was measurably affected by GLP-1-(9-36) amide. In a second set of experiments, 10 healthy subjects had glucose-stimulated insulin secretion measured during an infusion of GLP-1-(7-36)NH(2) alone or with a simultaneous infusion of GLP-1-(9-36)NH(2) that increased plasma levels approximately 10-fold over those produced by unmetabolized GLP-1. Augmentation of glucose-stimulated insulin secretion by GLP-1-(7-36)NH(2) was not altered by the coadministration of GLP-1-(9-36)NH(2). Based on these results we conclude that GLP-1-(9-36)NH(2) does not regulate insulin release or glucose metabolism in healthy humans.


School-based Prevention for Adolescent Internet Addiction: Prevention is the Key. A Systematic Literature Review.
Adolescents' media use represents a normative need for information, communication, recreation and functionality, yet problematic Internet use has increased. Given the arguably alarming prevalence rates worldwide and the increasingly problematic use of gaming and social media, the need for an integration of prevention efforts appears to be timely. The aim of this systematic literature review is (i) to identify school-based prevention programmes or protocols for Internet Addiction targeting adolescents within the school context and to examine the programmes' effectiveness, and (ii) to highlight strengths, limitations, and best practices to inform the design of new initiatives, by capitalizing on these studies' recommendations. The findings of the reviewed studies to date presented mixed outcomes and are in need of further empirical evidence. The current review identified the following needs to be addressed in future designs to: (i) define the clinical status of Internet Addiction (IA) more precisely, (ii) use more current psychometrically robust assessment tools for the measurement of effectiveness (based on the most recent empirical developments), (iii) reconsider the main outcome of Internet time reduction as it appears to be problematic, (iv) build methodologically sound evidence-based prevention programmes, (v) focus on skill enhancement and the use of protective and harm-reducing factors, and (vi) include IA as one of the risk behaviours in multi-risk behaviour interventions. These appear to be crucial factors in addressing future research designs and the formulation of new prevention initiatives. Validated findings could then inform promising strategies for IA and gaming prevention in public policy and education.


Older patients are more likely to breach the 4-hour target in Scotland.
To determine if age is a factor in a patients' likelihood of breaching the 4 hour time target to admission/discharge in emergency departments (EDs) within NHS Scotland. We used data from the Information Service Division Scotland to analyse all ED attendances in Scotland between January 2015 and September 2018 (n=5 596 642). We assessed the likelihood of time to admission/discharge being within 4 hours, 8 hours and 12 hours for all age categories (reference category 20 to 24 years). Univariable logistic regressions were carried out for sex, Scottish Index of Multiple Deprivation level and both major (potentially life threatening) and minor (not immediately life threatening) incidences. The likelihood of breaching the 4-hour target increased linearly with age from 15 to 19 years upward. Patients ≥85 years were significantly (p<0.001) more likely to have breached than patients aged 20 to 24 years (OR 3.80, 95% CI: 3.73 to 3.86). When considering major incidents, patients aged ≥85 years were more likely to have breached than those aged 20 to 24 years (OR 2.05, 95% CI: 2.01 to 2.09, p<0.001). The same was true of minor incidents (OR 2.85, 95% CI: 2.73 to 2.98, p<0.001). Older age is associated with a higher probability of breaching waiting time targets in a linear fashion within NHS Scotland, which is consistent with previous single hospital or regional studies. This association may be due to the higher proportion of elderly patients being admitted or a more systemic issue, but regardless, the elderly are being put more at risk.


Low background mould-prepared gelatine standards for reproducible quantification in elemental bio-imaging.
Standard preparation for elemental bio-imaging by laser ablation-inductively coupled plasma-mass spectrometry is confounded by the chemical and physical differences between standard and sample matrices. These differences lead to variable ablation, aerosol generation and transportation characteristics and must be considered when designing matrix-matched standards for reliable calibration and quantification. The ability to precisely mimic sample matrices is hampered due to the complexity and heterogeneity of biological tissue and small variabilities in standard matrices and sample composition often negatively impact accuracy, precision and robustness. Furthermore, cumbersome preparation protocols may limit reproducibility and traceability. This work presents novel facile methods for the preparation of gelatine standards using both commercial and laboratory-made moulds. Surface roughness, thickness and robustness of the mould-prepared standards were compared against cryo-sectioned gelatine and homogenised brain tissue standards. The mould-prepared standards had excellent thickness accuracy and signal precision which allowed robust quantification, were easier to prepare and therefore easier to reproduce. We also compared gelatine standards prepared from a variety of animal sources and discuss their suitability to calibrate low level elemental concentrations. Finally, we present a simple method to remove background metals in gelatine using various chelating resins to increase the dynamic calibration range and to improve limits of analysis.


A piecewise-constant Markov model and the effects of study design on the estimation of life expectancies in health and ill health.
Multi-state models are frequently applied to describe transitions over time between three states: healthy, not healthy and death. The three-state model can be used to estimate life expectancies in health and ill health. In this article, continuous-time Markov models are specified for the transitions between the three states. Transition intensities are regressed on age as a time-dependent covariate. The covariate is handled in a piecewise-constant fashion where the time interval between two consecutive observations is divided into subintervals of fixed and equal lengths. Study design choices such as sample size, length of follow-up, and time intervals between observations are investigated in a simulation study. The effects on parameter estimation are discussed as well as the effects on the estimation of life expectancies. In addition, data taken from the UK Cognitive Functioning and Ageing Study are analysed.


Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates.
Shiga-like toxin-producing Escherichia coli O157 isolates were characterised by random amplification of polymorphic DNA by PCR (RAPD-PCR) analysis developed to allow robust epidemiological typing of E. coli. Amplification with primer 1247 or 1290 generated a reproducible profile, but was not capable of distinguishing sufficiently between epidemiologically unrelated strains. Subsequent digestion of the amplicons with selected restriction endonucleases improved the discriminatory ability of this method for strains showing limited differentiation following RAPD-PCR analysis alone. Restriction endonuclease analysis of RAPD-PCR fragments generated from closely related strains has the potential to provide additional discriminatory information without loss of specificity.


Role of progesterone in human astrocytomas growth.
Progesterone (P) participates in the regulation of several reproductive processes such as ovulation and sexual behavior, however, this hormone also participates in non-reproductive processes, such as neural excitability, learning and memory, and pathological processes such as cancer. P mainly elicits its effects by interaction with its intracellular receptor (PR), which is a ligand-activated transcription factor that modifies the expression of genes involved in the control of cell growth and proliferation, such as vascular endothelial growth factor and epidermal growth factor receptor. Two PR isoforms have been reported: PR-B and PR-A, which present different function and regulation. PR isoforms are expressed in U373 and D54 cell lines, which are derived from grades III and IV of human astrocytomas, respectively. In both cells lines P increases the number of astrocytomas cells. The PR antagonist, RU486, blocked P effects and its treatment alone significantly reduced human astrocytomas cell growth in vitro. The over-expression of PR-A in U373 cells significantly reduced P effects. These data suggest that P regulates human astrocytomas cell proliferation through the interaction with PR.


Hybrid supervisory control using recurrent fuzzy neural network for tracking periodic inputs.
A hybrid supervisory control system using a recurrent fuzzy neural network (RFNN) is proposed to control the mover of a permanent magnet linear synchronous motor (PMLSM) servo drive for the tracking of periodic reference inputs. First, the field-oriented mechanism is applied to formulate the dynamic equation of the PMLSM. Then, a hybrid supervisory control system, which combines a supervisory control system and an intelligent control system, is proposed to control the mover of the PMLSM for periodic motion. The supervisory control law is designed based on the uncertainty bounds of the controlled system to stabilize the system states around a predefined bound region. Since the supervisory control law will induce excessive and chattering control effort, the intelligent control system is introduced to smooth and reduce the control effort when the system states are inside the predefined bound region. In the intelligent control system, the RFNN control is the main tracking controller which is used to mimic a idea control law and a compensated control is proposed to compensate the difference between the idea control law and the RFNN control. The RFNN has the merits of fuzzy inference, dynamic mapping and fast convergence speed, In addition, an online parameter training methodology, which is derived using the Lyapunov stability theorem and the gradient descent method, is proposed to increase the learning capability of the RFNN. The proposed hybrid supervisory control system using RFNN can track various periodic reference inputs effectively with robust control performance.


A longitudinal study into the perceptions of caring among student nurses using multivariate analysis of the Caring Dimensions Inventory.
A longitudinal study involving student nurses' responses to the Caring Dimensions Inventory (CDI) was carried out to investigate the development of perceptions of caring. This study continues previously reported work in which the development of the CDI was described. The current study was designed to see if the CDI could detect changes in perceptions of caring among student nurses as they progressed through their training. Data were collected on the same subjects at three points, each a year apart, throughout the study. A cohort of 168 student nurses entering training was recruited into the present study and data from the CDI was analysed using exploratory factor analysis. A four factor structure for perceptions of caring throughout the study was supported, with some evidence for a five factor structure at 24 months into the programme of nurse education. A professional and technical factor increased in congruence with a similar factor, identified in a previous study, throughout the present study and was used to provide factor scores for individual students. There was a statistically significant and moderately sized correlation between the score on this factor and the age of the student nurses. The five factor solution yielded a factor of accountability. Professional and educational consequences of the study are discussed along with the problem of attrition in longitudinal studies.


Assessment of macro- and micro-oxygenation parameters during fractional fluid infusion: A pilot study.
The main goal of this study was to assess whether maximal fluid infusion improves both oxygen delivery (DO2) and micro-circulatory parameters during hemodilution. The secondary objective was to assess the ability of baseline micro-circulatory parameters to predict oxygen consumption (VO2) response following fluid infusion. In a postoperative cardiac ICU, patients received repeated fluid infusion until stroke volume (SV) was maximized. Before and after each fluid expansion, macro- (DO2, VO2) and micro-circulatory oxygenation parameters were recorded [central venous oxygen saturation (ScVO2), blood lactate, difference in veno-arterial carbon dioxide tension (P(v-a)CO2), somatic and cerebral oxygen saturation (rSO2)]. Patients were classified as VO2-Responders or VO2-Non-Responders according to an increase in VO2 above or below 15%, respectively. After maximal fluid infusion, all patients showed improved macro- and micro-circulatory oxygenation parameters, but VO2-Responders had lower values (especially for ScVO2 and cerebral rSO2). Only baseline ScVO2 and cerebral rSO2 were useful to predict the VO2 response to maximal fluid infusion (ROCAUC 0.80 (95% CI: 0.54-0.95, P=0.012) and 0.83 (95% CI: 0.57-0.96, P=0.001). Maximal fluid infusion improves macro- and micro-circulatory oxygenation parameters. For VO2-Responders, only ScVO2 and cerebral rSO2 could serve as markers of tissue hypoxia.


A Novel Five-Node Feed-Forward Loop Unravels miRNA-Gene-TF Regulatory Relationships in Ischemic Stroke.
The complex and interlinked cascade of events regulated by microRNAs (miRNAs), transcription factors (TF), and target genes highlight the multifactorial nature of ischemic stroke pathology. The complexity of ischemic stroke requires a wider assessment than the existing experimental research that deals with only a few regulatory components. Here, we assessed a massive set of genes, miRNAs, and transcription factors to build a miRNA-gene-transcription factor regulatory network to elucidate the underlying post-transcriptional mechanisms in ischemic stroke. Feed-forward loops (three-node, four-node, and novel five-node) were converged to establish regulatory relationships between miRNAs, TFs, and genes. The synergistic function of miRNAs in ischemic stroke was predicted and incorporated into a novel five-node feed-forward loop. Significant miRNA-TF pairs were identified using cumulative hypergeometric distribution. Two subnetworks were derived from the extensive miRNA-TF regulatory network and analyzed to predict the molecular mechanism relating the regulatory components. NFKB and STAT were identified to be the chief regulators of innate inflammatory and neuronal survival mechanisms, respectively. Exclusive novel interactions between miR-9 and miR-124 with TLX, BCL2, and HDAC4 were identified to explain the post-stroke induced neurogenesis mechanism. Therefore, this network-based approach to delineate miRNA, TF, and gene interactions might promote the development of effective therapeutics against ischemic stroke.


Skeletal Muscle Cell Induction from Pluripotent Stem Cells.
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD). Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.


[Pharmacokinetics of carbapenems].
Carbapenems, beta-lactam antibiotics, are ideal candidates for the treatment of serious nosocomial infections including sepsis for their exceptionally broad antibacterial spectrum and high efficiency. They are administered parenterally by intravenous infusion. Carbapenems penetrate well and rapidly into many different tissue compartments and the interstitial fluid. They are metabolized by renal dihydropeptidase-1. Therefore, imipenem must be co-administered with an inhibitor of dihydropeptidase-1. Other carbapenems registered in the Czech Republic (meropenem, ertapenem and doripenem) are more stable to this enzyme. Carbapenems are mainly eliminated via the kidneys and dose adjustment in patients with renal impairment is necessary. The elimination half-life of most carbapenems is around 1 hour with the exception of ertapenem, with 3.8-hour half-life, which allows its once-daily use. Carbapenems are a group of antibiotics with time-dependent effect. Their typical pharmaceutical property is a limited stability in solution after dilution. Administration in the prolonged infusion appears to be a convenient strategy to achieve higher efficiency. Pharmacokinetic parameters of carbapenems may vary individually, especially in critically ill patients and those treated by renal replacement therapy. Therefore, individualization of dosing regimens based on knowledge of pharmacokinetic parameters of individual patients may be useful.


Models and algorithms for efficient multiresolution topology estimation of measured 3-D range data.
In this paper, we propose a new efficient topology estimation algorithm to construct a multiresolution polygonal mesh from measured three-dimensional (3-D) range data. The topology estimation problem is defined under the constraints of cognition, compactness, and regularity, and the algorithm is designed to be applied to either a cloud of points or a dense mesh. The proposed algorithm initially segments the range data into a finite number of Voronoi patches using the K-means clustering algorithm. Each patch is then approximated by an appropriate polygonal and eventually a triangular mesh model. In order to improve the equiangularity of the mesh, we employ a dynamic mesh model, in which the mesh finds its equilibrium state adaptively, according to the equiangularity constraint. Experimental results demonstrate that satisfactory equiangular triangular mesh models can be constructed rapidly at various resolutions, while yielding tolerable modeling error.


Lethality of suicide methods.
To (a) quantify the lethality of suicide methods used in Australia in the period 1 July 1993 to 30 June 2003, (b) examine method-specific case fatality by age and sex, and (c) identify changes in case fatality during the study period. Two sources of data on episodes of self-harm in Australia were used, mortality and hospital separation data. Double counting of cases recorded in both sources was controlled by omitting fatal hospital cases from estimates of episodes of self-harm. Overall case fatality was 12%. For each suicide method, case fatality was higher in males and older age groups. Firearms were the most lethal suicide means (90%) followed by hanging (83%). Rates of suicide involving firearms declined over time, and those involving hanging rose. Case fatality for firearm cases changed little over time, but declined for self-harm by hanging/suffocation, poisoning, sharp objects, and crashing a motor vehicle. This study (Australia) and two others (USA) show differences in method-specific lethality by gender and age. This study adds the finding of changes in lethality over time. Understanding of suicidality in populations, on which prevention efforts depend, requires explanation of these findings.


Delta-9-tetrahydrocannabinol modulates pain sensitivity among persons receiving opioid agonist therapy for opioid use disorder: A within-subject, randomized, placebo-controlled laboratory study.
The opioid and cannabinoid receptor systems are inextricably linked-overlapping at the anatomical, functional and behavioural levels. Preclinical studies have reported that cannabinoid and opioid agonists produce synergistic antinociceptive effects. Still, there are no experimental data on the effects of cannabinoid agonists among humans who receive opioid agonist therapies for opioid use disorder (OUD). We conducted an experimental study to investigate the acute effects of the delta-9-tetrahydrocannabinol (THC) among persons receiving methadone therapy for OUD. Using a within-subject, crossover, human laboratory design, 25 persons on methadone therapy for OUD (24% women) were randomly assigned to receive single oral doses of THC (10 or 20 mg, administered as dronabinol) or placebo, during three separate 5-h test sessions. Measures of experimental and self-reported pain sensitivity, abuse potential, cognitive performance and physiological effects were collected. Mixed-effects models examined the main effects of THC dose and interactions between THC (10 and 20 mg) and methadone doses (low-dose methadone defined as <90 mg/day; high dose defined as >90 mg/day). Results demonstrated that, for self-reported rather than experimental pain sensitivity measures, 10 mg THC provided greater relief than 20 mg THC, with no substantial evidence of abuse potential, and inconsistent dose-dependent cognitive adverse effects. There was no indication of any interaction between THC and methadone doses. Collectively, these results provide valuable insights for future studies aiming to evaluate the risk-benefit profile of cannabinoids to relieve pain among individuals receiving opioid agonist therapy for OUD, a timely endeavour amidst the opioid crisis.


A comparison of substance use disorder severity and course in American Indian male and female veterans.
The purpose of this analysis was to compare substance use disorder (SUD) diagnoses, severity, comorbidity, and course in 362 American Indian veterans. The sample was drawn from communities in the north central and southwestern areas of the United States, structured to over-sample women and to include half-rural/half-urban residents. Instruments used in the study included current demography, military history, the Diagnostic Interview Schedule/Quick Version, Michigan Alcoholism Screening Test modified to include alcohol and drugs (MAST/AD), Brief Symptom Inventory, Posttraumatic Checklist, and a treatment algorithm. Univariate analyses showed that women had lower scores on the MAST/AD, reported lower symptom levels on the Posttraumatic Checklist, and were less apt to use VA mental health services, but were more willing to seek mental health treatment if needed (with probabilities of .01 to .001). At borderline probabilities (.02 to .05), women were younger and had more education, whereas men had more drug-related diagnoses and more combat exposure. On binary logistic regression, women were less apt to have a drug diagnosis and had lower MAST/AD scores; the other differences were not significant. Female American Indian veterans with SUD manifested many of the same gender-related differences as women in the population at large, but with some exceptions (eg, comorbidity). Trauma, PTSD, and continuing posttraumatic symptoms occurred frequently in both male and female veterans of American Indian heritage. VA facilities should out-reach to American Indian women, who report a willingness to seek mental health services but may avoid VA care.


The pathophysiology of non-steroidal anti-inflammatory drug (NSAID)-induced mucosal injuries in stomach and small intestine.
Non-steroidal anti-inflammatory drugs are the most commonly prescribed drugs for arthritis, inflammation, and cardiovascular protection. However, they cause gastrointestinal complications. The pathophysiology of these complications has mostly been ascribed to non-steroidal anti-inflammatory drugs' action on the cyclooxygenase inhibition and the subsequent prostaglandin deficiency. However, recent clinical demonstrated the prevalence of non-steroidal anti-inflammatory drugs-induced small intestinal mucosal injury is more often than previously expected. In this review, we discuss the defense mechanisms of stomach, and the pathophysiology of non-steroidal anti-inflammatory drugs-induced injury of stomach and small intestine, especially focused on non-steroidal anti-inflammatory drugs' action on mitochondria.


Pharmacological differentiation of allergic and classically conditioned asthma in the guinea pig.
A whole-body plethysmographic technique was developed and then used to detect experimentally induced asthma in guinea pigs and to assess pharmacological treatments of allergic and classically conditioned attacks. Inhalation of a beta adrenergic compound (isoproterenol) controlled both forms of attack. Atropine and methscopolamine, parasympathetic blocking agents, prevented conditional but not allergic attacks; diphenhydramine, an antihistamine, prevented allergic attacks; and methysergide, which blocks serotonin (which is believed to trigger human asthma), prevented neither. The guinea pig's allergic reaction is probably the result of a bronchospasm induced by histamine released in tissue of the airway by a local combination of allergen and antibody. The conditional attack is believed to be a constriction of the airway mediated by parasympathetic fibers of central origin.


A Rare Case of Cor Triatriatum Leading to Respiratory Failure and Shock in an 8-Month-Old Boy.
An 8-month-old boy presents with 1 week of gradually worsening respiratory distress. After chest radiograph shows bilateral airspace opacities, he is treated for pneumonia with antibiotics and intravenous fluids. Shortly after this, he decompensates requiring intubation, followed by cardiopulmonary support with pressors and eventually extracorporeal membrane oxygenation. Bedside ultrasound then shows an additional congenital membrane in the left atrium obstructing pulmonary venous return and thought to have caused his original presentation and subsequent decompensation. After surgical correction, he is decannulated from extracorporeal membrane oxygenation, makes a quick recovery, and is discharged from the hospital in good condition within 2 weeks. This rare case of cor triatriatum highlights the importance of considering congenital heart disease even with common presentations in the pediatric population.


Casas Maternas in the Rural Highlands of Guatemala: A Mixed-Methods Case Study of the Introduction and Utilization of Birthing Facilities by an Indigenous Population.
An international NGO, with financial and managerial support from "partner" communities, established Casas Maternas (birthing facilities) in 3 municipalities in the isolated northwestern highlands of the department of Huehuetenango in Guatemala-an area with high maternal mortality ratio (338 maternal deaths per 100,000 live births). Traditional birth attendants are encouraged to bring patients for delivery at the Casas Maternas, where trained staff are present and access to referral care is facilitated. We conducted a mixed-methods study in San Sebastian Coatán municipality to assess the contribution of 2 Casas Maternas to health facility deliveries among partner and non-partner communities, with particular emphasis on equity in access. We surveyed all women who delivered in the study area between April 2013 and March 2014, the first full year in which both Casas Maternas in the study area were operating. In addition, using purposive sampling, we conducted in-depth interviews with 22 women who delivered and 6 focus group discussions with 42 community leaders, traditional birth attendants, and Casas Maternas staff members. We analyzed the quantitative data using descriptive statstics and the qualitative data with descriptive content analysis. Of the 321 women eligible for inclusion in the study, we surveyed 275 women (14.3% could not be located or refused to participate). Between April 2013 and March 2014, 70% of women living in partner communities delivered in a health facility (54% in a Casa Materna) compared with 30% of women living in non-partner communities (17% in a Casa Materna). There was no statistically significant difference in uptake of the Casa Materna by maternal education and only a weak effect by household wealth. In contrast, distance from the Casa Materna had a pronounced effect. Traditional birth attendants were strong advocates for utilization of the Casa Materna and played an important role in the decision regarding where the birth would take place. In addition, the program's outreach component, in which peer volunteers visit homes to promote healthy behaviors and appropriate use of health facilities, was identified as a key factor in encouraging mothers to deliver in facilities. The Casa Materna approach to strengthening maternity care as developed by Curamericas has potential to increase health facility utilization in isolated mountainous areas inhabited by an indigenous population where access to government services is limited and where maternal mortality is high. The approach shows promise for broader application in Guatemala and beyond.


Arthropod fossil data increase congruence of morphological and molecular phylogenies.
The relationships of major arthropod clades have long been contentious, but refinements in molecular phylogenetics underpin an emerging consensus. Nevertheless, molecular phylogenies have recovered topologies that morphological phylogenies have not, including the placement of hexapods within a paraphyletic Crustacea, and an alliance between myriapods and chelicerates. Here we show enhanced congruence between molecular and morphological phylogenies based on 753 morphological characters for 309 fossil and Recent panarthropods. We resolve hexapods within Crustacea, with remipedes as their closest extant relatives, and show that the traditionally close relationship between myriapods and hexapods is an artefact of convergent character acquisition during terrestrialisation. The inclusion of fossil morphology mitigates long-branch artefacts as exemplified by pycnogonids: when fossils are included, they resolve with euchelicerates rather than as a sister taxon to all other euarthropods.


[Borderline mental disorders in chronic diseases of the digestive system in children and adolescents].
Based on studies into the mental sphere of gastroenterological patients aged 6 to 16 years, the author systematized borderline neurotic and personality disorders on the clinical and etiopathogenetic basis. The primarily psychogenic origin of the psychopathological symptomatology was recorded in 39.3% of the patients, the primarily somatogenic one in 33.2%. The syndrome of the neurotic phantom of somatic disease, which formed in 27.2% of neurotic patients is described. A concept of the mental constituent of a chronic disease is worded. A system of measures aimed at early diagnosis, correction, treatment and psychoprophylaxis of borderline conditions and psychosocial readaptation of patients is scientifically based.


Genetic and molecular analysis of the proximal region of the mouse t-complex using new molecular probes and partial t-haplotypes.
The t-complex is located on the proximal third of chromosome 17 in the house mouse. Naturally occurring variant forms of the t-complex, known as complete t-haplotypes, are found in wild mouse populations. The t-haplotypes contain at least four nonoverlapping inversions that suppress recombination with the wild-type chromosome, and lock into strong linkage disequilibrium loci affecting normal transmission of the chromosome, male gametogenesis and embryonic development. Partial t-haplotypes derived through rare recombination between t-haplotypes and wild-type homologs have been critical in the analysis of these properties. Utilizing two new DNA probes. Au3 and Au9, and several previously described probes, we have analyzed the genetic structure of several partial t-haplotypes that have arisen in our laboratory, as well as several wild-type chromosomes deleted for loci in this region. With this approach we have been able to further our understanding of the structural and dynamic characteristics of the proximal region of the t-complex. Specifically, we have localized the D17Tul locus as most proximal known in t-haplotypes, achieved a better structural analysis of the partial t-haplotype t6, and defined the structure and lethal gene content of partial t-haplotypes derived from the lethal tw73 haplotype.


Feedback of the third interlaboratory exercise organised on wheat in the framework of the OBT working group.
Organically bound tritium (OBT) has become of increasing interest within the last decade, with a focus on its behaviour and also its analysis, which are both important to assess tritium distribution in the environment and dose consequences. After the first OBT International Workshop which was held in France in May 2012, an international working group was created. The expected benefits are the following: remove or reduce uncertainty in OBT analysis results, provide better OBT model validation data and better public dose results, increase the number of potential measuring OBT laboratories, validate all of the stages of the procedures based on a larger population and more statistically significant results, and investigate the feasibility of CRM's and RM's production. In this framework, three OBT exercises were organised; the 1st one on potatoes was conducted in 2013 by the Canadian National Laboratories (former AECL) with about 20 participating labs from around the world, the 2nd one on a sediment was organised in 2014 by GAU Radioanalytical (University of Southampton) on a sediment with again about 20 participating labs and the third one on wheat was organised in 2015 by the Commissariat à l'énergie atomique (CEA) with about 25 participating labs.


Two histone/protein acetyltransferases, CBP and p300, are indispensable for Foxp3+ T-regulatory cell development and function.
T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells.


Risk perception, decision-making, and risk communication in the time of COVID-19.
After first being declared a pandemic by the World Health Organization [WHO], (2020) in March 2020, coronavirus disease (COVID-19) spread rapidly and in the process altered our very way of life. At the same time, it became increasingly clear that a wide range of new behavioral science research was necessary to understand fully how people comprehend and respond to such an unprecedented and long lasting health threat as COVID-19. One of the primary aims for this Special Issue was to gather and publish that research. The studies contained in this Special Issue, conducted between April 2020 and March 2021, were selected to represent experimental research that is relevant to this unique situation and that also inform and extend existing theory. These studies investigate three broad topics: Risk perception, decision-making under risk, and risk communication in the context of COVID-19. Collectively, they advance our knowledge of risk calibration, health communication interventions, and decisions about behaviors that address risk in the context of a global health threat. Perhaps most importantly they also make a practical contribution to how we approach these issues going forward. (PsycInfo Database Record (c) 2022 APA, all rights reserved).


Feedback of Antibiotic Prescribing in Primary Care (FAPPC) trial: results of a real-world cluster randomized controlled trial in Scotland, UK.
To evaluate the effect of general practice-level prescribing feedback on antibiotic prescribing in a real-world pragmatic cluster randomized controlled trial. Three hundred and forty general practices in four territorial Health Boards in NHS Scotland were randomized in Quarter 1, 2016 to receive four quarterly antibiotic-prescribing feedback reports or not, from Quarter 2, 2016 to Quarter 1, 2017. Reports included different clinical topics, benchmarking against national and health board rates, and behavioural messaging with improvement actions. The primary outcome was total antibiotic prescribing rate. There were 16 secondary prescribing outcomes and 5 hospital admission outcomes (potential adverse effects of reduced prescribing). The main evaluation timepoint was 1 year after the final report (Quarter 1, 2018), with an additional evaluation in the quarter after the final report (Quarter 2, 2017). Routine administrative NHS data were used to generate the feedback reports and analyse the effects. Total antibiotic prescribing rates were lower at the main evaluation timepoint in both intervention (1.83 versus baseline 1.93 prescriptions/1000 patients/day) and control (1.90 versus baseline 1.98) practices, with no evidence of intervention effect [adjusted rate ratio (ARR) 0.98 (95% CI 0.94-1.02; P = 0.35)]. At the additional timepoint, adjusted total antibiotic prescribing rates were 1.67 and 1.73 prescriptions/1000 patients/day, with evidence of a small intervention effect, ARR 0.99 (0.98-1.00; P = 0.03). This well-designed, practice-level antibiotic-prescribing feedback had limited evidence of additional effects in the context of decreasing antibiotic prescribing and an established national stewardship programme.


Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye.
RPE65, a protein expressed in cells of the retinal pigment epithelium of the eye, is essential for the synthesis by isomerohydrolase of 11-cis-retinal, the chromophore of rod and cone opsins. Recent work has established that RPE65 is a retinyl ester binding protein, and as all-trans-retinyl esters are the substrate for isomerohydrolase activity, the hypothesis has emerged that RPE65 serves to deliver substrate to this enzyme or complex. We bred mice with five distinct combinations of the RPE65 Leu450/Met450 variants (Leu/Leu, Met/Met, Leu/Met, Leu/-, and Met/-), measured in mice of each genotype the mole quantity of RPE65 per eye, and measured the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin to estimate the maximum rate of 11-cis-retinal synthesis in vivo. The quantity of RPE65 per eye ranged from 5.7 pmol (Balb/c) to 0.32 pmol (C57BL/6N x Rpe65(-)(/)(-)); the initial rate of rhodopsin regeneration was a Michaelis function of RPE65, where V(max) = 18 pmol/min per eye and K(m) = 1.7 pmol, and not dependent on the Leu450/Met450 variant. At RPE65 levels well below the K(m), the rate of production of 11-cis-retinal per RPE65 molecule was approximately 10 min(-)(1). Thus, the results imply that as a chaperone each RPE65 molecule can deliver retinyl ester to the isomerohydrolase at a rate of 10 molecules/min; should RPE65 itself be identified as the isomerase, each copy must be able to produce at least 10 molecules of 11-cis-retinal per minute.


Transthyretin (prealbumin) gene in human primary hepatic cancer.
From a subtracting cDNA library constructed from normal liver versus human primary hepatic cancer (PHC) a cDNA clone pG8 was isolated. Using it as a probe, RNA extracted from one human liver and 9 PHC samples were analyzed by Northern hybridization. As expected, its mRNA was highly expressed in liver; however, the expression was strikingly suppressed in PHC. Only weak signal was observed in 2 out of 9 PHC, while no signal was detectable in the other 7 samples. Utilizing pG8 as a probe, DNA from the same PHC specimens was analyzed after MspI digestion and Southern hybridization. Deletion of DNA fragment was observed in 4 out of 9 samples. In further study of cancer and non-cancerous liver from other 7 PHC patients, similar deletion of DNA fragments in cancer was observed in 4 out of 7 samples. After sequencing of the clone of 572 bp, it was unexpectedly found that pG8 was completely homologous to the coding sequence of transthyretin, TTR gene, as TTR (or prealbumin) gene has been known to be linked to a hereditary disorder, familial amyloidosis (FAP), and related to thyroxine transport and binding to retinol-RBP (the retinol binding protein) complex. This is the first report of a study on TTR in human primary hepatic cancer. Since TTR gene was strikingly suppressed in mRNA expression and possibly defective in its gene structure, it was strongly implicated that TTR might be an important gene marker or a candidate of anti-oncogene for human PHC. The biological activity of TTR gene is under study.


Cyclable Condensation and Hierarchical Assembly of Metastable Reflectin Proteins, the Drivers of Tunable Biophotonics.
Reversible changes in the phosphorylation of reflectin proteins have been shown to drive the tunability of color and brightness of light reflected from specialized cells in the skin of squids and related cephalopods. We show here, using dynamic light scattering, electron microscopy, and fluorescence analyses, that reversible titration of the excess positive charges of the reflectins, comparable with that produced by phosphorylation, is sufficient to drive the reversible condensation and hierarchical assembly of these proteins. The results suggest a two-stage process in which charge neutralization first triggers condensation, resulting in the emergence of previously cryptic structures that subsequently mediate reversible, hierarchical assembly. The extent to which cyclability is seen in the in vitro formation and disassembly of complexes estimated to contain several thousand reflectin molecules suggests that intrinsic sequence- and structure-determined specificity governs the reversible condensation and assembly of the reflectins and that these processes are therefore sufficient to produce the reversible changes in refractive index, thickness, and spacing of the reflectin-containing subcellular Bragg lamellae to change the brightness and color of reflected light. This molecular mechanism points to the metastability of reflectins as the centrally important design principle governing biophotonic tunability in this system.


Combined effects of dietary yeast supplementation and methoprene treatment on sexual maturation of Queensland fruit fly.
Yeast hydrolysate supplements promote maturation of many tephritid flies targeted for control using the sterile insect technique (SIT), including Queensland fruit fly (Bactrocera tryoni; 'Q-fly'). Recently, application of the juvenile hormone analogue methoprene has been demonstrated to further promote maturation in some species. We here investigate the separate and combined effects of yeast hydrolysate and methoprene treatment on sexual maturation of sterile male and female Q-flies. Two methods of applying methoprene solution were used; topical application to adults and dipping of pupae. Consistent with previous studies, access to yeast hydrolysate greatly increased maturation of both male and female Q-flies. Maturation was further promoted by methoprene treatment, with similar effects evident for males and females and for both application methods. For flies provided access to yeast hydrolysate supplements, methoprene treatment advanced maturation by approximately 2days. No effects of diet or methoprene treatment were found on timing of copulation or copula duration. Countering the positive effects on sexual maturation, dipping of pupae in methoprene/acetone solution did diminish emergence rates and flight ability indices, and increased rates of wing deformity. Promising results of the present study encourage further investigation of treatment methods that maximise maturation while minimising detrimental effects on other aspects of fly quality.


Dynamics of dental implants and orthodontics in today's periodontal prosthesis.
Periodontal prosthesis refers to multidisciplinary efforts to stop disease progression, correct deformities created by dental diseases, and establish a therapeutic occlusion to restore the form and function of the masticatory system. These efforts are critical for the long-term prognosis and maintenance of the guarded teeth and the overall prosthesis. This article presents a functionally and esthetically challenging case, which illustrates a multidisciplinary approach--specifically implantology and orthodontics--and how they impact on each other during treatment. The role and importance of dental implants and orthodontics in setting up a case is illustrated in this article.


Theory of spatial-frequency filtering by the human visual system. II. Performance limited by video noise.
A detailed theory of the detection of sinusoidal gratings displayed with suprathreshold luminous fluctuations is developed by employing a previous model of the visual and decision-making systems. An important feature of the model is the organization of the photoreceptors and decision-making system into a set of parallel, independent photoreceptive field (PRF)-decision center channels that function like a set of parallel spatial-frequency filters, each associated with an independent threshold detector. A technique is proposed for determining the modulation sensitivity functions (MSFs) of single detection channels by obtaining threshold modulation (MTN) data at a fixed sinusoidal grating frequency (v) while varying the center frequency (vc) of narrow-band luminous fluctuations caused by video noise (VN). The theory predicts that the ratio, at a given v, of MTN obtained as a function of vc to the MTN obtained without VN is proportional to the MSF of the particular channel for which the widths of the excitatory and inhibitory regions of the PRF equal a half-period of v. Good agreement between theoretical curves and experimental data appearing in the literature provides strong corroboration of the theory.


Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1.
Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARgamma agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARgamma agonist rosiglitazone on human podocytes. Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARgamma agonists may improve podocyte function in diabetic nephropathy.


Inhibition of akt/protein kinase B signaling by naltrindole in small cell lung cancer cells.
The phosphatidylinositol 3-kinase-Akt/protein kinase B (PKB) survival signaling is very important for cancer cell survival and growth. Constitutively active phosphatidylinositol 3-kinase-Akt/PKB signaling in small cell lung cancer (SCLC) is a major factor for the survival of SCLC cells. Inhibitors of this signaling pathway would be potential antitumor agents, particularly for SCLC. Here we report that naltrindole, which has been used as a classic delta opioid antagonist, inhibited growth and induced apoptosis in the three characteristic SCLC cell lines, NCI-H69, NCI-H345, and NCI-H510. Naltrindole treatment reduced constitutive phosphorylation of Akt/PKB on serine 473 and threonine 308 in cells. We found that the levels of constitutive phosphorylation of Akt/PKB on serine 473 correlate with the sensitivity of the three cell lines to naltrindole treatment. Furthermore, naltrindole treatment not only reduced the phosphorylation of the Akt/PKB upstream kinase phosphoinositide-dependent kinase-1, but also its downstream effectors glycogen synthase kinase-3beta and the Forkhead transcription factors AFX and FKHR. DNA array analysis of 205 apoptosis-related genes indicated that some Akt/PKB-dependent genes were either up- or down-regulated by naltrindole. Flow cytometric and microscopic analyses clearly showed that naltrindole induced apoptosis in SCLC cells. RNA interference experiments confirmed that naltrindole-induced cell death was associated with the Akt/PKB survival pathway. Together, these results show that naltrindole is a new inhibitor of the Akt/PKB signaling pathway, suggesting that naltrindole could be a potential lead for the development of a new type of inhibitors that target the constitutively active Akt/PKB signaling-dependent SCLC cells.


Accurate prediction of the electronic properties of low-dimensional graphene derivatives using a screened hybrid density functional.
Over the last several years, low-dimensional graphene derivatives, such as carbon nanotubes and graphene nanoribbons, have played a central role in the pursuit of a plausible carbon-based nanotechnology. Their electronic properties can be either metallic or semiconducting depending purely on morphology, but predicting their electronic behavior has proven challenging. The combination of experimental efforts with modeling of these nanometer-scale structures has been instrumental in gaining insight into their physical and chemical properties and the processes involved at these scales. Particularly, approximations based on density functional theory have emerged as a successful computational tool for predicting the electronic structure of these materials. In this Account, we review our efforts in modeling graphitic nanostructures from first principles with hybrid density functionals, namely the Heyd-Scuseria-Ernzerhof (HSE) screened exchange hybrid and the hybrid meta-generalized functional of Tao, Perdew, Staroverov, and Scuseria (TPSSh). These functionals provide a powerful tool for quantitatively studying structure-property relations and the effects of external perturbations such as chemical substitutions, electric and magnetic fields, and mechanical deformations on the electronic and magnetic properties of these low-dimensional carbon materials. We show how HSE and TPSSh successfully predict the electronic properties of these materials, providing a good description of their band structure and density of states, their work function, and their magnetic ordering in the cases in which magnetism arises. Moreover, these approximations are capable of successfully predicting optical transitions (first and higher order) in both metallic and semiconducting single-walled carbon nanotubes of various chiralities and diameters with impressive accuracy. This versatility includes the correct prediction of the trigonal warping splitting in metallic nanotubes. The results predicted by HSE and TPSSh provide excellent agreement with existing photoluminescence and Rayleigh scattering spectroscopy experiments and Green's function-based methods for carbon nanotubes. This same methodology was utilized to predict the properties of other carbon nanomaterials, such as graphene nanoribbons. Graphene nanoribbons may be viewed as unrolled (and passivated) carbon nanotubes. However, the emergence of edges has a crucial impact on the electronic properties of graphene nanoribbons. Our calculations have shown that armchair nanoribbons are predicted to be nonmagnetic semiconductors with a band gap that oscillates with their width. In contrast, zigzag graphene nanoribbons are semiconducting with an electronic ground state that exhibits spin polarization localized at the edges of the carbon nanoribbon. The spatial symmetry of these magnetic states in graphene nanoribbons can give rise to a half-metallic behavior when a transverse external electric field is applied. Our work shows that these properties are enhanced upon different types of oxidation of the edges. We also discuss the properties of rectangular graphene flakes, which present spin polarization localized at the zigzag edges.


[Optimisation of possible success in an IVF program].
The success of an IVF (in vitro fertilisation) programme is mainly dependent an the quality of the IVF laboratory. Beside this the quality of ovarian stimulation play another, but not as important role. In the 4 (th) quarter of 2003 we have changed the culture media for oocytes and embryos in the IVF laboratory of ENDOKRINOLOGIKUM Hamburg from simple to complex media (G-1 version 3). As protein source we used HSA-solution. Before embryo transfer equilibration for 1 hour in EmbryoGlue was performed, which contains hyaluran. Hyaluran is said to improve implantation rates by different ways. The media were bought from Vitrolife Schweden AB (Kungsbacka, Schweden) via a German distributor. These data were compared to those of two previous 3-month-periods (3rd quarter 2003 und 4th quarter 2002). The study design was prospective, with two historical control groups. In the three study periods 255, 343 and 503 patients were treated by either conventional IVF or ICSI (intracytoplasmic sperm injection). The cohorts were comparable regarding their anamnestic data. The number of patients treated which the GnRH antagonist cetrorelix (Cetrotide) significantly increased from 24.9 % to 49.5 % and 60.9 %. Under this protocol significantly less oocytes were retrieved (10.23 vs. 8.73 vs. 8.88), leading to slightly less embryos (1.94 vs. 1.91 vs. 1.85, not significant, n. s.). The cumulative embryo score significantly decreased from 28.58 to 24.10. It was significantly higher in the study cohort as compared to the two control cohorts (32.44). The clinical pregnancy rate significantly increased to 25.29 % in the study cohort as compared to the two historical control cohorts (15.02 % and 18.75 %). We could achieve a significant improvement in the success rates of our IVF program by switch from simple to more complex culture media in the IVF laboratory. The use of the GnRH antagonist protocol with cetrorelix (Cetrotide) lead to more comfort to the patients but did not influence the results.


Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50).
Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes beta-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a beta-arrestin-independent but dynamin-dependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.


Invisible inventors. A historical overview of creative midwives and nurses.
This historical overview documents women's inventions for providing nursing care dating from 1608 to 1928. The word invention is broadly defined and includes ideas that created therapeutic activities, caregiving environments, and specific devices for care. It focuses on the creative contributions of early outstanding midwives and other practicing nurses around the late 19th and early 20th centuries. Sources include illustrations of patented items, practical hints published in early issues of the American Journal of Nursing (AJN), published translations of original documents from Europe, original historical research on women in Europe and America, and records from the U.S. Government Patent Office. The role of nurses in creating and developing tools and methods for providing nursing care has gone unrecognized. It is important to clarify the record of women's and nurses' inventiveness.


[Fecal calprotectin in complex diagnostics of intestinal diseases].
The review of evidential base is presented in article about a place of a noninvasive laboratory method--dough on Calprotectin in complex diagnostics of diseases of intestines.


Prevalence of factitious disorder with psychological symptoms in hospitalized patients.
Factitious disorder is characterized by the invention, production or falsification of physical and psychological symptoms that feign a physical or mental illness. Although it is not rare to find symptoms that seem to be factitious among psychiatric patients (both outpatients and inpatients), we have never been able to confirm this suspicion. Once we had established the suspicion criteria for factitious disorder with psychological symptoms, we discovered that 8% of the patients admitted to an inpatient psychiatric unit had factitious symptoms. The patients were mostly women with a mean age of 36.2 years. The most frequent symptoms were non-consistent response to treatment, worsening of the symptoms when faced with the perspective of a discharge plan, disappearance of the symptoms just after being admitted and intense relationship with other patients or staff during the hospitalization. Although it is not possible to determine the method used in the factitious production of the psychological systems accurately, close observation of the patients helps to infer that 75% of the patients exaggerate the psychological symptoms currently present or those occurring in the past and thus experienced at some time during their lifespan. Invention of psychological symptoms never felt before by the patient and deliberate intervention in the psychiatric treatment to modify the evolution of the illness were less frequent. A total of 25% of the patients with suspicion criteria for factitious disorder with psychological symptoms also had physical symptoms considered to be factitious during the hospitalization and 62% of patients with factitious symptoms developed intense relationships with other patients.


In Vitro Susceptibility Testing of a Novel Benzimidazole, SPR719, against Nontuberculous Mycobacteria.
Nontuberculous mycobacterium (NTM) infections are increasing globally. The Mycobacterium avium complex (MAC) and Mycobacterium abscessus are the most frequently encountered NTM among clinical laboratories, and treatment options are extremely limited. In this study, the in vitro potency of a novel benzimidazole, SPR719, the microbiologically active form of the orally available prodrug SPR720, was tested against several species of NTM. MICs were determined for 161 isolates of NTM of 13 taxa (seven species, three subspecies, and three groups/complexes) in cation-adjusted Mueller-Hinton Broth, as described and recommended by the Clinical and Laboratory Standards Institute (CLSI M24-A2). Comparator antimicrobials included amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, minocycline, moxifloxacin, tigecycline, and trimethoprim-sulfamethoxazole (TMP-SMX) for the rapidly growing mycobacteria (RGM), amikacin and clarithromycin for the MAC, and amikacin, ciprofloxacin, clarithromycin, doxycycline, linezolid, moxifloxacin, rifabutin, rifampin, and TMP-SMX for the other slowly growing NTM. SPR719 was found to be potent against multiple clinical strains of NTM with an MIC50 range of 0.25 to 4 μg/ml for several species of NTM. These findings support the further advancement of SPR720 for the treatment of NTM disease.


The Tp38 (TpMglB-2) lipoprotein binds glucose in a manner consistent with receptor function in Treponema pallidum.
A 38-kDa lipoprotein of Treponema pallidum (Tp38) was predicted to be a periplasmic sugar-binding protein based on its sequence similarity to the glucose/galactose-binding (MglB) protein of Escherichia coli (P. S. Becker, D. R. Akins, J. D. Radolf, and M. V. Norgard, Infect. Immun. 62:1381-1391, 1994). Inasmuch as glucose is believed to be the principal, if not sole, carbon and energy source for T. pallidum and is readily available to the spirochete during its obligate infection of humans, we hypothesized that Tp38 may serve as the organism's requisite glucose receptor. For the present study, a nonacylated recombinant form of Tp38 was coexpressed with GroES and GroEL in E. coli to facilitate the isolation of soluble, properly folded Tp38. The highly sensitive method of intrinsic fluorescence spectroscopy, predicated on the manner in which tryptophan residues reside and move within protein microenvironments, was then used to assess sugar binding to Tp38. The intrinsic fluorescence of Tp38 was essentially unaltered when it was exposed to D-mannose, D-fucose, D-ribose, L-glucose, or L-galactose, but it changed markedly in the presence of D-glucose, and to a lesser extent, D-galactose, indicating binding. The K(d) values for D-glucose and D-galactose binding to Tp38 were 152.2 +/- 20.73 nM and 251.2 +/- 55.25 nM, respectively. Site-directed mutagenesis of Trp-145, a residue postulated to contribute to the sugar-binding pocket in a manner akin to the essential Trp-183 in E. coli MglB, abolished Tp38's conformational change in response to D-glucose. The combined data are consistent with Tp38 serving as a glucose receptor for T. pallidum. These findings potentially have important implications for syphilis pathogenesis, particularly as they may pertain to glucose-mediated chemotactic responses by T. pallidum.


Detection of nitric oxide production from a perfused organ by a luminol-H2O2 system.
Nitric oxide, thought to be the endothelium-derived relaxing factor (EDRF), is involved in intra- and intercellular signalling in various tissues. A system for the continuous detection of NO in the picomolar range from a perfused organ is described. The detection is based upon the chemiluminescence reaction between NO and the luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-H2O2 system. The chemiluminescence is due to the formation of peroxynitrite from NO and H2O2. The luminol-H2O2 system is specifically reactive to NO, so that other nitrogen-containing compounds, (organic nitrite, organic nitrate, and thio-nitroso compounds) or endothelium-derived compounds do not interfere. The limit of determination was approximately 100 fM. This system has been used to measure continuous NO release from isolated perfused rat kidney and the simultaneous changes of perfusion pressure. In Wistar rats basal NO release was 85 +/- 11 fmol/min-1 (g of kidney weight)-1 (39 pM in the perfusate), and acetylcholine increased NO release dose dependently with a concomitant pressure reduction. The changes in NO release were always associated with mirror image changes in the perfusion pressure. Simple pressure reduction did not interfere with the chemiluminescence. Precise titration data as well as results of some preliminary experiments using this method are presented.


Metabolic engineering of Pichia pastoris for malic acid production from methanol.
The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by-product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.


Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination.
Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.