Food supplements and herbal medicines.
Over-the-counter food supplements and herbal medicines are a growth industry in the UK and are now regulated under European Union (EU) directives 2002/46/EC, 2004/24/EC and 2004/27/EC (European Parliament and Council of the European Union 2002, 2004a, 2004b). In practice, the use of food supplements and herbal medicines should be considered in association with the individual's diet, medical history and prescribed treatments, especially drug therapy, since interactions with drugs can be dangerous and even fatal in some cases. As members of the interprofessional team, nurses can play a key role in identifying problems through careful assessment and prevention by health education.


Polydim-I antimicrobial activity against MDR bacteria and its model membrane interaction.
The rapid spread of multi-drug resistant pathogens represents a serious threat to public health, considering factors such as high mortality rates, treatment restrictions and high prevalence of multi-drug resistant bacteria in the hospital environment. Antimicrobial peptides (AMPs) may exhibit powerful antimicrobial activity against different and diverse microorganisms, also presenting the advantage of absence or low toxicity towards animal cells. In this study, the evaluation of the antimicrobial activity against multi-drug resistant bacteria of a recently described AMP from wasp, Polydim-I, was performed. Polydim-I presented activity against standard strains (non-carriers of multi-resistant genes) that are susceptible to commercial antimicrobials, and also against multi-drug resistant strains at concentrations bellow 1μg/ml (0.41 μM). This is a rather low concentration among those reported for AMPs. At this concentration we found out that Polydim-I inhibits almost 100% of the tested pathogens growth, while with the ATCC strains the minimum inhibitory concentration (MIC100) is 400 times higher. Also, in relation to in vitro activity of conventional drugs against multi-drug resistant bacteria strains, Polydim-I is almost 10 times more efficient and with broader spectrum. Cationic AMPs are known as multi-target compounds and specially for targeting the phospholipid matrix of bacterial membranes. Exploring the interactions of Polydim-I with lipid bilayers, we have confirmed that this interaction is involved in the mechanism of action. Circular dichroism experiments showed that Polydim-I undergoes a conformational transition from random coil to a mostly helical conformation in the presence of membrane mimetic environments. Zeta potential measurements confirmed the binding and partial charge neutralization of anionic asolectin vesicles, and also suggested a possible aggregation of peptide molecules. FTIR experiments confirmed that some peptide aggregation occurs, which is minimized in the presence of strongly anionic micelles of sodium dodecyl sulfate. Also, Polydim-I induced channel-like structures formation to asolectin lipid bilayers, as demonstrated in the electrophysiology experiments. We suggest that cationic Polydim-I targets the membrane lipids due to electrostatic attraction, partially accumulates, neutralizing the opposite charges and induces pore formation. Similar mechanism of action has already been suggested for other peptides from wasp venoms, especially mastoparans.


α-Hydroxycyclopentanones via one-pot oxidation of the trimethylaluminum-mediated Nazarov reaction with triplet oxygen.
Trimethylaluminum and molecular oxygen are used in tandem to interrupt the Nazarov cyclization. α-Hydroxycyclopentanones are produced in moderate to good yield as a mixture of epimers. This sequence is the first example of combined nucleophilic/electrophilic trapping of the Nazarov oxyallyl intermediate.


Disparities in Emergency Department Visits for Opioid and Stimulant Overdoses in Florida During COVID-19.
Emergency department (ED) visits for drug overdoses increased nationally during COVID-19 despite declines in all-cause ED visits. The study purpose was to compare characteristics of ED visits for opioid and stimulant overdoses before and during COVID-19 in Florida. This study tested for disparities in ED visits for opioid and stimulant overdoses by race/ethnicity, age, and insurance status. The study identified ED visits for opioid and stimulant overdose in Florida during quarters two and three of 2019 and compared them with quarters two and three of 2020. Overall, there was an increase in the number of opioid and stimulant overdoses during COVID-19. Combined with the decline in the number of all-cause ED visits, drug overdoses represented a larger share of ED visits during COVID-19 compared with before COVID-19. The study did not find evidence of disparities by race/ethnicity, as each group experienced similar increases in the likelihood of ED visits involving drug overdoses during COVID-19. Differences emerged according to age and insurance status. ED visits involving those under age 18 were more likely to involve opioid or stimulant overdose, and ED visits among those over age 65 were less likely to involve opioid overdose during COVID-19. ED visits among those with vulnerable insurance status were more likely to involve opioid overdose during COVID-19. Patterns of behavior change during periods of restricted activity due to a pandemic. These changes in behavior change the mix of risks that people face, suggesting the need for a reallocation of population health management resources during pandemics.


Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.
To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.


Depression as a risk factor for the onset of type 2 diabetes mellitus. A meta-analysis.
Evidence strongly suggests that depression and type 2 diabetes are associated, but the direction of the association is still unclear. Depression may occur as a consequence of having diabetes, but may also be a risk factor for the onset of type 2 diabetes. This study examined the latter association by reviewing the literature and conducting a meta-analysis of longitudinal studies on this topic. Medline and PsycInfo were searched for articles published up to January 2005. All studies that examined the relationship between depression and the onset of type 2 diabetes were included. Pooled relative risks were calculated using fixed and random effects models. To explore sources of heterogeneity between studies, subgroup analyses and meta-regression analyses were performed. Nine studies met our inclusion criteria for this meta-analysis. The pooled relative risk was 1.26 (1.13-1.39) using the fixed effects model and 1.37 (1.14-1.63) using the random effects model. Heterogeneity between studies could not be explained by (1) whether studies controlled for undetected diabetes at baseline; (2) the method of diabetes assessment at follow-up; (3) the baseline overall risk of diabetes in the study population; and (4) follow-up duration. Depressed adults have a 37% increased risk of developing type 2 diabetes mellitus. The pathophysiological mechanisms underlying this relationship are still unclear and warrant further research. A randomised controlled study is needed to test whether effective prevention or treatment of depression can reduce the incidence of type 2 diabetes and its health consequences.


A Scandinavian experience of register collaboration: the Nordic Arthroplasty Register Association (NARA).
The Nordic (Scandinavian) countries have had working arthroplasty registers for several years. However, the small numbers of inhabitants and the conformity within each country with respect to preferred prosthesis brands and techniques have limited register research. A collaboration called NARA (Nordic Arthroplasty Register Association) was started in 2007, resulting in a common database for Denmark, Norway, and Sweden with regard to hip replacements in 2008 and primary knee replacements in 2009. Finland joined the project in 2010. A code set was defined for the parameters that all registers had in common, and data were re-coded, within each national register, according to the common definitions. After de-identification of the patients, the anonymous data were merged into a common database. The first study based on this common database included 280,201 hip arthroplasties and the second, 151,814 knee arthroplasties. Kaplan-Meier and Cox multiple regression analyses, with adjustment for age, sex, and diagnosis, were used to calculate prosthesis survival, with any revision as the end point. In later studies, specific reasons for revision were also used as end points. We found differences among the countries concerning patient demographics, preferred surgical approaches, fixation methods, and prosthesis brands. Prosthesis survival was best in Sweden, where cement implant fixation was used more commonly than it was in the other countries. As the comparison of national results was one of the main initial aims of this collaboration, only parameters and data that all three registers could deliver were included in the database. Compared with each separate register, this combined register resulted in reduced numbers of parameters and details. In future collaborations of registers with a focus on comparing the performances of prostheses and articulations, we should probably include only the data needed specifically for the predetermined purposes, from registers that can deliver these data, rather than compiling all data from all registers that are willing to participate.


Effects of neighbourhood-level educational attainment on HIV prevalence among young women in Zambia.
Investigations of the association between socio-economic position indicators and HIV in East, Central and Southern Africa have chiefly focused on factors that pertain to individual-level characteristics. This study investigated the effect of neighbourhood educational attainment on HIV prevalence among young women in selected urban and rural areas in Zambia. This study re-analysed data from a cross-sectional population survey conducted in Zambia in 2003. The analyses were restricted to women aged 15-24 years (n = 1295). Stratified random cluster sampling was used to select 10 urban and 10 rural clusters. A measure for neighbourhood-level educational attainment was constructed by aggregating individual-level years-in-school. Multi-level mixed effects regression models were run to examine the neighbourhood-level educational effect on HIV prevalence after adjusting for individual-level underlying variables (education, currently a student, marital status) and selected proximate determinants (ever given birth, sexual activity, lifetime sexual partners). HIV prevalence among young women aged 15-24 years was 12.5% in the urban and 6.8% in the rural clusters. Neighbourhood educational attainment was found to be a strong determinant of HIV infection in both urban and rural population, i.e. HIV prevalence decreased substantially by increasing level of neighbourhood education. The likelihood of infection in low vs. high educational attainment of neighbourhoods was 3.4 times among rural women and 1.8 times higher among the urban women after adjusting for age and other individual-level underlying variables, including education. However, the association was not significant for urban young women after this adjustment. After adjusting for level of education in the neighbourhood, the effect of the individual-level education differed by residence, i.e. a strong protective effect among urban women whereas tending to be a risk factor among rural women. The findings suggested structural effects on HIV prevalence. Future research should include more detailed mapping of neighbourhood factors of relevance to HIV transmission as part of the effort to better understand the causal mechanisms involved.


The Interactive Visualization Gap in Initial Exploratory Data Analysis.
Data scientists and other analytic professionals often use interactive visualization in the dissemination phase at the end of a workflow during which findings are communicated to a wider audience. Visualization scientists, however, hold that interactive representation of data can also be used during exploratory analysis itself. Since the use of interactive visualization is optional rather than mandatory, this leaves a "visualization gap" during initial exploratory analysis that is the onus of visualization researchers to fill. In this paper, we explore areas where visualization would be beneficial in applied research by conducting a design study using a novel variation on contextual inquiry conducted with professional data analysts. Based on these interviews and experiments, we propose a set of interactive initial exploratory visualization guidelines which we believe will promote adoption by this type of user.


A sympathetic pressor reflex in the conscious dog.
1. The effects of mechanical activation of aortic sympathetic sensory fibres have been studied in conscious dogs. 2. A cannula covered by an inflatable rubber balloon, previously implanted in the descending thoracic aorta, was used to stretch the aortic walls without obstructing blood flow. 3. Aortic stretch significantly increased mean arterial blood pressure and heart rate from control values in the absence of pseudo-affective reactions. 4. This response indicates that positive feedback sympathetic reflexes are operative in the conscious, fully innervated animal.


A Ketogenic Diet Reduces Central Obesity and Serum Insulin in Women with Ovarian or Endometrial Cancer.
The glycolytic nature of cancer cells presents a potential treatment target that may be addressed by a ketogenic diet (KD). We hypothesized that a KD would improve body composition and lower serum insulin and insulin-like growth factor-I (IGF-I) in women with ovarian or endometrial cancer. In this randomized controlled trial, women with ovarian or endometrial cancer [age: ≥19 y; body mass index (kg/m2): ≥18.5] were randomly assigned to a KD (70:25:5 energy from fat, protein, and carbohydrate) or the American Cancer Society diet (ACS; high-fiber, low-fat). Body composition (DXA) and fasting serum insulin, IGF-I, and β-hydroxybutyrate were obtained at baseline and at 12 wk; urinary ketones were also measured throughout the intervention. We assessed differences between the diets with ANCOVA and independent t tests. We used correlation analyses to estimate associations between changes in serum analytes and body composition. After 12 wk, the KD (compared with ACS) group had lower adjusted total (35.3 compared with 38.0 kg, P < 0.05) and android (3.0 compared with 3.3 kg, P < 0.05) fat mass. Percentage of change in visceral fat was greater in the KD group (compared with the ACS group; -21.2% compared with -4.6%, P < 0.05). Adjusted total lean mass did not differ between the groups. The KD (compared with ACS) group had lower adjusted fasting serum insulin (7.6 compared with 11.2 µU/mL, P < 0.01). There was a significant inverse association between the changes in serum β-hydroxybutyrate and IGF-I concentrations (r = -0.57; P < 0.0001). In women with ovarian or endometrial cancer, a KD results in selective loss of fat mass and retention of lean mass. Visceral fat mass and fasting serum insulin also are reduced by the KD, perhaps owing to enhanced insulin sensitivity. Elevated serum β-hydroxybutyrate may reflect a metabolic environment inhospitable to cancer proliferation. This trial was registered at www.clinicaltrials.gov as NCT03171506.


Akt targeting as a strategy to boost chemotherapy efficacy in non-small cell lung cancer through metabolism suppression.
Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo, we further revealed that the chronic administration of low-doses of paclitaxel - i.e. metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors.


Application of decision tree for prediction of cutaneous leishmaniasis incidence based on environmental and topographic factors in Isfahan Province, Iran.
Cutaneous Leishmaniasis (CL) is a neglected tropical disease that continues to be a health problem in Iran. Nearly 350 million people are thought to be at risk. We investigated the impact of the environmental factors on CL incidence during the period 2007- 2015 in a known endemic area for this disease in Isfahan Province, Iran. After collecting data with regard to the climatic, topographic, vegetation coverage and CL cases in the study area, a decision tree model was built using the classification and regression tree algorithm. CL data for the years 2007 until 2012 were used for model construction and the data for the years 2013 until 2015 were used for testing the model. The Root Mean Square error and the correlation factor were used to evaluate the predictive performance of the decision tree model. We found that wind speeds less than 14 m/s, altitudes between 1234 and 1810 m above the mean sea level, vegetation coverage according to the normalized difference vegetation index (NDVI) less than 0.12, rainfall less than 1.6 mm and air temperatures higher than 30°C would correspond to a seasonal incidence of 163.28 per 100,000 persons, while if wind speed is less than 14 m/s, altitude less than 1,810 m and NDVI higher than 0.12, then the mean seasonal incidence of the disease would be 2.27 per 100,000 persons. Environmental factors were found to be important predictive variables for CL incidence and should be considered in surveillance and prevention programmes for CL control.


Trend Analysis of 20 Years of FIM Road Racing Grand Prix Motorcycle World Championship.
The technological advancements in motorcycle road racing have ensured the evolution of motorcycle performance; however, it is unknown whether these advancements have resulted in increased speed and therefore risk. To better understand the top level of this sport and inform future regulations, performance-related strategies, and safety procedures, this study aimed to (1) analyze and describe how the performance characteristics of the top class of the Fédération Internationale de Motocyclisme Grand Prix motorcycle world championship (GPWC) have changed with time and (2) quantify potential interactions between performance data (ie, crashes, speed, and environmental conditions). Variables such as top 10 riders' speed of racing, crashes, starting position of winner, participants, and environmental conditions were collected from official race reports from 1997 to 2016. Data standardization was also ensured by including only dry competitions in the analysis. The mean racing speed ranged from a minimum of 155 (7.27) km·h-1 (year 2000) to a maximum of 165 (6.48) km·h-1 (year 2015). Linear mixed-model analysis revealed that the variables year and class significantly influenced changes in mean speed (faster racing). Per race, 12-14% of starters (95% confidence interval) suffered a crash. No significant associations were found between crashes and mean speed, ground temperature, air temperature, or air humidity. The speed of dry racing of the top class of the GPWC has increased over a 20-y span. Motorcycle circuit racing riders should consider the importance of being prepared to cope with crashing events to minimize chances of injury.


De novo assembly of genomes from long sequence reads reveals uncharted territories of Propionibacterium freudenreichii.
Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species. We used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm. The complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.


Occupational Therapy in Postacute Care for Survivors of COVID-19: Research Gaps We Need to Fill.
This column discusses issues in the delivery of postacute care (PAC) rehabilitation services for coronavirus disease 2019 (COVID-19) survivors and gaps in the current research. Occupational therapy practitioners must not only better understand factors that influence the type of PAC rehabilitation a COVID-19 survivor will receive but also comprehend how wide variations in delivery of PAC occupational therapy have affected important outcomes for survivors. The COVID-19 Rehabilitation Research Framework, developed by Cochrane Rehabilitation and the World Health Organization Rehabilitation Program, offers a guide for occupational therapy research priorities that may fill two important gaps: (1) the need for high-quality PAC rehabilitation studies and (2) the need for research on activity and participation assessments and outcomes for COVID-19 survivors. Using electronic health records and other data sources, occupational therapy practitioners and researchers can help build the evidence base to support and guide PAC rehabilitation for survivors of COVID-19 and, perhaps, future pandemics.


Transbronchial needle aspiration "by the books".
Training for advanced bronchoscopic procedures is acquired during the interventional pulmonology (IP) Fellowship. Unfortunately a number of such programs are small, limiting dissemination of formal training. We studied success of conventional transbronchial needle aspiration (C-TBNA) in the hands of physicians without formal IP training. A technique of C-TBNA was learned solely from the literature, videos and practicing on inanimate models at "Hands-On" courses. Conventional TBNA with 21 and/or 19 gauge Smooth Shot Needles (Olympus(®), Japan) was performed on consecutive patients with undiagnosed mediastinal lymphadenopathy. Thirty-four patients (male 23), mean age 54.9 ± 11.8 years underwent C-TBNA. Twenty-two patients had nodes larger than 20 mms. Suspected diagnoses were malignancy in 20 and nonmalignant conditions in 14. Final diagnoses were malignancy 17, sarcoidosis 4, reactive lymph nodes 12, and tuberculosis 1. Final diagnosis was established by C-TBNA in 14 (11 malignancy, 3 sarcoidosis; yield 41.1%), mediastinoscopy in 14, transthoracic needle aspiration in 3, peripheral lymph node biopsies in 2 and by endobronchial biopsy in 1. Nodal size had an impact on outcome (P = 0.000) while location did not (P = 0.33). C-TBNA was positive in 11/20 when malignancy was suspected (yield 55%), while 3/14 when benign diagnosis was suspected (yield 21.4%) (P = 0.05). Sensitivity, specificity, PPV, NPV, and diagnostic accuracy were 66.6%, 100%, 100%, 65%, and 79.4%, respectively. There were no complications or scope damage. Conventional-TBNA can be learned by the books and by practicing on inanimate models without formal training and results similar to those published in the literature could be achieved.


Parental influence on human germline de novo mutations in 1,548 trios from Iceland.
The characterization of mutational processes that generate sequence diversity in the human genome is of paramount importance both to medical genetics and to evolutionary studies. To understand how the age and sex of transmitting parents affect de novo mutations, here we sequence 1,548 Icelanders, their parents, and, for a subset of 225, at least one child, to 35× genome-wide coverage. We find 108,778 de novo mutations, both single nucleotide polymorphisms and indels, and determine the parent of origin of 42,961. The number of de novo mutations from mothers increases by 0.37 per year of age (95% CI 0.32-0.43), a quarter of the 1.51 per year from fathers (95% CI 1.45-1.57). The number of clustered mutations increases faster with the mother's age than with the father's, and the genomic span of maternal de novo mutation clusters is greater than that of paternal ones. The types of de novo mutation from mothers change substantially with age, with a 0.26% (95% CI 0.19-0.33%) decrease in cytosine-phosphate-guanine to thymine-phosphate-guanine (CpG>TpG) de novo mutations and a 0.33% (95% CI 0.28-0.38%) increase in C>G de novo mutations per year, respectively. Remarkably, these age-related changes are not distributed uniformly across the genome. A striking example is a 20 megabase region on chromosome 8p, with a maternal C>G mutation rate that is up to 50-fold greater than the rest of the genome. The age-related accumulation of maternal non-crossover gene conversions also mostly occurs within these regions. Increased sequence diversity and linkage disequilibrium of C>G variants within regions affected by excess maternal mutations indicate that the underlying mutational process has persisted in humans for thousands of years. Moreover, the regional excess of C>G variation in humans is largely shared by chimpanzees, less by gorillas, and is almost absent from orangutans. This demonstrates that sequence diversity in humans results from evolving interactions between age, sex, mutation type, and genomic location.


Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells.
Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases. We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium. Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood-derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells. When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression. CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.


Effects of the sintering conditions of dental zirconia ceramics on the grain size and translucency.
This study aimed to identify the effects of the sintering conditions of dental zirconia on the grain size and translucency. Ten specimens of each of two commercial brands of zirconia (Lava and KaVo) were made and sintered under five different conditions. Microwave sintering (MS) and conventional sintering (CS) methods were used to fabricate zirconia specimens. The dwelling time was 20 minutes for MS and 20 minutes, 2, 10, and 40 hours for CS. The density and the grain size of the sintered zirconia blocks were measured. Total transmission measurements were taken using a spectrophotometer. Two-way analysis of variance model was used for the analysis and performed at a type-one error rate of 0.05. There was no significant difference in density between brands and sintering conditions. The mean grain size increased according to sintering conditions as follows: MS-20 min, CS-20 min, CS-2 hr, CS-10 hr, and CS-40 hr for both brands. The mean grain size ranged from 347-1,512 nm for Lava and 373-1,481 nm for KaVo. The mean light transmittance values of Lava and KaVo were 28.39-34.48% and 28.09-30.50%, respectively. Different sintering conditions resulted in differences in grain size and light transmittance. To obtain more translucent dental zirconia restorations, shorter sintering times should be considered.


APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA.
Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G) have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown. To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y(40)RHHY(44), were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA), and the levels of G-to-A mutations in virion RNA (vRNA) were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA. These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual infection and complementation can result in the packaging of hypermutated genomes which, through recombination with wild-type genomes, could increase viral genetic variation and contribute to evolution.


Experience of devolution in district health system of Pakistan: perspectives regarding needed reforms.
To identify the strengths and weaknesses of the devolved district health system from the experiences of different stakeholders, and recommend direction for reforms in the existing system. Using qualitative exploratory design, the study was conducted in 3 cities of the province of Sindh in Pakistan--Karachi, Khairpur and Larkana--from January to March 2010. Nine in-depth interviews were conducted with multiple stakeholders (District Coordination Officer, Executive District Officer, Medical Superintendent, Medical officers, Health system experts) of the district health system. Interviews included questions on autonomy in decision-making at the district level and the effectiveness of the devolved health system. Data transcripts were made from the recorded tapes and notes taken during the interviews. Thematic analysis was done and the data was classified into 3 broad themes of governance, financing and factors related to resources and service delivery. The main strengths identified included formation of District Health Management Team for wider inter-sectoral collaboration, creation of new posts at sub-district level for close monitoring and supervision, and greater financial autonomy to prioritise according to needs. The reported weaknesses included lack of team work, limited autonomy, lack of capacity, nepotism and poor accountability. While devolution has been scrapped in most parts of the country, the findings of the study provide recommendations for the delegation of further powers at sub-district and union council level, enhanced capacity and increased transparency and accountability to make the system work.


Primary amelanotic melanoma of the male urethra: A rare entity and diagnostic challenge.
Malignant melanoma (melanoma) is a tumor of melanocytes that usually presents as cutaneous lesions. While melanoma can infrequently appear as a primary tumor elsewhere in the body, it is extremely rare in the urethra and even rarer as amelanotic malignant melanoma. We report the case of a 66-year-old male who presented with painless gross hematuria and lower urinary tract obstructive symptoms in the recent 2 weeks prior to his visit to our clinic. History and physical examination, including external genital examination, abdominopelvic sonography, and urine culture, were not conclusive. Cystourethroscopy revealed a creamy pink fragile mass located in the anterior proximal urethra that extended to the mid portion. Pathological examination of this lesion confirmed the diagnosis of amelanotic malignant melanoma using immunohistochemistry. Radical cystourethrectomy with ileal conduit was subsequently conducted. Although this tumor is extremely rare, urologists and pathologists should consider malignant melanoma as a diagnosis in patients with urethral tumor because of the likelihood of early metastasis and, consequently, poor prognosis. Complete surgical removal of the tumor and use of effective therapies can improve outcomes in these patients.


Microwave-Assisted Protein Digestion in a Plate Well for Facile Sampling and Rapid Digestion.
Protein digestion is one of the most important processes in proteomic analysis. Here, we report microwave-assisted protein digestion in a plate well, which allows for facile sampling as well as rapid protein digestion based on the combination of highly stable enzyme immobilization and 3D printing technologies. Trypsin (TR) was immobilized on polystyrene-based nanofibers via an enzyme coating (EC) approach. The EC with stabilized TR activity was assembled with the 3D-printed structure in the plate well (EC/3D), which provides two separated compartments for the solution sampling and the TR-catalyzed protein digestion, respectively. EC/3D can effectively prevent the interference of sampling by accommodating EC in the separated compartment from the sampling hole in the middle. EC/3D in the plate well maintained its protein digestion performance under shaking over 160 days. Microwave irradiation enabled the digestion of bovine serum albumin within 10 min, generating the MALDI-TOF MS results of 75.0% sequence coverage and 61 identified peptides. EC/3D maintained its protein digestion performance under microwave irradiation after 30 times of recycled uses. EC/3D in the plate well has demonstrated its potential as a robust and facile tool for the development of an automated protein digestion platform. The combination of stable immobilized enzymes and 3D-printed structures can be potentially utilized not only for the protein digestion, but also for many other enzyme applications, including bioconversion and biosensors.


Load fatigue of compromised teeth: a comparison of 3 luting cements.
This study compared the number of cycles to failure of central incisors restored with full cast crowns and then cemented with 3 different luting cements. Fifteen human maxillary central incisors received cast post-and-core restorations. These were cemented with zinc phosphate. The teeth were then divided into 3 groups of 5 samples each. Each tooth had a ferrule length of 1.0 mm and was prepared for a full crown. A waxing jig was used to standardize the load application point on all waxed crowns. Complete cast crowns were cemented to the compromised teeth using 3 different luting cements: a zinc phosphate cement (control group), a resin-modified glass-ionomer cement, and a resin cement with a dentin bonding agent. A fatigue load of 1.5 kg was applied at a rate of 72 cycles per minute until failure of the cement layer occurred between the crown and the tooth (preliminary failure). The independent variable was the number of load cycles required to create preliminary failure. An electrical resistance strain gauge was used to provide evidence of preliminary failure. The resin cement samples had a significantly higher number of load cycles to preliminary failure than both the zinc phosphate and the resin-modified glass ionomer (P < or = 0.05). There was no significant difference between the zinc phosphate and the resin-modified glass-ionomer cements.


The evolution, impact and properties of exonic splice enhancers.
In humans, much of the information specifying splice sites is not at the splice site. Exonic splice enhancers are one of the principle non-splice site motifs. Four high-throughput studies have provided a compendium of motifs that function as exonic splice enhancers, but only one, RESCUE-ESE, has been generally employed to examine the properties of enhancers. Here we consider these four datasets to ask whether there is any consensus on the properties and impacts of exonic splice enhancers. While only about 1% of all the identified hexamer motifs are common to all analyses we can define reasonably sized sets that are found in most datasets. These consensus intersection datasets we presume reflect the true properties of exonic splice enhancers. Given prior evidence for the properties of enhancers and splice-associated mutations, we ask for all datasets whether the exonic splice enhancers considered are purine enriched; enriched near exon boundaries; able to predict trends in relative codon usage; slow evolving at synonymous sites; rare in SNPs; associated with weak splice sites; and enriched near longer introns. While the intersect datasets match expectations, only one original dataset, RESCUE-ESE, does. Unexpectedly, a fully experimental dataset identifies motifs that commonly behave opposite to the consensus, for example, being enriched in exon cores where splice-associated mutations are rare. Prior analyses that used the RESCUE-ESE set of hexamers captured the properties of consensus exonic splice enhancers. We estimate that at least 4% of synonymous mutations are deleterious owing to an effect on enhancer functioning.


Differential in vivo activation of monocyte subsets during low-grade inflammation through experimental endotoxemia in humans.
Human monocytes are a heterogeneous cell population, which can be divided into a classical (CD14++CD16-), a non-classical (CD14+CD16+), and an intermediate (CD14++CD16+) subset. We hypothesized that low-grade inflammation may differentially affect monocyte subsets. We used a human lipopolysaccharide (LPS) infusion model to mimic low-grade inflammation to identify, which monocyte subsets are preferentially activated under these conditions. Monocyte subsets were identified by staining for CD14 and CD16, activation status of monocytes was analyzed by staining for CD11b and a novel in situ mRNA hybridization approach to detect IL-6 and IL-8 specific mRNA at the single-cell level by flow cytometry. After LPS challenge, cell numbers of monocyte subsets dropped after 2 h with cell numbers recovering after 6 h. Distribution of monocyte subsets was skewed dramatically towards the intermediate subset after 24 h. Furthermore, intermediate monocytes displayed the largest increase of CD11b expression after 2 h. Finally, IL-6 and IL-8 mRNA levels increased in intermediate and non-classical monocytes after 6 h whereas these mRNA levels in classical monocytes changed only marginally. In conclusion, our data indicates that the main responding subset of monocytes to standardized low-grade inflammation induced by LPS in humans is the CD14++CD16+ intermediate subset followed by the CD14+CD16+ non-classical monocyte subset. Circulating classical monocytes showed comparably less reaction to LPS challenge in vivo.


Impulsivity is associated with uric acid: evidence from humans and mice.
The ability to control impulses varies greatly, and difficulty with impulse control can have severe consequences; in the extreme, it is the defining feature of many psychiatric disorders. Evidence from disparate lines of research suggests that uric acid is elevated in psychiatric disorders characterized by high impulsivity, such as attention-deficit/hyperactivity disorder and bipolar disorder. The present research tests the hypothesis that impulsivity is associated with higher uric acid in humans and mice. Using two longitudinal, nonclinical community samples (total n = 6883), we tested whether there is an association between uric acid and normal variation in trait impulsivity measured with the Revised NEO Personality Inventory. We also examined the effect of uric acid on behavior by comparing wild-type mice, which naturally have low levels of uric acid, with mice genetically modified to accumulate high levels of uric acid. In both human samples, the emotional aspects of trait impulsivity, specifically impulsiveness and excitement seeking, were associated with higher levels of uric acid concurrently and when uric acid was measured 3 to 5 years later. Consistent with the human data, the genetically modified mice displayed significantly more exploratory and novelty-seeking behavior than the wild-type mice. Higher uric acid was associated with impulsivity in both humans and mice. The identification of biological markers of impulsivity may lead to a better understanding of the physiological mechanisms involved in impulsivity and may suggest potential targets for therapeutic intervention.


Quantitative structure-permeation relationships for solute transport across silicone membranes.
The purpose of this work was to assess the molecular properties that influence solute permeation across siliconemembranes and to compare the results with transport across human skin. The permeability coefficients (log Kp) of a series of model solutes across silicone membranes were determined from the analysis of simple transport experiments using a pseudosteady-state mathematical model of the diffusion process. Subsequently, structure permeation relationships were constructed and examined, focusing in particular on the difference between solute octanol/water and 1,2 dichloroethane/water partition coefficients (deltalog P(oct-dce)), which re ported upon H-bond donor activity, and the computationally derived molecular hydrogen-bonding potential. The hydrogen-bond donor acidity and the lipophilicity of the compounds examined greatly influenced their permeation across sil cone membranes. Furthermore, for a limited dataset, a significant correlation was identified between solute permeation across silicone membranes and that through human epidermis. The key molecular properties that control solute perme ation across silicone membranes have been identified. For the set of substituted phenols and other unrelated compounds examined here a similar structure-permeation relationship has been derived for their transport through human epidermis, suggesting application of the results to the prediction of flux across biological barriers.


Influence of cations in lithium and magnesium polysulphide solutions: dependence of the solvent chemistry.
In order to gain a deeper understanding of Li and Mg polysulphides in Li/S and Mg/S batteries, respectively, this work investigates the impact of the two different cations as well as the influence of the electrolyte solvents' relative dielectric permittivity and Gutmann's donor number on the solubility and relative stability of different Li and Mg polysulphide species. Therefore, the disproportionation and dissociation equilibria of chemically prepared "Li2S8" and "MgS8" solutions in DMSO, DMF, ACN, THF, DME, TEGDME, and Pyr14TFSI are characterized by UV/Vis spectroscopy. Varying the cation and the solvent reveals their mutual interplay in stabilizing different polysulphide species. To our knowledge, this is the first time that chemically synthesized Mg polysulphides in solutions are studied. The results of this work provide essential knowledge for further development of the economically, ecologically, and also in terms of energy density and safety, attractive Mg/S batteries.


Restriction Factors: From Intrinsic Viral Restriction to Shaping Cellular Immunity Against HIV-1.
Antiviral restriction factors are host cellular proteins that constitute a first line of defense blocking viral replication and propagation. In addition to interfering at critical steps of the viral replication cycle, some restriction factors also act as innate sensors triggering innate responses against infections. Accumulating evidence suggests an additional role for restriction factors in promoting antiviral cellular immunity to combat viruses. Here, we review the recent progress in our understanding on how restriction factors, particularly APOBEC3G, SAMHD1, Tetherin, and TRIM5α have the cell-autonomous potential to induce cellular resistance against HIV-1 while promoting antiviral innate and adaptive immune responses. Also, we provide an overview of how these restriction factors may connect with protein degradation pathways to modulate anti-HIV-1 cellular immune responses, and we summarize the potential of restriction factors-based therapeutics. This review brings a global perspective on the influence of restrictions factors in intrinsic, innate, and also adaptive antiviral immunity opening up novel research avenues for therapeutic strategies in the fields of drug discovery, gene therapy, and vaccines to control viral infections.


An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death.
Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.


Quantitative PCR analysis of CYP1A induction in Atlantic salmon (Salmo salar).
Environmental pollutants are hypothesized to be one of the causes of recent declines in wild populations of Atlantic salmon (Salmo salar); across Eastern Canada and the United States. Some of these pollutants, such as polychlorinated biphenyls and dioxins, are known to induce expression of the CYP1A subfamily of genes. We applied a highly sensitive technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), for measuring the levels of CYP1A induction in Atlantic salmon. This assay was used to detect patterns of CYP1A mRNA levels, a direct measure of CYP1A expression, in Atlantic salmon exposed to pollutants under both laboratory and field conditions. Two groups of salmon were acclimated to 11 and 17 degrees C, respectively. Each subject then received an intraperitoneal injection (50 mg kg(-1)) of either beta-naphthoflavone (BNF) in corn oil (10 mg BNF ml(-1) corn oil) or corn oil alone. After 48 h, salmon gill, kidney, liver, and brain were collected for RNA isolation and analysis. All tissues showed induction of CYP1A by BNF. The highest base level of CYP1A expression (2.56 x 10(10) molecules/microg RNA) was found in gill tissue. Kidney had the highest mean induction at five orders of magnitude while gill tissue showed the lowest mean induction at two orders of magnitude. The quantitative RT-PCR was also applied to salmon sampled from two streams in Massachusetts, USA. Salmon liver and gill tissue sampled from Millers River (South Royalston, Worcester County), known to contain polychlorinated biphenyls (PCBs), showed on average a two orders of magnitude induction over those collected from a stream with no known contamination (Fourmile Brook, Northfield, Franklin County). Overall, the data show CYP1A exists and is inducible in Atlantic salmon gill, brain, kidney, and liver tissue. In addition, the results obtained demonstrate that quantitative PCR analysis of CYP1A expression is useful in studying ecotoxicity in populations of Atlantic salmon in the wild.


A model for the evolution of developmental arrest in male orangutans.
Male Sumatran orangutans (Pongo abelii) may delay for many years the acquisition of the full array of secondary sexual traits, including their characteristic cheek flanges. Such flexible developmental arrest is unique among male primates. Among male Bornean orangutans (Pongo pygmaeus) such long delays appear less common. Here, we develop a simple model to identify the conditions under which developmental arrest can be adaptive. We show that the baseline strategy (i.e., males are not susceptible to arrest) cannot be invaded by the flexible strategy (i.e., males can arrest their development when the conditions are unfavorable) when the potential for high-ranking unflanged or flanged males to monopolize sexual access to females is low. In contrast, at high monopolization potential, the flexible strategy is the evolutionarily stable strategy. We also derive the proportion of flanged males in the population for each combination of monopolization values. This model concurs with field data that found a different monopolization potential between Bornean and Sumatran flanged males and a lower proportion of flanged males in the population in Sumatran orangutans. Pronounced developmental arrest is linked to very low adult mortality, which explains why it is so limited in its taxonomic distribution.


Three-dimensional ocular kinematics underlying binocular single vision.
We have analyzed the binocular coordination of the eyes during far-to-near refixation saccades based on the evaluation of distance ratios and angular directions of the projected target images relative to the eyes' rotation centers. By defining the geometric point of binocular single vision, called Helmholtz point, we found that disparities during fixations of targets at near distances were limited in the subject's three-dimensional visual field to the vertical and forward directions. These disparities collapsed to simple vertical disparities in the projective binocular image plane. Subjects were able to perfectly fuse the vertically disparate target images with respect to the projected Helmholtz point of single binocular vision, independent of the particular location relative to the horizontal plane of regard. Target image fusion was achieved by binocular torsion combined with corrective modulations of the differential half-vergence angles of the eyes in the horizontal plane. Our findings support the notion that oculomotor control combines vergence in the horizontal plane of regard with active torsion in the frontal plane to achieve fusion of the dichoptic binocular target images.


Structural insights into cooperative DNA recognition by the CCAAT-binding complex and its bZIP transcription factor HapX.
The heterotrimeric CCAAT-binding complex (CBC) is a fundamental eukaryotic transcription factor recognizing the CCAAT box. In certain fungi, like Aspergilli, the CBC cooperates with the basic leucine zipper HapX to control iron metabolism. HapX functionally depends on the CBC, and the stable interaction of both requires DNA. To study this cooperative effect, X-ray structures of the CBC-HapX-DNA complex were determined. Downstream of the CCAAT box, occupied by the CBC, a HapX dimer binds to the major groove. The leash-like N terminus of the distal HapX subunit contacts the CBC, and via a flexible polyproline type II helix mediates minor groove interactions that stimulate sequence promiscuity. In vitro and in vivo mutagenesis suggest that the structural and functional plasticity of HapX results from local asymmetry and its ability to target major and minor grooves simultaneously. The latter feature may also apply to related transcription factors such as yeast Hap4 and distinct Yap family members.


LC-MS analysis reveals biological and metabolic processes essential for Candida albicans biofilm growth.
Candidiasis is the most common fungal infection associated with high morbidity and mortality among immunocompromised patients. The ability to form biofilm is essential for Candida albicans pathogenesis and drug resistance. In this study, the planktonic cell and biofilm proteomes of C. albicans SC5314 strain analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) were compared. In total, 280 and 449 proteins are annotated from the planktonic cell and biofilm proteomes, respectively. The biofilm proteome demonstrated significantly higher proportion of proteins associated with the endomembrane system, mitochondrion and cytoplasm than planktonic proteome. Among proteins detected, 143 and 207 biological processes are annotated, of which, 38 and 102 are specific to the planktonic cell and biofilm proteomes, respectively, while 105 are common biological processes. The specific biological processes of C. albicans planktonic cell proteome are associated with cell polarity, energy metabolism and nucleotide (purine) metabolism, oxido-reduction coenzyme metabolic process, monosaccharide and amino acid (methionine) biosynthesis, regulation of anatomical structure morphogenesis and cell cycling, and single organism reproduction. Meanwhile, regulation of cellular macromolecule biosynthesis and metabolism, transcription and gene expression are major biological processes specifically associated with C. albicans biofilm proteome. Biosynthesis of leucine, isoleucine, and thiocysteine are highlighted as planktonic-related pathways, whereas folate metabolism, fatty acid metabolism and biosynthesis of amino acids (lysine, serine and glycine) are highlighted as biofilm-related pathways. In summary, LC-MS-based proteomic analysis reveals different adaptative strategies of C. albicans via specific biological and metabolic processes for planktonic cell and biofilm lifestyles. The mass spectrometry data are available via ProteomeXchange with identifiers PXD007830 (for biofilm proteome) and PXD007831 (for planktonic cell proteome).


VARUN: discovering extensible motifs under saturation constraints.
The discovery of motifs in biosequences is frequently torn between the rigidity of the model on one hand and the abundance of candidates on the other hand. In particular, motifs that include wild cards or "don't cares" escalate exponentially with their number, and this gets only worse if a don't care is allowed to stretch up to some prescribed maximum length. In this paper, a notion of extensible motif in a sequence is introduced and studied, which tightly combines the structure of the motif pattern, as described by its syntactic specification, with the statistical measure of its occurrence count. It is shown that a combination of appropriate saturation conditions and the monotonicity of probabilistic scores over regions of constant frequency afford us significant parsimony in the generation and testing of candidate overrepresented motifs. A suite of software programs called Varun is described, implementing the discovery of extensible motifs of the type considered. The merits of the method are then documented by results obtained in a variety of experiments primarily targeting protein sequence families. Of equal importance seems the fact that the sets of all surprising motifs returned in each experiment are extracted faster and come in much more manageable sizes than would be obtained in the absence of saturation constraints.


The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme.
The identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli was investigated and is suggested to be Fe(II). The metal content of whole cells in the absence and presence of expression of the type I MetAP from E. coli was determined by inductively coupled plasma (ICP) emission analysis. The observed change in whole cell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible. On the other hand, significant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in iron concentration being 4.4 and 6.2 times greater than that of manganese and zinc, respectively. Activity assays of freshly lysed BL21(DE3) cells containing the pMetAAP plasmid revealed detectable levels (>2 units/mg) of MetAP activity. Control experiments with BL21(DE3) without the MetAP plasmid showed no detectable enzymatic activity. Since MetAP is active upon expression, these data strongly suggest that cobalt is not the in vivo metal ion for the MetAP from E. coli. The MetAP from E. coli as purified was found to be catalytically inactive (</=2 units/mg). ICP emission analysis of the as-purified enzyme revealed no catalytically relevant metal ions. Both the Co(II)- and Fe(II)-MetAP enzymes are susceptible to autoxidation, so strict care must be taken to remove all dissolved oxygen. Enzymatic assays performed under anaerobic conditions indicated that of the di- and trivalent metal cations tested to date, only Co(II) (37.3 units/mg), Fe(II) (29.7 units/mg), Mn(II) (7.0 units/mg), and Zn(II) (3.3 units/mg) provided detectable levels of enzymatic activity. In each case, excess metal ions were found to be inhibitory. The observed specific activity of Co(II)-MetAP is more than 3 times greater than that previously reported for the MetAP from E. coli [Ben-Bassat, A., et al. (1987) J. Bacteriol. 169, 751-757]. This increase in activity is likely due to the strict exclusion of air from reaction samples. Oxidation of either the Fe(II) or Co(II) form of the enzyme resulted in the complete loss of catalytic activity. The substrate binding constants (K(m)) for Met-Gly-Met-Met binding to the Co(II)- or Fe(II)-substituted MetAP enzymes, under anaerobic conditions, were found to be 3.16 and 1.95 mM, respectively. The combination of these data suggests that the in vivo metal ions for the MetAP enzyme from E. coli are likely Fe(II) ions.


Intima-media thickness at the near or far wall of the common carotid artery in cardiovascular risk assessment.
Current guidelines recommend measuring carotid intima-media thickness (IMT) at the far wall of the common carotid artery (CCA). We aimed to precisely quantify associations of near vs. far wall CCA-IMT with the risk for atherosclerotic cardiovascular disease (CVD, defined as coronary heart disease or stroke) and their added predictive values. We analysed individual records of 41 941 participants from 16 prospective studies in the Proof-ATHERO consortium {mean age 61 years [standard deviation (SD) = 11]; 53% female; 16% prior CVD}. Mean baseline values of near and far wall CCA-IMT were 0.83 (SD = 0.28) and 0.82 (SD = 0.27) mm, differed by a mean of 0.02 mm (95% limits of agreement: -0.40 to 0.43), and were moderately correlated [r = 0.44; 95% confidence interval (CI): 0.39-0.49). Over a median follow-up of 9.3 years, we recorded 10 423 CVD events. We pooled study-specific hazard ratios for CVD using random-effects meta-analysis. Near and far wall CCA-IMT values were approximately linearly associated with CVD risk. The respective hazard ratios per SD higher value were 1.18 (95% CI: 1.14-1.22; I² = 30.7%) and 1.20 (1.18-1.23; I² = 5.3%) when adjusted for age, sex, and prior CVD and 1.09 (1.07-1.12; I² = 8.4%) and 1.14 (1.12-1.16; I²=1.3%) upon multivariable adjustment (all P < 0.001). Assessing CCA-IMT at both walls provided a greater C-index improvement than assessing CCA-IMT at one wall only [+0.0046 vs. +0.0023 for near (P < 0.001), +0.0037 for far wall (P = 0.006)]. The associations of near and far wall CCA-IMT with incident CVD were positive, approximately linear, and similarly strong. Improvement in risk discrimination was highest when CCA-IMT was measured at both walls.


Developments with maximum likelihood X-ray computed tomography.
An approach to the maximum-likelihood estimation of attenuation coefficients in transmission tomography is presented as an extension of earlier theoretical work by K. Lange and R. Carson (J. Comput. Assist. Tomography, vol.8, p.306-16, 1984). The reconstruction algorithm is based on the expectation-maximization (EM) algorithm. Several simplifying approximations are introduced which make the maximization step of the algorithm available. Computer simulations are presented using noise-free and Poisson randomized projections. The images obtained with the EM-type method are compared to those reconstructed with the EM method of Lange and Carson and with filtered backprojection. Preliminary results show that there are potential advantages in using the maximum likelihood approaches in situations where a high-contrast object, such as bone, is embedded in low-contrast soft tissue.


Neovascularization in intimal hyperplasia is associated with vein graft failure after coronary artery bypass surgery.
Vein graft atherosclerosis is the major limitation of arterial bypass surgery. This study was carried out to determine if the number of microvessels per area of intimal hyperplasia correlated with vein graft disease. Vein grafts (n=24, graft age range 2-19 years) were taken from 22 patients undergoing redo-coronary artery bypass surgery. Mean age of the patients was 68 +/- 9 years; 92% were males. Samples were divided into three groups (n=8 per group): in group I segments were from grafts without angiographic or histologic disease, in groups II and III segments were from grafts with significant angiographic stenosis, without (group II) and with (group III) atheroma. Intimal hyperplasia was identified by Masson staining, morphometric analysis was performed with NIH image analysis software. Microvessels in the intimal hyperplasia were identified using immunohistochemical techniques. Significance was determined by single-factor ANOVA p < 0.05. The mean area of intimal hyperplasia was similar in groups I and II at 1.06 +/- 0.25 mm2 and 0.97 +/- 0.37 mm2, respectively. The extent of intimal hyperplasia was significantly greater in group III, 1.70 +/- 0.62 mm2 (p < 0.01). In group I, the microvessel count in the intimal hyperplasia was 5.62 +/- 3.89 vessels/mm2, while in group III it was 15.26 +/- 3.66/mm2 (p < 0.01 versus group I). Interestingly the number of microvessels per area of intimal hyperplasia in group II was similar to that in group III). In this study, the extent of neovascularization in intimal hyperplasia correlated with stenoses in human vein grafts. Strategies designed to limit neovascularization in intimal hyperplasia may lead to novel therapies to prevent vein graft failure.


Sensitising effects of genetically modified enzymes used in flavour, fragrance, detergence and pharmaceutical production: cross-sectional study.
The use of genetically engineered enzymes in the synthesis of flavourings, fragrances and other applications has increased tremendously. There is, however, a paucity of data on sensitisation and/or allergy to the finished products. We aimed to review the use of genetically modified enzymes and the enormous challenges in human biomonitoring studies with suitable assays of specific IgE to a variety of modified enzyme proteins in occupational settings and measure specific IgE to modified enzymes in exposed workers. Specific IgE antibodies against workplace-specific individual enzymes were measured by the specific fluorescence enzyme-labelled immunoassay in 813 exposed workers seen in cross-sectional surveys. Twenty-three per cent of all exposed workers showed type I sensitisation with IgE antibodies directed against respective workplace-specific enzymes. The highest sensitisation frequencies observed were for workers exposed enzymes derived from α-amylase (44%), followed by stainzyme (41%), pancreatinin (35%), savinase (31%), papain (31%), ovozyme (28%), phytase (16%), trypsin (15%) and lipase (4%). The highest individual antibody levels (up to 110 kU/L) were detected in workers exposed to phytase, xylanase and glucanase. In a subgroup comprising 134 workers, detailed clinical diagnostics confirmed work-related symptoms. There was a strong correlation (r=0.75, p<0.0001) between the symptoms and antibody levels. Workers with work-related respiratory symptoms showed a higher prevalence for the presence of specific IgE antibodies against workplace-specific enzymes than asymptomatic exposed workers (likelihood ratio 2.32, sensitivity 0.92, specificity 0.6). Our data confirm the previous findings showing that genetically engineered enzymes are potent allergens eliciting immediate-type sensitisation. Owing to lack of commercial diagnostic tests, few of those exposed receive regular surveillance including biomonitoring with relevant specific IgE assays.


Paracoccidioidomycosis in the state of Maranhão, Brazil: geographical and clinical aspects.
The study aimed to show the situation of paracoccidioidomycosis in the state of Maranhão, Brazil. This study is a descriptive case series developed in two stages. First, a survey of cases originating from the state of Maranhão at the Instituto de Doenças Tropicais Natan Portela, Piauí (IDTNP) from 1997 to 2007, and second, the clinical description of 29 cases diagnosed in the Centro de Referências em Doenças Infecciosas e Parasitárias, Maranhão (CREDIP) from 2004 to 2010. Two hundred and sixteen cases have been cataloged at the IDTNP. West, east, and central regions of the state of Maranhão recorded 90.3% of cases proving to be important areas for study. The western region, with a prevalence of 10.8/100,000 inhabitants, has a significantly higher proportion of cases than the northern, southern, and eastern regions (p < 0.05). The occurrence was higher in men with 89.3% of cases, and the male-to-female ratio was 8.4:1. The majority of patients were older than 20 years, lived in rural areas, and had farming or soil management as main occupation (73.8%). At CREDIP, 29 cases were diagnosed, of which 26 (89.6%) had multifocal manifestations. Mucous tissues were involved more (75.8%) frequently, followed by lymph nodes, skin, and lungs with 65.5%, 39% and 37.9 %, respectively. The diagnosis was made by combining direct examination, culture, and histopathology. The study shows the geographical distribution and the epidemiological and clinical aspects of paracoccidioidomycosis, revealing the significance of the disease to the state of Maranhão.


Theoretical Basis of the Test-Negative Study Design for Assessment of Influenza Vaccine Effectiveness.
Influenza viruses undergo frequent antigenic changes. As a result, the viruses circulating change within and between seasons, and the composition of the influenza vaccine is updated annually. Thus, estimation of the vaccine's effectiveness is not constant across seasons. In order to provide annual estimates of the influenza vaccine's effectiveness, health departments have increasingly adopted the "test-negative design," using enhanced data from routine surveillance systems. In this design, patients presenting to participating general practitioners with influenza-like illness are swabbed for laboratory testing; those testing positive for influenza virus are defined as cases, and those testing negative form the comparison group. Data on patients' vaccination histories and confounder profiles are also collected. Vaccine effectiveness is estimated from the odds ratio comparing the odds of testing positive for influenza among vaccinated patients and unvaccinated patients, adjusting for confounders. The test-negative design is purported to reduce bias associated with confounding by health-care-seeking behavior and misclassification of cases. In this paper, we use directed acyclic graphs to characterize potential biases in studies of influenza vaccine effectiveness using the test-negative design. We show how studies using this design can avoid or minimize bias and where bias may be introduced with particular study design variations.


Coffee consumption and risk of prostate cancer: a meta-analysis of prospective cohort studies.
Observational studies and animal evidence suggest an association between coffee consumption and the risk of prostate cancer. However, the results are inconsistent. We evaluated the association by conducting a meta-analysis of prospective cohort studies. PubMed and Embase were searched through June 2013 to identify studies that met predetermined inclusion criterion. A random-effects model was used to calculate the pooled risk estimates. Ten prospective cohort studies involving 8973 patients with prostate cancer and 206 096 participants were included in this systematic review. Compared with individuals who seldom or never drink coffee, the pooled relative risk of prostate cancer was 0.88 (95% confidence interval: 0.82-0.95) for regular coffee drinkers. Exclusion of any single study did not materially alter the combined risk estimate. Visual inspection of a funnel plot and Begg's and Egger's tests did not indicate evidence of publication bias. In summary, integrated evidence from prospective cohort studies supports the hypothesis that coffee consumption may decrease the risk of prostate cancer.


Determinants of glycemic control among diabetes mellitus patients in a tertiary clinic in Gaborone, Botswana: findings and implications.
Glycemic control among patients with diabetes mellitus is associated with a marked reduction of both macrovascular and microvascular complications; however, glycemic control remains an elusive goal worldwide. The aim of this study was to determine factors associated with glycemic control among patients attending a tertiary clinic in Botswana as limited information to date. Cross-sectional study in a tertiary clinic in Gaborone, Botswana. Patients were recruited between 21 July 2015 and 21 September 2015. The majority of the randomly recruited patients (368/380-96.8%) had documentation of glycemic control (HbA1c) within three months of study recruitment and were subsequently included in the analysis. Glycemic control was categorized as desirable, suboptimal and poor if HbA1c was <7%, 7-9% and >9%, respectively. Data were analyzed using SPSS for descriptive statistics including both bivariate and multinomial logistic regression. A value of p < 0.05 was considered statistically significant. The analyzed study population consisted of 258/368 (70.1%) females with a mean age (SD) of 56.7 ± 13.6 years. Means (SDs) for diabetes duration and glycated hemoglobin were 7.2 ± 7.1 years and 7.97 ± 2.02%, respectively. Of the 368 patients, 136 (36.95%) and 132/368 (35.86%) had desirable and suboptimal glycemic control, respectively. Older age, attending the clinic for more or equal to 3 years and not being on insulin were associated with both desirable and suboptimal glycemic control whereas duration of diabetes between 5 and 10 years was associated with poor glycemic control. The majority of patients had poor glycemic control. Older age and not being on insulin were associated with better glycemic control. The fact that patients on insulin had poor glycemic control calls for more research to determine the timing of insulin initiations and dosing schedule factors as these will help to improve overall glycemic control in Botswana and elsewhere.


Evaluation of antigen-specific T-cell responses with a miniaturized and automated method.
The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-microl volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-gamma) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 10(4) PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20- to 50-fold fewer PBMCs than other analytical methods.


Factors impacting short and long-term kidney graft survival: modification by single intra-operative -high-dose induction with ATG-Fresenius.
A majority of recipients benefited from the intra-operative single high-dose induction (HDI) with ATG-Fresenius (ATG-F) still leaving a group of recipients who did not profit from this kind of induction. Therefore the aim of this retrospective analysis was 1st to identify the risk factors impacting short and long-term graft survival, and 2nd to assess the efficacy of this type of induction in kidney graft recipients with or without these risk factors. A total of 606 recipients receiving two different immunosuppressive treatment regimens (1st: Triple drug therapy [TDT, n=196] consisting mainly of steroids, azathioprine and cyclosporine; 2nd: TDT + 9 mg/kg ATG-F intra-operatively [HDI, n=410]) were included in this analysis and grouped according to their kidney graft survival time (short GST: ≤1 yr, n=100 and long GST: >5 yrs, n=506). The main risk factors associated with a shortened graft survival were pre-transplant sensitization, re-transplantation, rejections (in particular vascular or mixed ones) and the necessity of a long-term anti-rejection therapy. Adding ATG-F single high dose induction to TDT was more efficient in prolonging kidney graft survival than TDT alone not only in recipients without any risk factors (p<0.005) but also in recipients with at least one risk factor (p<0.021). Only in 4.6% of recipients having two or more risk factors this effect could not be demonstrated. The intra-operative single high-dose induction with ATG-F significantly improves the kidney graft survival in recipients with or without risk factors and can therefore be recommended.


Tissue Regeneration Enhancer Elements: A Way to Unlock Endogenous Healing Power.
Regenerative capacity is widespread throughout almost all animal phyla. However, the distribution pattern remains incompletely understood. Various examples show that very closely related species display different regenerative capacities. Why and how have diverse regenerative capacities evolved across species? One prevailing thought in the field of regeneration is that most regeneration-associated factors are evolutionarily conserved, suggesting the existence of an innate tissue regeneration ability in all species. However, its regulation is differentially controlled in distinct species, resulting in heterogeneous regenerative capabilities. In this review, we discuss regeneration-associated enhancers, the key cis-regulatory elements controlling gene expression, their underlying molecular mechanisms, and their influence on regenerative capacity. Understanding the regulatory mechanisms of regeneration enhancers can provide fundamental insights into tissue regeneration and further help us develop therapeutic strategies to unlock latent healing powers in humans. Developmental Dynamics 248:34-42, 2019. © 2018 Wiley Periodicals, Inc.


Inhibition of VEGF blocks TGF-beta1 production through a PI3K/Akt signalling pathway.
Vascular endothelial growth factor (VEGF) is a mediator of airway inflammation and remodelling in asthma. Transforming growth factor (TGF)-beta(1) plays pivotal roles in diverse biological processes, including tissue remodelling and repair in a number of chronic lung diseases. However, there are few studies elucidating the interactions between VEGF and TGF-beta(1) in allergic airway disease. A murine model of allergic airway disease was used to define the mechanism by which VEGF induces subepithelial fibrosis and to investigate a potential relationship between VEGF and TGF-beta(1) and the mechanisms by which VEGF signalling regulates TGF-beta(1) expression in allergic airway disease. The ovalbumin (OVA)-inhaled murine model revealed the following typical pathophysiological features of allergic airway disease in the lungs: increased numbers of inflammatory cells of the airways, airway hyperresponsiveness, increased peribronchial fibrosis, and increased levels of VEGF and TGF-beta(1). Administration of VEGF inhibitors reduced the pathophysiological signs of allergic airway disease and decreased the increased TGF-beta(1) levels and peribronchial fibrosis, including phosphoinositide 3-kinase (PI3K) activity after OVA inhalation. In addition, the increased TGF-beta(1) levels and collagen deposition after OVA inhalation were decreased by administration of PI3K inhibitors. These results suggest that inhibition of vascular endothelial growth factor attenuates peribronchial fibrosis, at least when mediated by regulation of transforming growth factor-beta(1) expression through phosphoinositide 3-kinase/Akt pathway in a murine model of allergic airway disease.


Tumour tropism and anti-cancer efficacy of polymer-based doxorubicin prodrugs in the treatment of subcutaneous murine B16F10 melanoma.
Doxorubicin (5 mg kg-1) was administered intravenously to C57 mice bearing subcutaneous B16F10 melanomas, distributing into the tumour with an area under the concentration-time curve (0-48 h; AUC) of 8.7 micrograms h g-1. Injection of doxorubicin-N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate, containing 5 mg of doxorubicin equivalent per kg, mediated an AUC for free doxorubicin (i.e. doxorubicin released from the conjugate) of 15.2 micrograms h g-1 and for total doxorubicin (i.e. free plus conjugated) of 149.1 micrograms h g-1. An increased dose of doxorubicin-HPMA copolymer conjugate (18 mg of doxorubicin equivalent per kg) produced AUC values of 40.1 micrograms h g-1 and 671.7 micrograms h g-1 for free and total doxorubicin respectively. Hence administration of doxorubicin-HPMA copolymer conjugate achieved rises of 1.7- to 4.6-fold in tumour AUC (free doxorubicin) and 17.19 to 77.0-fold in tumour AUC (total doxorubicin). HPMA copolymers bearing fluorescein isothiocyanate accumulated in vascularised stromal regions, particularly in new growth sites at the tumour periphery. Treatment of mice with doxorubicin-HPMA copolymer conjugate achieved treated/control lifespans up to 320% (three doses of 27 mg of doxorubicin equivalent per kg) compared with only 133% using aggressive regimens of free doxorubicin (3 x 5 mg kg-1).


Lumps and bumps on the head in children: use of CT and MR imaging in solving the clinical diagnostic dilemma.
Lumps and bumps of the scalp are a common presenting complaint in children and often pose a diagnostic dilemma. These lesions can be difficult to image, with evaluation confounded by their small size. However, accuracy in diagnosis is critical because the diagnostic and therapeutic implications can vary significantly. The clinical examination can be helpful in developing the differential diagnosis and the imaging strategy. Often, however, a single imaging study is insufficient, and the radiologist finds it necessary to image with more than one modality to correctly diagnose a lesion and provide adequate information for the surgeon. Radiography and ultrasonography are often the initial screening diagnostic tests, followed by magnetic resonance (MR) imaging or computed tomography (CT) for more detail. Multidetector thin-section CT and thin-section MR imaging with surface coils are beneficial in the work-up of these small lesions of the head and neck. The use of newer MR imaging sequences such as heavily T2-weighted single-shot turbo spin-echo imaging and diffusion-weighted imaging can improve the characterization of difficult lesions. Familiarity with the variety of new imaging tools and techniques that are available can help characterize pediatric head and neck lesions and guide clinical management.


Using Personality and Temperament to Predict Exercise Behavior: A Pilot Study of the Braverman Nature Assessment.
The Braverman Nature Assessment (BNA) is intended to determine the dominant monoamine neurochemical that drives an individual's temperament and behavior. The measure has been colloquially praised for the ability to determine the most effective exercise protocols for an individual based on their "dominant nature." This study seeks to examine the proposed relationship between the Braverman Natures and exercise behavior. Seventy-three adults (57 females) between ages 18-65 (mean = 26 years) completed an online survey consisting of the BNA, Big Five Personality Inventory (BFI), and Aerobics Center Longitudinal Study Physical Activity Questionnaire (ACLSPAQ). All Natures showed significant correlations to unique sets of personality traits (BFI). Dopamine and Serotonin Nature scores (via BNA) showed positive correlations to total physical activity (PA)measures. Serotonin Nature had positive correlation with participation in resistance exercise (r = .36, p < .01) and showed the strongest correlations to PA participation. Dopamine was not associated with Extraversion as predicted; however, showed positive correlations to vigorous intensity exercise (r = .26, p < .05). The Natures demonstrate some low to moderate correlations between neurochemical scores and exercise behaviors such as preference for various exercise modalities. There is preliminary evidence to suggest that the BNA may be a useful tool for exercise prescription based on correlations between personality and exercise behavior found in this study. The results do not support colloquial literature on the use of BNA in exercise prescription.


Who Should Be Investigated for Haematuria? Results of a Contemporary Prospective Observational Study of 3556 Patients.
There remains a lack of consensus among guideline relating to which patients require investigation for haematuria. We determined the incidence of urinary tract cancer in a prospective observational study of 3556 patients referred for investigation of haematuria across 40 hospitals between March 2016 and June 2017 (DETECT 1; ClinicalTrials.gov: NCT02676180) and the appropriateness of age at presentation in cases with visible (VH) and nonvisible (NVH) haematuria. The overall incidence of urinary tract cancer was 10.0% (bladder cancer 8.0%, renal parenchymal cancer 1.0%, upper tract transitional cell carcinoma 0.7%, and prostate cancer 0.3%). Patients with VH were more likely to have a diagnosis of urinary tract cancer compared with NVH patients (13.8% vs 3.1%). Older patients, male gender, and smoking history were independently associated with urinary tract cancer diagnosis. Of bladder cancers diagnosed following NVH, 59.4% were high-risk cancers, with 31.3% being muscle invasive. The incidence of cancer in VH patients <45 yr of age was 3.5% (n=7) and 1.0% (n=4) in NVH patients <60 yr old. Our results suggest that patients with VH should be investigated regardless of age. Although the risk of urinary tract cancer in NVH patients is low, clinically significant cancers are detected below the age threshold for referral for investigation. This study highlights the requirement to investigate all patients with visible blood in the urine and an age threshold of ≥60 yr, as recommended in some guidelines, as the investigation of nonvisible blood in the urine will miss a significant number of urinary tract cancers. Patient preference is important, and evidence that patients are willing to submit to investigation should be considered in reaching a consensus recommendation for the investigation of haematuria. International consensus to guide that patients will benefit from investigation should be developed.


The 'sticky business' of cleaning gas-phase membrane proteins: a detergent oriented perspective.
In recent years the properties of gas-phase detergent clusters have come under close scrutiny due in part to their participation in the analysis of intact membrane protein complexes by mass spectrometry. The detergent molecules that cover the protein complex are removed in the gas-phase by thermally agitating the ions by collision-induced dissociation. This process however, is not readily controlled and can frequently result in the disruption of protein structure. Improved methods of releasing proteins from detergent clusters are clearly required. To facilitate this the structural properties of detergent clusters along with the mechanistic details of their dissociation need to be understood. Pivotal to understanding the properties of gas-phase detergent clusters is the technique of ion mobility mass spectrometry. This technique can be used to assign polydisperse detergent clusters and provide information about their geometries and packing densities. In this article we consider the shapes of detergent clusters and show that these clusters possess geometries that are inconsistent with those in solution. We analyse the distributions of clusters in detail using tandem mass spectrometry and suggest that the mean charge of clusters formed from certain detergents is governed by electrostatic repulsion. We discuss the dissociation of detergent clusters and propose that detergent evaporation it a key process in the protection of protein complexes during high energy collisions in the gas-phase.


Effect of Asiatic Acid on the Treatment of Spinal Cord Injury: An Experimental Study in Rats.
Spinal cord injury (SCI) is a devastating condition of the central nervous system. There is no proven therapeutic agent for the treatment of this complex disorder. Asiatic acid (AA) has been used as an anti-inflammatory and anti-oxidant agent in Eastern countries for many years. The aim of this study was to investigate the effectiveness of AA on the treatment of traumatic SCI in rats. Thirty-two adult male Sprague-Dawley rats were divided into 4 groups as laminectomy, laminectomy+trauma, vehicle, and AA treatment groups. SCI was created by the modified Allen"s weight-drop technique. After the injury, the levels of pro-inflammatory cytokines (IL-6, IL1-Β, TNF-α) and lipid peroxidation products (MDA) were measured. Tarlov functional recovery scores were also determined for each rat. The One-way ANOVA test was used for the analysis of difference between 4 experimental groups and the groups were compared individually by Tukey-LSD post hoc analysis test (p=0.001). AA administration just after SCI attenuated the levels of lipid peroxidation products (MDA) and pro-inflammatory cytokines (TNF-α, IL1Β). It also increased the Tarlov functional recovery scores of the rats. AA administration could attenuate a number of deleterious reactions after traumatic SCI. Further studies are needed to elucidate the pathways of neuroprotective effects of AA after spinal trauma.


Low T3 syndrome predicts severe neurological deficits of cerebral infarction inpatients with large artery artherosclerosis in internal carotid artery system.
The low triiodothyronine (T3) syndrome indicates poor prognosis for patients with cerebral infarction. It is unknown, however, whether basic conditions or severities in the patients with the low T3 syndrome are different compared to those without the low T3 syndrome. We compared the risk factors and the severity of the disease using the National Institutes of Health stroke scale (NIHSS) score at the worst condition for cerebral infarction in patients with or without the low T3 syndrome in order to better understand the characteristics underlying the worse prognosis in patients with the low T3 syndrome. We found that cerebral infarction patients with the low T3 syndrome were significantly older (p<0.001) and significantly more likely to be female (p=0.002) and had hypertension (p=0.04) or homocystinemia (p=0.001), but less likely to smoke (p=0.008), compared to patients without the low T3 syndrome. The proportion of NIHSS score ≥8 in the patients with LAA-ICA-associated cerebral infarction accompanied by the low T3 syndrome was significantly higher than in those without the low T3 syndrome (p=0.001). We concluded that increased numbers of risk factors for cerebral infarction and more severe neurological deficits may be important causes for worse prognosis in the patients with the low T3 syndrome which may more likely occur in patients with LAA-ICA cerebral infarction. Intense secondary prevention in cerebral infarction especially in older women are needed.


Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle.
Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi, and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation to release of Pi Because the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein-protein interactions during the Fe protein cycle.


Perception of postgraduate students in pharmacology toward animal simulation model.
The objective of the study is to evaluate the perception of postgraduate pharmacology students toward computer-simulated method (CSM) in comparison to the prevalent isolated live tissue-based bioassay method. A questionnaire-based survey was conducted in 30 postgraduate pharmacology students who had used the animal simulation software and had completed at least five isolated tissue experiments. Students' opinions on the usage, logistics, advantages, disadvantages, and usefulness of CSM compared to live animal experiments (LAE) were analyzed. Four tissues were used for LAE, whereas with CSM, students could perform experiments using 11 different tissues. Of the total nine bioassay methods, students had performed six assay methods using both LAE and CSM. Majority of the students (23/30) agreed that CSM reduces anxiety, technical errors and is less time consuming when used before LAE. Most of the students agreed that CSM can be used for difficult, lengthy experiments (19/30), and for UG/PG teaching (19/30). However, opinions regarding replacing LAE with CSM in PG teaching were divided (agree: 7, neutral: 12, and disagree: 12). CSM should be integrated alongside LAE to complement, reinforce, and enhance learning from other techniques.


A new design method for the complex-valued multistate Hopfield associative memory.
A method to store each element of an integral memory set M subset {1,2,...,K}/sup n/ as a fixed point into a complex-valued multistate Hopfield network is introduced. The method employs a set of inequalities to render each memory pattern as a strict local minimum of a quadratic energy landscape. Based on the solution of this system, it gives a recurrent network of n multistate neurons with complex and symmetric synaptic weights, which operates on the finite state space {1,2,...,K}/sup n/ to minimize this quadratic functional. Maximum number of integral vectors that can be embedded into the energy landscape of the network by this method is investigated by computer experiments. This paper also enlightens the performance of the proposed method in reconstructing noisy gray-scale images.