Effect of Long-Term Metformin and Lifestyle in the Diabetes Prevention Program and Its Outcome Study on Coronary Artery Calcium.
Despite the reduced incidence of coronary heart disease with intensive risk factor management, people with diabetes mellitus and prediabetes remain at increased coronary heart disease risk. Diabetes prevention interventions may be needed to reduce coronary heart disease risk. This approach was examined in the DPP (Diabetes Prevention Program) and the DPPOS (Diabetes Prevention Program Outcome Study), a long-term intervention study in 3234 subjects with prediabetes (mean±SD age, 64±10 years) that showed reduced diabetes risk with lifestyle and metformin compared with placebo over 3.2 years. The DPPOS offered periodic group lifestyle sessions to all participants and continued metformin in the originally randomized metformin group. Subclinical atherosclerosis was assessed in 2029 participants with coronary artery calcium (CAC) measurements after an average of 14 years of follow-up. The CAC scores were analyzed continuously as CAC severity and categorically as CAC presence (CAC score >0) and reported separately in men and women. There were no CAC differences between lifestyle and placebo intervention groups in either sex. CAC severity and presence were significantly lower among men in the metformin versus the placebo group (age-adjusted mean CAC severity, 39.5 versus 66.9 Agatston units, P=0.04; CAC presence, 75% versus 84%, P=0.02), but no metformin effect was seen in women. In multivariate analysis, the metformin effect in men was not influenced by demographic, anthropometric, or metabolic factors; by the development of diabetes mellitus; or by use/nonuse of statin therapy. Metformin may protect against coronary atherosclerosis in prediabetes and early diabetes mellitus among men. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00038727.


Chloroplast genome characterization in the red alga Griffithsia pacifica.
It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes. In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoechst-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.


Severity of Curvularia Stem Blight Disease of Cassava in West Africa.
In diagnostic surveys, Curvularia stem blight affected 9, 13, and 38% of cassava fields, respectively, in Benin, Ghana, and Nigeria. Disease incidence (number of plants with visible symptoms per total sampled) ranged between 0 and 80%, and severity (number of lesions) between 2 and 25 lesions per stem. In greenhouse studies, the fungus inhibited shoot growth depending on the degree of bud colonization, such that when buds were completely colonized, they failed to sprout. Partially colonized buds sprouted, but depending on genotype, overall growth was reduced 20 to 50% compared with healthy stems. Shoot growth for all artificially inoculated cultivars was consistently lower than for the respective noninoculated plants, and they suffered up to 50% leaf abscission. In two field localities, shoot sprouting for cultivars TMS 30572 and Odongbo was reduced 4 to 18% and 26 to 58% compared with noninoculated stems.


Changes on Walking During Street Crossing Situations and on Dorsiflexion Strength of Older Caribbean Americans After an Exercise Program: A Pilot Study.
The objective of this pilot study was to evaluate a 6-month exercise program completed by 10 older Caribbean Americans. Assessments were done at baseline and 3 and 6 months, and included walks on an instrumented mat at preferred speed, and during street crossing simulations with regular (10 s) and reduced time (5 s). There were no significant differences on preferred walking speed over time. Differences between the street crossing conditions were found only at 6 months. Significant changes over time among the assessments were found only during street crossing with reduced time. Street crossing with reduced time was the only walking condition sensitive to capture changes associated with participating in the exercise program. There was a significant increase in dorsiflexion strength overtime. At 6 months it was significantly higher than at baseline and 3 months. The program was feasible, acceptable, and had some positive effects on walking, knee flexion, and dorsiflexion strength.


Rhizoctonia solani AG-13 Isolated from Corn in Mississippi.
Rhizoctonia solani K¨hn anastomosis group (AG) 13 was isolated from asymptomatic root tissue of a corn (Zea mays L.) seedling grown at the Black Belt Branch Experiment Station, Brooksville, MS. Rhizoctonia solani AG-13 was recently reported from cotton grown in Georgia (2). Rhizoctonia solani isolate MS-168 was successfully anastomosed with tester isolate AG-13 (courtesy of D. E. Carling, University of Alaska). The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. These results were confirmed by D. E. Carling and are consistent with C2 anastomosis hyphal reactions (1). Rhizoctonia solani isolate MS-168 was cultured on potato dextrose agar (PDA) and incubated at 27 ± °C with a 12-h photoperiod for 28 days. Mycelium was buff-brown to beige with diurnal zonation throughout the colony. Aggregates of bulbils developed in the center of the colony that were dark brown, dome shaped, and accompanied by brown exudate. Bulbils were submerged in the medium and scattered across the surface of the colony. The optimal growing temperature of MS-168 was 27°C. Two pathogenicity evaluations were conducted on 170 corn seedlings planted into soilless potting medium. Four-day-old corn seedlings were inoculated with 7-day-old PDA hyphal plugs (2 mm in diameter) of R. solani isolate MS-168 by placing the mycelium side of the hyphal plug in contact with the mesocotyl tissue beneath the soil surface. The hyphal plugs were covered with soil. The nontreated corn seedlings were inoculated with PDA plugs minus the fungus. Corn seedlings were maintained under environmentally controlled conditions at 27 ±2 °C with a 12-h photoperiod and watered to prevent wilting. Disease symptoms on mesocotyl tissue were rated from 1 to 4 in which 1 = no symptoms, 2 = a few pinpoint lesions and diffuse discolored areas, 3 = distinct necrotic lesions, and 4 = girdling lesions (3). Fourteen days postinoculation, treated seedlings had a significantly higher disease rating (1.5) than the nontreated control (1.0). Thirty of eighty-seven corn seedlings inoculated with MS-168 expressed symptoms of discoloration and pinpoint and necrotic lesions on the mesocotyl tissue at the site of inoculation. On the basis of the results of the pathogenicity evaluation, MS-168 can be characterized as weakly virulent on seedling corn when grown under controlled environmental conditions. The identification of R. solani isolate MS-168 (AG-13) from corn in Mississippi broadens the natural distribution of occurrence and host range of this anastomosis group. References: (1) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. Pages 37-47 in: Rhizoctonia Species: Taxonomy, Molecular, Biological, Ecological, Pathology, and Disease Control. B. Sneh et al., eds. Kluwer Academic Publishers, The Netherlands, 1996. (2) D. E. Carling et al. Phytopathology 92:893, 2002. (3) C. S. Rothrock. Report of the cottonseed treatment committee for 1993. Page 216-217 in: Proc. Beltwide Cotton Conf. Natl. Cotton Counc. Am., Memphis, TN, 1994.


Identification of protective linear B-cell epitopes on the subolesin/akirin orthologues of Ornithodoros spp. soft ticks.
Subolesin/akirin is a protective antigen that is highly conserved across hematophagous vector species and is therefore potentially useful for the development of a universal vaccine for vector control, including soft ticks. Recent results have shown that in Ornithodoros erraticus and O. moubata soft ticks, RNAi-mediated subolesin gene knockdown inhibits tick oviposition and fertility by more than 90%; however, vaccination with recombinant subolesins resulted in remarkably low protective efficacies (5-24.5% reduction in oviposition). Here we report that vaccination with subolesin recombinants induces non-protective antibodies mainly directed against immunodominant linear B-cell epitopes located on highly structured regions of the subolesin protein, probably unrelated to its biological activity, while leaving the unstructured/disordered regions unrecognized. Accordingly, for a new vaccine trial we designed four synthetic peptides (OE1, OE2, OM1 and OM2) from the unrecognized/disordered regions of the Ornithodoros subolesin sequences and coupled them to keyhole limpet haemocyanin (KLH). These KLH-peptide conjugates induced the synthesis of antibodies that recognized linear B-cell epitopes located on the unstructured loops of the subolesin protein and provided up to 70.1% and 83.1% vaccine efficacies in O. erraticus and O. moubata, respectively. These results show that the protective effect of subolesin-based vaccines is highly dependent on the particular epitope recognized by antibodies on the subolesin sequence and strongly suggest that the biological activity of subolesin is exerted through its unstructured regions. The results reported here contribute to our understanding of the mechanism of protection of subolesin-based vaccines and reveal novel protective peptides that could be included among the array of candidate antigens useful for developing anti-vector vaccines based on subolesin/akirin.


Case-Control Study of acute diarrhea in Children.
To determine the risk factor for acute diarrhea disease in children. In the survey, 220 cases and 220 controls were involved. Cases were children with loose watery feces, pathologically diagnosed as acute diarrhea by the physician, and enrolled from the Emergency Ward as the Incident Cases in Dr. Sheykh Hospital, Mashhad, northeast of Iran. Control group too, was enrolled from the same cases and the members were children whose risk factors disease were not the risk factor for acute diarrhea, and were enrolled from emergency ward of burn patients, accidents, acute respiratory diseases and eligible patients. By using univariate logistic regression models, variables consisting used milk up to 6 months age, antibiotic usage during the last 15 days, mother's employment, mother's acquaintance with ORS, occupancy, and water slack during the week before illness, were significantly related with acute diarrhea. In a multiple logistic regression, the relationship between the said factors as the independent variables and acute diarrhea was studied. Use of formulas before 6 month age with (95% CI 1.77- 5.79) OR=3.21, and tenantry with (95% CI 1.24-2.71) OR=1.83, and mother's employment with (95%CI 1.2-5.06) OR= 2.48 were factors affecting acute diarrhea which remained in the multiple logistic model and had significant relationship with the disease. During this study, important environmental risk factors were recognized. Endangered groups and mothers with children under 5 years old should be aware of these risk factors in order to prevent their children's diseases. Prevention of acute diarrhea in children can reduce massive detriments to health system.


Curcumin inhibits activation of TRPM2 channels in rat hepatocytes.
Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca(2+) homeostasis, resulting in a sustained elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca(2+) entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca(2+)]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.


Demethylation of the human eotaxin-3 gene promoter leads to the elevated expression of eotaxin-3.
DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. In this article, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue. The demethylation of a specific CpG site (designated CpG 2), which is juxtaposed to a key cAMP-responsive element site, was significantly demethylated in patient-derived compared with normal control tissue-derived epithelial cells. Levels of methylation at CpG 2 inversely correlated with basal and IL-13-induced eotaxin-3 gene expression. Conversely, global inhibition of methylation with 5-azacytidine promoted eotaxin-3 production in association with decreasing CpG 2 methylation. In addition, the basal and IL-13-induced eotaxin-3 transcriptional activity was suppressed by promoter methylation using a methylation-free in vitro system. Furthermore, EMSAs demonstrated that the attachment of CREB binding protein and activating transcription factor 2 (ATF-2) to the cAMP-responsive element site was methylation dependent. Taken together, these data identify a contributory role for DNA methylation in regulating eotaxin-3 production in human allergic inflammation.


A randomized study comparing three groups of vein harvesting methods for coronary artery bypass grafting: endoscopic harvest versus standard bridging and open techniques.
The use of an open vein harvesting (OVH) technique for saphenous vein harvesting (SVH) is associated with wound complications and delayed patient mobilization. This has led to the development of minimally invasive vein harvesting (MIVH) techniques, such as standard bridging and endoscopic SVH (EVH). This randomized trial was established to assess immediate clinical outcome and patient satisfaction in our centre. A total of 150 consecutive patients were prospectively randomized into three groups. Group 1 consisted of 50 patients who underwent OVH, Group 2 consisted of 50 patients who underwent a standard bridging technique (SBT) and Group 3 consisted of 50 patients who underwent EVH. Each group was assessed for the incidence of wound infection, postoperative pain and satisfaction and the number of vein repairs using previously validated scoring systems. The MIVH techniques reduced the pain at hospital (P < 0.001) and at 6 weeks (P < 0.001), and improved cosmesis (P < 0.001), compared with the OVH group. Patient satisfaction was greatest in the EVH group followed by the SBT and then the OVH group. The clinical markers of inflammation were reduced with an MIVHt. There were more vein repairs in the EVH compared with the OVH (P < 0.001) and the SBT (P = 0.04) groups. This study demonstrates that MIVH reduces wound morbidity. We believe that each technique has advantages and disadvantages, which should be considered during the selection of a harvesting procedure by both the patient and the surgeon.


Failure to inhibit endotoxin-induced uveitis with antibodies that neutralize tumor necrosis factor.
Many of the effects of bacterial endotoxin are attributed to the cytokines that it induces. Tumor necrosis factor alpha (TNF alpha) may be responsible for many of the pathophysiologic effects of endotoxin because antibodies that neutralize TNF alpha reduce endotoxin-induced mortality in experimental animals and many of the activities of endotoxin can be mimicked by the infusion of TNF alpha. Accordingly, we tested the ability of an antibody that neutralizes TNF alpha in a well characterized rat model, endotoxin-induced uveitis. Rats receiving subcutaneous endotoxin had a prompt rise in serum TNF alpha that was eliminated by the monoclonal antibody. Small quantities of TNF alpha were detectable in aqueous humor after either subcutaneous or intravitreal injection of endotoxin. Endotoxin-induced uveitis as quantitated by cells and protein in aqueous humor was, however, not affected by the antibody. Similarly, intraocular inflammation was not reduced by the intravitreal injection of the neutralizing antibody. Although TNF alpha may mediate many of the systemic effects of endotoxin, its role in the intraocular inflammatory effects of endotoxin should be questioned.


Effect of prosthesis number and position on rima glottidis area in equine laryngeal specimens.
To assess the effect of 1 or 2 laryngeal prosthetic sutures on rima glottidis areas in equine laryngeal specimens. Experimental, randomized design. Cadaveric equine larynges (n=16). Larynges were collected from 10 horses; 2 sutures each were preplaced in the right and left sides of each larynx. A dorsal suture (DS) was placed through the caudal rim of the dorsal midline of the cricoid cartilage, under the cricopharyngeus muscle and through the proximal and rostral aspect of the muscular process. A lateral suture (LS) was placed 1.5 cm lateral to the DS and through the muscular process more distal and caudal to the 1st suture. Larynges were positioned in a customized stand and the rima glottidis photographed after each suture (LS or DS) or suture combination (CS) was tied in random sequence. An additional 6 larynxes were used to determine whether the tension applied to the sutures was repeatable. Sutures were preplaced in both the right and left side of each larynx as described above and each suture and CS was tied and released 3 times in each larynx. Photographs were taken of the rima glottidis after each suture or CS was tied generating 3 replicates for each suture configuration on each side of the 6 larynges. Mean rima glottidis area was not different between DS and LS when tied alone (P=.85); however, mean area after CS (DS+LS) was greater than DS (P<.001) and LS (P<.001) alone. The coefficient of variation for the 6 suture patterns were low (1-7%) and the intraclass correlation coefficient estimates were very high (0.997-0.998) demonstrating excellent repeatability between replicates for each of the 3 suture configurations. Our results suggest that laryngoplasty using 2 prostheses; 1 placed dorsally in the cricoid and through the rostral and proximal muscular process and 1 placed 1.5 cm lateral to the 1st and more caudal and distal in the muscular process results in a greater cross sectional area of the rima glottidis than either suture used alone. Seemingly prosthetic sutures contribute independently to each other in determining the contour of the rima glottidis. Use of 2 prosthetic sutures improves crosssectional area of the rima glottidis compared with each suture alone and may improve surgical outcome in laryngoplasty.


In vitro efficacy of a buffered chelating solution as an antimicrobial potentiator for antifungal drugs against fungal pathogens obtained from horses with mycotic keratitis.
To determine whether a novel third-generation chelating agent (8 mM disodium EDTA dehydrate and 20 mM 2-amino-2-hydroxymethyl-1, 3-propanediol) would act as an antimicrobial potentiator to enhance in vitro activity of antifungal medications against fungal isolates obtained from horses with mycotic keratitis. Fungal isolates (3 Aspergillus isolates, 5 Fusarium isolates, 1 Penicillium isolate, 1 Cladosporium isolate, and 1 Curvularia isolate) obtained from horses with mycotic keratitis and 2 quality-control strains obtained from the American Type Culture Collection (ATCC; Candida albicans ATCC 90028 and Paecilomyces variotii ATCC 36257). Minimum inhibitory concentrations (MICs) against fungal isolates for 4 antifungal drugs (miconazole, ketoconazole, itraconazole, and natamycin) were compared with MICs against fungal isolates for the combinations of each of the 4 antifungal drugs and the chelating agent. The Clinical and Laboratory Standards Institute microdilution assay method was performed by use of reference-grade antifungal powders against the fungal isolates and quality-control strains of fungi. Values for the MIC at which the antifungal drugs decreased the growth of an organism by 50% (MIC50) and 90% (MIC90) were decreased for the control strains and ophthalmic fungal isolates by 50% to 100% when the drugs were used in combination with the chelating agent at a concentration of up to 540 microg/mL. The chelating agent increased in vitro activity of antifungal drugs against common fungal pathogens isolated from eyes of horses with mycotic keratitis.


Deconstructing breast cancer heterogeneity: clinical implications for women with Basal-like tumors.
To compare and contrast the molecular and environmental factors contributing to basal-like breast cancer and highlight the clinical implications for women with this phenotype. CINAHL® and PubMed databases, journals, and citation indices were searched using the key word basal-like in combination with breast cancer, epigenetic, treatment, subtype, risk factor, and BRCA1 to synthesize the literature on the multiple underpinnings of basal-like breast cancer. Research findings related to the molecular foundation of basal-like breast cancer were integrated with knowledge of nongenetic contributing risk factors. Approved therapies and those under development were summarized with the goal of improving understanding for research and practice. Of the five subtypes of breast cancer, the basal-like subtype has the shortest survival and poorest prognosis. The development of gene expression assays with epigenetic studies has enabled reliable identification of the basal-like subtype and has shed light on novel therapeutic possibilities. Clinical trials for basal-like breast cancer are underway, and the potential for individualized treatments for women with this subtype show promise. The main difficulties with basal-like breast cancer are its aggressive course, treatment refractory nature, and complex biology, all of which pose real challenges for clinical management and patient education. Oncology nurses play a pivotal role in providing holistic care and patient support. Therefore, nurses must understand the complexity of the clinical presentation and the underlying biology of this cancer subtype.


Parental judgements of infant pain: importance of perceived cognitive abilities, behavioural cues and contextual cues.
Despite blatant indications, such as behavioural and contextual cues, infant pain is often undermanaged by adult caretakers. The belief that infants are limited in their abilities to comprehend the meaning of an experience or recall that experience has been used to minimize or deny the need for intervention in this vulnerable population. This investigation explored parental beliefs regarding the impact of infant cognitive capabilities, behavioural cues and contextual cues to their pain judgments. Particular interest was focused on their beliefs regarding the general cognitive capabilities of infants of different ages. Forty-nine parents viewed videotapes of healthy infants, aged two, four, six, 12 and 18 months, receiving routine immunization injections and provided judgements of the severity of pain on a 100 mm Visual Analogue Scale. Upon completion of their pain judgements for each of the five age groups (two infants per age group; 10 infants total), parents completed questionnaires regarding their beliefs about the capabilities of infants in that age group and then reported the importance of the various cues utilized to formulate their pain judgements. Parents attributed substantial pain to infants in all age groups, almost twice the amount they hypothesized an adult undergoing a similar injection would experience. The cues rated as most important for judgements were similar for infants of varying ages. Overall, facial expressions, sounds and body movements were consistently reported to be most important. Parents acknowledged the development of memory and understanding of pain throughout infancy. However, these beliefs were not deemed by parents as important to their pain ratings, nor were their importance ratings directly related to the pain ratings. Parents judged that infants undergoing a routine immunization were experiencing clinically significant levels of pain. However, despite generally acknowledging a developing trajectory for memory and understanding across the five age groups, parents did not indicate that a child's ability to remember and understand pain were essential features of their pain judgements. The results indicated that memory and understanding did not influence parental judgements of infant pain demonstrating the validity of the parents' self-assessments.


Angiotensin II stimulates superoxide production by nitric oxide synthase in thick ascending limbs.
Angiotensin II (Ang II) causes nitric oxide synthase (NOS) to become a source of superoxide (O2 (-)) via a protein kinase C (PKC)-dependent process in endothelial cells. Ang II stimulates both NO and O2 (-) production in thick ascending limbs. We hypothesized that Ang II causes O2 (-) production by NOS in thick ascending limbs via a PKC-dependent mechanism. NO production was measured in isolated rat thick ascending limbs using DAF-FM, whereas O2 (-) was measured in thick ascending limb suspensions using the lucigenin assay. Consistent stimulation of NO was observed with 1 nmol/L Ang II (P < 0.001; n = 9). This concentration of Ang II-stimulated O2 (-) production by 50% (1.77 ± 0.26 vs. 2.62 ± 0.36 relative lights units (RLU)/s/μg protein; P < 0.04; n = 5). In the presence of the NOS inhibitor L-NAME, Ang II-stimulated O2 (-) decreased from 2.02 ± 0.29 to 1.10 ± 0.11 RLU/s/μg protein (P < 0.01; n = 8). L-arginine alone did not change Ang II-stimulated O2 (-) (2.34 ± 0.22 vs. 2.29 ± 0.29 RLU/s/μg protein; n = 5). In the presence of Ang II plus the PKC α/β1 inhibitor Gö 6976, L-NAME had no effect on O2 (-) production (0.78 ± 0.23 vs. 0.62 ± 0.11 RLU/s/μg protein; n = 7). In the presence of Ang II plus apocynin, a NADPH oxidase inhibitor, L-NAME did not change O2 (-) (0.59 ± 0.04 vs. 0.61 ± ×0.08 RLU/s/μg protein; n = 5). We conclude that: (1) Ang II causes NOS to produce O2 (-) in thick ascending limbs via a PKC- and NADPH oxidase-dependent process; and (2) the effect of Ang II is not due to limited substrate.


Randomized phase 2 trial of intracoronary nitrite during acute myocardial infarction.
Preclinical evidence demonstrates that inorganic nitrite, after its in situ conversion to nitric oxide, attenuates consequent myocardial reperfusion injury. We investigated whether intracoronary injection of nitrite during primary percutaneous coronary intervention might improve infarct size in ST-elevated myocardial infarction. Patients undergoing primary percutaneous coronary intervention (n=80) were randomized to receive intracoronary (10 mL) sodium nitrite (1.8 μmol) or NaCl (placebo) before balloon inflation. The primary end point was infarct size assessed by measuring creatine kinase release. Secondary outcomes included infarct size assessed by troponin T release and by cardiac MRI on day 2. Baseline characteristics were similar between the groups. No evidence of differences in creatine kinase release (P=0.92), troponin T (P=0.85), or cardiac MRI-assessed infarct size (P=0.254) were evident. In contrast, there was an improvement [corrected] in myocardial salvage index (P=0.05) and reduction in [corrected] major adverse cardiac event at 1 year (2.6% versus 15.8%; P=0.04) in the nitrite group. In a 66-patient subgroup with thrombolysis in myocardial infarction ≤1 flow, there was reduced serum creatine kinase (P=0.030) and a 19% reduction in cardiac MRI-determined infarct size (P=0.034) with nitrite. No adverse effects of nitrite were detected. In this phase II study, intracoronary nitrite infusion did not alter infarct size, although a trend to improved myocardial salvage index and a significant reduction in major adverse cardiac event was evident. In a subgroup of patients with thrombolysis in myocardial infarction flow ≤1, nitrite reduced infarct size and major adverse cardiac event and improved myocardial salvage index, indicating that a phase III clinical trial assessing intracoronary nitrite administration as an adjunct to percutaneous coronary intervention in ST-elevated myocardial infarction patients is warranted. http://www.clinicaltrials.gov. Unique identifier: NCT01584453.


In vivo measurement of energy substrate contribution to cold-induced brown adipose tissue thermogenesis.
The present study was designed to investigate the effects of cold on brown adipose tissue (BAT) energy substrate utilization in vivo using the positron emission tomography tracers [(18)F]fluorodeoxyglucose (glucose uptake), 14(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid [nonesterified fatty acid (NEFA) uptake], and [(11)C]acetate (oxidative activity). The measurements were performed in rats adapted to 27°C, which were acutely subjected to cold (10°C) for 2 and 6 hours, and in rats chronically adapted to 10°C for 21 days, which were returned to 27°C for 2 and 6 hours. Cold exposure (acutely and chronically) led to increases in BAT oxidative activity, which was accompanied by concomitant increases in glucose and NEFA uptake. The increases were particularly high in cold-adapted rats and largely readily reduced by the return to a warm environment. The cold-induced increase in oxidative activity was meaningfully blunted by nicotinic acid, a lipolysis inhibitor, which emphasizes in vivo the key role of intracellular lipid in BAT thermogenesis. The changes in BAT oxidative activity and glucose and NEFA uptakes were paralleled by inductions of genes involved in not only oxidative metabolism but also in energy substrate replenishment (triglyceride and glycogen synthesis). The capacity of BAT for energy substrate replenishment is remarkable.


Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRASG13D mutation.
Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies. To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRASG13D allele (mtKRAS). RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling. We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.


Possible sources and spreading routes of highly pathogenic avian influenza virus subtype H5N1 infections in poultry and wild birds in Central Europe in 2007 inferred through likelihood analyses.
Recurrent outbreaks of H5N1 HPAIV occurred in several Central European countries in 2007. In-depth phylogenetic analyses which included full-length genomic sequences of the viruses involved were performed to elucidate possible origins of incursions and transmission pathways. Tree reconstructions as well as host-shift and ancestral area inferences were conducted in a maximum likelihood framework. All viruses belonged to a separate subgroup (termed "EMA-3") within clade 2.2, and, thus, were distinct from two lineages of HPAIV H5N1 viruses (termed "EMA-1" and "EMA-2") present in the same geographic area in 2006. Analysis of concatenated coding regions of all eight genome segments significantly improved resolution and robustness of the reconstructed phylogenies as compared to single gene analyses. At the same time, the methodological limits to establish retrospectively transmission networks in a comparatively small geographic region and spanning a short period of time became evident when only few corroborating field-epidemiological data are available. Ambiguities remained concerning the origin of the EMA-3 viruses from a region covering Southeast Germany and the Czech Republic as well as routes of spread to other European countries. AIV monitoring programmes in place for wild birds and poultry in these countries did not reveal presence of these viruses in either population. Host switches between domestic poultry and wild bird populations occurred several times. Analysis of outbreaks in Northeast Germany and nearby Northern Poland in December 2007 demonstrated that geographic and even temporal vicinity of outbreaks does not necessarily indicate a common source of incursion.


Enhanced immigration enforcement in the USA and the transnational continuity of HIV care for Latin American immigrants in deportation proceedings.
In our work as clinicians, researchers, and immigrant rights advocates, we have noted increased anxiety about the possibility of deportation and disruptions in care among immigrants with HIV. Before the 2016 US elections, patients rarely asked about HIV treatment in their home countries. However, since the increase in anti-immigrant rhetoric and arrests by US Immigration and Customs Enforcement, patients have voiced concerns about the availability of HIV treatment in their home countries much more frequently. Although antiretroviral therapy is available throughout Latin America, access depends on economic, social, and political circumstances. Maintaining uninterrupted continuity of care among immigrants held in detention or deported to their home countries is challenging. In this Viewpoint, we identify periods of particular vulnerability for immigrants during deportation proceedings, from initial detention to deposition in their country of origin. We discuss the effect of enhanced immigration enforcement on the health and wellbeing of HIV-infected immigrants, and on public health. Finally, we also discuss recommendations for clinicians, immigration authorities, and public health institutions in the USA and in receiving countries.


Effect of type 2 diabetes on risk for malignancies includes hepatocellular carcinoma in chronic hepatitis C.
The aim of this retrospective cohort study was to assess the cumulative development incidence and predictive factors for malignancies after the termination of interferon (IFN) therapy in Japanese patients for hepatitis C virus (HCV). A total of 4,302 HCV-positive patients treated with IFN were enrolled. The mean observation period was 8.1 years. The primary outcome was the first onset of malignancies. Evaluation was performed using the Kaplan-Meier method and Cox proportional hazard analysis. A total of 606 patients developed malignancies: 393 developed hepatocellular carcinoma (HCC) and 213 developed malignancies other than HCC. The cumulative development rate of HCC was 4.3% at 5 years, 10.5% at 10 years, and 19.7% at 15 years. HCC occurred significantly (P<0.05) when the following characteristics were present: advanced histological staging, sustained virological response not achieved, male sex, advanced age of ≥50 years, total alcohol intake of ≥200 kg, and presence of type 2 diabetes (T2DM). T2DM caused a 1.73-fold enhancement in HCC development. In patients with T2DM, HCC decreased when patients had a mean hemoglobin A1c (HbA1c) level of <7.0% during follow-up (hazard ratio, 0.56; 95% confidence interval, 0.33-0.89; P=0.015). The cumulative development rate of malignancy other than HCC was 2.4% at 5 years, 5.1% at 10 years, and 9.8% at 15 years. Malignancies other than HCC occurred significantly when patients were of advanced age of ≤50 years, smoking index (package per day×year) was ≥20, and T2DM was present. T2DM caused a 1.70-fold enhancement in the development of malignancies other than HCC. T2DM causes an approximately 1.7-fold enhancement in the development of HCC and malignancies other than HCC in HCV-positive patients treated with IFN. In T2DM patients, maintaining a mean HbA1c level of <7.0% reduces the development of HCC.


Exploring volatile general anesthetic binding to a closed membrane-bound bacterial voltage-gated sodium channel via computation.
Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics "flooding" simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the "fenestrations" present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.


Dataset for solving a hybrid flexibility strategy on personnel scheduling problem in the retail industry.
This data article describes datasets from a home improvement retail store located in Santiago, Chile. The datasets have been developed to simultaneously solve a staffing and tour scheduling problem that incorporates flexible contracts and multiskilled staff. This Data in Brief article is related to the published article "Hybrid flexibility strategy on personnel scheduling: Retail case study" [1]. The datasets contain real, processed, and simulated data. Regarding the real and processed datasets, they are presented for three different store sizes (4, 5 or 6 departments). Real datasets include information about the employment-contract characteristics, cost parameters, and a forecast of the number of employees required in each department for each day of the week and each time period into which the operating day is divided. As regards the data processed for the case study, they include the set of skill sets considering that the employees can be trained in a maximum of two store departments. Regarding the simulated datasets, they include information about the random parameter of staff demand in each store department. The simulated data are presented in 90 text files classified by: (i) Store size (4, 5 or 6 departments). (ii) Coefficient of variation (10, 20, 30%). (iii) Instance identification number (10 instances per scenario that resulted from combining the store sizes and coefficients of variation). Researchers can use the datasets for benchmarking the performance of different approaches with the one presented by Porto et al. [1], and in consequence, they can find solutions to the same (or similar) type of personnel scheduling problem. The dataset includes an Excel workbook that can be used to randomly generate staff demand instances according to a chosen coefficient of variation.


Effects of maternal undernutrition during late gestation on the lung surfactant system and morphometry in rats.
Intrauterine growth restriction (IUGR) is associated with reduced lung function during infancy and throughout adulthood. We investigated the effects of maternal undernutrition (50% rations of the control food intake) during the last week of gestation on the pulmonary surfactant system and lung morphometry in postnatal rats. IUGR rats exhibited a significantly lower body weight, lower lung weight, lower lung/body weight ratio, lower lung volume, and lower lung volume/body weight ratio on some postnatal days. IUGR rats had a significantly lower lung saturated phosphatidylcholine and lower plasma corticosterone levels on postnatal d 1 only, and values were comparable between control and IUGR rats in the ensuing weeks. Lung surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNA expressions were similar between control and IUGR rats. Volume fractions of the alveolar airspace were significantly lower in IUGR rats on postnatal d 7, 14, and 42. Alveolar surface areas were significantly lower in IUGR rats during the study period. The alveolar surface area/body weight ratio reached a peak on postnatal d 7, and values were significantly lower in IUGR rats on postnatal d 1, 14, 28, and 42. We conclude that maternal undernutrition during late gestation decreases lung surfactant lipid levels in the immediate postnatal period and alters the development of lung structure during the postnatal period. Alteration of lung surfactant and structure may be important in the pathogenesis of impaired pulmonary function in IUGR infants and children.


Successful autologous haematopoietic stem cell transplantation for refractory myasthenia gravis - a case report.
Myasthenia gravis (MG) is an autoimmune disease, with immune reactivity against the post-synaptic endplate of the neuromuscular junction. Apart from symptomatic treatment with choline esterase blockers, many patients also require immunomodulatory treatment. Despite existing treatment options, some patients are treatment refractory. We describe a patient with severe MG refractory to corticosteroids, four oral immunosuppressants, cyclophosphamide, rituximab and bortezomib who was treated with autologous haematopoietic stem cell transplantation. Two years after this, the patient has significantly improved in objective tests and in quality of life and leads an active life. Diplopia is her only remaining symptom and she is completely free of medication for MG. We believe that autologous haematopoietic stem cell transplantation can be an effective therapeutic option for carefully selected cases of severe, treatment refractory MG.


A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation.
Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.


Challenges in linking health research to policy: a commentary on developing a multi-stakeholder response to orphans and vulnerable children in Ghana.
The Research and Development Division (RDD) of the Ghana Health Service (GHS) has a remit to build research capacity and conduct policy relevant research. By being situated within the GHS, RDD has good access to directors and programme managers, within and beyond the Ministry of Health. This structure has been facilitating collaboration through research cycles for 20 years, from agenda setting to discussions on policy relevance.This approach has been applied to research activities within the Addressing the Balance of Burden in AIDS (ABBA) Research Programme Consortium to tackle the challenges facing HIV affected orphans and vulnerable children (OVCs). The government strategy on OVCs recommends they should be encouraged to live in their home communities rather than in institutions. We present lessons here on efforts to use research to build a response across different agencies to address the problems that communities and families face in caring for these children in their communities.This approach to building consensus on research priorities points to the value of collaboration and dialogue with multiple stakeholders as a means of fostering ownership of a research process and supporting the relevance of research to different groups. Our experience has shown that if the context within which researchers, policy makers and stakeholders work were better understood, the links between them were improved and research were communicated more effectively, then better policy making which links across different sectors may follow. At the same time, collaboration among these different stakeholders to ensure that research meets social needs, must also satisfy the requirements of scientific rigour.


Prognostic indicators of life-threatening malaria are associated with distinct parasite variant antigen profiles.
PfEMP1 is a family of cytoadhesive surface antigens expressed on erythrocytes infected with Plasmodium falciparum, the parasite that causes the most severe form of malaria. These surface antigens play a role in immune evasion and are thought to contribute to the pathogenesis of the malaria parasite. Previous studies have suggested a role for a specific subset of PfEMP1 called "group A" in severe malaria. To explore the role of group A PfEMP1 in disease, we measured the expression of the var genes that encode them in parasites from clinical isolates collected from children suffering from malaria. We also looked at the ability of these clinical isolates to induce rosetting of erythrocytes, which indicates a cytoadhesion phenotype that is thought to be important in pathogenesis. These two sets of data were correlated with the presence of two life-threatening manifestations of severe malaria in the children: impaired consciousness and respiratory distress. Using regression analysis, we show that marked rosetting was associated with respiratory distress, whereas elevated expression of group A-like var genes without elevated rosetting was associated with impaired consciousness. The results suggest that manifestations of malarial disease may reflect the distribution of cytoadhesion phenotypes expressed by the infecting parasite population.


Oxidative stress in fetal distress: potential prospects for diagnosis.
Our aim was to investigate the relation between fetal distress and oxidative stress. Fetal distress was associated with increased concentration of superoxide in the fetal blood and with significant increase of the level of H2O2 in both maternal and fetal blood. The activity of superoxide dismutase was increased roughly sixfold (p<0.01) in the maternal (7330 +/- 2240 U/g of hemoglobin in controls (C) and 36811 +/- 16862 U/g in fetal distress (FD)) and fetal blood (C: 5930 +/- 2641 U/g; FD: 41912 +/- 17133 U/g). In contrast, fetal distress was related to a considerable decrease of catalase activity in both maternal (C: 26011 +/- 8811 U/g; FD: 7212 +/- 1270 U/g) and fetal blood (C: 37194 +/- 9191 U/g; FD: 6173 +/- 1965 U/g). From this we concluded that in fetal distress, the maternal and fetal bloods are exposed to superoxide- and H2O2-mediated oxidative stress, which could be initiated by hypoxic conditions in the fetal blood and placenta. A tremendous increase/decrease of the activities of superoxide dismutase/catalase in the blood of women bearing a distressed fetus in comparison to healthy subjects implies that the assessment of superoxide dismutase/catalase activity could be of use for establishing a timely and accurate ante- or intrapartum diagnosis of fetal distress.


Submicrometer-wide amorphous and polycrystalline anatase TiO2 waveguides for microphotonic devices.
We demonstrate amorphous and polycrystalline anatase TiO(2) thin films and submicrometer-wide waveguides with promising optical properties for microphotonic devices. We deposit both amorphous and polycrystalline anatase TiO(2) using reactive sputtering and define waveguides using electron-beam lithography and reactive ion etching. For the amorphous TiO(2), we obtain propagation losses of 0.12 ± 0.02 dB/mm at 633 nm and 0.04 ± 0.01 dB/mm at 1550 nm in thin films and 2.6 ± 0.5 dB/mm at 633 nm and 0.4 ± 0.2 dB/mm at 1550 nm in waveguides. Using single-mode amorphous TiO(2) waveguides, we characterize microphotonic features including microbends and optical couplers. We show transmission of 780-nm light through microbends having radii down to 2 μm and variable signal splitting in microphotonic couplers with coupling lengths of 10 μm.


Prevalence of multimorbidity in a geographically defined American population: patterns by age, sex, and race/ethnicity.
To describe the prevalence of multimorbidity involving 20 selected chronic conditions in a geographically defined US population, emphasizing age, sex, and racial/ethnic differences. Using the Rochester Epidemiology Project records linkage system, we identified all residents of Olmsted County, Minnesota, on April 1, 2010, and electronically extracted the International Classification of Diseases, Ninth Revision codes associated with all health care visits made between April 1, 2005, and March 31, 2010 (5-year capture frame). Using these codes, we defined the 20 common chronic conditions recommended by the US Department of Health and Human Services. We counted only persons who received at least 2 codes for a given condition separated by more than 30 days, and we calculated the age-, sex-, and race/ethnicity-specific prevalence of multimorbidity. Of the 138,858 study participants, 52.4% were women (n=72,732) and 38.9% had 1 or more conditions (n=54,012), 22.6% had 2 or more conditions (n=31,444), and 4.9% had 5 or more conditions (n=6853). The prevalence of multimorbidity (≥2 conditions) increased steeply with older age and reached 77.3% at 65 years and older. However, the absolute number of people affected by multimorbidity was higher in those younger than 65 years. Although the prevalence of multimorbidity was similar in men and women overall, the most common dyads and triads of conditions varied by sex. Compared with white persons, the prevalence of multimorbidity was slightly higher in black persons and slightly lower in Asian persons. Multimorbidity is common in the general population; it increases steeply with older age, has different patterns in men and women, and varies by race/ethnicity.


Clinical trial methodology of pain treatment studies: selection and measurement of self-report primary outcomes for efficacy.
The past century has seen immense progress in the advancement of methodology to evaluate efficacy of treatment interventions for acute and chronic pain. Continuing challenges revolve around how to best select and measure primary efficacy outcomes for a given analgesic trial. Recognizing the complex, multidimensional, sensory and emotional nature of pain and applying psychometric techniques have facilitated the development of several valid and reliable self-report measures that evaluate pain intensity, pain relief, and other important outcome domains relevant to pain treatment. In the setting of emerging new pain treatment strategies, careful consideration must be given to match current or novel outcome measures to the specific goals of a proposed trial. Future research is needed to directly compare current methods with newer measurement approaches for the critical goal of maximizing validity, reliability, and utility of different outcome measures in clinical trials of pain treatment.


Dental infection and vascular disease.
Periodontitis is a chronic inflammatory response to bacterial plaque in which the anchoring bone and soft tissues supporting teeth are destroyed, resulting in tooth mobility and loss. Dental caries involves the spread of infection from the dentine to the vascular dental pulp and periapical bony tissues, before involvement of adjacent soft tissues and spreading sepsis. Several case-controlled, cross-sectional, and cohort studies report correlation between periodontitis and increased cardiovascular, cerebrovascular, and peripheral artery disease, as determined by clinical disease, angiography, ultrasonography, and reduced flow-mediated dilation. Some studies report a similar relationship of atherosclerosis with periapical infection and potentially also with coronal caries, and this review identifies the need to investigate these associations further. Smoking and cadmium exposure are epidemiologically confounding environmental risk factors shared by atherosclerosis and periodontitis. Further complicating epidemiological studies are the risk factors for both atherosclerosis and periodontitis, with which periodontitis appears to have separate positive feedback relationships. These include diabetes, increased plasma lipid levels, hypertension, and white blood cell count. Animal and human intervention studies provide some direct support of a causal role for periodontitis in atherosclerosis, and possible mechanisms include bacterial invasion of arteries, specific atherogenic properties of oral bacteria, the acute phase response, and cytokine polymorphisms.


Systems Biology and Biomarkers of Early Effects for Occupational Exposure Limit Setting.
In a recent National Research Council document, new strategies for risk assessment were described to enable more accurate and quicker assessments. This report suggested that evaluating individual responses through increased use of bio-monitoring could improve dose-response estimations. Identification of specific biomarkers may be useful for diagnostics or risk prediction as they have the potential to improve exposure assessments. This paper discusses systems biology, biomarkers of effect, and computational toxicology approaches and their relevance to the occupational exposure limit setting process. The systems biology approach evaluates the integration of biological processes and how disruption of these processes by chemicals or other hazards affects disease outcomes. This type of approach could provide information used in delineating the mode of action of the response or toxicity, and may be useful to define the low adverse and no adverse effect levels. Biomarkers of effect are changes measured in biological systems and are considered to be preclinical in nature. Advances in computational methods and experimental -omics methods that allow the simultaneous measurement of families of macromolecules such as DNA, RNA, and proteins in a single analysis have made these systems approaches feasible for broad application. The utility of the information for risk assessments from -omics approaches has shown promise and can provide information on mode of action and dose-response relationships. As these techniques evolve, estimation of internal dose and response biomarkers will be a critical test of these new technologies for application in risk assessment strategies. While proof of concept studies have been conducted that provide evidence of their value, challenges with standardization and harmonization still need to be overcome before these methods are used routinely.


Population genetics of Trypanosoma brucei rhodesiense: clonality and diversity within and between foci.
African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda) and Southern (Malawi) Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.


A conserved RING finger protein required for histone H2B monoubiquitination and cell size control.
Monoubiquitination of histone H2B is required for methylation of histone H3 on lysine 4 (K4), a modification associated with active chromatin. The identity of the cognate ubiquitin ligase is unknown. We identify Bre1 as an evolutionarily conserved RING finger protein required in vivo for both H2B ubiquitination and H3 K4 methylation. The RING domain of Bre1 is essential for both of these modifications as is Lge1 (Large 1), a protein required for cell size control that copurifies with Bre1. In cells lacking the euchromatin-associated histone variant H2A.Z, BRE1, RAD6, and LGE1 are each essential for cell viability, supporting redundant functions for H2B ubiquitination and H2A substitution in the formation of active chromatin. Notably, analysis of mutants demonstrates a function for Bre1/Lge1-dependent H2B monoubiquitination in the control of cell size.


The MEK5/ERK5 signalling pathway in cancer: a promising novel therapeutic target.
Conventional mitogen-activated protein kinase (MAPK) family members are among the most sought-after oncogenic effectors for the development of novel human cancer treatment strategies. MEK5/ERK5 has been the less-studied MAPK subfamily, despite its increasingly demonstrated relevance in the growth, survival, and differentiation of normal cells. MEK5/ERK5 signalling has already been proposed to have pivotal roles in several cancer hallmarks, and to mediate the effects of a range of oncogenes. Accumulating evidence indicates the contribution of MEK5/ERK5 signalling to therapy resistance and the benefits of using MEK5/ERK5 inhibitory strategies in the treatment of human cancer. Here, we explore the major known contributions of MEK5/ERK5 signalling to the onset and progression of several types of cancer, and highlight the potential clinical relevance of targeting MEK5/ERK5 pathways.


Panax ginseng: a role in cancer therapy?
Panax ginseng is a plant that has been used in traditional medicine in China for thousands of years. It is used as a general tonic or adaptogen with chronically ill patients and is frequently featured in traditional medicine prescriptions from China, Japan, and Korea used by cancer patients. The putative active compounds are the ginsenosides, of which there are more than two dozen. These compounds are found in both Panax ginseng and in other Panax species that are used in herbal medicine. Analysis of ginsenosides is being used in developing quality control assessments for ginseng, which has frequently been adulterated due to its high cost; many currently available standardized extracts do appear to contain the amounts of ginsenosides listed on package labeling. The toxicity of ginseng appears to be low: some of the reports of toxic episodes of ginseng may actually pertain to other components of multicomponent preparations. Very low incidence of toxicity has been observed in ginseng clinical trials using well-characterized preparations. Numerous pharmacological activities of ginseng and the ginsenosides have been explored: the authors review here the activities relating to cancer. Immune system modulation, antistress activities, and antihyperglycemic activities are among the most notable features of ginseng noted in laboratory and clinical analyses. Much testing has been done in humans to explore ginseng's purported antifatigue properties, but this area remains controversial. A number of investigations point to antitumor properties and other pharmacological activities related to cancer, but no trials have yet confirmed a clinically significant anticancer activity. Cancer patients may empirically find ginseng to be useful when they are fatigued, although clinical trials should be conducted to confirm its benefits.


Inhibition of L-type calcium currents in guinea pig ventricular myocytes by the kappa-opioid agonist U50488H does not involve binding to opiate receptors.
In guinea pig ventricular myocytes the kappa-opioid agonist trans(+/-)-3,3-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate salt (U50488H) inhibited L-type calcium currents (ICa) as measured by the patch clamp technique in a dose-dependent fashion. The basal, as well as the isoprenaline and 8-Br-cAMP-stimulated, ICa were found to be reduced by U50488H. The inhibition was reversible and was not prevented on preincubation with pertussis toxin or the receptor antagonists naloxone and naltrexone. Naltrexone alone also caused an inhibition of ICa. Leucine enkephaline, a peptide opiate agonist, had no effect on ICa. U50488H did not alter the current/voltage relationship of the calcium current. The inhibition was independent of the cytosolic calcium concentration because it was also observed in the presence of 10 mM BAPTA in the pipette. If the compound was applied intracellularly via a perfused patch pipette there was no inhibition of ICa. The calcium current stimulated by the dihydropyridine calcium agonist Bay K 8644 was also reduced by U50488H. Conversely the inhibition of ICa by U50488H could be antagonized by Bay K 8644. In conclusion, these results demonstrate that binding to specific membrane receptors is not involved in the inhibition of L-type calcium current by U50488H and other nonpeptide opioid agonists. A direct interaction with the channel molecule at the exterior of the cell is probably involved.


The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection.
The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.


Individualistic and time-varying tree-ring growth to climate sensitivity.
The development of dendrochronological time series in order to analyze climate-growth relationships usually involves first a rigorous selection of trees and then the computation of the mean tree-growth measurement series. This study suggests a change in the perspective, passing from an analysis of climate-growth relationships that typically focuses on the mean response of a species to investigating the whole range of individual responses among sample trees. Results highlight that this new approach, tested on a larch and stone pine tree-ring dataset, outperforms, in terms of information obtained, the classical one, with significant improvements regarding the strength, distribution and time-variability of the individual tree-ring growth response to climate. Moreover, a significant change over time of the tree sensitivity to climatic variability has been detected. Accordingly, the best-responder trees at any one time may not always have been the best-responders and may not continue to be so. With minor adjustments to current dendroecological protocol and adopting an individualistic approach, we can improve the quality and reliability of the ecological inferences derived from the climate-growth relationships.


Tctex-1 controls ciliary resorption by regulating branched actin polymerization and endocytosis.
The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain. Furthermore, Tctex-1, originally identified as a cytoplasmic dynein light chain, has a dynein-independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex-1, actin, and dynamin. Mechanistically, Tctex-1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex-1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin-dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex-1-regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.


Clopidogrel and warfarin pharmacogenetic tests: what is the evidence for use in clinical practice?
To review the most promising genetic markers associated with the variability in the safety or efficacy of warfarin and clopidogrel and highlight the verification and validation initiatives for translating clopidogrel and warfarin pharmacogenetic tests to clinical practice. Rapid advances in pharmacogenetics, continuous decrease in genotyping cost, development of point-of-care devices and the newly established clinical genotyping programs at several institutions hold the promise of individualizing clopidogrel and warfarin based on genotype. Guidelines have been established to assist clinicians in prescribing clopidogrel or warfarin dose based on genotype. However, the clinical utility of clopidogrel and warfarin is still limited. Accordingly, large randomized clinical trials are underway to define the role of clopidogrel and warfarin pharmacogenetics in clinical practice. Pharmacogenetics has offered compelling evidence toward the individualization of clopidogrel and warfarin therapies. The rapid advances in technology make the clinical implementation of clopidogrel and warfarin pharmacogenetics possible. The clinical genotyping programs and the ongoing clinical trials will help in overcoming some of the barriers facing the clinical implementation of clopidogrel and warfarin pharmacogenetics.


Structure and function of the Zika virus full-length NS5 protein.
The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.


Modification of linear (β1→3)-linked gluco-oligosaccharides with a novel recombinant β-glucosyltransferase (trans-β-glucosidase) enzyme from Bradyrhizobium diazoefficiens.
Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (β1→3) linkages preferably and to a lesser extent (β1→6) or (β1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (β1→3)(β1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic β-glucans in B. diazoefficiens.


Comparative neuroscience holds promise for quiet revolutions.
The brain has diversified and advanced in evolution more than any other organ; the variety of nervous systems and behaviors among animal species is thus available for our exploitation. Comparative neuroscience is likely to reach insights so novel as to constitute revolutions in understanding the structure, functions, ontogeny, and evolution of nervous systems. This promise requires pursuit on a wide front, in respect to disciplines and in respect to the species, stages, and states compared. It also requires deliberate concentration on the differences among animals, in addition to the prevailing concern for the basic and common. Neglect of these challenges would be costly. Without due consideration of the neural and behavioral correlates of differences between higher taxa and between closely related families, species, sexes, and stages, we cannot expect to understand our nervous systems or ourselves.


Collaborative Reflection Under the Microscope: Using Conversation Analysis to Study the Transition From Case Presentation to Discussion in GP Residents' Experience Sharing Sessions.
PHENOMENON: In higher education, reflection sessions are often used when participants learn in the workplace. In the Netherlands, all General Practitioner training programs include regular meetings called Exchange of Experiences, in which General Practitioner trainees are expected to learn collaboratively from their own and one another's experiences. Despite this being common practice, we found little research into the structure and process of these sessions. The purpose of this study is to describe the structure and characteristics of group reflection by describing transitions in interactions. We aim to describe the tutor's role in some detail, as this could lead to faculty development. In medical education, reflection is often approached from a cognitive perspective. However, learning in a group is also an interactional achievement. It is therefore relevant to study the sequential nature of group interaction in collaborative reflective practice. We have used conversation analysis to study the reflection meetings, zooming in on the transition between case presentation and discussion, focusing on the role of each of the participants in these transitions. The transitions were conversationally complex. Three interactional aspects recurred in the meetings. First, the transitions can be characterized as ambiguous, as there is ambiguity about what will happen next and the floor is open. Second, transitions are an arena for negotiations between case presenter, participants, and tutors, in which knowledge and the right to take the floor (epistemics) play an important part. Third, the tutor can have different interactional roles, namely, that of teacher, expert, facilitator, and active participant. The role of the tutor is important as the tutor's interactional behavior is part of the hidden curriculum. Insights: Conversation analysis focuses on the interaction in group learning and shows how the interaction is part of what is learned and how learning takes place. Transitions are the "messy" moments in interaction yet can tell a lot about the way in which group participants relate to one another. Being conscious of how the floor is taken, the tutor's roles, and the way negotiations take place could help medical educators in the way they shape collaborative learning sessions.


A protocol to assess the impact of adding nursing support workers to ward staffing.
To assess the impact of adding nursing support workers to ward staffing. Nurses' capacity to provide safe care is compromised by increased workloads and nursing shortages. Use of unregulated workers is an alternative to increasing the number of regulated nurses. The impact of adding nursing support workers on patient, nurse and system outcomes has not been systematically evaluated. A mixed longitudinal and cross-sectional design using administrative data sets and prospective data from a sample of wards. Payroll data will identify wards on which unregulated staff work. To assess the impact on nursing-sensitive outcomes, retrospective analysis of morbidity and mortality data of all patients admitted to Western Australia hospitals for over 24 hours across 4 years will be undertaken. For the cross-sectional study, a sample of 20 pairs of matched wards will be selected: 10 with unregulated workers added and 10 where they have not. From this sample the impact on patients will be assessed using the Patient Evaluation of Emotional Care during Hospitalisation survey. The impact on nurses will be assessed by a nurse survey used extensively which includes variables such as job satisfaction and intention to leave. The impact on system outcomes will be explored using work sampling of staff activities and the Practice Environment Scale. Interviews will determine nurses' experience of working with nursing support workers. The study aims to provide evidence about the impact of adding nursing support workers to ward staffing for patients, staff and the work environment.


Psychobiological influences on maternal sensitivity in the context of adversity.
This study evaluated prospective longitudinal relations among an index of poverty-related cumulative risk, maternal salivary cortisol, child negative affect, and maternal sensitivity across the first 2 postpartum years. Participants included 1,180 biological mothers residing in rural and predominantly low-income communities in the United States. Multilevel growth curve analyses indicated that an index of cumulative risk was positively associated with maternal cortisol across the postpartum (study visits occurring at approximately 7, 15, and 24 months postpartum) over and above effects for African American ethnicity, time of day of saliva collection, age, parity status, having given birth to another child, contraceptive use, tobacco smoking, body mass index, and breastfeeding. Consistent with a psychobiological theory of mothering, maternal salivary cortisol was negatively associated with maternal sensitivity observed during parent-child interactions across the first 2 postpartum years over and above effects for poverty-related cumulative risk, child negative affect, as well as a large number of covariates associated with cortisol and maternal sensitivity. Child negative affect expressed during parent-child interactions was negatively associated with observed maternal sensitivity at late (24 months) but not early time points of observation (7 months) and cumulative risk was negatively associated with maternal sensitivity across the postpartum and this effect strengthened over time. Results advance our understanding of the dynamic, transactional, and psychobiological influences on parental caregiving behaviors across the first 2 postpartum years. (PsycINFO Database Record


Sugar intake and body weight in Cambodian and Japanese children.
Because of the tastiness of sugars, it is easy to consume more than an adequate amount. There are many research reports that excess sugar intake contributes to dental decay, obesity, diabetes etc. Continuing economic development in Cambodia has made it easier than before for people to consume sugars in their daily life. Currently, isomerized sugar (a mixture of glucose and fructose) made from starches is commonly used in commercial beverages because of its low price. However, in Cambodia and Japan, sugar composition tables that include not only sucrose but also glucose, fructose, lactose and maltose have not been available. Prior to the present nutrition surveys, we made sugar composition tables for both countries. In this study we tried to estimate the intakes of various sugars by children in Cambodia and Japan and to determine the relationship between intake and body weight. Nutrition surveys of children aged 7, 10 and 13 years old were conducted for 3 nonconsecutive days by the 24 h recall method in 89 Cambodian children living in the capital city of Cambodia, Phnom Penh, and 151 Japanese children living in 3 prefectures from north to south. Height and weight of children in Cambodia and Japan were similar until 10 years old but at 13 years old, the Cambodians were shorter and lighter than the Japanese. We could not observe any differences in BMI in either country. The sugar intakes from beverages and snacks were not different among the different gender and age. Thus we combined the mean total sugar intake for Cambodian and Japanese, 28.42 ± 25.28 g and 25.69 ± 16.16 g respectively. These were within the range of WHO recommendations (less than 10% of energy intakes). Cambodian children consumed about 46% of sugars from commercial beverages and snacks and Japanese children 26%. This means that for Cambodians half of the sugars came from isomerized sugar made from starches. Relationships between sugar intake and body weight were not observed in both countries. In conclusion, the Cambodian children consumed about 46% of sugar from glucose and fructose (probably in the form of isomerized sugar), while the Japanese children took 26%; however, the intakes in both countries met the WHO recommendation and there was no relationship to body weight.


Evaluation of benefit to patients of training mental health professionals in suicide guidelines: cluster randomised trial.
Randomised studies examining the effect on patients of training professionals in adherence to suicide guidelines are scarce. To assess whether patients benefited from the training of professionals in adherence to suicide guidelines. In total 45 psychiatric departments were randomised (Dutch trial register: NTR3092). In the intervention condition, all staff in the departments were trained with an e-learning supported train-the-trainer programme. After the intervention, patients were assessed at admission and at 3-month follow-up. Primary outcome was change in suicide ideation, assessed with the Beck Scale for Suicide Ideation. For the total group of 566 patients with a positive score on the Beck Scale for Suicide Ideation at baseline, intention-to-treat analysis showed no effects of the intervention on patient outcomes at 3-month follow-up. Patients who were suicidal with a DSM-IV diagnosis of depression (n = 154) showed a significant decrease in suicide ideation when treated in the intervention group. Patients in the intervention group more often reported that suicidality was discussed during treatment. Overall, no effect of our intervention on patients was found. However, we did find a beneficial effect of the training of professionals on patients with depression.


Modelling of optimal vaccination strategies in response to a bioterrorism associated smallpox outbreak.
The reemergence of smallpox as a bioterrorism attack is now an increasing and legitimate concern. Advances in synthetic biology have now made it possible for the virus to be synthesized in a laboratory, with methods publicly available. Smallpox introduction into a susceptible population, with increased immunosuppression and an aging population, raises questions of how vaccination should be used in an epidemic situation when supply may be limited. We constructed three modified susceptible-latent-infectious-recovered (SEIR) models to simulate targeted, ring and mass vaccination in response to a smallpox outbreak in Sydney, Australia. We used age-specific distributions of susceptibility, infectivity, contact rates, and tested outputs under different assumptions. The number of doses needed of second- and third-generation vaccines are estimated, along with the total number of deaths at the end of the epidemic. We found a faster response is the key and ring vaccination of traced contacts is the most effective strategy and requires a smaller number of doses. However if public health authorities are unable to trace a high proportion of contacts, mass vaccination with at least 125,000 doses delivered per day is required. This study informs a better preparedness and response planning for vaccination in a case of a smallpox outbreak in a setting such as Sydney.


Effects of the offset term in experimental simulation on afterglow decay curve.
This study examines the effect of the offset term in a multiple single exponential equation that fits into experimental afterglow decay curve data for material applications. For afterglow materials applied and attached to structures, the inclusion of this offset term may reduce the values of the calculated decay times, τ i , and enlarge the time invariant constants, A i , in the associated equation compared to theoretically perfect test conditions. Using a set of experimental data obtained from a lab under dim light, adjustments can be made to calculate the required parameters for an equation without the offset term. This study uses mathematical simulations and lab tests to support our thesis and crosslink test results generated from different ambient light conditions. This paper defines the offset ratio as the ratio of the offset value, I 0, versus the initial light intensity in an equation. This ratio can be used to evaluate possible effects on the calculated parameters of an equation in an associated numerical simulation. The most reliable parameters will have consistent results from the use of multiple single exponential equations, with and without the offset term, in simulations to obtain them in an equation to model a set of data.


Kinetic discrimination of self/non-self RNA by the ATPase activity of RIG-I and MDA5.
The cytoplasmic RIG-like receptors are responsible for the early detection of viruses and other intracellular microbes by activating the innate immune response mediated by type I interferons (IFNs). RIG-I and MDA5 detect virus-specific RNA motifs with short 5'-tri/diphosphorylated, blunt-end double-stranded RNA (dsRNA) and >0.5-2 kb long dsRNA as canonical agonists, respectively. However, in vitro, they can bind to many RNA species, while in cells there is an activation threshold. As SF2 helicase/ATPase family members, ATP hydrolysis is dependent on co-operative RNA and ATP binding. Whereas simultaneous ATP and cognate RNA binding is sufficient to activate RIG-I by releasing autoinhibition of the signaling domains, the physiological role of the ATPase activity of RIG-I and MDA5 remains controversial. A cross-analysis of a rationally designed panel of RNA binding and ATPase mutants and truncated receptors, using type I IFN promoter activation as readout, allows us to refine our understanding of the structure-function relationships of RIG-I and MDA5. RNA activation of RIG-I depends on multiple critical RNA binding sites in its helicase domain as confirmed by functional evidence using novel mutations. We found that RIG-I or MDA5 mutants with low ATP hydrolysis activity exhibit constitutive activity but this was fully reverted when associated with mutations preventing RNA binding to the helicase domain. We propose that the turnover kinetics of the ATPase domain enables the discrimination of self/non-self RNA by both RIG-I and MDA5. Non-cognate, possibly self, RNA binding would lead to fast ATP turnover and RNA disassociation and thus insufficient time for the caspase activation and recruitment domains (CARDs) to promote downstream signaling, whereas tighter cognate RNA binding provides a longer time window for downstream events to be engaged. The exquisite fine-tuning of RIG-I and MDA5 RNA-dependent ATPase activity coupled to CARD release allows a robust IFN response from a minor subset of non-self RNAs within a sea of cellular self RNAs. This avoids the eventuality of deleterious autoimmunity effects as have been recently described to arise from natural gain-of-function alleles of RIG-I and MDA5.


Adenosine diphosphate involvement in THP-1 maturation triggered by the contact allergen 1-fluoro-2,4-dinitrobenzene.
Dendritic cells' (DC) activation is considered a key event in the adverse outcome pathway for skin sensitization elicited by covalent binding of chemicals to proteins. The mechanisms underlying DC activation by contact sensitizers are not completely understood. However, several "danger signals" are pointed as relevant effectors. Among these extra-cellular early danger signals, purines may be crucial for the development of xenoinflammation and several reports indicate their involvement in contact allergic reactions. In the present work we used the DC-surrogate monocytic cell line THP-1, cultured alone or co-cultured with the human keratinocyte cell line HaCaT, to explore the contribution of extracellular adenine nucleotides to THP-1 maturation triggered by the extreme contact sensitizer, 1-fluoro-2,4-dinitrobenzene (DNFB). We found that THP-1 maturation induced by DNFB is impaired after purinergic signaling inhibition, and that the transcription of the purinergic metabotropic receptors P2Y2 and P2Y11 is modulated by the sensitizer. We also detected that THP-1 cells only partially hydrolyse extracellular adenosine triphosphate, leading to accumulation of the mono-phosphate derivative, AMP. We detected different and non-overlapping activation patterns of mitogen activated protein kinases by DNFB and extracellular nucleotides. Overall, our results indicate that THP-1 maturation induced by DNFB is strongly modulated by extracellular adenine nucleotides through metabotropic purinergic receptors. This knowledge unveils a molecular toxicity pathway evoked by sensitizers and involved in THP-1 maturation, a DC-surrogate cell line thoroughly used in in vitro tests for the identification of skin allergens.


Estrogen receptor-alpha and endothelial nitric oxide synthase nuclear complex regulates transcription of human telomerase.
We report that in endothelial cells, the angiogenic effect of 17beta-estradiol (E2) is inhibited by the estrogen receptor (ER) antagonist ICI or the NO synthase (NOS) inhibitor 7-nitroindazole via downregulation of hTERT, the telomerase catalytic subunit, suggesting that E2 and NO are involved in controlling hTERT transcription. Quantitative Real-Time PCR and chromatin immunoprecipitations in E2-treated human umbilical vein endothelial cells, showed recruitment of ERs on the hTERT promoter and concomitant enrichment in histone 3 methylation at Lysine 79, a modification associated with transcription-competent chromatin. Confocal microscopy and re-chromatin immunoprecipitations revealed that on E2 induction, endothelial (e)NOS rapidly localized into the nucleus and associated with ERalpha on the hTERT promoter. Transfections of a constitutively active eNOS mutant (S1177D) strongly induced the hTERT promoter, indicating a direct role of the protein in hTERT transcriptional regulation. Mutation of the estrogen response element in the promoter abolished response to both ERs and active eNOS, demonstrating that the estrogen response element integrity is required for hTERT regulation by these factors. To investigate this novel regulation in a reduced NO environment, pulmonary endothelial cells were isolated from eNOS(-/-) mice and grown with/without E2. In wild-type cells, E2 significantly increased telomerase activity. In eNOS(-/-) cells, basal telomerase activity was rescued by exogenous eNOS or an NO donor, whereas responsiveness to E2 demanded the active protein. In conclusion, we document the novel findings of a combinatorial eNOS/ERalpha complex at the hTERT estrogen response element site and that active eNOS and ligand-activated ERs cooperate in regulating hTERT expression in the endothelium.


Elucidation of the conformational dynamics of multi-body systems by construction of Markov state models.
Constructing Markov State Models (MSMs) based on short molecular dynamics simulations is a powerful computational technique to complement experiments in predicting long-time kinetics of biomolecular processes at atomic resolution. Even though the MSM approach has been widely applied to study one-body processes such as protein folding and enzyme conformational changes, the majority of biological processes, e.g. protein-ligand recognition, signal transduction, and protein aggregation, essentially involve multiple entities. Here we review the attempts at constructing MSMs for multi-body systems, point out the challenges therein and discuss recent algorithmic progresses that alleviate these challenges. In particular, we describe an automatic kinetics based partitioning method that achieves optimal definition of the conformational states in a multi-body system, and discuss a novel maximum-likelihood approach that efficiently estimates the slow uphill kinetics utilizing pre-computed equilibrium populations of all states. We expect that these new algorithms and their combinations may boost investigations of important multi-body biological processes via the efficient construction of MSMs.


Progression of cellular adaptations in medial prefrontal and orbitofrontal cortex in response to repeated amphetamine.
Recent theories on addiction implicate adaptive changes in prefrontal cortex (PFC) neurons in reinforcing and psychotomimetic properties of psychostimulants, yet little is known about how neuronal responses to these drugs change over time. Here we describe electrophysiological evidence for a progressive and sustained change in the response of PFC neurons to amphetamine during repeated exposure. In spontaneously behaving rats and in rats engaged in an instrumental responding task, we followed the activity of medial PFC (mPFC) and orbitofrontal cortex (OFC) neurons during daily exposure to amphetamine and after a post-withdrawal challenge. Repeated amphetamine increased the number of responsive neurons and the magnitude of responses and modified spontaneous burst patterns. These changes were apparent after a few exposures to amphetamine, were amplified after withdrawal, and were region specific in that repeated amphetamine increasingly produced inhibitory responses in mPFC and excitatory responses in OFC. In behaviorally engaged animals, the gradual enhancement in mPFC inhibition and OFC overactivation correlated with a progressive impairment of instrumental responding. Furthermore, these changes were evident predominately in neurons that displayed phasic responses during task-related events. These rapid-onset and sustained cellular adaptations suggest that even limited exposure to psychostimulants may reduce the influence of mPFC neurons on behavior while at the same time exaggerating information encoded by OFC neurons.


Nucleotide sequence of an Escherichia coli chromosomal hemolysin.
We determined the DNA sequence of an 8,211-base-pair region encompassing the chromosomal hemolysin, molecularly cloned from an O4 serotype strain of Escherichia coli. All four hemolysin cistrons (transcriptional order, C, A, B, and D) were encoded on the same DNA strand, and their predicted molecular masses were, respectively, 19.7, 109.8, 79.9, and 54.6 kilodaltons. The identification of pSF4000-encoded polypeptides in E. coli minicells corroborated the assignment of the predicted polypeptides for hlyC, hlyA, and hlyD. However, based on the minicell results, two polypeptides appeared to be encoded on the hlyB region, one similar in size to the predicted molecular mass of 79.9 kilodaltons, and the other a smaller 46-kilodalton polypeptide. The four hemolysin gene displayed similar codon usage, which is atypical for E. coli. This reflects the low guanine-plus-cytosine content (40.2%) of the hemolysin DNA sequence and suggests the non-E. coli origin of the hemolysin determinant. In vitro-derived deletions of the hemolysin recombinant plasmid pSF4000 indicated that a region between 433 and 301 base pairs upstream of the putative start of hlyC is necessary for hemolysin synthesis. Based on the DNA sequence, a stem-loop transcription terminator-like structure (a 16-base-pair stem followed by seven uridylates) in the mRNA was predicted distal to the C-terminal end of hlyA. A model for the general transcriptional organization of the E. coli hemolysin determinant is presented.