DockoMatic: automated peptide analog creation for high throughput virtual screening.
The purpose of this manuscript is threefold: (1) to describe an update to DockoMatic that allows the user to generate cyclic peptide analog structure files based on protein database (pdb) files, (2) to test the accuracy of the peptide analog structure generation utility, and (3) to evaluate the high throughput capacity of DockoMatic. The DockoMatic graphical user interface interfaces with the software program Treepack to create user defined peptide analogs. To validate this approach, DockoMatic produced cyclic peptide analogs were tested for three-dimensional structure consistency and binding affinity against four experimentally determined peptide structure files available in the Research Collaboratory for Structural Bioinformatics database. The peptides used to evaluate this new functionality were alpha-conotoxins ImI, PnIA, and their published analogs. Peptide analogs were generated by DockoMatic and tested for their ability to bind to X-ray crystal structure models of the acetylcholine binding protein originating from Aplysia californica. The results, consisting of more than 300 simulations, demonstrate that DockoMatic predicts the binding energy of peptide structures to within 3.5 kcal mol(-1), and the orientation of bound ligand compares to within 1.8 Å root mean square deviation for ligand structures as compared to experimental data. Evaluation of high throughput virtual screening capacity demonstrated that Dockomatic can collect, evaluate, and summarize the output of 10,000 AutoDock jobs in less than 2 hours of computational time, while 100,000 jobs requires approximately 15 hours and 1,000,000 jobs is estimated to take up to a week.


The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties.
TET aminopeptidases assemble as large homo-dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.


In vivo efficacy of artemether-lumefantrine and artesunate-amodiaquine for uncomplicated Plasmodium falciparum malaria in Malawi, 2014.
Malaria causes significant morbidity in Malawi, with an estimated 5 million cases in 2014. Artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) are the first- and second-line treatments for uncomplicated malaria, respectively, but emerging resistance threatens their efficacy. In order to understand whether AL and ASAQ remain efficacious for the treatment of uncomplicated Plasmodium falciparum malaria in Malawi, a therapeutic efficacy trial was conducted. During March-July 2014, febrile children aged 6-59 months with microscopy-confirmed uncomplicated P. falciparum malaria (1000-200,000 parasites/μL) were enrolled in a 28-day randomized in vivo efficacy trial at three sites: one each in northern (Karonga), central (Nkhotakota) and southern (Machinga) Malawi. The study was powered to estimate site-specific efficacy for AL and overall efficacy for ASAQ, with 3:1 randomization to AL or ASAQ. Blood was collected for malaria microscopy and molecular testing on days 0-3, 7, 14, 21, and 28. Recrudescence and reinfection were differentiated using polymerase chain reaction (PCR) genotyping of merozoite surface protein. The primary outcome was the PCR-corrected day 28 Kaplan-Meier cumulative success rate. A total of 452 children were enrolled; 303/338 (89 %) and 98/114 (86 %) reached a study endpoint in AL and ASAQ arms, respectively. All treatment failures occurred after day 3. The day 28 uncorrected cumulative success rate was 97.1 % (95 % confidence interval [CI]: 93.9-100 %) for ASAQ and 76.8 % (95 % CI 72.1-81.5 %) for AL, with 82.5 % (95 % CI 75.4-89.7 %), 69 % (95 % CI 59.9-78.1 %), and 78.2 % (95 % CI 70.2-86.3 %) success in the northern, central, and southern regions, respectively. The day 28 PCR-corrected cumulative success rate was 99 % (95 % CI 97.2-100 %) in the ASAQ arm and 99.3 % (95 % CI 98.3-100 %) in the AL arm, with 98-100 % efficacy in each site. As evidenced by the day 28 PCR-corrected cumulative success rates, both AL and ASAQ remain efficacious treatments for uncomplicated malaria in Malawi. The lower uncorrected efficacy in the AL arm compared to ASAQ may be explained by the shorter half-life of lumefantrine (3-6 days) compared to amodiaquine (9-18 days). The high reinfection rate suggests that there is a continued need to scale-up effective malaria prevention interventions.


Swollen hexagonal liquid crystals as smart nanoreactors: implementation in materials chemistry for energy applications.
Materials are the key roadblocks for the commercialization of energy conversion devices in fuel cells and solar cells. Significant research has focused on tuning the intrinsic properties of materials at the nanometer scale. The soft template mediated controlled fabrication of advanced nanostructured materials is attracting considerable interest due to the promising applications of these materials in catalysis and electrocatalysis. Swollen hexagonal lyotropic liquid crystals (SLCs) consist of oil-swollen surfactant-stabilized 1D, 2D or 3D nanometric assemblies regularly arranged in an aqueous solvent. Interestingly, the characteristic size of the SLCs can be controlled by adjusting the volume ratio of oil to water. The non-polar and/or polar compartments of the SLCs can be doped with guest molecules and used as nanoreactors for the synthesis of various metals (Pt, Pd, Au, etc.), conducting polymers and composite nanostructures with controlled size and shape. 1D, 2D and 3D mono- and bimetallic nanostructures of controlled composition and porosity can also be fabricated. These materials have demonstrated impressive enhancements of their electrochemical properties as compared to their bulk counterparts and have been identified as promising for further implementation in energy harvesting applications. In this review article, recent research materials are described regarding the development of functional materials with much improved performances for catalysis applications. This review addresses a brief overview of swollen hexagonal mesophases as nanoreactors, describes examples of nanostructured materials synthesized in these nanoreactors, shows several examples of the energy conversion applications in solar light harvesting, fuel cells etc. and also summarizes the associated reaction mechanisms developed in the recent literature for enhanced catalytic activity.


Absence of Escherichia coli, Listeria monocytogenes, and Klebsiella pneumoniae antigens within inflammatory bowel disease tissues.
Escherichia coli, listeria, and streptococcal antigens have been found in Crohn's disease tissues. Antibodies to Klebsiella pneumoniae have been found in patients with inflammatory bowel disease and ankylosing spondylitis. The presence of these bacterial antigens in Crohn's granulomas would be of aetiological interest, while their presence in ulcers alone would be more likely to indicate secondary infection. To investigate inflammatory bowel disease tissues for the presence of these bacteria. Formalin fixed, paraffin processed sections from 53 patients (19 ulcerative colitis, 23 Crohn's disease; 11 normal tissues from cancer resections) were studied by immunohistochemistry. Control tissue consisted of normal human small bowel injected submucosally with either E coli, Listeria monocytogenes, Proteus mirabilis, or Klebsiella pneumoniae serotypes K2, 3, 17, 21, 26, 36, and 50, and colonic biopsies from a child with E coli 0114 infection. Tissues were stained by Gram-Twort, and with specific antibodies for E coli (Dako B357), L monocytogenes (Difco 2302-50), and K pneumoniae (Biogenesis 5580-5208) using an immunoperoxidase technique. Positive staining for E coli was observed on the luminal surface epithelium and in ulcers in 35% of Crohn's disease patients, 26% of ulcerative colitis patients, and no normal controls. Superficial staining for L monocytogenes was observed in one case of ulcerative colitis only. Staining for K pneumoniae was observed in one case of ulcerative colitis and one of Crohn's disease. No granulomas, giant cells, or germinal centres stained positively for any of the three bacterial antigens. These data do not support a primary role for E coli, L monocytogenes, and K pneumoniae in inflammatory bowel disease. The presence of E coli antigens in ulcers suggests secondary infection in these lesions.


Hexavalent chromium reduction and energy recovery by using dual-chambered microbial fuel cell.
Microbial fuel cell (MFC) technology is utilized to treat hexavalent chromium (Cr(VI)) from wastewater and to generate electricity simultaneously. The Cr(VI) is bioelectrochemically reduced to non-toxic Cr(III) form in the presence of an organic electron donor in a dual-chambered MFC. The Cr(VI) as catholyte and artificial wastewater inoculated with anaerobic sludge as anolyte, Cr(VI) at 100 mg/L was completely removed within 48 h (initial pH value 2.0). The total amount of Cr recovered was 99.87% by the precipitation of Cr(III) on the surface of the cathode. In addition to that 78.4% of total organic carbon reduction was achieved at the anode chamber within 13 days of operation. Furthermore, the maximum power density of 767.01 mW/m² (2.08 mA/m²) was achieved by MFCs at ambient conditions. The present work has successfully demonstrated the feasibility of using MFCs for simultaneous energy production from wastewater and reduction of toxic Cr(VI) to non-toxic Cr(III).


Blood pressure and metabolic changes during dietary L-arginine supplementation in humans.
Dietary L-arginine supplementation has been proposed to reverse endothelial dysfunction in such diverse pathophysiologic conditions as hypercholesterolemia, coronary heart disease, and some forms of animal hypertension. In particular, chronic oral administration of L-arginine prevented the blood pressure rise induced by sodium chloride loading in salt-sensitive rats. To investigate the effects of L-arginine-rich diets on blood pressure and metabolic and coagulation parameters we performed a single-blind, controlled, crossover dietary intervention in six healthy volunteers. The subjects (aged 39+/-4 years, body mass index [BMI] 26+/-1 kg/m2, mean +/- SEM) received, in random sequence, three different isocaloric diets, each for a period of 1 week (Diet 1: control; Diet 2: L-arginine enriched by natural foods; Diet 3: identical to Diet 1 plus oral L-arginine supplement). Sodium intake was set at a constant level (about 180 mmol/day) throughout the three study periods. A blood pressure decrease was observed with both L-arginine-rich diets (Diet 2 v 1, SBP: -6.2 mm Hg [95% CI: -0.5 to -11.8], DBP: -5.0 mm Hg [-2.8 to -7.2]; Diet 3 v 1, SBP: -6.2 mm Hg [-1.8 to -10.5], DBP: -6.8 mm Hg [-3.0 to -10.6]). A slight increase in creatinine clearance (P = .07) and a fall in fasting blood glucose (P = .008) occurred after Diet 3 and, to a lesser extent, after Diet 2. Serum total cholesterol (P = .06) and triglyceride (P = .009) decreased and HDL cholesterol increased (P = .04) after Diet 2, but not after Diet 3. These results indicate that a moderate increase in L-arginine significantly lowered blood pressure and affected renal function and carbohydrate metabolism in healthy volunteers.


High-performance reconstruction of microscopic force fields from Brownian trajectories.
The accurate measurement of microscopic force fields is crucial in many branches of science and technology, from biophotonics and mechanobiology to microscopy and optomechanics. These forces are often probed by analysing their influence on the motion of Brownian particles. Here we introduce a powerful algorithm for microscopic force reconstruction via maximum-likelihood-estimator analysis (FORMA) to retrieve the force field acting on a Brownian particle from the analysis of its displacements. FORMA estimates accurately the conservative and non-conservative components of the force field with important advantages over established techniques, being parameter-free, requiring ten-fold less data and executing orders-of-magnitude faster. We demonstrate FORMA performance using optical tweezers, showing how, outperforming other available techniques, it can identify and characterise stable and unstable equilibrium points in generic force fields. Thanks to its high performance, FORMA can accelerate the development of microscopic and nanoscopic force transducers for physics, biology and engineering.


Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy.
Two-dimensional (2D) (13)C-(13)C NMR correlation spectra were collected on (13)C-enriched dragline silk fibers produced from Nephila clavipes spiders. The 2D NMR spectra were acquired under fast magic-angle spinning (MAS) and dipolar-assisted rotational resonance (DARR) recoupling to enhance magnetization transfer between (13)C spins. Spectra obtained with short (150 ms) recoupling periods were utilized to extract distinct chemical shifts for all carbon resonances of each labeled amino acid in the silk spectra, resulting in a complete resonance assignment. The NMR results presented here permit extraction of the precise chemical shift of the carbonyl environment for each (13)C-labeled amino acid in spider silk for the first time. Spectra collected with longer recoupling periods (1 s) were implemented to detect intermolecular magnetization exchange between neighboring amino acids. This information is used to ascribe NMR resonances to the specific repetitive amino acid motifs prevalent in spider silk proteins. These results indicate that glycine and alanine are both present in two distinct structural environments: a disordered 3(1)-helical conformation and an ordered beta-sheet structure. The former can be ascribed to the Gly-Gly-Ala motif while the latter is assigned to the poly(Ala) and poly(Gly-Ala) domains.


Early post-anaesthesia recovery parameters - a prospective observational study.
To evaluate the patients' baseline condition upon arrival in the PACU as a method of assessment of the quality of anaesthesia, and to establish a model for future comparisons. Prospective observational study. Surgical PACU in an academic tertiary hospital. All patients (n = 11,241) arriving in our hospital's recovery units after elective surgery. In this observational study, clinical data, vital signs and comfort parameters were collected from surgical patients who arrived in the PACU. For each parameter, its frequency distribution or percentage of occurrence was determined. The incidence of anaesthesia associated side effects such as hypoxemia, cold extremities, shivering and/or vomiting was 5%. The incidence of nausea, sore throat, headache and/or pruritus was 9%. Sore throat occurred in 4.8% of intubated patients, in 4% after laryngeal mask insertions and in 3.6% with no usage of any airway device. From all patients 48% had no pain at all (VAS = 0), 31% had low pain scores (VAS 1-3) and 16% had moderate (VAS 4-6) pain levels, while 5% indicated severe pain (VAS 7-10). 97.5% of patients were normothermic (35.0-37.3°C), 77% had normal heart rate (60-100 beats per minute) and 74% had normal systolic blood pressure (90-140 mmHg). After use of neuromuscular relaxants, moderate clinical signs of residual curarisation (2 of 3 clinical criteria positive) were observed in 1% and slight clinical signs of residual curarisation (1 of 3 clinical criteria positive) were observed in 22.4% of patients. These findings were collected in all patients, independently whether they had reversal of neuromuscular relaxation. Compared with the figures published in the literature, we report a lower incidence and severity of anaesthesia related side effects, measured as baseline data, upon patients' arrival in the post anaesthesia care unit. The baseline data may serve as a model to trigger specific interventions aimed at improving the quality of anaesthetic care, which could be assessed in future investigations.


Comparison of Heterosubtypic Protection in Ferrets and Pigs Induced by a Single-Cycle Influenza Vaccine.
Influenza is a major health threat, and a broadly protective influenza vaccine would be a significant advance. Signal Minus FLU (S-FLU) is a candidate broadly protective influenza vaccine that is limited to a single cycle of replication, which induces a strong cross-reactive T cell response but a minimal Ab response to hemagglutinin after intranasal or aerosol administration. We tested whether an H3N2 S-FLU can protect pigs and ferrets from heterosubtypic H1N1 influenza challenge. Aerosol administration of S-FLU to pigs induced lung tissue-resident memory T cells and reduced lung pathology but not the viral load. In contrast, in ferrets, S-FLU reduced viral replication and aerosol transmission. Our data show that S-FLU has different protective efficacy in pigs and ferrets, and that in the absence of Ab, lung T cell immunity can reduce disease severity without reducing challenge viral replication.


Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction.
Hepatitis A virus (HAV) differs from other members of the family Picornaviridae in that the cleavage of the polyprotein at the 2A/2B junction, commonly considered to be the primary polyprotein cleavage by analogy with other picornaviruses, is mediated by 3C(pro), the only proteinase encoded by the virus. However, it has never been formally demonstrated that the 2A/2B junction is the site of primary cleavage, and the actual function of the 2A sequence, which lacks homology with sequence of other picornaviruses, remains unknown. To determine whether 2A functions in cis as a precursor with the nonstructural proteins, we constructed dicistronic HAV genomes in which a heterologous picornaviral internal ribosome entry site was inserted at the 2A/2B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possible cis replication function spanning the 2A/2B junction. However, infectious virus was recovered from recombinant HAV genomes containing exogenous protein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C(pro) cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not dependent upon the length of the inserted sequence. An HAV recombinant containing a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were deleted from replicating viruses, but this did not result from homologous recombination at the flanking 3C(pro) cleavage sites, since the 5' and 3' segments of the inserted sequence were retained in the deletion mutants. These results indicate that the HAV polyprotein can tolerate an insertion at the 2A/2B junction and that the 2A polypeptide does not function in cis as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding sequences inserted at the 2A/2B junction are novel and potentially useful protein expression vectors.


Hair as a long-term retrospective cortisol calendar in orang-utans (Pongo spp.): new perspectives for stress monitoring in captive management and conservation.
This study examined whether the method of hair cortisol analysis is applicable to orang-utans (Pongo spp.) and can help to advance the objective monitoring of stress in non-human primates. Specifically, we examined whether fundamental prerequisites for hair cortisol analysis are given in orang-utans and, subsequently, whether segmental hair analysis may provide a retrospective calendar of long-term cortisol levels. For this, hair samples were examined from 71 zoo-living orang-utans (38 males, mean age=22.5years; 33 females, mean age=24years) for which detailed records of past living conditions were available. Hair samples were cut from defined body regions and were analyzed either in full length or in segments. Results showed that hair cortisol concentrations (HCC) were unrelated to age or sex of the individual animal. HCC were found to be higher in orang-utans, with perceived long-term stressful periods (mean HCC=43.6±26.5pg/mg, n=13) compared to animals without perceived stressful periods (19.3±5.5pg/mg, n=55, P<0.001). In non-stressed animals, segmental hair analyses revealed that HCC was stable along the hair shaft even when hair reached >40cm. The possibility of obtaining a retrospective calendar of stress-related cortisol changes through hair analysis was further supported by data of three case studies showing close correspondence between the segmental HCC results and keeper reports of stress exposure during the respective time periods. Finally, low within-animal variation in HCC from different body regions (CV%: 14.3) suggested that this method may also be applicable to naturally shed hair, e.g., as found in nests of wild orang-utans and other great apes. Therefore, using HCC may provide an ideal non-invasive tool for both captive management as well as conservation in orang-utans and potentially other great apes.


Phase I trial of intravenous peptide-pulsed dendritic cells in patients with metastatic melanoma.
Sixteen patients with metastatic stage IV melanoma were treated with use of intravenous infusions of dendritic cells (DC) derived by incubation of plastic-adherent peripheral blood mononuclear cells (PBMC) with IL-4 and GM-CSF for 8 days in serumless AIM-V medium, followed by overnight pulsing with peptides. The tyrosinase368-376 (370D) and gp100(209-217 (210M)) peptides restricted to HLA class I A*0201 each differed from wild type by one amino acid modified to increase HLA binding. Median age was 49, with nine men and seven women. All patients, except one, had visceral disease. Patients received escalating doses of peptide-pulsed DCs at 10e7, 3 x 10e7, and 10e8 cells/dose twice at 2 weeks apart, with toxicity and clinical and immune responses as the principal endpoints. The first infusion of DCs was fresh, and frozen DCs were given for the second infusion of each cycle. Mean DC purity by flow cytometry was 49%, with a mean HLA-DR level of 57%, CD86 of 41%, CD58 of 46%, and mean CD14 cells of 0.9%. Toxicity was minimal, with two patients having transient grade III DC-related toxicity. Ten patients received one cycle of treatment and six patients received two cycles of treatment. One patient had a complete remission (CR) of lung and pleural disease after two cycles of DC therapy. Two additional patients had stable disease and two patients had mixed responses. Overall immunity was assessed by recall skin testing with peptides, gamma interferon ELISA assays of peptide specific cytolytic T cell (CTL) stimulated twice with peptide, IL-2, and IL-7 over 24 days, and peptide-specific tetramer assays performed before and after vaccination. Five of 16 patients had an immune response to gp100 or tyrosinase by gamma interferon ELISA assay; four of five were clinically stable or had tumor regression. These data suggest that melanoma antigen peptide-pulsed DC given intravenously are not toxic, and regression or stability of tumor appeared to correlate with the detection of a peptide-specific immune response in the peripheral blood.


Genomic organization of the rat alpha 2u-globulin gene cluster.
The alpha 2u-globulin are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many alpha 2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 alpha 2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the alpha 2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the alpha 2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing alpha 2u-globulin genes indicated that the alpha 2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the alpha 2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes.


Experiments in long-distance holographic imagery.
Long-range holographic experiments have been performed over distances as large as 12 km. Holograms and images of retroreflector and Scotchlite objects were obtained. The reference wave was supplied by a retroreflector placed next to the object, and the source of illumination was a Q-switched ruby laser. Resolutions obtained varied with atmospheric seeing conditions, ranging from 3 see of arc to 0.5 see of arc. The primary practical problem encountered was detector saturation, a consequence of the limited dynamic range of photographic film. The use of a low contrast developer only partially alleviates this problem.


Discovery and characterization of ACE2 - a 20-year journey of surprises from vasopeptidase to COVID-19.
Angiotensin-converting enzyme (ACE) is a zinc membrane metallopeptidase that plays a key role in regulating vasoactive peptide levels and hence cardiovascular activity through its conversion of angiotensin I (Ang I) to Ang II and its metabolism of bradykinin. The discovery of its homologue, ACE2, 20 years ago has led to intensive comparisons of these two enzymes revealing surprising structural, catalytic and functional distinctions between them. ACE2 plays multiple roles not only as a vasopeptidase but also as a regulator of amino acid transport and serendipitously as a viral receptor, mediating the cellular entry of the coronaviruses causing severe acute respiratory syndrome (SARS) and, very recently, COVID-19. Catalytically, ACE2 functions as a monocarboxypeptidase principally converting the vasoconstrictor angiotensin II to the vasodilatory peptide Ang-(1-7) thereby counterbalancing the action of ACE on the renin-angiotensin system (RAS) and providing a cardioprotective role. Unlike ACE, ACE2 does not metabolise bradykinin nor is it inhibited by classical ACE inhibitors. However, it does convert a number of other regulatory peptides in vitro and in vivo. Interest in ACE2 biology and its potential as a possible therapeutic target has surged in recent months as the COVID-19 pandemic rages worldwide. This review highlights the surprising discoveries of ACE2 biology during the last 20 years, its distinctions from classical ACE and the therapeutic opportunities arising from its multiple biological roles.


Nephrogenic diabetes insipidus in mice caused by deleting COOH-terminal tail of aquaporin-2.
In mammals, the hormonal regulation of water homeostasis is mediated by the aquaporin-2 water channel (Aqp2) of the collecting duct (CD). Vasopressin induces redistribution of Aqp2 from intracellular vesicles to the apical membrane of CD principal cells, accompanied by increased water permeability. Mutations of AQP2 gene in humans cause both recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. In this study, we generated a line of mice with the distal COOH-terminal tail of the Aqp2 deleted (Aqp2(Delta230)), including the protein kinase A phosphorylation site (S256), but still retaining the putative apical localization signal (221-229) at the COOH-terminal. Mice heterozygous for the truncation appear normal. Homozygotes are viable to adulthood, with reduced urine concentrating capacity, increased urine output, decreased urine osmolality, and increased daily water consumption. Desmopressin increased urine osmolality in wild-type mice but had no effect on Aqp2(Delta230/Delta230) mice. Kidneys from affected mice showed CD and pelvis dilatation and papillary atrophy. By immunohistochemical and immunoblot analyses using antibody against the NH(2)-terminal region of the protein Aqp2(Delta230/Delta230) mice had a markedly reduced protein abundance. Expression of the truncated protein in MDCK cells was consistent with a small amount of functional expression but no stimulation. Thus we have generated a mouse model of NDI that may be useful in studying the physiology and potential therapy of this disease.


Vertex balding, plasma insulin-like growth factor 1, and insulin-like growth factor binding protein 3.
A recent report suggested that men with vertex balding have higher levels of plasma insulin-like growth factor 1 (IGF-1). The association of its major carrier protein, insulin-like growth factor binding protein 3 (IGFBP-3), with male pattern hair loss has not been examined. We evaluated the relations of plasma concentrations of IGF-1 and IGFBP-3 with vertex balding in middle-aged and elderly men. Participants were 431 male members of the Health Professionals Follow-up Study who responded to a question in 1992 on their hair pattern at 45 years of age and who were 47 to 81 years old when they provided a blood specimen in 1993-1994. Odds ratios (ORs) of vertex balding associated with IGF-1 and IGFBP-3 were estimated from logistic regression models mutually adjusting for each other and controlling for age at blood draw. Of the 431 men, 128 had vertex balding at age 45. Compared with men who were not balding, for a 1 standard deviation increase in plasma IGF-1 level (72.4 ng/mL), the OR for vertex balding was 1. 31 (95% CI, 0.95-1.81). For a 1 standard deviation increase in plasma IGFBP-3 (957 ng/mL), the OR for vertex balding was 0.62 (95% CI, 0.44-0.88). Older men with vertex balding have lower circulating levels of IGFBP-3 and higher levels of IGF-1 when controlling for IGFBP-3 level.


Angiotensin-converting enzyme inhibitor-induced angioedema--a dangerous new epidemic.
Angioedema is a sudden localised and often asymmetric swelling of the skin or mucous membranes caused by transient increased endothelial permeability causing plasma extravasation. In the last decades the incidence of severe angioedema involving the upper airways and even fatal outcome due to asphyxia has increased. This is mainly due to pharmaceuticals such as angiotensin converting enzyme-inhibitors, which are extensively used worldwide. Some aspects of the pathophysiology have been elucidated and the vasoactive molecule bradykinin is shown to be one of the main causative agents. The diagnosis is often delayed and traditional treatment usually ineffective. Complement C1 inhibitor concentrate and bradykinin receptor antagonists, normally used to treat patients with hereditary angioedema, have shown good results when used in patients with bradykinin-mediated angioedema. This review discusses the disease, prognosis and treatment options.


Transient occlusion of bilateral internal iliac arteries facilitates bloodless operative field in subcapsular prostatectomy.
Transurethral resection of the prostate is the gold standard of surgical treatment for benign prostatic hyperplasia (BPH). Nevertheless, open subcapsular prostatectomy is still performed for large BPH. While enucleation of prostatic adenoma is being performed, unneglectable bleeding can occur and surgeons need to rush to remove adenomas, often using fingers and in a blinded fashion. The blood supply to the prostatic capsule and adenoma can be reduced to a marked extent in subcapsular prostatectomy if the bilateral internal iliac arteries are transiently occluded. Thus, a bloodless operative field is reasonably acquired during enucleation of adenoma, which would, otherwise, be a cause for concern to surgeons due to bleeding. It is not always applicable, but it could be an option if the estimated volume of BPH is more than 100 mL. In two cases, bilateral internal iliac arteries were occluded with Bulldog clamps, and then adenomas of 159 and 97 g were enucleated.


Imaging of acute subarachnoid hemorrhage with a fluid-attenuated inversion recovery sequence in an animal model: comparison with non-contrast-enhanced CT.
Fluid-attenuated inversion recovery (FLAIR) MR imaging sequences have been previously described in the evaluation of acute subarachnoid hemorrhage (SAH) in human subjects and have demonstrated good sensitivity. The purpose of this study was to evaluate a FLAIR sequence in an animal model of SAH and to compare the results with those obtained with non-contrast-enhanced CT. SAH was experimentally induced in 18 New Zealand rabbits by injecting autologous arterial blood into the subarachnoid space of the foramen magnum. Nine animals had high-volume (1-2 mL) injections, and nine animals had low-volume (0.2-0.5 mL) injections. Four control animals were injected with 0.5 mL of saline. The animals were imaged with a FLAIR sequence and standard CT 2-5 hours after injection. Gross pathologic evaluation of seven of the animals was performed. Four blinded readers independently evaluated the CT and FLAIR images for SAH and graded the probability of SAH on a scale of 1 to 5 (1 = no hemorrhage, 5 = definite hemorrhage). Overall, the sensitivity of FLAIR was 89%, and the sensitivity of CT was 39% (P <.01). In animals with a high volume of SAH, the sensitivity of FLAIR was 100%, and the sensitivity of CT was 56%. In animals with a low volume of SAH, the sensitivity of FLAIR was 78%, and the sensitivity of CT was 22%. The specificity of FLAIR in animals without SAH was 100%, and the specificity of CT was 100%. The average reader score for FLAIR was 3.8, and that for CT was 2.2 (P <.001). Reader scores for FLAIR were higher than those for CT in 94% (P <.01) of animals with SAH and in 25% of animals without SAH (P >.05). Seven animals underwent gross pathologic examination, and all had blood in the subarachnoid space around the brain stem. FLAIR was more sensitive than CT in the evaluation of acute SAH in this model, especially when a high volume of SAH was present. This study provides a model for further experimentation with MR imaging in the evaluation of SAH. These findings are consistent with those of current clinical literature, which show FLAIR to be an accurate MR sequence in the diagnosis of SAH.


Effects of fumonisin-containing culture material on pulmonary clearance in swine.
To determine the potential effects of feeding tumonisin-containing culture material on the pulmonary clearance of blood-borne particulates and bacteria in swine. 21 healthy male pigs randomly assigned to control and treated groups. Control pigs were fed a standard grower ration while culture material containing fumonisins (20 mg of hydrolyzed fumonisin B1/kg of body weight/d) was added to the feed of treated pigs for 7 days. On day 8, pigs were anesthetized with halothane and catheterized, using a sterile cut-down procedure. 18 hours after recovery from anesthesia, Monastral Blue or Pseudomonas aeruginosa was infused into the right atrium of treated and control pigs for 30 minutes and pulmonary clearance was determined. Pigs that were fed fumonisin-containing culture material had a significantly (P < 0.05) decreased ability to clear Monastral Blue and P aeruginosa. Ultrastructural examination of the lung indicated that uptake of copper pigment by pulmonary intravascular macrophages was decreased in treated pigs. Fumonisins, even when fed to pigs at sub-lethal concentrations, can inhibit pulmonary intravascular macrophages from removing particulate matter and bacteria from the circulation, thus potentially predisposing swine to infectious disease.


Klippel-Trenaunay and Parkes-Weber syndromes: two case reports.
Parkes-Weber syndrome is a congenital vascular disease that comprises capillary, venous, lymphatic, and arteriovenous malformations. Although Parkes-Weber syndrome is a clinically distinct entity with serious complications, it is still frequently misdiagnosed as Klippel-Trenaunay syndrome, which consists of a triad of malformations involving the capillary, venous, and lymphatic vessels, without arteriovenous fistulas. Both syndromes are generally diagnosed with Doppler ultrasound and confirmed by magnetic resonance angiography. The aim of this study is to describe one case of Klippel-Trenaunay syndrome, in a 36-year-old patient, and one case of Parkes-Weber syndrome, in a 21-year-old patient. We review the literature in order to discuss the possible causes and consequences of these diseases related to venous hypertension and angiodysplasia, taking a clearer approach to their differences, and discussing their treatment.


Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site.
The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase (GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step of the closely related human ABO(H) blood group A and B antigens. The catalytic mechanism of these model retaining enzymes remains under debate, where Glu303 has been suggested to act as a putative nucleophile in a double displacement mechanism, a local dipole stabilizing the intermediate in an orthogonal associative mechanism or a general base to stabilize the reactive oxocarbenium ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes having nearly identical sequences, the corresponding mutants of GTA/GTB have up to a 13-fold difference in their residual activities relative to wild type. High-resolution single crystal X-ray diffraction studies reveal, surprisingly, that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å further from the anomeric carbon of donor substrate compared to wild type. However, complicating the analysis is the observation that Glu303 itself plays a critical role in maintaining the stability of a strained "double-turn" in the active site through several hydrogen bonds, and any mutation other than E303Q leads to significantly higher thermal motion or even disorder in the substrate recognition pockets. Thus, there is a remarkable juxtaposition of the mutants E303C and E303D, which retain significant activity despite disrupted active site architecture, with GTB/E303Q, which maintains active site architecture but exhibits zero activity. These findings indicate that nucleophilicity at position 303 is more catalytically valuable than active site stability and highlight the mechanistic elasticity of these enzymes.


The correlation of substitution effects across populations and generations in the presence of nonadditive functional gene action.
Allele substitution effects at quantitative trait loci (QTL) are part of the basis of quantitative genetics theory and applications such as association analysis and genomic prediction. In the presence of nonadditive functional gene action, substitution effects are not constant across populations. We develop an original approach to model the difference in substitution effects across populations as a first order Taylor series expansion from a "focal" population. This expansion involves the difference in allele frequencies and second-order statistical effects (additive by additive and dominance). The change in allele frequencies is a function of relationships (or genetic distances) across populations. As a result, it is possible to estimate the correlation of substitution effects across two populations using three elements: magnitudes of additive, dominance, and additive by additive variances; relationships (Nei's minimum distances or Fst indexes); and assumed heterozygosities. Similarly, the theory applies as well to distinct generations in a population, in which case the distance across generations is a function of increase of inbreeding. Simulation results confirmed our derivations. Slight biases were observed, depending on the nonadditive mechanism and the reference allele. Our derivations are useful to understand and forecast the possibility of prediction across populations and the similarity of GWAS effects.


Identifying strategies early intervention occupational therapists use to teach caregivers.
This study investigated early intervention occupational therapists' use of strategies to teach caregivers. A sample of 40 videotapes made by early intervention occupational therapists was randomly selected from an archival videotape data set of provider home visits. The sample included 20 videotapes illustrating traditional services and 20 videotapes illustrating therapists providing participation-based services. Videotapes were rated using the Teaching Caregivers Scale, which rates three variables on 30-s intervals: (1) routine, (2) provider role, and (3) strategies used to teach caregivers during early intervention home visits. Regardless of the model of service, explicit teaching strategies were rarely used during home visits.


Treatment-based strategy for the management of post-kala-azar dermal leishmaniasis patients in the Sudan.
Post-kala-azar dermal leishmaniasis (PKDL) is a dermatosis that affects more than 50% of successfully treated visceral leishmaniasis (VL) patients in Sudan. PKDL is considered an important reservoir for the parasite and its treatment may help in the control of VL. Currently, treatment is mainly with sodium stibogluconate (SSG), an expensive and fairly toxic drug and without universally in treatment protocols used. A literature review, a consensus of a panel of experts, and unpublished data formed the basis for the development of guidelines for the treatment of PKDL in the Sudan. Six treatment modalities were evaluated. Experts were asked to justify their choices based on their experience regarding of drug safety, efficacy, availability, and cost. The consensus was defined by assigning a categorical rank (first line, second line, third line) to each option. Regarding the use of AmBisome the presence of the drug in the skin was confirmed in smears from PKDL lesions. AmBisome at 2.5 mg/kg/day/20 days or SSG at 20 mg/kg/day/40 days plus four/weekly intradermal injection of alum-precipitated autoclave L. major vaccine are suggested as first- and second-treatment options for PKDL in the Sudan, respectively. SSG at 20 mg/Kg/day/60 or more days can be used if other options are not available.


Deep lateral transurethral incisions for recurrent bladder neck contracture: promising 5-year experience using a standardized approach.
To evaluate our 5-year experience with deep lateral transurethral incisions of bladder neck contracture (TUIBNC), identify risk factors associated with failure of procedure, and assess outcomes of men subsequently treated for concomitant or de novo stress urinary incontinence (SUI). We reviewed 50 consecutive patients who underwent TUIBNC between June 2007 and January 2012. TUIBNC was standardized to include initial dilation followed by deep transurethral incisions. All patients were evaluated cystoscopically after a 2-month interval; those with recurrent bladder neck contracture (RBNC) underwent a second TUIBNC. For patients with concomitant SUI, we assessed the timing between TUIBNC and subsequent artificial urinary sphincter (AUS) placement and its outcome. Perioperative characteristics were reviewed to identify factors associated with failure. Of the 50 patients analyzed, most were refractory, 78% having failed previous TUIBNC. After TUIBNC, 72% required no further surgery for obstruction at a mean follow-up of 12.9 months. Of the 14 who failed initial TUIBNC, 7 underwent repeat TUIBNC with success, representing an overall success rate of 86% after 2 procedures. Significant factors associated with treatment failure were >10 pack/year smoking history (P = .039) and ≥ 2 previous endoscopic BNC procedures (P = .03). Of 39 men (78%) with concomitant SUI, two-thirds underwent AUS placement after an average of 2.9 months after TUIBNC. Only 2 of 26 (8%) patients required repeat transurethral procedures after AUS placement for RBNC. Deep lateral TUIBNC alone is a highly effective treatment modality for RBNC. Smokers and those having 2 or more previous transurethral procedures appear to have greater risk for failure. Subsequent AUS placement can be safely performed with >90% long-term urethral patency.


DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.
Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.


Presynaptic Boutons That Contain Mitochondria Are More Stable.
The addition and removal of presynaptic terminals reconfigures neuronal circuits of the mammalian neocortex, but little is known about how this presynaptic structural plasticity is controlled. Since mitochondria can regulate presynaptic function, we investigated whether the presence of axonal mitochondria relates to the structural plasticity of presynaptic boutons in mouse neocortex. We found that the overall density of axonal mitochondria did not appear to influence the loss and gain of boutons. However, positioning of mitochondria at individual presynaptic sites did relate to increased stability of those boutons. In line with this, synaptic localization of mitochondria increased as boutons aged and showed differing patterns of localization at en passant and terminaux boutons. These results suggest that mitochondria accumulate locally at boutons over time to increase bouton stability.


Strategies for prevention of varicella pneumonia: a cost-effectiveness analysis.
The objective of this study was to compare the cost-effectiveness of 3 strategies of serologic enzyme-linked immunosorbent assay (ELISA) testing and post-exposure varicella zoster immune globulin (VZIG) prophylaxis for the prevention of maternal varicella pneumonia during pregnancy in patients with negative or uncertain histories of varicella infection. A decision tree was constructed to compare the following strategies: 1) routine serologic testing for varicella immunity followed by targeted post-exposure VZIG prophylaxis, 2) post-exposure serologic testing followed by targeted VZIG prophylaxis, and 3) untargeted post-exposure VZIG administration. The probabilities for the model were obtained from the medical literature and supplemented by expert opinion. The costs were obtained by a review of inpatient hospitalizations for varicella pneumonia. All costs were converted to 1995 dollars. Routine serologic testing followed by targeted post-exposure VZIG prophylaxis was the most costly strategy ($37.22/person), with no demonstrable increase in benefit compared with the other 2 strategies. The disutility of this strategy compared with the others was stable across a wide range of values for the probabilities and costs utilized in the sensitivity analysis. We were unable to differentiate between the cost-effectiveness of the other 2 strategies. Routine serologic testing for varicella immunity in patients with negative or uncertain histories of varicella infection should not be performed. The remaining options of screening and prophylaxis appear to be reasonable alternatives for dealing with varicella exposures.


New Data on Vaccine Antigen Deficient Bordetella pertussis Isolates.
Evolution of Bordetella pertussis is driven by natural and vaccine pressures. Isolates circulating in regions with high vaccination coverage present multiple allelic and antigenic variations as compared to isolates collected before introduction of vaccination. Furthermore, during the last epidemics reported in regions using pertussis acellular vaccines, isolates deficient for vaccine antigens, such as pertactin (PRN), were reported to reach high proportions of circulating isolates. More sporadic filamentous hemagglutinin (FHA) or pertussis toxin (PT) deficient isolates were also collected. The whole genome of some recent French isolates, deficient or non-deficient in vaccine antigens, were analyzed. Transcription profiles of the expression of the main virulence factors were also compared. The invasive phenotype in an in vitro human tracheal epithelial (HTE) cell model of infection was evaluated. Our genomic analysis focused on SNPs related to virulence genes known to be more likely to present allelic polymorphism. Transcriptomic data indicated that isolates circulating since the introduction of pertussis vaccines present lower transcription levels of the main virulence genes than the isolates of the pre-vaccine era. Furthermore, isolates not producing FHA present significantly higher expression levels of the entire set of genes tested. Finally, we observed that recent isolates are more invasive in HTE cells when compared to the reference strain, but no multiplication occurs within cells.


Neuroprotective effect of Indian propolis in β-amyloid induced memory deficit: Impact on behavioral and biochemical parameters in rats.
The study aimed at the investigation of neuroprotective activity of macerated ethanolic extract of Indian propolis (MEEP) against β-Amyloid 25-35 (Aβ25-35) induced memory impairment in Alzheimer's disease. MEEP was administrated orally to Wistar rats at doses of 100, 200 and 300mg/kg. Behavioral performances were evaluated using morris water maze and radial arm maze. At the end of behavioral study, the brains were removed and antioxidant parameters and brain monoamines were estimated. Further acetylcholinesterase (AchE) inhibition and brain-derived neurotropic factor (BDNF) were evaluated. In addition hematological parameters and histopathological tests were also carried out. In behavioral models, MEEP significantly (P<0.05) reversed the cognitive impairment of β amyloid-induced rats. The antioxidant potential was significantly increased (P<0.05) after administration of MEEP. Malondialdehyde levels were significantly (P<0.01) decreased in brain homogenate after treatment with MEEP extract as compared with diseased control group (group III). MEEP showed dose-dependent AChE inhibition and increased the levels of brain monoamines (P<0.05) as compared with group III. MEEP improved memory deficits by increasing BDNF in plasma (P<0.05). The study concludes that MEEP has anti-Alzheimer potential in rats through multiple mechanisms and further studies are ongoing for fractionation and biological screening.


A 10-item Fugl-Meyer Motor Scale Based on Machine Learning.
The Fugl-Meyer motor scale (FM) is a well-validated measure for assessing upper extremity and lower extremity motor functions in people with stroke. The FM contains numerous items (50), which reduces its clinical usability. The purpose of this study was to develop a short form of the FM for people with stroke using a machine-learning methodology (FM-ML) and compare the efficiency (ie, number of items) and psychometric properties of the FM-ML with those of other FM versions, including the original FM, the 37-item FM, and the 12-item FM. This observational study with follow-up used secondary data analysis. For developing the FM-ML, the random lasso method of ML was used to select the 10 most informative items (in terms of index of importance). Next, the scores of the FM-ML were calculated using an artificial neural network. Finally, the concurrent validity, predictive validity, responsiveness, and test-retest reliability of all FM versions were examined. The FM-ML used fewer items (80% fewer than the FM, 73% fewer than the 37-item FM, and 17% fewer than the 12-item FM) to achieve psychometric properties comparable with those of the other FM versions (concurrent validity: Pearson r = 0.95-0.99 vs 0.91-0.97; responsiveness: Pearson r = 0.78-0.91 vs 0.33-0.72; and test-retest reliability: intraclass correlation coefficient = 0.88-0.92 vs 0.93-0.98). The findings preliminarily support the efficiency and psychometric properties of the 10-item FM-ML. The FM-ML has potential to substantially improve the efficiency of motor function assessments in patients with stroke.


High spatial resolution distributed sensing in optical fibers by Brillouin gain-profile tracing.
A novel BOTDA technique for distributed sensing of the Brillouin frequency in optical fibers with cm-order spatial resolution is proposed. The technique is based upon a simple modulation scheme, requiring only a single long pump pulse for acoustic excitation, and no subsequent interrogating pulse. Instead, the desired spatial mapping of the Brillouin response is extracted by taking the derivative of the probe signal. As a result, the spatial resolution is limited by the fall-time of the pump modulation, and the phenomena of secondary "echo" signals, typically appearing in BOTDA sensing methods based upon pre-excitation, is mitigated. Experimental demonstration of the detection of a Brillouin frequency variation significantly smaller than the natural Brillouin linewidth, with a 2cm spatial resolution, is presented.


De novo assembly and characterization of the complete chloroplast genome of radish (Raphanus sativus L.).
Radish (Raphanus sativus L.) is an edible root vegetable crop that is cultivated worldwide and whose genome has been sequenced. Here we report the complete nucleotide sequence of the radish cultivar WK10039 chloroplast (cp) genome, along with a de novo assembly strategy using whole genome shotgun sequence reads obtained by next generation sequencing. The radish cp genome is 153,368 bp in length and has a typical quadripartite structure, composed of a pair of inverted repeat regions (26,217 bp each), a large single copy region (83,170 bp), and a small single copy region (17,764 bp). The radish cp genome contains 87 predicted protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Sequence analysis revealed the presence of 91 simple sequence repeats (SSRs) in the radish cp genome. Phylogenetic analysis of 62 protein-coding gene sequences from the 17 cp genomes of the Brassicaceae family suggested that the radish cp genome is most closely related to the cp genomes of Brassica rapa and Brassicanapus. Comparisons with the B. rapa and B. napus cp genomes revealed highly divergent intergenic sequences and introns that can potentially be developed as diagnostic cp markers. Synonymous and nonsynonymous substitutions of cp genes suggested that nucleotide substitutions have occurred at similar rates in most genes. The complete sequence of the radish cp genome would serve as a valuable resource for the development of new molecular markers and the study of the phylogenetic relationships of Raphanus species in the Brassicaceae family.


Evidence against an early signalling role for ceramide in Fas-mediated apoptosis.
We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.


A home-based walking program using rhythmic auditory stimulation improves gait performance in patients with multiple sclerosis: a pilot study.
Few interventions have been successful in improving gait dysfunction in patients with multiple sclerosis (MS). Rhythmic auditory stimulation (RAS) has demonstrated positive results on gait performance in other neurologically impaired populations. To measure the effects of RAS on quantitative walking parameters in ambulatory patients with MS. Ten MS patients with gait disturbance were randomly assigned to receive RAS versus no intervention for 2 weeks. All participants received RAS for another 2 weeks. Between weekly clinic visits, they were provided with MP3 players containing songs whose tempo was 10% above the participant's spontaneous cadence and were instructed to walk to the music 20 minutes daily. Quantitative gait parameters were measured using the GAITRite system. A statistically significant decrease between groups was found for change in double-support time (left, P = .0176; right, P = .0247), whereas trends with medium to high effect sizes were found for other gait parameters, including walking speed. A pooled within-group analysis showed significant improvement of cadence, stride length, step length, velocity, and normalized velocity after 1 week of treatment. Satisfaction level with RAS was high. These results in a convenience sample of MS patients demonstrate the feasibility and safety of RAS when used at home and suggest a potential benefit on gait parameters.


Trends in prostate cancer survival in Sweden, 1960 through 1988: evidence of increasing diagnosis of nonlethal tumors.
The incidence of prostate cancer has increased during the past 30 years but has been paralleled by increases in survival rates from this disease, despite the absence of documented major improvement in curative treatment. Since a high prevalence of microscopic prostate cancer has been observed in autopsied men and because many prostate cancers may never surface clinically, increased diagnostic activities might have led to increased detection of less aggressive tumors. This study was conducted to elucidate whether the trends in prostate cancer incidence and patient survival may be due to increasing diagnoses of nonlethal tumors. We analyzed a population-based cohort comprising all cases of prostate cancer (n = 80,901) detected in Sweden during the period of 1960 through 1988. Five hundred eighteen patients (0.64% of the total number) who could not be followed because of emigration or an incomplete national registration number were excluded. Observed and relative survival rates were calculated for the entire cohort of 80,383 assessable patients per 5-year age group in 5-year periods of diagnosis and according to diagnostic method and were compared between geographic areas with differences in incidence rates. To estimate the independent effects of these determinants, multivariate analyses were performed. For the 80,383 patients with complete follow-up, the 10- and 20-year observed survival rates were 17.5% (95% confidence interval [CI] = 17.2%-17.9%) and 3.5% (95% CI = 3.2%-3.7%), and the relative survival rates were 41.1% (95% CI = 40.3%-41.9%) and 28.6% (95% CI = 26.5%-30.1%), respectively. Relative survival rates improved markedly over time; 10-year relative survival rates increased from 29% (95% CI = 27%-31%) among case patients diagnosed in 1960 through 1964 to 45% (95% CI = 43%-46%) among those diagnosed in 1975 through 1979. Relative survival rates leveled off after about 18 years at 18% (95% CI = 15%-20%) among patients diagnosed in 1960 through 1964 and at 31% (95% CI = 28%-34%) among those diagnosed in 1970 through 1974. An even more favorable outlook was observed in those case patients diagnosed later. In areas with a high or low incidence of prostate cancer, the 10-year relative survival rates were 45% (95% CI = 44%-47%) and 36% (95% CI = 34%-38%), respectively. In the early 1960s, the calculated loss of life expectancy after diagnosis varied from about 68% (95% CI = 61%-75%) of the expected length of life in the youngest age group to about 48% (95% CI = 46%-50%) in the oldest age group. From 1960 through 1964 to 1985 through 1988, the loss of life expectancy decreased by more than 50% in all age groups. The differences in relative survival rates between age groups were small, with a gradual decrease in age groups more than 60-64 years of age. Most of the great temporal improvement and geographic variation in survival rates are quantitatively consistent, with likely increases in the rate of detection of nonlethal tumors. The increase in relative survival rates must be taken into consideration when evaluating the outcome of treatment of prostate cancer, since nonrandomized comparisons may be confounded by time trends. Diagnosis of nonlethal tumors raises concerns because the individual would suffer from the psychologic burden of a cancer diagnosis without any therapeutic benefit.


Calculation of ophthalmic viscoelastic device-induced focus shift during femtosecond laser-assisted cataract surgery.
To assess if a change in refractive index of the anterior chamber during femtosecond laser-assisted cataract surgery can affect the laser beam focus position. The index of refraction and chromatic dispersion of six ophthalmic viscoelastic devices (OVDs) was measured with an Abbe refractometer. Using the Gullstrand eye model, the index values were used to predict the error in the depth of a femtosecond laser cut when the anterior chamber is filled with OVD. Two sources of error produced by the change in refractive index were evaluated: the error in anterior capsule position measured with optical coherence tomography biometry and the shift in femtosecond laser beam focus depth. The refractive indices of the OVDs measured ranged from 1.335 to 1.341 in the visible light (at 587 nm). The error in depth measurement of the refilled anterior chamber ranged from -5 to +7 μm. The OVD produced a shift of the femtosecond laser focus ranging from -1 to +6 μm. Replacement of the aqueous humor with OVDs with the densest compound produced a predicted error in cut depth of 13 μm anterior to the expected cut. Our calculations show that the change in refractive index due to anterior chamber refilling does not sufficiently shift the laser beam focus position to cause the incomplete capsulotomies reported during femtosecond laser-assisted cataract surgery.


Effects of jejunoileal bypass on meal patterns in genetically obese and lean rats.
Jejunoileal bypass surgery, designed to reproduce the human operation (long bypass) was contrasted with a short bypass of only a few centimeters of ileum in both genetically obese rats and their lean littermates. In comparison with the short-bypassed rats, long-bypassed rats of both genotypes lost weight and reduced food intake. When adjustments were made for group differences before surgery, no significant differences were detected in the effectiveness of the operation on the two genotypes. The reduction in food intake was accomplished by a decrease in meal size and an increase in intermeal interval. Food intake gradually returned in the long-bypassed rats toward its level in the short-bypassed rats. This increase was accomplished by an increase in meal size alone. Intermeal intervals remained prolonged. In the long-bypassed rats, compared with the short, meal duration was initially elevated and gradually increased more than did meal size, indicating a great slowing of eating rate. Water intake was unaffected, but water-to-food ratio was elevated initially and gradually declined in the long-bypassed rats. Learned food aversion, dehydration, and gastrointestinal and metabolic alterations due to malabsorption are mentioned as possible contributors to the reduction in food intake, but none of these alone can account for all the changes in meal pattern by which intake reduction is accomplished.


Maternal and child outcomes for pregnant women with pre-existing multiple long-term conditions: protocol for an observational study in the UK.
One in five pregnant women has multiple pre-existing long-term conditions in the UK. Studies have shown that maternal multiple long-term conditions are associated with adverse outcomes. This observational study aims to compare maternal and child outcomes for pregnant women with multiple long-term conditions to those without multiple long-term conditions (0 or 1 long-term conditions). Pregnant women aged 15-49 years old with a conception date between 2000 and 2019 in the UK will be included with follow-up till 2019. The data source will be routine health records from all four UK nations (Clinical Practice Research Datalink (England), Secure Anonymised Information Linkage (Wales), Scotland routine health records and Northern Ireland Maternity System) and the Born in Bradford birth cohort. The exposure of two or more pre-existing, long-term physical or mental health conditions will be defined from a list of health conditions predetermined by women and clinicians. The association of maternal multiple long-term conditions with (a) antenatal, (b) peripartum, (c) postnatal and long-term and (d) mental health outcomes, for both women and their children will be examined. Outcomes of interest will be guided by a core outcome set. Comparisons will be made between pregnant women with and without multiple long-term conditions using modified Poisson and Cox regression. Generalised estimating equation will account for the clustering effect of women who had more than one pregnancy episode. Where appropriate, multiple imputation with chained equation will be used for missing data. Federated analysis will be conducted for each dataset and results will be pooled using random-effects meta-analyses. Approval has been obtained from the respective data sources in each UK nation. Study findings will be submitted for publications in peer-reviewed journals and presented at key conferences.


Factors contributing to delay in diagnosis and start of treatment of leprosy: analysis of help-seeking narratives in northern Bangladesh and in West Bengal, India.
The objective of our research was to identify factors contributing to delay in diagnosis and start of treatment in leprosy, focussing on patients' narratives of help-seeking behaviour. Our research took place in Purulia, West Bengal, India and in Nilphamari, northern Bangladesh. Between January and August 2000, we conducted semi-structured interviews with 104 patients that explored each individual's narrative of help-seeking behaviour and the context of beliefs and attitudes towards leprosy. Subsequently we surveyed 356 patients currently receiving treatment for leprosy and recorded specific aspects of each help-seeking action and their reports of local beliefs and attitudes towards leprosy. Delay was estimated from time of first symptoms through to start of effective treatment (mean 18 months, median 9 months in Purulia and mean 20 months, median 12 months in Nilphamari). The number of help-seeking actions ranged from 1 to 7. Time committed to first actions contributed 86% (Nilphamari) and 79% (Purulia) to total delay. The most important contributor to delay in the first action occurred when people simply monitored or ignored first symptoms, 80% in Nilphamari and 67% in Purulia. With delay longer than 12 months as outcome, logistic regression analyses identified age over 35 years, multiple visits to practitioners in traditional medicine and multiple visits to health service practitioners as predictive of delay. Attending a nearby clinic and exposure to health education materials were predictive of early presentation reduced delay.


A comparative study on cannabidiol-induced apoptosis in murine thymocytes and EL-4 thymoma cells.
It has been shown that leukemia and glioma cells are sensitive to cannabidiol (CBD)-induced apoptosis, whereas primary monocytes and glia cells are relatively insensitive. In the current study, the cellular events and sensitivity to CBD-induced apoptosis between murine thymocytes and EL-4 thymoma cells were compared. Cannabidiol markedly induced apoptosis in a time- and concentration-related manner in both cells. The efficacy of CBD to induce apoptosis was comparable between the 2 types of T cells, whereas CBD induced apoptosis in thymocytes with a slightly greater potency than in EL4 cells. Time-course analyses revealed CBD-mediated apoptosis occurred earlier in EL-4 cells than that in thymocytes. An increased level of cellular reactive oxygen species (ROS) was detected in both cells with the peak response at 2 h post CBD treatment. Concordantly, CBD triggered a gradual diminishment in the cellular thiols. The presence of N-acetyl-L-cysteine (NAC), a precursor of glutathione, markedly attenuated the induction of apoptosis, and restored the diminished levels of cellular thiols. The results demonstrated that both thymocytes and EL-4 thymoma cells were susceptible to CBD-induced apoptosis and that ROS played a critical role in the apoptosis induction.


Audiotactile temporal order judgments.
We report a series of three experiments in which participants made unspeeded 'Which modality came first?' temporal order judgments (TOJs) to pairs of auditory and tactile stimuli presented at varying stimulus onset asynchronies (SOAs) using the method of constant stimuli. The stimuli were presented from either the same or different locations in order to explore the potential effect of redundant spatial information on audiotactile temporal perception. In Experiment 1, the auditory and tactile stimuli had to be separated by nearly 80 ms for inexperienced participants to be able to judge their temporal order accurately (i.e., for the just noticeable difference (JND) to be achieved), no matter whether the stimuli were presented from the same or different spatial positions. More experienced psychophysical observers (Experiment 2) also failed to show any effect of relative spatial position on audiotactile TOJ performance, despite having much lower JNDs (40 ms) overall. A similar pattern of results was found in Experiment 3 when silent electrocutaneous stimulation was used rather than vibrotactile stimulation. Thus, relative spatial position seems to be a less important factor in determining performance for audiotactile TOJ than for other modality pairings (e.g., audiovisual and visuotactile).


A Genetic Variant of the CD14 C-159T in Patients with Functional Dyspepsia (FD) in Japanese Subjects.
Inflammatory changes in the gastric mucosa are commonly observed in Japanese patients with functional dyspepsia (FD). However, detailed data regarding the relationship between the genetic regulatory factors of inflammation and FD are not available. CD14 is an important mediator of the inflammatory response in the first line of host defense by recognition of Lipopolysaccharide (LPS). We aimed to investigate the association between CD14 promoter C-159T polymorphism and FD in a Japanese population. 108 patients with FD and 99 non-dyspeptic subjects enrolled in this study. Dyspeptic symptoms were divided according to Rome III criteria. CD14 gene C-159T polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism. In the non-dyspeptics, the CD14 genotype distribution was 28CC (28.3%), 51CT (51.5%), 21TT (21.2%). Meanwhile, the CD14 genotype distribution in FD was 31CC (28.4%), 56CT (51.4%), 22TT (20.2%). The genotype distribution was not significantly different. There was no significant difference between two groups in the genotype distribution. We did not found any association between CD14 genotypes and dyspeptic patients in different gender and Helicobacter pylori infection status. No significant association was also found between CD14 polymorphism and any of different subtypes of FD according to Rome III while there was a weak correlation between TT genotype and PDS in male subjects (TT vs others, OR = 3.18, 95% CI = 0.98-10.26, p = 0.06). In conclusion, our results suggest that CD14 polymorphism is unlikely to associate with susceptibility of dyspeptic symptoms. The role of inflammation related-gene polymorphisms to the development of dyspepsia needs to further evaluation.


The Large Omnidirectional Child (LODC) ATD: Biofidelity Comparison with the Hybrid III 10 Year Old.
When the Hybrid III 10-year old (HIII-10C) anthropomorphic test device (ATD) was adopted into Code of Federal Regulations (CFR) 49 Part 572 as the best available tool for evaluating large belt-positioning booster seats in Federal Motor Vehicle Safety Standard (FMVSS) No. 213, NHTSA stated that research activities would continue to improve the performance of the HIII-10C to address biofidelity concerns. A significant part of this effort has been NHTSA's in-house development of the Large Omnidirectional Child (LODC) ATD. This prototype ATD is comprised of (1) a head with pediatric mass properties, (2) a neck that produces head lag with Zaxis rotation at the atlanto-occipital joint, (3) a flexible thoracic spine, (4) multi-point thoracic deflection measurement capability, (5) skeletal anthropometry representative of a seated child, and (6) an abdomen that can directly measure belt loading. The objective of this study was to evaluate the LODC by comparing its body region and full-body responses to both standard HIII-10C responses and pediatric biomechanical data. In body region tests, the LODC (BioRank = 1.21) showed improved biofidelity over the HIII-10C (BioRank = 2.70). The LODC also exhibited kinematics more similar to pediatric PMHS kinematics in a reconstruction test. In FMVSS No. 213 tests, the LODC was observed to have lower HIC values with the absence of hard chin-to-chest contacts, indicating that chin-to-chest contact severity is mitigated in the LODC design. LODC abdomen pressures and belt penetrations discriminated between restraint conditions. These results suggest the LODC has biofidelic characteristics that make it a candidate for improved assessment of injury risk in restraint system development.


Time course of congener uptake and elimination in rats after short-term inhalation exposure to an airborne polychlorinated biphenyl (PCB) mixture.
Despite the continued presence of PCBs in indoor and ambient air, few studies have investigated the inhalation route of exposure. While dietary exposure has declined, inhalation of the semivolatile, lower-chlorinated PCBs has risen in importance. We measured the uptake, distribution, and time course of elimination of inhaled PCB congeners to characterize the pulmonary route after short-term exposure. Vapor-phase PCBs were generated from Aroclor 1242 to a nose-only exposure system and characterized for congener levels and profiles. Rats were exposed via inhalation acutely (2.4 mg/m3 for 2 h) or subacutely (8.2 mg/m3, 2 hx10 days), after which pulmonary immune responses and PCB tissue levels were measured. Animals acutely exposed were euthanized at 0, 1, 3, 6, and 12 h post exposure to assess the time course of PCB uptake and elimination. Following rapid absorption and distribution, PCBs accumulated in adipose tissue but decayed in other tissues with half-lives increasing in liver (5.6 h)<lung (8.2 h)<brain (8.5 h)<blood (9.7 h). PCB levels were similar in lung, liver, and adipose tissue, lower in brain, and lowest in blood. Inhalation of the airborne PCB mixture contributed significantly to the body burden of lower-chlorinated congeners. Congeners detected in tissue were mostly ortho-substituted ranging from mono- to pentachlorobiphenyls. Selective uptake and elimination led to accumulation of a distinct congener spectrum in tissue. Minimal evidence of toxicity was observed.


Effect of multiple perfusion components on pseudo-diffusion coefficient in intravoxel incoherent motion imaging.
The aim of this work was to investigate the effect of multiple perfusion components on the pseudo-diffusion coefficient D * in the bi-exponential intravoxel incoherent motion (IVIM) model. Simulations were first performed to examine how the presence of multiple perfusion components influences D *. The real data of livers (n = 31), spleens (n = 31) and kidneys (n = 31) of 31 volunteers was then acquired using DWI for in vivo study and the number of perfusion components in these tissues was determined together with their perfusion fraction and D *, using an adaptive multi-exponential IVIM model. Finally, the bi-exponential model was applied to the real data and the mean, standard variance and coefficient of variation of D * as well as the fitting residual were calculated over the 31 volunteers for each of the three tissues and compared between them. The results of both the simulations and the in vivo study showed that, for the bi-exponential IVIM model, both the variance of D * and the fitting residual tended to increase when the number of perfusion components was increased or when the difference between perfusion components became large. In addition, it was found that the kidney presented the fewest perfusion components among the three tissues. The present study demonstrated that multi-component perfusion is a main factor that causes high variance of D * and the bi-exponential model should be used only when the tissues under investigation have few perfusion components, for example the kidney.


Phosphorylated Akt expression is a prognostic marker in early-stage non-small cell lung cancer.
To determine the prognostic significance of pAkt expression in order to identify high-risk stage IB patients with non-small cell lung cancer (NSCLC) in an exploratory study. We identified 471 consecutive patients with stage IB primary NSCLC according to the American Joint Commission on Cancer 6th edition tumour-node-metastasis (TNM) staging system, who underwent surgical resection between 1990 and 2008. Patients who received neoadjuvant or adjuvant treatments were excluded. Pathology reports were reviewed, and pathological characteristics were extracted. Expression of phosphorylated Akt (pAkt) in both cytoplasmic and nuclear locations was assessed by immunohistochemistry, and clinicopathological factors were analysed against 10-year overall survival using Kaplan-Meier and Cox proportional hazards model. 455 (96.6%) cancers were adequate for pAkt immunohistochemical analysis. The prevalence of pAkt expression in the cytoplasm and nucleus of the cancers was 60.7% and 43.7%, respectively. Patients whose cancers expressed higher levels of cytoplasmic pAkt had a trend towards longer overall survival than those with lower levels (p=0.06). Conversely, patients whose cancers expressed higher levels of nuclear pAkt had a poorer prognosis than those with lower levels of expression (p=0.02). Combined low cytoplasmic/high nuclear expression of pAkt was an independent predictor of overall survival (HR=2.86 (95% CI 1.35 to 6.04); p=0.006) when modelled with age (HR=1.05 (95% CI 1.03 to 1.07); p<0.001), extent of operation (HR=2.11 (95% CI 1.48 to 3.01); p<0.001), visceral pleural invasion (HR=1.63 (95% CI 1.24 to 2.15); p<0.001), gender, tumour size, histopathological type and grade (p>0.05). Level of expression of pAkt in the cytoplasm and nucleus is an independent prognostic factor that may help to select patients with high-risk disease.


Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants.
The mitochondrial DNA (mtDNA) encompasses two classes of functionally important sequence variants: recent pathogenic mutations and ancient adaptive polymorphisms. To rapidly and cheaply evaluate both classes of single nucleotide variants (SNVs), we have developed an integrated system in which mtDNA SNVs are analyzed by multiplex primer extension using the SNaPshot system. A multiplex PCR amplification strategy was used to amplify the entire mtDNA, a computer program identifies optimal extension primers, and a complete global haplotyping system is also proposed. This system genotypes SNVs on multiplexed mtDNA PCR products or directly from enriched mtDNA samples and can quantify heteroplasmic variants down to 0.8% using a standard curve. With this system, we have developed assays for testing the common pathogenic mutations in four multiplex panels: two genotype the 13 most common pathogenic mtDNA mutations and two genotype the 10 most common Leber Hereditary Optic Neuropathy mutations along with haplogroups J and T. We use a hierarchal system of 140 SNVs to delineate the major global mtDNA haplogroups based on a global phylogenetic tree of coding region polymorphisms. This system should permit rapid and inexpensive genotyping of pathogenic and lineage-specific mtDNA SNVs by clinical and research laboratories.


Conservation of function between mammalian and plant steroid 5alpha-reductases.
Arabidopsis det2 mutants are small dark-green dwarfs displaying pleiotropic defects in light-regulated development during multiple stages of the plant life cycle. The DET2 gene encodes a protein that shares approximately 40% sequence identity with mammalian steroid 5alpha-reductases and is implicated in the synthesis of a class of plant steroids, the brassinosteroids. Here we show that the DET2 protein, when expressed in human embryonic kidney 293 cells, catalyzes the 5alpha-reduction of several animal steroid substrates and has similar kinetic properties to the mammalian steroid 5alpha-reductase enzymes. Moreover, human steroid 5alpha-reductases expressed in det2 mutant plants can substitute for DET2 in brassinosteroid biosynthesis. These data indicate that DET2 is an ortholog of the mammalian steroid 5alpha-reductases and provide further evidence that brassinosteroids play an essential role in light-regulated plant development. The structural and functional conservation between DET2 and human steroid 5alpha-reductases raise interesting issues concerning the evolutionary origin of the steroid hormone signaling system.


Development of a unique epigenetic signature during in vivo Th17 differentiation.
Activated naive CD4(+) T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4(+) T cells, Th1 and Th17 cells. We could demonstrate that naive CD4(+) T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.


Impact of efflux in the development of multidrug resistance phenotypes in Staphylococcus aureus.
Efflux has been recognized as a resistance mechanism to antimicrobials in Staphylococcus aureus; however its role on the development of clinically relevant resistance is still poorly characterized. This study aimed to examine the impact of efflux on development of resistance to fluoroquinolones and other antimicrobials in S. aureus strains representing relevant phenotypes in terms of antibiotic susceptibility and efflux activity. Two closely related methicillin- and ciprofloxacin-resistant Staphylococcus aureus clinical strains, with different efflux capacity and the pan-susceptible strain ATCC25923 were exposed to constant concentrations of the efflux pump (EP) substrates ciprofloxacin, ethidium bromide and cetrimide. Parental and exposed strains were tested regarding their susceptibility towards antibiotics, biocides and ethidium bromide, efflux capacity and levels of EP gene expression. Occurrence of resistance-associated mutations was screened by sequencing. Multidrug resistance phenotypes emerged upon exposure, independently of the substrate or its concentration, which were correlated with increased efflux capacity of the exposed strains. The temporal pattern of EP gene expression disclosed an early-response with high expression of several genes, followed by a late-response, characterized by overexpression of specific genes. The overall cell response was more pronounced for strains with an initial basal efflux activity. Remarkably, detection of the IS256 element in the promoter regions of mgrA and norA, in some cases associated with increased gene expression, suggests that these genes may be hot spots for IS256 insertion events. The results obtained with exposure of ATCC25923 to ciprofloxacin were particularly striking, revealing a step-wise development of fluoroquinolone resistance, with a first efflux-mediated response, followed by the occurrence of a mutation in grlA that resulted in phenotypic resistance. Additionally, challenge by non-fluoroquinolone agents, particularly cetrimide, promoted cross resistance to fluoroquinolones, revealing the potential role of biocides as selective pressure for the emergence of resistance to these antibiotics. This study reveals efflux as a significant component of S. aureus resistance to fluoroquinolones and biocides and as a primary mechanism to withstand stress imposed by antimicrobials. This efflux-mediated response can result in the emergence of multidrug resistance in healthcare environments and should be taken into account in the management of this major pathogen.


Parenteral medroxyprogesterone for the management of luteinizing hormone releasing hormone induced hot flashes in men with advanced prostate cancer.
Luteinizing hormone releasing hormone (LHRH) agonist therapy for advanced prostate cancer can manifest significant side effects affecting quality of life, most notably hot flashes. This study evaluated the effectiveness of parenteral medroxyprogesterone acetate (MPA) in reducing the frequency and severity of these hot flashes. A multi-institutional retrospective review of hot flashes from LHRH therapy for prostate cancer was conducted. The hot flashes were quantified and the severity was graded (3-point analogue scale) before and after treatment with MPA. Two doses of MPA (400 or 150 mg intramuscularly) were administered. Statistical analysis (Student's t test) evaluated the quantity of hot flashes, the quality of hot flashes, and dose effectiveness. A total of 48 men (40 at 400 mg, 8 at 150 mg) with a mean age of 71.4 years (range 54 to 87) from 3 institutions were evaluated. There were 91% with symptomatic improvement with MPA, and half (46%) had a complete response defined as total elimination of hot flashes. The median number of the hot flashes per day decreased from 4 to 1 and the median severity score decreased from 2 to 1 (p <0.05). Significance was not achieved comparing the 2 doses. Complete responders were not noted with the 150 mg dose. Anticipated response to MPA did not correlate with the number or severity of the hot flashes. This study is the first multi-institutional evaluation of hot flashes demonstrating significant reduction in quantity and severity with MPA. Based on these data we now manage hot flashes associated with LHRH analogues with 400 mg of MPA.


Gene Expression Profiling of Pseudomonas aeruginosa Upon Exposure to Colistin and Tobramycin.
Pseudomonas aeruginosa (Pae) is notorious for its high-level resistance toward clinically used antibiotics. In fact, Pae has rendered most antimicrobials ineffective, leaving polymyxins and aminoglycosides as last resort antibiotics. Although several resistance mechanisms of Pae are known toward these drugs, a profounder knowledge of hitherto unidentified factors and pathways appears crucial to develop novel strategies to increase their efficacy. Here, we have performed for the first time transcriptome analyses and ribosome profiling in parallel with strain PA14 grown in synthetic cystic fibrosis medium upon exposure to polymyxin E (colistin) and tobramycin. This approach did not only confirm known mechanisms involved in colistin and tobramycin susceptibility but revealed also as yet unknown functions/pathways. Colistin treatment resulted primarily in an anti-oxidative stress response and in the de-regulation of the MexT and AlgU regulons, whereas exposure to tobramycin led predominantly to a rewiring of the expression of multiple amino acid catabolic genes, lower tricarboxylic acid (TCA) cycle genes, type II and VI secretion system genes and genes involved in bacterial motility and attachment, which could potentially lead to a decrease in drug uptake. Moreover, we report that the adverse effects of tobramycin on translation are countered with enhanced expression of genes involved in stalled ribosome rescue, tRNA methylation and type II toxin-antitoxin (TA) systems.


Regulation of human chondrocyte function through direct inhibition of cartilage master regulator SOX9 by microRNA-145 (miRNA-145).
Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator transcription factor SOX9. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how SOX9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of SOX9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses SOX9 expression in human cells through a unique binding site in its 3'-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.


Semi-nested RT-PCR enables sensitive and high-throughput detection of SARS-CoV-2 based on melting analysis.
Asymptomatic transmission was found to be the Achilles' heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors. A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples. The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83-100) and a specificity of 99.87% (95% CI: 99.55-99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure. The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity. .


Epitaxial stabilization versus interdiffusion: synthetic routes to metastable cubic HfO2 and HfV2O7 from the core-shell arrangement of precursors.
Metastable materials that represent excursions from thermodynamic minima are characterized by distinctive structural motifs and electronic structure, which frequently underpins new function. The binary oxides of hafnium present a rich diversity of crystal structures and are of considerable technological importance given their high dielectric constants, refractory characteristics, radiation hardness, and anion conductivity; however, high-symmetry tetragonal and cubic polymorphs of HfO2 are accessible only at substantially elevated temperatures (1720 and 2600 °C, respectively). Here, we demonstrate that the core-shell arrangement of VO2 and amorphous HfO2 promotes outwards oxygen diffusion along an electropositivity gradient and yields an epitaxially matched V2O3/HfO2 interface that allows for the unprecedented stabilization of the metastable cubic polymorph of HfO2 under ambient conditions. Free-standing cubic HfO2, otherwise accessible only above 2600 °C, is stabilized by acid etching of the vanadium oxide core. In contrast, interdiffusion under oxidative conditions yields the negative thermal expansion material HfV2O7. Variable temperature powder X-ray diffraction demonstrate that the prepared HfV2O7 exhibits pronounced negative thermal expansion in the temperature range between 150 and 700 °C. The results demonstrate the potential of using epitaxial crystallographic relationships to facilitate preferential nucleation of otherwise inaccessible metastable compounds.