Sumatriptan-naproxen and butalbital: a double-blind, placebo-controlled crossover study.
The primary objective was to compare the efficacy of a sumatriptan and naproxen combination medication (SumaRT/Nap-85mg sumatriptan and 500mg naproxen sodium), a butalbital-containing combination medication (BCM-50mg butalbital, 325mg acetaminophen, 40mg caffeine), and placebo when used to treat moderate to severe migraine headache pain in subjects who used BCMs in the past. Despite the lack of Food and Drug Administration approval and the absence of placebo-controlled trials to demonstrate efficacy, butalbital-containing medications are among the most commonly prescribed acute migraine treatments in the United States. Butalbital-containing medications are associated with serious and undesirable side effects, and have been linked to the chronification of migraine and development of medication-overuse headaches. This study compares the relative efficacy, safety, and tolerability of a fixed dose SumaRT/Nap versus a BCM and placebo. Enrolled subjects were required to have treated at least 1 migraine with a butalbital medication in the past. Enrolled subjects treated 3 moderate to severe migraines using each of the 3 study treatments once in a randomized sequence. The primary endpoint compared SumaRT/Nap versus BCM for sustained pain freedom at 2-24 hours without the use of any rescue medication. This study combines data from 2 identical outpatient, randomized, multicenter, double-blind, double-dummy, 3 attack crossover studies in adult migraineurs (International Classification of Headache Disorders, 2nd edition). A total of 442 subjects treated at least 1 attack with study medication. The majority of the treated subjects were female (88%) with a mean age 43 years, who reported that their migraines had a severe impact on their lives (78% with Headache Impact Test-6 of >59). At screening, 88% of subjects reported current butalbital use; 68% had used butalbital for more than 6 weeks; and 82% reported satisfaction with butalbital. Across treatment groups, 28-29% of subjects took study medication within 15 minutes of migraine onset, 34-37% of subjects took study medication >15 minutes to 2 hours after onset, and 32-36% of subjects took study medication more than 2 hours after onset. This study did not detect a difference at the nominal 0.05 level in percent sustained pain-free between SumaRT/Nap (8%), BCM (6%), and placebo (3%). SumaRT/Nap was superior to BCM for pain free at 2, 4, 6, 8, 24, 48 hours (P≤.044); pain relief (mild or no pain) at 2, 4, 6, 8, 24, 48 hours (P≤.01); sustained pain relief 2-24 hours (P<.001); migraine free (pain free with no nausea, photophobia, or phonophobia) at 4, 6, 8, 24, 48 hours (P≤.046); and complete symptom free (migraine free with no neck/sinus pain) at 4, 6, 8, 48 hours (P≤.031). Adverse event incidence was similar for all treatments (10%, 12%, and 9% for placebo, SumaRT/Nap, and BCM, respectively). Nausea was the most frequent adverse event (2%, 2%, and <1% for placebo, SumaRT/Nap, and BCM, respectively). Five serious adverse events were reported by 3 subjects: viral meningitis and colon neoplasm (placebo); chest pain and hypertension 17 days postdose (SumaRT/Nap); and breast cancer (BCM). Investigators judged no serious adverse events related to study medication. This study primarily included subjects whose migraines significantly impacted their lives. Before the study, these subjects used butalbital-containing medications as part of their current migraine treatment regimen and were satisfied with it, suggesting they were butalbital responders who had found a workable treatment strategy for themselves. When treated with SumaRT/Nap versus BCM in this study, however, a significant proportion of subjects reported better treatment outcomes for themselves for both migraine pain and associated symptoms. Use of SumaRT/Nap was also associated with less rescue medication use and a longer time before use of rescue medication compared with both BCM and placebo.


Calpain-mediated activation of NO synthase in human neuroblastoma SK-N-BE cells.
In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.


In vitro fatigue behavior of the equine third metacarpus: remodeling and microcrack damage analysis.
We studied remodeling and microcrack damage in specimens of Thoroughbred racehorse third metacarpal bone that had been subjected to monotonic or fatigue failure. We asked three questions. What effects does mechanical loading have on histologically observable microcrack damage? Are there regional variations in remodeling of the equine cannon bone, and do these variations correlate with mechanical properties? To what extent are remodeling and microcrack damage age-dependent? Machined beams from the medial, lateral, and dorsal cortices were loaded to fracture in four-point bending monotonically, or cyclically at a load initially producing 10,000 microstrain. Specimens were then bulk-stained in basic fuchsin, and cross sections were prepared from loaded and load-free regions of each beam. Current and past remodeling, porosity, and microcrack density and length were determined histomorphometrically. Strained and unstained microcracks were observed. Unstained cracks were associated with regions of woven bone and appeared to be damaged Sharpey's fibers. Their density (approximately 30/mm2) did not increase after failure, but their length (approximately 25 microns) did, especially near the surfaces of the beam. Stained cracks were wider and longer than unstained cracks and were located primarily near the fracture surface and on the compressed side of the beam. Stained cracks after failure were more numerous in those beams having a higher elastic modulus, a shorter fatigue life, or greater deformation at failure. The extent of past remodeling increased with age, especially in the medial region; the rate of current remodeling generally declined with age, but not in the dorsal region, which has the best fatigue resistance. In summary, while remodeling varied with age and region, its effects on bone structure did not appear to influence microdamage. Basic fuchsin staining of damage in fractured equine bone was independent of age and region and confined to near the fracture surfaces. Distributed microdamage consisted only of what appeared to be subtle disruptions of Sharpey's fibers.


Comparison of nebulized glycopyrrolate and metaproterenol in chronic obstructive pulmonary disease.
We assessed the bronchodilating effect of glycopyrrolate (GP) and compared it with that of metaproterenol (MP), alone and in combination (GP+MP), in patients with chronic obstructive pulmonary disease (COPD). In a double-blind study, 11 patients (aged (mean +/- SD) 69 +/- 6 yrs; forced expiratory volume in one second (FEV1) 1.2 +/- 0.4 L) with stable COPD inhaled nebulized GP 1.0 mg, MP 15 mg, or GP 1.0 mg+MP 15 mg. Spirometry was performed before inhalation and at hourly intervals for 8 h after inhalation. It was found that GP produced a bronchodilating effect that was about equal to that of MP but lasted longer (8 vs 5 h). The combination of GP and MP produced a bronchodilating effect that was greater than that of either drug alone and was evident mostly during the effect of MP. The mean peak percentage improvement in FEV1 over baseline was 35% for GP+MP and 25% for either drug alone. These data suggest that nebulized glycopyrrolate is an effective bronchodilator in some patients with chronic obstructive pulmonary disease. Concurrent administration of glycopyrrolate and metaproterenol produces additional bronchodilation that is primarily apparent during the bronchodilator effect of metaproterenol.


Parents' beliefs and knowledge about the management of acute otitis media: a qualitative study.
Acute otitis media is a common reason for antibiotic prescribing, despite strong evidence that antibiotics provide minimal benefit. Studies have demonstrated that patients' (or parents') expectations of antibiotics often influence general practitioners' (GPs) decision to prescribe antibiotics, but few have explored parents' expectations of the management of infections in children, or which factors influence the development of these expectations. This study aimed to explore parents' knowledge and beliefs about the management of acute otitis media in children. Individual semi-structured interviews were conducted with 15 parents of children who had recently presented to their GP with acute otitis media. Parents were recruited at childcare centres or playgroups in Brisbane, Australia. Many parents did not have an accurate understanding of what causes acute otitis media. GPs were primarily consulted for the management of symptoms such as pain and fever. Others specifically wanted reassurance or were concerned about hearing loss. Most parents assumed that antibiotics were the best treatment option. Parents' perceptions about the best treatment were mainly based on their previous experience and the advice of the GP. Pain relief medications, such as paracetamol and non-steroidal anti-inflammatory drugs, were not considered by parents to be sufficient treatment on their own. There is discrepancy between parents' beliefs and expectations of management of acute otitis media and the evidence-based recommendations. This study provides insights into parents' expectations of management of acute otitis media, which may help inform clinicians about perceptions and misperceptions that may be valuable to elicit and discuss.


Treatment of infants and toddlers with cystic fibrosis-related pancreatic insufficiency and fat malabsorption with pancrelipase MT.
Pancreatic enzyme replacement therapy (PERT) improves nutritional status and growth in patients with cystic fibrosis (CF) with pancreatic insufficiency (PI). The current recommendation for infants and young children, who are not able to swallow the whole capsule, is to open the capsule and mix the beads in a spoon with some applesauce; however, the efficacy and safety data of this approach are currently lacking. The aim of this study was to assess the efficacy, palatability (ease of swallowing), and safety of 4 dose levels of pancrelipase microtablets (Pancrease MT) in infants and young children with CF-related PI. This study was a phase II randomized, investigator-blinded, parallel-group pilot study in DNA-proven infants with CF and PI. The study design included a run-in period (days 1-5) and an experimental period (days 6-11). Pancrelipase microtablets (2-mm, enteric coated) were provided orally. Sixteen subjects, 6 to 30 months of age, were provided 500 U lipase/kg/meal for 5 days (baseline period). Subsequently, subjects were randomly assigned to 1 of 4 treatment groups (each n = 4), receiving 500, 1000, 1500, or 2000 U (Ph. EUR) of lipase/kg/meal, respectively, for 5 days (experimental period). The primary endpoint was medication efficacy assessed by the 72-hour fecal fat excretion, expressed as coefficient of fecal fat absorption (CFA), and 13C mixed triglyceride breath test. Secondary endpoints were safety and palatability. Overall compliance, defined as used study medication, was 89% to 99% for the entire study. None of the 4 dose regimens significantly influenced the CFA, relative to the baseline period (median range 83%-93%). During the run-in period the median cumulative % 13C was 11 (range -8 to 59). After randomization the median cumulative % 13C was 18 (range 14-23) in the 500-U, 14 (range -1 to 17) in the 1000-U, 10 (range 10-27) in the 1500-U, and 3 (range 1-49) in the 2000-U groups. Palatability was scored fair to good by the parents in each of the treatment groups. Gastrointestinal symptoms were reported in some patients, including common adverse events reported in clinical trials involving pancreatic enzyme therapy. No serious or other adverse events were reported. Treatment with Pancrease MT at a dosage of 500 U lipase/kg/meal resulted in a CFA of approximately 89% in pediatric subjects ages 6 to 30 months with PI resulting from CF. Pancrease MT doses were well tolerated and mean palatability was scored as fair to good. Present results do not indicate that a dosage higher than 500 U (Ph. EUR) lipase/kg/meal increases the coefficient of fat absorption in a cohort of infants 6 to 30 months of age.


Changes in tobacco industry advertising around high schools in Greece following an outdoor advertising ban: a follow-up study.
As tobacco advertising bans are enacted in accordance with the Framework Convention on Tobacco Control, it is essential to assess enforcement and how the industry may circumvent such measures. During this longitudinal study, we compared the characteristics of points-of-sale (POS) advertising within 300 m of all high schools in Heraklion, Greece before (n=101 POS and 44 billboards in 2007) and after (n=106 POS in 2011) an outdoor advertising ban was implemented in 2009. Cigarette advertisements in all retailers near all high schools were assessed. Following the ban, tobacco industry billboards around schools were eradicated (from 44 to 0). The proportion of POS that had external advertisements dropped from 98% to 66% (p<0.001), more so in regulated convenience stores (from 97% to 35%, p<0.001) than in kiosks (98% to 92%, p=0.192), which were exempt from the ban. The proportion of convenience stores that had advertisements on the door (79.5% to 20.4%, p<0.001), ads that could be seen from the street (92.3% to 22.4%, p<0.001) or illuminated exterior ads (46.2% to 10.2%, p<0.001) was also significantly reduced. Overall, the average number of exterior advertisements per POS fell from 7.4 to 3.9 (p<0.05). This reduction was noted in regulated convenience stores (4.8±3.0 vs 0.9±2.1, p<0.001) and in unregulated kiosks (9.0±6.7 vs 6.5±4.5, p=0.019). The outdoor advertising restriction in Greece has led to a reduced number of tobacco advertisements per POS, and the eradication of billboard advertising. Nevertheless, there is a need to regulate kiosks, which were identified as a key vector for tobacco advertising, and to increase compliance among regulated convenience stores.


Causal evidence for lateral prefrontal cortex dynamics supporting cognitive control.
The lateral prefrontal cortex (LPFC) is essential for higher-level cognition, but the nature of its interactions in supporting cognitive control remains elusive. Previously (Nee and D'Esposito, 2016), dynamic causal modeling (DCM) indicated that mid LPFC integrates abstract, rostral and concrete, caudal influences to inform context-appropriate action. Here, we use continuous theta-burst transcranial magnetic stimulation (cTBS) to test this model causally. cTBS was applied to three LPFC sites and a control site in counterbalanced sessions. Behavioral modulations resulting from cTBS were largely predicted by information flow within the previously estimated DCM. However, cTBS to caudal LPFC unexpectedly impaired processes that are presumed to involve rostral LPFC. Adding a pathway from caudal to mid-rostral LPFC significantly improved the model fit and accounted for the observed behavioral findings. These data provide causal evidence for LPFC dynamics supporting cognitive control and demonstrate the utility of combining DCM with causal manipulations to test and refine models of cognition.


A histological study of toxic effects of aluminium sulfate on rat hippocampus.
Aluminium has toxic effects on many organ systems of the human body. Aluminium toxicity also is a factor in many neurodegenerative diseases. We investigated changes in numbers of hippocampal neurons in rats exposed to aluminium using an optical fractionator and we investigated aluminium-induced apoptosis using the transferase mediated dUTP nick end labeling (TUNEL) assay. Twenty-four female rats were divided equally into control, sham and aluminium-exposed groups. The control group received no treatment. The two treatment groups were injected intraperitoneally with 1 ml 0.9% saline without (sham) and with 3 mg/ml aluminium sulfate every day for two weeks. Following the treatments, the brains were removed, the left hemisphere was used for hippocampal neuron counting using an optical fractionator and the right hemisphere was investigated using hippocampal TUNEL assay to determine the apoptotic index. The number of neurons in the stratum pyramidale of the hippocampus was significantly less in the aluminium group than in the control and sham groups; there was no significant difference between the control and sham groups. The apoptotic index also was significantly higher in the aluminium group than in the other two groups. We quantified the toxic effects of aluminium on the rat hippocampus and determined that apoptosis was the mechanism of aluminium-induced neuron death in the hippocampus.


Is the perceived neighborhood built environment associated with domain-specific physical activity in Latin American adults? An eight-country observational study.
Characteristics of the neighborhood built environment are associated with physical activity (PA). However, few studies with representative samples have examined environmental correlates of domain-specific PA in Latin America. We examined the associations of the perceived neighborhood built environment with domain-specific PA in a large sample of adults from eight Latin American countries. This study examined data from 8185 adults (aged 18-65 years) from eight Latin American countries. The Neighbourhood Environment Walkability Survey - Abbreviated (NEWS-A) scale was used to assess perceptions of land use mix-diversity, land use mix-access, street connectivity, walking/cycling facilities, aesthetics, safety from traffic, and safety from crime. Perceived proximity from home to public open spaces (metropolitan parks, playgrounds, public squares) and to shopping centers was also measured. Transport-related and leisure-time PA were assessed using the long form of the International Physical Activity Questionnaire. Both logistic and linear regression models were estimated on pooled data. Perceptions of higher land use mix-access (OR: 1.40; 95% CI: 1.22,1.61), the existence of many alternative routes in the neighbourhood (1.12; 1.04,1.20), slow speed of traffic (1.19; 1.03,1.35) and few drivers exceeding the speed limits (1.09; 1.03,1.15) were associated with greater odds of reporting at least 10 min/week of transport-related PA. Perceptions of higher levels of land use mix-diversity, better aesthetics and greater safety from crime, the presence of crosswalks and pedestrian signals, and greater proximity of shopping centers were associated with more min/week of transport-related PA. Perceptions of higher land use mix-diversity (1.12; 1.05,1.20), higher land use mix-access (1.27; 1.13,1.43), more walking/cycling facilities (1.18; 1.09,1.28), and better aesthetics (1.10; 1.02,1.18) were associated with greater odds of engaging in at least 10 min/week of leisure-time PA versus none. Perceptions of higher land use mix-diversity were associated with more min/week of leisure PA. Different perceived neighborhood built environment characteristics were associated with domain-specific PA among adults from Latin America countries. Interventions designed to modify perceptions of the neighbourhood built environment might influence initiation or maintenance of domain-specific PA. ClinicalTrials. Gov NCT02226627 . Retrospectively registered on August 27, 2014.


The advantages of using traditional Chinese medicine as an adjunctive therapy in the whole course of cancer treatment instead of only terminal stage of cancer.
Recent studies indicate that Traditional Chinese medicine (TCM) can play an important role in the whole course of cancer treatment such as recovery stages of post-operative, radiotherapy or chemotherapy stages instead of only terminal stage of cancer. In this review, we have summarized current evidence for using TCM as adjuvant cancer treatment in different stages of cancer lesions. Some TCMs (e.g., TJ-41, Liu-jun-zi-tang, PHY906, Coumarin, and Aescine) are capable of improving the post-operative symptoms such as fatigue, pain, appetite, diarrhea, nausea, vomiting, and lymphedema. Some TCMs (e.g., Ginseng, Huang-Qi, BanZhiLian, TJ-48, Huachansu injection, Shenqi fuzheng injection, and Kanglaite injection) in combination with chemo- or radio-therapy are capable of enhancing the efficacy of and diminishing the side effects and complications caused by chemo- and radiotherapy. Taken together, they have great advantages in terms of suppressing tumor progression, relieving surgery complications, increasing the sensitivity of chemo- and radio- therapeutics, improving an organism's immune system function, and lessening the damage caused by surgery, chemo- or radio-therapeutics. They have significant effects on relieving breast cancer-related lymphedema, reducing cancer-related fatigue and pain, improving radiation pneumonitis and gastrointestinal side effects, protecting liver function, and even ameliorating bone marrow suppression. This review of those medicines should contribute to an understanding of Chinese herbal medicines as an adjunctive therapy in the whole course of cancer treatment instead of only terminal stage of cancer, by providing useful information for development of more effective anti-cancer drugs and making more patients "survival with cancer" for a long time.


An update on less invasive and endoscopic techniques mimicking the effect of bariatric surgery.
Obesity (BMI 30-35 kg/m(2)) and its associated disorders such as type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular disease have reached pandemic proportions worldwide. For the morbidly obese population (BMI 35-50 kg/m(2)), bariatric surgery has proven to be the most effective treatment to achieve significant and sustained weight loss, with concomitant positive effects on the metabolic syndrome. However, only a minor percentage of eligible candidates are treated by means of bariatric surgery. In addition, the expanding obesity epidemic consists mostly of relatively less obese patients who are not (yet) eligible for bariatric surgery. Hence, less invasive techniques and devices are rapidly being developed. These novel entities mimic several aspects of bariatric surgery either by gastric restriction (gastric balloons, gastric plication), by influencing gastric function (gastric botulinum injections, gastric pacing, and vagal nerve stimulation), or by partial exclusion of the small intestine (duodenal-jejunal sleeve). In the last decade, several novel less invasive techniques have been introduced and some have been abandoned again. The aim of this paper is to discuss the safety, efficacy, complications, reversibility, and long-term results of these latest developments in the treatment of obesity.


Tranexamic Acid Reduces Transfusion Rates in Obese Patients Undergoing Total Joint Arthroplasty.
While tranexamic acid (TXA) has been well shown to reduce blood loss after joint replacement surgery, little is known regarding its effectiveness in obese patients. The aim of this study was to evaluate the effect of TXA changes in hematocrit and hemoglobin levels as well as incidence of packed red blood cell (pRBC) transfusions in obese patients undergoing total joint arthroplasty (TJA). Between January 2014 and May 2015, 420 consecutive primary joint replacements were performed by two surgeons at our institution. One-hundred-fifty-seven patients (total hip arthroplasty [THA]=29; total knee arthroplasty [TKA]=128) were obese with a body mass index (BMI) greater than or equal to 30 kg/m2. Medical records were reviewed and identified that TXA was utilized in 85 (54.1%) arthroplasties (study group) and was compared to a consecutive series of 72 (45.9%) TJAs (control group). TXA was given intravenously (IV) in two doses: (1) one gram prior to incision and (2) one gram at the time of femoral preparation in THA or prior to cementation in TKA. Changes in hemoglobin and hematocrit levels, number of pRBC transfusions, and occurrence of thrombolytic events were recorded. The changes in hematocrit (7.2% vs. 8.1%) and hemoglobin levels (3.0 g/dl vs. 3.3 g/dl) were less in the group that received TXA than the control group, albeit not significantly (p=0.100 and p=0.278, respectively). Within the control group, 26 (36.1%) patients required a pRBC transfusion with a mean of 2.0 units per patient (range:1-5); whereas, only eight (9.4%) patients with TXA required a mean of 1.6 units per patient (range: 1-2). The use of TXA significantly reduced the incidence of pRBC transfusions, especially in TKA (p<0.001). Sub-analyses revealed that transfusion rates were even more significantly reduced by TXA in obesity type II and III. Two pulmonary emboli were reported in the group that did not receive TXA; whereas, no thrombolytic events were reported in the group that did receive TXA. Utilization of TXA significantly reduced the rate of pRBC transfusions in obese patients.


A youth violence prevention program. Description and preliminary evaluation.
In response to growing violence, primary prevention programs have been launched, but scientific rationale and credible evaluations have been lacking. Fifth and seventh-grade students in three inner-city schools (n = 135) participated in a violence prevention program. Controls consisted of students from the same schools and grades during the following school year (n = 115). Students were taught social problem-solving skills and risk factors for violence. Multivariate analyses were performed on posttest measures while controlling for base-line differences. Program participants were much less likely to define social problems in adversarial ways, were less likely to provide violent solutions in hypothetical conflict situations, listed more negative consequences to using violence, and were less inclined to legitimatize violence. Risk factor knowledge also was significantly increased. No increase was shown in the students' abilities to identify viable nonviolent solutions. The program produced immediate influences on knowledge and some attitudes and social skills shown to be related to aggressive behavior.


Risk of elective major noncardiac surgery after coronary stent insertion: a population-based study.
Guidelines recommend that noncardiac surgery be delayed until 30 to 45 days after bare-metal stent implantation and 1 year after drug-eluting stent implantation. We used linked registry data and population-based administrative health care databases to conduct a cohort study of 8116 patients (≥40 years of age) who underwent major elective noncardiac surgery in Ontario, Canada between 2003 and 2009, and received coronary stents within 10 years before surgery. Approximately 34% (n=2725) underwent stent insertion within 2 years before surgery, of whom 905 (33%) received drug-eluting stents. For comparison, we assembled a separate cohort of 341 350 surgical patients who had not undergone coronary revascularization. The primary outcome was 30-day major adverse cardiac events (mortality, readmission for acute coronary syndrome, or repeat coronary revascularization). The overall rate of 30-day events in patients with coronary stents was 2.1% (n=170). When the interval between stent insertion and surgery was <45 days, event rates were high for bare-metal (6.7%) and drug-eluting (20.0%) stents. When the interval was 45 to 180 days, the event rate for bare-metal stents was 2.6%, approaching that of intermediate-risk nonrevascularized individuals. Adjusted analyses suggested that event rates were increased if this interval exceeded 180 days. For drug-eluting stents, the event rate was 1.2% once the interval exceeded 180 days, approaching that of intermediate-risk nonrevascularized individuals. The earliest optimal time for elective surgery is 46 to 180 days after bare-metal stent implantation or >180 days after drug-eluting stent implantation.


Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers.
To determine the correlation between polymorphisms in the IL-4 gene cluster and resistance to HIV-1 infection. : A cross-sectional genetic analysis of polymorphisms within the IL-4 gene cluster was conducted in a well-described female sex worker cohort from Nairobi, Kenya, known to exhibit differential susceptibility to HIV-1 infection. Microsatellite genotyping was used to screen six microsatellite markers in the IL-4 gene cluster for associations with HIV-1 resistance. Further analysis of the interferon regulatory factor 1 (IRF-1) gene was conducted by genomic sequencing. Associations between IRF-1 gene polymorphisms and the HIV-1 resistance phenotype were determined using the chi-square test and Kaplan-Meier survival analysis. The functional consequence of IRF-1 polymorphism was conducted by quantitative Western blot. Three polymorphisms in IRF-1, located at 619, the microsatellite region and 6516 of the gene, showed associations with resistance to HIV-1 infection. The 619A, 179 at IRF-1 microsatellite and 6516G alleles were associated with the HIV-1-resistant phenotype and a reduced likelihood of seroconversion. Peripheral blood mononuclear cells from patients with protective IRF-1 genotypes exhibited significantly lower basal IRF-1 expression and reduced responsiveness to exogenous IFN-gamma stimulation. Polymorphisms in the IRF-1 gene are associated with resistance to infection by HIV-1 and a lowered level of IRF-1 protein expression. This study adds IRF-1, a transcriptional immunoregulatory gene, to the list of genetic correlates of altered susceptibility to HIV-1. This is the first report suggesting that a viral transcriptional regulator might contribute to resistance to HIV-1. Further functional analysis on the role of IRF-1 polymorphisms and HIV-1 resistance is underway.


Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats.
Pomegranate (Punica granatum L.) seed oil (PGO) contains more than 70% cis(c)9,trans(t)11,c13-18:3 as conjugated linolenic acids (CLN). Our previous short-term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in c9,t11,t13-CLN, inhibited the occurrence of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM). In this study, we investigated the effect of dietary PGO on the development of AOM-induced colonic malignancies and compared it with that of conjugated linoleic acid (CLA). To induce colonic tumors, 6-week old male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks. One week before the AOM treatment they were started on diet containing 0.01%, 0.1%, or 1% PGO or 1% CLA for 32 weeks. Upon termination of the bioassay (32 weeks) colon tumors were evaluated histopathologically. AOM exposure produced colonic adenocarcinoma with an incidence of 81% and multiplicity of 1.88 +/- 1.54 at week 32. Administration of PGO in the diet significantly inhibited the incidence (AOM + 0.01% PGO, 44%, P < 0.05; AOM + 0.1% PGO, 38%, P < 0.01; AOM + 1% PGO, 56%) and the multiplicity (AOM + 0.01% PGO, 0.56 +/- 0.73, P < 0.01; AOM + 0.1% PGO, 0.50 +/- 0.73, P < 0.005; AOM + 1% PGO, 0.88 +/- 0.96, P < 0.05) of colonic adenocarcinomas, although a clear dose-response relationship was not observed at these dose levels. CLA feeding also slightly, but not significantly, reduced the incidence and multiplicity of colonic adenocarcinomas. The inhibition of colonic tumors by PGO was associated with an increased content of CLA (c9,t11-18:2) in the lipid fraction of colonic mucosa and liver. Also, administration of PGO in the diet elevated expression of peroxisome proliferator-activated receptor (PPAR) gamma protein in the non-tumor mucosa. These results suggest that PGO rich in c9,t11,c13-CLN can suppress AOM-induced colon carcinogenesis, and the inhibition is associated in part with the increased content of CLA in the colon and liver and/or increased expression of PPARgamma protein in the colon mucosa.


Effects of a high-fat diet and bamboo extract supplement on anxiety- and depression-like neurobehaviours in mice.
High-fat diet is a major causative factor of overweight and obesity, which are associated with an increased risk of neuropsychiatric diseases, such as anxiety and depression. In the present study, we investigated the protective effects of bamboo extract (BEX) on anxiety- and depression-like neurobehaviours in mice treated with a high-fat diet. Male mice with CD-1 genetic background were treated for 2 months with either a standard or a high-fat diet (10 or 45 % energy from fat, respectively), with or without the BEX supplement (11 g dry mass per 17 MJ). The anxiety levels of mice were evaluated using open-field and hole-board tests, and depression was measured using the force-swimming test. The anxiety responses of the animals were found significantly increased after the high-fat diet treatment, and this elevation was effectively abolished by the BEX supplement. The high-fat diet seemed to have an anti-depressive effect in mice at the tested time point, but the effect of the BEX supplement on the depression level of the animals was not conclusive. The high-fat diet significantly decreased total glutathione content in the blood while the BEX supplement increased glutathione oxidation. In summary, the present study shows that decreased total glutathione concentration in the blood co-occurred with a high-fat treatment, high anxiety level and low depression level in mice, and when supplemented in a high-fat diet, BEX had an anxiolytic effect in mice.


Survival in fatal road crashes: body mass index, gender, and safety belt use.
This study evaluated the associations of body mass index (BMI), gender, and use of safety belts with the survival of drivers involved in fatal road crashes. The census data of all U.S. fatal crashes that did not involve pedestrians, bicyclists, or motorcyclists were examined for an 11-year period. If involved in a crash with one or more fatalities, the odds of female drivers being among the fatalities are 1.28 times higher than those of male drivers, and the odds of unbelted drivers being among the fatalities are 5.43 times higher than those of belted drivers. The relationship of survivability to BMI depends on the gender and safety belt use of the driver. For male drivers, increased BMI appears beneficial when safety belts are used but detrimental when not used. For belted female drivers, normal BMI is associated with the lowest odds of being killed, and both increased and decreased BMIs increase the odds. For unbelted female drivers, no reliable trends were present among the BMI categories.


Exploring evidence-policy linkages in health research plans: a case study from six countries.
The complex evidence-policy interface in low and middle income country settings is receiving increasing attention. Future Health Systems (FHS): Innovations for Equity, is a research consortium conducting health systems explorations in six Asian and African countries: Bangladesh, India, China, Afghanistan, Uganda, and Nigeria. The cross-country research consortium provides a unique opportunity to explore the research-policy interface. Three key activities were undertaken during the initial phase of this five-year project. First, key considerations in strengthening evidence-policy linkages in health system research were developed by FHS researchers through workshops and electronic communications. Four key considerations in strengthening evidence-policy linkages are postulated: development context; research characteristics; decision-making processes; and stakeholder engagement. Second, these four considerations were applied to research proposals in each of the six countries to highlight features in the research plans that potentially strengthen the research-policy interface and opportunities for improvement. Finally, the utility of the approach for setting research priorities in health policy and systems research was reflected upon. These three activities yielded interesting findings. First, developmental consideration with four dimensions - poverty, vulnerabilities, capabilities, and health shocks - provides an entry point in examining research-policy interfaces in the six settings. Second, research plans focused upon on the ground realities in specific countries strengthens the interface. Third, focusing on research prioritized by decision-makers, within a politicized health arena, enhances chances of research influencing action. Lastly, early and continued engagement of multiple stakeholders, from local to national levels, is conducive to enhanced communication at the interface. The approach described has four main utilities: first, systematic analyses of research proposals using key considerations ensure such issues are incorporated into research proposals; second, the exact meaning, significance, and inter-relatedness of these considerations can be explored within the research itself; third, cross-country learning can be enhanced; and finally, translation of evidence into action may be facilitated. Health systems research proposals in low and middle income countries should include reflection on transferring research findings into policy. Such deliberations may be informed by employing the four key considerations suggested in this paper in analyzing research proposals.


Distribution of EGFR mutations commonly observed in primary lung adenocarcinomas in Pakistan as predictors for targeted therapy.
Acquired genetic alterations and presence of sensitizing mutations in the tyrosine kinase domain of EGFR and other signaling molecules have been found in different subsets of primary lung adenocarcinoma. The commonest EGFR mutations are small in frame deletions of exon 19 and a point mutation (L858R) in exon 21, having a combined occurrence of around 90%. The objective of this study was to determine the frequency and types of EGFR mutations in primary lung adenocarcinomas in Pakistan. EGFR mutations in tumor samples were screened by multiplex real time PCR. Briefly, DNA from formalin fixed paraffin-embedded tissue was amplified with primers and probes specific to 43 different EGFR mutations in a Cobas z 480 instrument. The assay detects mutations in four exons (18-21) of the EGFR gene. Out of 94 patients, 65 were males and 29 females with a M:F ratio of 2.2: 1. The median age was 62 years (range, 28 - 85 years). In our biopsy samples 70 (74%) cases were of primary lung adenocarcinoma, whereas 24 (26%) were confirmed metastatic adenocarcinoma of primary lung origin. EGFR mutation was positive in 29% of the patients. The highest frequency of L858R was observed in 48% of these, followed by deletion in exon 19 (44%). In addition, other rare mutations such as compound G718X:S768I and insertions in exon 20 insertion were detected in approximately 4% of the patients. This study showed that Del 19 and L858R are the most frequent mutations in Pakistani lung adenocarcinoma patients and around 29% of the patients were found eligible for erlotinib therapy.


[Hyperuricemia and Gout].
Already known to ancient Egyptians, gout is one of the first diseases which have been described as a clinical entity. To date, gout is the most common form of inflammatory arthritis. Gout is defined by the deposition of monosodium urate crystals within tissues, causing episodes of acute arthritis and the development of tophi, nephrolithiasis, and urate nephropathy. Hyperuricemia, i.e. levels of serum uric acid above 6.8 mg / dL(404mol/L), is a condition necessary, yet not sufficient for gout to develop. The increasing incidence of risk factors such as hypertension, obesity, and renal failure together with an ever-growing life expectancy has led in recent decades to a significant increase in gout prevalence, which has more than doubled when compared to the 1960s. This article addresses the issue of gout by highlighting the role played by the kidneys in uric acid homeostasis; the clinical effect of crystal deposition in tissues, including the kidney; the more recent guidelines on diagnosis and management strategies, with special regard to the use of old and new drugs in renal patients.


The Latino paradox in neighborhood context: the case of asthma and other respiratory conditions.
Evidence indicates that foreign-born Latinos have a health advantage compared with US-born persons of the same socioeconomic status. An explanation for this paradox has remained elusive. We examined the extent to which this paradox exists for the prevalence of asthma and other respiratory conditions. We then explored the role of neighborhood social context in understanding any observed advantage. We invoked theories of social organization, collective efficacy, and the urban ethnic enclave. We combined data from the Project on Human Development in Chicago Neighborhoods Community Survey with 2 other data sources and used hierarchical generalized linear modeling techniques. We found a distinctly graded effect for asthma and other breathing problems among foreign-born Latinos, depending on community composition. Foreign-born Latinos embedded in a neighborhood that had a high percentage of foreign-born residents experienced a significantly lower prevalence of asthma and other breathing problems; those in communities that had a low percentage of foreign-born residents had the highest prevalence overall (even when compared with African Americans). Foreign-born Latinos have a respiratory health advantage only in enclave-like settings. Contexts such as these may provide the cohesiveness critical for effective prevention.


Time-dependent rates of molecular evolution.
For over half a century, it has been known that the rate of morphological evolution appears to vary with the time frame of measurement. Rates of microevolutionary change, measured between successive generations, were found to be far higher than rates of macroevolutionary change inferred from the fossil record. More recently, it has been suggested that rates of molecular evolution are also time dependent, with the estimated rate depending on the timescale of measurement. This followed surprising observations that estimates of mutation rates, obtained in studies of pedigrees and laboratory mutation-accumulation lines, exceeded long-term substitution rates by an order of magnitude or more. Although a range of studies have provided evidence for such a pattern, the hypothesis remains relatively contentious. Furthermore, there is ongoing discussion about the factors that can cause molecular rate estimates to be dependent on time. Here we present an overview of our current understanding of time-dependent rates. We provide a summary of the evidence for time-dependent rates in animals, bacteria and viruses. We review the various biological and methodological factors that can cause rates to be time dependent, including the effects of natural selection, calibration errors, model misspecification and other artefacts. We also describe the challenges in calibrating estimates of molecular rates, particularly on the intermediate timescales that are critical for an accurate characterization of time-dependent rates. This has important consequences for the use of molecular-clock methods to estimate timescales of recent evolutionary events.


Hand Robotic Therapy in Children with Hemiparesis: A Pilot Study.
The aim of this study was to understand the impact of training with a hand robotic device on hand paresis and function in a population of children with hemiparesis. Twelve children with hemiparesis (mean age, 9 [SD, 3.64] years) completed participation in this prospective, experimental, pilot study. Participants underwent clinical assessments at baseline and again 6 weeks later with instructions to not initiate new therapies. After these assessments, participants received 6 weeks of training with a hand robotic device, consisting of 1-hour sessions, 3 times weekly. Assessments were repeated on completion of training. Results showed significant improvements after training on the Assisting Hand Assessment (mean difference, 2.0 Assisting Hand Assessment units; P = 0.011) and on the upper-extremity component of the Fugl-Meyer scale (raw score mean difference, 4.334; P = 0.001). No significant improvements between pretest and posttest were noted on the Jebsen-Taylor Test of Hand Function, the Quality of Upper Extremity Skills Test, or the Pediatric Evaluation of Disability Inventory after intervention. Total active mobility of digits and grip strength also failed to demonstrate significant changes after training. Participants tolerated training with the hand robotic device, and significant improvements in bimanual hand use, as well as impairment-based scales, were noted. Improvements were carried over into bimanual skills during play. Complete the self-assessment activity and evaluation online at http://www.physiatry.org/JournalCME CME OBJECTIVES: Upon completion of this article, the reader should be able to: (1) Understand key components of neuroplasticity; (2) Discuss the benefits of robotic therapy in the recovery of hand function in pediatric patients with hemiplegia; and (3) Appropriately incorporate robotic therapy into the treatment plan of pediatric patients with hemiplegia. Advanced ACCREDITATION: The Association of Academic Physiatrists is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.The Association of Academic Physiatrists designates this activity for a maximum of 1.5 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.


An unusually large (CA)n repeat in the region of divergence between subtelomeric alleles of human chromosome 16p.
Previous work has demonstrated discontinuous length variation at the tip of the short arm of human chromosome 16 (16pter) due to polymorphism of the subtelomeric region. We have now analyzed the zone where the two most common subtelomeric alleles (A and B) diverge. This lies 145 kb distal to the alpha-globin genes and comprises a complex segment of approximately 4 kb where there is partial loss of homology between the alleles, preceding the final point of divergence. Most notably, there is an imperfect (CA)n repeat that differs in length with different 16pter alleles and is exceptionally large (n = 250-350) in the case of the A allele and homologous sequences on Xqter and Yqter. Both the (CA)n expansion and the genetic exchange between chromosomes 16, X, and Y seem to have occurred since the divergence of man from other great apes. The occurrence of long (CA)n tracts may be related to the biology of subtelomeric regions.


Evaluation of two types of sponges used to collect cervical secretions and assessment of antibody extraction protocols for recovery of neutralizing anti-human papillomavirus type 16 antibodies.
Immunogenicity evaluations in human papillomavirus (HPV) vaccine trials have relied on serological samples, yet cervical antibodies are likely to be most relevant for protection against infection. In order to assess functional antibody levels at the cervix, the secreted-alkaline-phosphatase neutralization assay (SEAPNA) was used to measure HPV-neutralizing activity. We assessed the variability of the SEAPNA with serum samples after vaccination with an HPV type 16 (HPV16) L1 virus-like particle vaccine and whether the SEAPNA can be used to monitor neutralizing activity at the cervix. The SEAPNA has an overall coefficient of variation of 29.3%. Recovery from ophthalmic sponges was assessed by spiking V5 (mouse anti-HPV16) antibody onto and extracting it from sterile Merocel and Ultracell sponges and sponges used to collect specimens from participants. V5 recovery from sterile Merocel sponges was complete, yet that from Ultracell sponges was null. The mean V5 recoveries from participant Ultracell and Merocel sponges were 61.2% and 93.5%, respectively, suggesting that Merocel sponges are more appropriate for specimen collection. The SEAPNA can be applied to determine the surrogates of protection and to examine the durability of protection at the cervix.


Contribution of lipids in honeybee (Apis mellifera) royal jelly to health.
Honeybee (Apis mellifera) royal jelly (RJ) has a long history in human medicine because of its health-protecting properties. To develop a fundamental and comprehensive understanding of lipids in RJ, this article reviews the available literature on lipid compounds identified from RJ extracts and in vitro pharmacological effects of 10-hydroxy-2-decenoic acid in RJ and other closely related compounds, some of which are also identified as lipid compounds in RJ. Overall, the lipids in RJ are composed of mostly (aliphatic) fatty acids, almost all of which are present as free fatty acids and scarcely any as esters. Most fatty acids in RJ are medium-chain fatty acids, whether hydroxylated in terminal and/or internal positions, terminated with mono- or dicarboxylic acid groups, and saturated or monounsaturated at the 2-position. Besides these fatty acids, lipids in RJ contain sterols in minor amounts. Lipids in RJ are useful as preventive and supportive medicines with functionalities that include potential inhibitors of cancer growth, immune system modulators, alternative therapies for menopause, skin-aging protectors, neurogenesis inducers, and more. Taken together, the evidence suggests that health-protecting properties of RJ can be, in part, ascribed to actions of lipids in RJ.


Stroma: the forgotten cells of innate immune memory.
All organisms are exposed constantly to a variety of infectious and injurious stimuli. These induce inflammatory responses tailored to the threat posed. While the innate immune system is the front line of response to each stimulant, it has been considered traditionally to lack memory, acting in a generic fashion until the adaptive immune arm can take over. This outmoded simplification of the roles of innate and acquired arms of the immune system has been challenged by evidence of myeloid cells altering their response to subsequent encounters based on earlier exposure. This concept of 'innate immune memory' has been known for nearly a century, and is accepted among myeloid biologists. In recent years other innate immune cells, such as natural killer cells, have been shown to display memory, suggesting that innate immune memory is a trait common to several cell types. During the last 30 years, evidence has slowly accumulated in favour of not only haematopoietic cells, but also stromal cells, being imbued with memory following inflammatory episodes. A recent publication showing this also to be true in epithelial cells suggests innate immune memory to be widespread, if under-appreciated, in non-haematopoietic cells. In this review, we will examine the evidence supporting the existence of innate immune memory in stromal cells. We will also discuss the ramifications of memory in long-lived tissue-resident cells. Finally, we will pose questions we feel to be important in the understanding of these forgotten cells in the field of innate memory.


Development of congestive heart failure after treatment with metoprolol in acute myocardial infarction.
In a double blind study of metoprolol in the treatment of suspected acute myocardial infarction 698 patients (study group) received metoprolol and 697 a placebo (control group). Metoprolol was given in an intravenous dose of 15 mg as soon as possible after admission to hospital followed by 50 g by mouth four times a day for two days and thereafter 100 mg twice a day for three months. A placebo was similarly given. Congestive heart failure occurred in a similar percentage of patients in both the study (27%) and the control groups (30%). Its severity was estimated by calculating the total dose of frusemide given during the first four days in hospital. Less frusemide was given to patients treated with metoprolol compared with those given a placebo in the total series. An appreciably lower total dose of frusemide was given to patients included in the trial less than or equal to 12 hours after the onset of pain and treated with metoprolol compared with a placebo, while no difference was seen among patients treated later. The initial heart rate, systolic blood pressure, and infarct site affected the results.


The Effects of Breakdown Set Resistance Training on Muscular Performance and Body Composition in Young Men and Women.
Breakdown (BD) training has been advocated by multiple commercial and academic publications and authors, seemingly as a result of the acute hormonal and muscle activation responses it produces. However, there is a relative dearth of research that has empirically considered this advanced method of resistance training (RT) over a chronic intervention while appropriately controlling other RT variables. The present study considered 36 male and female participants divided into 3 groups: BD (n = 11), heavy-load breakdown (n = 14), and traditional (n = 11), performing full-body RT programs 2 times per week for 12 weeks. No significant between-group differences were identified for change in absolute muscular endurance for chest press, leg press, or pull-down exercises or for body composition changes. Effect sizes for absolute muscular endurance changes were large for all groups and exercises (0.86-2.74). The present study supports previous research that the use of advanced training techniques stimulates no greater muscular adaptations when compared with performing more simplified RT protocols to momentary muscular failure.


Role of mecA transcriptional regulation in the phenotypic expression of methicillin resistance in Staphylococcus aureus.
The gene required for methicillin resistance in staphylococci, mecA, encodes the low-affinity penicillin-binding protein 2a (PBP2a). Transcriptional regulation of mecA is accomplished in some isolates by mecR1 and mecI, cotranscribed chromosomal genes that encode a putative signal transducer and a transcriptional repressor, respectively. Two Staphylococcus aureus strains that have identical mecR1-mecI nucleotide sequences, BMS1 and N315P, both exhibit low-level, heterotypic expression of methicillin resistance and contain no beta-lactamase coregulatory sequences. mecR1-mecI was amplified from BMS1 by PCR and was shown to be functional on a high-copy-number plasmid when introduced into an S. aureus strain with a deleted mecR1-mecI locus. Cloned mecR1-mecI repressed phenotypic expression of methicillin resistance, mecA transcription and PBP2a production and mediated PBP2a induction in response to certain beta-lactam antibiotics. However, mecR1-mecI had different regulatory activities in its native chromosomal location in N315P compared with those in BMS1. Uninduced mecA transcription was markedly repressed in N315P, and mecI inactivation increased mecA transcription and PBP2a production 5- and 40-fold, respectively. Furthermore, the N315P phenotype changed from low-level, heterotypic resistance with intact mecI to high-level, homotypic resistance in strains with disrupted mecI. In contrast, uninduced BMS1 produced abundant mecA transcript and PBP2a, while the disruption of mecI had no effect on phenotype and little effect on mecA transcription or PBP2a production. Thus, mecI-mediated repression of mecA appears to be dysfunctional in BMS1 because of the presence or absence of additional regulatory cofactors. Furthermore, heterotypic resistance expression in this strain is independent of mecA transcriptional regulation.


CD8+ T cell responses against TAP-inhibited cells are readily detected in the human population.
Target cell recognition by CTLs depends on the presentation of peptides by HLA class I molecules. Tumors and herpes viruses have adopted strategies to greatly hamper this peptide presentation at the important bottleneck, the peptide transporter TAP. Previously, we described the existence of a CD8(+) CTL subpopulation that selectively recognizes such TAP-deficient cells in mouse models. In this study, we show that the human counterpart of this CTL subset is readily detectable in healthy subjects. Autologous PBMC cultures were initiated with dendritic cells rendered TAP-impaired by gene transfer of the viral evasion molecule UL49.5. Strikingly, specific reactivity to B-LCLs expressing one of the other viral TAP-inhibitors (US6, ICP47, or BNLF2a) was already observed after three rounds of stimulation. These short-term T cell cultures and isolated CD8(+) CTL clones derived thereof did not recognize the normal B-LCL, indicating that the cognate peptide-epitopes emerge at the cell surface upon an inhibition in the MHC class I processing pathway. A diverse set of TCRs was used by the clones, and the cellular reactivity was TCR-dependent and HLA class I-restricted, implying the involvement of a broad antigenic peptide repertoire. Our data indicate that the human CD8(+) T cell pool comprises a diverse reactivity to target cells with impairments in the intracellular processing pathway, and these might be exploited for cancers that are associated with such defects and for infections with immune-evading herpes viruses.


Challenges in Determining Genotypes for Pharmacogenetics in Allogeneic Hematopoietic Cell Transplant Recipients.
As part of a pharmacogenetic study, paired blood and oral fluid samples were tested for the IL28B polymorphism (rs12979860) before and after hematopoietic cell transplantation (HCT) to evaluate changes in the genotype and investigate the utility of genotyping in oral fluid in HCT recipients. In 54 patients with leukemia >18 years of age, samples were collected approximately 7 days before HCT and 60 days after HCT. IL28B polymorphism testing was performed using real-time PCR with allele-specific probes. Twenty-four patients had the same genotype as their donors. In 30 patients, the genotype was different from that of the donor. In the oral fluid samples, 4 retained the recipient's genotype, and 18 had a genotype that matched that of the donor. In the remaining 8 patients, the results could not be characterized and appeared to be a combination of both, suggesting mixed proportions of donor and recipient cells. The assumption was that the sloughed epithelial cells of the mouth are of recipient origin. However, oral fluid is a mixture that contains varying numbers of cells of the recipient and immunomodulatory cells from the donor. Therefore, the use of oral fluid after HCT for clinical pharmacogenetics purposes needs further investigation.


Recent progress in luminescence tuning of Ce(3+) and Eu(2+)-activated phosphors for pc-WLEDs.
Nowadays, phosphor converted white light-emitting diodes (pc-WLEDs) have been widely used in solid-state lighting and display areas due to their superior lifetime, efficiency, and reliability as well as significant reduction in power consumption. Phosphors are indispensable components of pc-WLED devices, and their luminescence properties determine the quality of WLED lighting and displays. In order to further achieve high luminous efficacy, chromatic stability, and color-rending properties in pc-WLEDs, much effort has been focused on improving current pc-WLED phosphors and developing novel pc-WLED phosphors recently. This review article concerns commonly used rare earth ion (Eu(2+) and Ce(3+)) activated inorganic phosphors, highlighting the important effect of spectral tuning via local structural variations on improving the luminescence performance of phosphors. The main spectral tuning strategies are discussed in detail and summarized, including (1) doping level control; (2) cationic substitution; (3) anionic substitution; (4) cationic-anionic substitution; (5) the crystal-site engineering approach; (6) mixing of nanophases.


Non-randomised feasibility study testing a primary care intervention to promote engagement in an online health community for adults with troublesome asthma: protocol.
In the UK, approximately 4.3 million adults have asthma, with one-third experiencing poor asthma control, affecting their quality of life, and increasing their healthcare use. Interventions promoting emotional/behavioural self-management can improve asthma control and reduce comorbidities and mortality. Integration of online peer support into primary care services to foster self-management is a novel strategy. We aim to co-design and evaluate an intervention for primary care clinicians to promote engagement with an asthma online health community (OHC). Our protocol describes a 'survey leading to a trial' design as part of a mixed-methods, non-randomised feasibility study to test the feasibility and acceptability of the intervention. Adults on the asthma registers of six London general practices (~3000 patients) will be invited to an online survey, via text messages. The survey will collect data on attitudes towards seeking online peer support, asthma control, anxiety, depression, quality of life, information on the network of people providing support with asthma and demographics. Regression analyses of the survey data will identify correlates/predictors of attitudes/receptiveness towards online peer support. Patients with troublesome asthma, who (in the survey) expressed interest in online peer support, will be invited to receive the intervention, aiming to reach a recruitment target of 50 patients. Intervention will involve a one-off, face-to-face consultation with a practice clinician to introduce online peer support, sign patients up to an established asthma OHC, and encourage OHC engagement. Outcome measures will be collected at baseline and 3 months post intervention and analysed with primary care and OHC engagement data. Recruitment, intervention uptake, retention, collection of outcomes, and OHC engagement will be assessed. Interviews with clinicians and patients will explore experiences of the intervention. Ethical approval was obtained from a National Health Service Research Ethics Committee (reference: 22/NE/0182). Written consent will be obtained before intervention receipt and interview participation. Findings will be shared via dissemination to general practices, conference presentations and peer-reviewed publications. NCT05829265.


Genome-Wide DNA Methylation Analysis of a Cohort of 41 Patients Affected by Oculo-Auriculo-Vertebral Spectrum (OAVS).
Oculo-auriculo-vertebral-spectrum (OAVS; OMIM 164210) is a rare disorder originating from abnormal development of the first and second branchial arch. The clinical phenotype is extremely heterogeneous with ear anomalies, hemifacial microsomia, ocular defects, and vertebral malformations being the main features. MYT1, AMIGO2, and ZYG11B gene variants were reported in a few OAVS patients, but the etiology remains largely unknown. A multifactorial origin has been proposed, including the involvement of environmental and epigenetic mechanisms. To identify the epigenetic mechanisms contributing to OAVS, we evaluated the DNA-methylation profiles of 41 OAVS unrelated affected individuals by using a genome-wide microarray-based methylation approach. The analysis was first carried out comparing OAVS patients with controls at the group level. It revealed a moderate epigenetic variation in a large number of genes implicated in basic chromatin dynamics such as DNA packaging and protein-DNA organization. The alternative analysis in individual profiles based on the searching for Stochastic Epigenetic Variants (SEV) identified an increased number of SEVs in OAVS patients compared to controls. Although no recurrent deregulated enriched regions were found, isolated patients harboring suggestive epigenetic deregulations were identified. The recognition of a different DNA methylation pattern in the OAVS cohort and the identification of isolated patients with suggestive epigenetic variations provide consistent evidence for the contribution of epigenetic mechanisms to the etiology of this complex and heterogeneous disorder.


Biologic modification of ligamentum flavum cells by marker gene transfer and recombinant human bone morphogenetic protein-2.
The study involves an in vitro experiment using human ligamentum flavum (LF), adenovirus lacZ construct (Ad/lacZ), and recombinant human bone morphogenetic protein-2 (BMP-2). To demonstrate the feasibility of marker gene transfer to human LF cells and the effect of BMP-2 on the osteogenic differentiation of human LF cells. BMP-2 is a widely known pivotal osteoinductive agent. Clinically and experimentally, BMP-2 has proven to be an effective in spinal fusion. Degenerated LF has only been implicated to be of pathophysiological significance in spinal stenosis. However, biologic modifications of LF to enhance osteogenesis have not been attempted previously. Human LF and cancellous bone from the ilium were harvested from patients with lumbar spinal stenosis. LF cells and osteoblasts were isolated and cultured, and adenovirus lacZ construct (Ad/ lacZ), luciferase construct (Ad/luciferase), and BMP-2 were designed and produced. LF cell cultures were then exposed to various concentrations of Ad/lacZ (25, 50, 75, 100, 150 multiplicity of infection) and BMP-2 (50, 100, 500, 1,000, and 1,500 ng/mL). Osteoblast cultures were used as a positive control for LF culture. LF cell cultures with Ad/luciferase served as viral controls for culture with Ad/ lacZ. The transgene expression of lacZ was assessed by X-gal stain and beta-galactosidase assay. Alkaline phosphatase, Von Kossa, and Alizarin red-S stains were used to confirm osteogenic differentiation and bone nodule formation. Immunocytochemical staining was also performed to detect osteocalcin expression. LF cell cultures transduced with Ad/lacZ showed extensive X-gal expression and increased beta-galactosidase activity compared to viral (Ad/luciferase) and saline controls. In LF cultures treated with BMP-2, robust alkaline phosphatase expression, and bone nodule formations were observed as evidenced by positive Von Kossa and Alizarin red-S staining, and the strong expression of osteocalcin. The osteogenic response of LF cells to BMP-2 was dose dependent. Human LF cells were found to be susceptible to adenovirus-mediated marker gene transfer, which offers the possibility of a new range of possible genetic modifications. In human LF cells, BMP-2 was found to markedly up-regulate the expression of osteogenic phenotypes and to induce bone nodule formation. The results of this study support the notion that biologically modified LF cells, i.e., LF cells treated with BMP-2, or with adenovirus-mediated BMP-2 cDNA gene transfer, may facilitate spinal fusion.


Prevention of eclampsia.
Eclampsia continues to be a major cause of maternal morbidity and mortality rates, especially in underdeveloped nations. Our data indicate that eclampsia may represent the end stage of at least two very different pathophysiological pathways: one in which cerebral perfusion is low because of vasospasm and another in which cerebral perfusion is increased because of abnormal autoregulation and a failure of the normal protective mechanisms. Magnesium sulfate has been extensively used in the management and prevention of eclamptic seizures in the United States and has been recently shown to be superior to both diphenylhydantoin and diazepam. We have shown that magnesium sulfate is a potent vasorelaxant and that its action may depend on improving cerebral perfusion. Nimodipine, a calcium channel blocker with selective cerebrovascular effect, is currently under investigation in severe preeclampsia. The data show that it is as effective as magnesium sulfate in preventing eclampsia, with less maternal and fetal side effects. Magnesium sulfate and nimodipine have opposite effects on the estimated cerebral perfusion pressure as determined with the Doppler ultrasound. We speculate that the estimated cerebral perfusion pressure may be used to determine the type of cerebrovascular abnormality and the most appropriate treatment in each individual patient with preeclampsia.


Design of a study evaluating the effects, health economics, and stakeholder perspectives of a multi-component occupational rehabilitation program with an added workplace intervention - a study protocol.
Recent research has suggested that interventions at the workplace might be the most potent ingredient in return to work interventions, but few studies have investigated the different effects of workplace interventions as part of occupational rehabilitation programs. The comprehensive design described in this article includes effect (on return to work and health outcomes), and health economic evaluations of a workplace intervention added to a multicomponent rehabilitation program. Qualitative and mixed method studies will investigate sick-listed persons', rehabilitation therapists' and employers' perspectives on the usability and outcomes of the rehabilitation program and the workplace intervention. The program and intervention are provided to patients with musculoskeletal, psychological or general and unspecified diagnoses. The program is multi-component and includes Acceptance and Commitment Therapy, physical exercise, patient education and creating a plan for increased work participation. Persons who are employed, aged from 18 to 60 years, with a current sick leave status of 50% or more and a diagnosis within the musculoskeletal, psychological or general and unspecified chapters of International Classification of Primary Care-2 (ICPC-2) will be recruited to a researcher-blinded parallel-group randomized controlled trial. All participants take part in an in-patient occupational rehabilitation program, while the intervention group also takes part in an intervention at the workplace. The effect and economic evaluation will investigate the effect of the added workplace intervention. The primary outcome measures will be time until full sustainable return to work and total number of sickness absence days in the 12 months after inclusion. Health economic evaluations will investigate the cost-effectiveness and cost-utility. Qualitative studies will investigate rehabilitation therapists' experiences with working towards return to work within an ACT-approach and stakeholders' experiences with the workplace intervention. A mixed methods study will combine quantitative and qualitative findings on the participants' expectations and motivation for return to work. The outline of this comprehensive study could represent an important addition to the standard designs of return to work evaluation. The mixed methods design, with qualitative approaches as well as a rigorous randomized controlled trial, might prove useful to shed light on contextual factors. ClinicalTrials.gov : NCT02541890 . September 4, 2015.


Outcomes following infliximab therapy for pediatric patients hospitalized with refractory colitis-predominant IBD.
Although randomized trials demonstrated the efficacy of infliximab for both pediatric Crohn disease and ulcerative colitis (UC), few patients in these studies exhibited colitis requiring hospitalization. The aims of this study were to determine the rate of subsequent infliximab failure and dose escalation in pediatric patients who started taking infliximab during hospitalization for colitis-predominant IBD, and to identify potential predictors of these endpoints. This is a single-center retrospective cohort study of patients admitted from 2005 to 2010 with Crohn colitis, UC, or IBD-unspecified (IBD-U) and initiated on infliximab. We identified 29 patients (12 Crohn colitis, 15 UC, and 2 IBD-U; median age 14 years) with a median follow-up of 923 days. Eighteen patients (62%) required infliximab dose escalation (increased dose or decreased infusion interval). Infliximab failure occurred in 18 patients (62%) because of ineffectiveness in 12 (67%) and adverse reactions in 6 (33%). Twelve patients (41%) underwent colectomy. Subsequent need for infliximab dose escalation was associated with lower body mass index z score (P = 0.01), lower serum albumin (P = 0.03), and higher erythrocyte sedimentation rate (ESR) (P = 0.002) at baseline. ESR predicted subsequent infliximab dose escalation with an area under the curve of 0.89 (95% confidence interval [CI] 0.72-1.00) and a sensitivity and specificity at a cutoff of 38 mm/hour of 0.79 (95% CI 0.49-0.95) and 0.88 (95% CI 0.47-0.99), respectively. Most hospitalized pediatric patients with colitis treated with infliximab require early-dose escalation and fail the drug long term. Low body mass index and albumin and high ESR, may identify patients who would benefit from a higher infliximab starting dose.


Peroxidase-dependent oxidation of sulfonamides by monocytes and neutrophils from humans and dogs.
The hydroxylamine and nitroso metabolites formed by N4-oxidation of sulfonamides are thought to be involved in the pathogenesis of idiosyncratic reactions to this class of drugs. Idiosyncratic reactions to sulfonamides are characterized by multisystemic toxicity, including hepatitis, nephritis, dermatitis, and blood dyscrasias (aplastic anemia, agranulocytosis). We have previously shown that cytochrome P-450 in the liver metabolizes sulfamethoxazole to its hydroxylamine metabolite. In this paper we report the N4-oxidation of sulfamethoxazole by activated monocytes and neutrophils (human and canine) to form sulfamethoxazole hydroxylamine and nitrosulfamethoxazole. The presumed nitroso intermediate was not detected. Purified myeloperoxidase and prostaglandin H synthase were also capable of mediating the oxidation of sulfamethoxazole. The present studies suggest that myeloperoxidase is responsible for the observed oxidation by phagocytic cells. Oxidation by neutrophils may play a role in agranulocytosis, and oxidation by monocytes may facilitate antigen presentation. Extrahepatic bioactivation of sulfonamides by peroxidases in phagocytic cells and other tissues may be important in determining the range of adverse reactions to sulfonamides that occur.


Persistent clonal expansions of peripheral blood CD4+ lymphocytes in chronic inflammatory bowel disease.
It is increasingly recognized that chronic Ag exposure may lead to clonal expansions of T cells, including those within the peripheral blood. Inflammatory bowel disease is a chronic, multisystemic disease of unknown origin that predominantly affects the intestine. We sought to determine whether clonal expansions of T cells are present in the peripheral blood of patients with inflammatory bowel disease by an examination of TCR usage. Positively selected CD4+ and CD8+ peripheral blood T cells were isolated from subjects with active ulcerative colitis, Crohn's disease, and diverticulitis and from normal controls. Analysis of complementarity determining region 3 lengths of 24 TCR-beta chain V region families from CD4+ and CD8+ peripheral blood T cells showed a skewed distribution in the three inflammatory groups, consistent with expansion of T cell clones, in comparison to the normally distributed pattern observed among the control donors. Random sequencing of the PCR amplification products of CD4+ peripheral blood T cells from the subjects with ulcerative colitis, Crohn's disease, and diverticulitis revealed reiterative TCR-beta chain sequences that were not found in the normal donors. In subjects with Crohn's disease, the reiterative TCR-beta chain sequences from the CD4+ peripheral blood T cells were persistent over at least a 1-yr period. The persistently expanded TCR-beta chain sequences of CD4+ peripheral blood T cells were identifiable in genomic DNA isolated from archival tissue of intestine from subjects with Crohn's disease and ulcerative colitis by Southern blotting and direct DNA sequencing. An identical twin pair, concordant for Crohn's disease, shared the same reiterative TCR-beta chain sequences in their CD4+ peripheral blood T cells. These studies show that chronic intestinal inflammation is associated with expansions of CD4+ peripheral blood T cells. Furthermore, in inflammatory bowel disease these T cell clonal expansions are persistent and shared among HLA-identical individuals, implicating a response to specific, persistent, and stimulating Ags in these diseases.


Managing psychosocial issues faced by young women with breast cancer at the time of diagnosis and during active treatment.
This review examines recent literature on the psychosocial needs of and interventions for young women. We focus on the active treatment period given the toxicity of treatment, the incidence of anxiety, and depressive symptoms in these women during treatment. This review summarizes research relevant to addressing their social and emotional concerns. Young women undergoing treatment for breast cancer remain understudied despite unique needs. Psychoeducational interventions help to relieve symptoms and emotional distress during treatment, but effects do not appear to persist over the longer term. In the clinical context, the performance of prognostic-risk prediction models in this population is poor. Surgical decision-making is often driven by fear of recurrence and body image rather than prognosis, and decision aids may help young women to synthesize information to preserve their role in the treatment process. First, shared decision-making, second, balancing body image, fear of recurrence, and recommended treatment, and third, palliative care for metastasis are essential research priorities for the clinical setting. In the larger social context, unique family/partner dynamics as well as financial and insurance concerns warrant particular attention in this population.


Retinoic acid prevents virus-induced airway hyperreactivity and M2 receptor dysfunction via anti-inflammatory and antiviral effects.
Inhibitory M(2) muscarinic receptors on airway parasympathetic nerves normally limit acetylcholine release. Viral infections decrease M(2) receptor function, increasing vagally mediated bronchoconstriction. Since retinoic acid deficiency causes M(2) receptor dysfunction, we tested whether retinoic acid would prevent virus-induced airway hyperreactivity and prevent M(2) receptor dysfunction. Guinea pigs infected with parainfluenza virus were hyperreactive to electrical stimulation of the vagus nerves, but not to intravenous acetylcholine, indicating that hyperreactivity was due to increased release of acetylcholine from parasympathetic nerves. The muscarinic agonist pilocarpine, which inhibits vagally mediated bronchoconstriction in control animals, no longer inhibited vagally induced bronchoconstriction, demonstrating M(2) receptor dysfunction. Treatment with all-trans retinoic acid (1 mg/kg) prevented virus-induced hyperreactivity and M(2) receptor dysfunction. However, retinoic acid also significantly reduced viral titers in the lungs and attenuated virus-induced lung inflammation. In vitro, retinoic acid decreased M(2) receptor mRNA expression in both human neuroblastoma cells and primary cultures of airway parasympathetic neurons. Thus, the protective effects of retinoic acid on airway function during viral infection appear to be due to anti-inflammatory and antiviral mechanisms, rather than to direct effects on M(2) receptor gene expression.


Omega-3 fatty acids and leukocyte-endothelium adhesion: Novel anti-atherosclerotic actions.
Endothelial cells (ECs) play a role in the optimal function of blood vessels. When endothelial function becomes dysregulated, the risk of developing atherosclerosis increases. Specifically, upregulation of adhesion molecule expression on ECs promotes the movement of leukocytes, particularly monocytes, into the vessel wall. Here, monocytes differentiate into macrophages and may become foam cells, contributing to the initiation and progression of an atherosclerotic plaque. The ability of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) to influence the expression of adhesion molecules by ECs and to modulate leukocyte-endothelial adhesion has been studied in cell culture using various types of ECs, in animal feeding studies and in human trials; the latter have tended to evaluate soluble forms of adhesion molecules that circulate in the bloodstream. These studies indicate that n-3 PUFAs (both eicosapentaenoic acid and docosahexaenoic acid) can decrease the expression of key adhesion molecules, such as vascular cell adhesion molecule 1, by ECs and that this results in decreased adhesive interactions between leukocytes and ECs. These findings suggest that n-3 PUFAs may lower leukocyte infiltration into the vascular wall, which could contribute to reduced atherosclerosis and lowered risk of cardiovascular disease.


Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices.
We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.


Application of a convolutional neural network to the quality control of MRI defacing.
Large-scale neuroimaging datasets present unique challenges for automated processing pipelines. Motivated by a large clinical trials dataset with over 235,000 MRI scans, we consider the challenge of defacing - anonymisation to remove identifying facial features. The defacing process must undergo quality control (QC) checks to ensure that the facial features have been removed and that the brain tissue is left intact. Visual QC checks are time-consuming and can cause delays in preparing data. We have developed a convolutional neural network (CNN) that can assist with the QC of the application of MRI defacing; our CNN is able to distinguish between scans that are correctly defaced and can classify defacing failures into three sub-types to facilitate parameter tuning during remedial re-defacing. Since integrating the CNN into our anonymisation pipeline, over 75,000 scans have been processed. Strict thresholds have been applied so that ambiguous classifications are referred for visual QC checks, however all scans still undergo an efficient verification check before being marked as passed. After applying the thresholds, our network is 92% accurate and can classify nearly half of the scans without the need for protracted manual checks. Our model can generalise across MRI modalities and has comparable performance when tested on an independent dataset. Even with the introduction of the verification checks, incorporation of the CNN has reduced the time spent undertaking QC checks by 42% during initial defacing, and by 35% overall. With the help of the CNN, we have been able to successfully deface 96% of the scans in the project whilst maintaining high QC standards. In a similarly sized new project, we would expect the model to reduce the time spent on manual QC checks by 125 h. Our approach is applicable to other projects with the potential to greatly improve the efficiency of imaging anonymisation pipelines.


Chemical reaction network designs for asynchronous logic circuits.
Chemical reaction networks (CRNs) are a versatile language for describing the dynamical behaviour of chemical kinetics, capable of modelling a variety of digital and analogue processes. While CRN designs for synchronous sequential logic circuits have been proposed and their implementation in DNA demonstrated, a physical realisation of these devices is difficult because of their reliance on a clock. Asynchronous sequential logic, on the other hand, does not require a clock, and instead relies on handshaking protocols to ensure the temporal ordering of different phases of the computation. This paper provides novel CRN designs for the construction of asynchronous logic, arithmetic and control flow elements based on a bi-molecular reaction motif with catalytic reactions and uniform reaction rates. We model and validate the designs for the deterministic and stochastic semantics using Microsoft's GEC tool and the probabilistic model checker PRISM, demonstrating their ability to emulate the function of asynchronous components under low molecular count.


Post-Traumatic Stress Disorder (PTSD) in Sexually Abused Children and Educational Status in Kenya: A Longitudinal Study.
Children who experience sexual abuse often meet the criteria of Post-Traumatic Stress Disorder and other psychiatric disorders. This article examines Post-Traumatic Stress Disorder and their educational status among children who have been sexually abused and its effects on the children's educational status. The study was carried out between June 2015 and July 2016. The study adopted a longitudinal study design. The study was conducted at Kenyatta National Teaching and Referral Hospital and Nairobi Women's Hospitals in Kenya. The children who had experienced sexual abuse and their parents/legal guardians were followed up for a period of one year after every four months interval. One hundred and ninety one children who had experienced sexual abuse and their parents/legal guardians were invited to participate in the study. Findings indicate that the children continued to experience PTSD one year after the sexual abuse incidence. PTSD was associated with the length of time taken to receive medical attention (p<0.005). Children with partial PTSD who had experienced sexual abuse were 2 times more likely to perform above average than children with full PTSD, OR=2.1 [95% CI of OR 1.2-3.8], p=0.01. Children who experience sexual abuse have negative mental health outcomes. These outcomes have detrimental effects to the normal development of children and educational status. There is need to screen for PTSD and offer psychosocial support and follow up to children who have been sexual abuse.


Opposite effects of pioglitazone and rosiglitazone on mitochondrial respiration in skeletal muscle of patients with type 2 diabetes.
Skeletal muscle insulin resistance has been linked to mitochondrial dysfunction. We examined how improvements in muscular insulin sensitivity following rosiglitazone (ROSI) or pioglitazone (PIO) treatment would affect muscle mitochondrial function in patients with type 2 diabetes mellitus (T2DM). Muscle biopsies were obtained from 21 patients with T2DM before and after 12 weeks on either ROSI (4 mg once daily) [n = 12; age, 59.2 +/- 2.2 years; body mass index (BMI), 29.6 +/- 0.7 kg/m(2)] or PIO (30 mg once daily) (n = 9; age, 56.3 +/- 2.4 years; BMI, 29.5 +/- 1.5 kg/m(2)). An age- and BMI-matched control group was also included (n = 8; age, 61.8 +/- 2.3 years; BMI, 28.4 +/- 0.6 kg/m(2)). Insulin sensitivity, citrate synthase- and beta-hydroxyacyl-CoA-dehydrogenase (HAD) activity, intramuscular triglyceride (IMTG) and protein content of complexes I-IV were measured, while mitochondrial respiration per milligram muscle was measured in saponin-treated skinned muscle fibres using high-resolution respirometry. Mitochondrial respiration per milligram muscle was lower in T2DM compared to controls at baseline and decreased during ROSI treatment but increased during PIO treatment. Citrate synthase activity and average protein content of complexes I-IV were unchanged in the ROSI group, but protein content of complexes II and III increased during PIO treatment. Insulin sensitivity improved in all patients, but IMTG levels were unchanged. We show opposite effects of ROSI and PIO on mitochondrial respiration, and also show that insulin sensitivity can be improved independently of changes in mitochondrial respiration. We confirm that mitochondrial respiration is reduced in T2DM compared to age- and BMI-matched control subjects.


The b subunits in the peripheral stalk of F1F0 ATP synthase preferentially adopt an offset relationship.
The peripheral stalk of F1F0 ATP synthase is essential for the binding of F1 to FO and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b' subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463-5471). The spatial arrangement of the chimeric b and b' subunits, abbreviated Tb and Tb', has been investigated by Cu2+-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F1F0 ATP synthase complexes. In both cases, disulfides were preferentially formed between TbA83C and Tb'A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist.


The challenge of applying Raman spectroscopy to monitor recombinant antibody production.
UV resonance Raman (UVRR) spectroscopy combined with chemometric techniques was investigated as a physiochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) cells. Due to the enhanced selectivity of the UVRR, spectral variations arising from protein, small molecule substrates, and nucleic acid medium components could be measured simultaneously and we have successfully determined antibody titre. Medium samples were taken during culture of three CHO cell lines: two antibody-producing cell lines and a non-producing cell line, and analysed by UVRR spectroscopy using an excitation laser of 244 nm. Principal component analysis (PCA) was applied to the spectral sets and showed a linear trend over time for the antibody-producing cell lines that was not observed in the non-producing cell line. Partial least squares regression (PLSR) was used to predict antibody titres, glucose utilization and lactate accumulation, and compared very favourably with gold standard data acquired with the much slower techniques of ELISA and liquid chromatography. Further analysis of the UVRR spectral sets using two-dimensional correlation moving windows also revealed that spectral variations due to protein and nucleic acid concentrations in the medium during cell culture varied between each of the three cell lines investigated.


In vitro selection of mutations in the human immunodeficiency virus type 1 reverse transcriptase that decrease susceptibility to (-)-beta-D-dioxolane-guanosine and suppress resistance to 3'-azido-3'-deoxythymidine.
Human immunodeficiency virus type 1 (HIV-1) isolates resistant to (-)-beta-D-dioxolane-guanosine (DXG), a potent and selective nucleoside analog HIV-1 reverse transcriptase (RT) inhibitor, were selected by serial passage of HIV-1(LAI) in increasing drug concentrations (maximum concentration, 30 microM). Two independent selection experiments were performed. Viral isolates for which the DXG median effective concentrations (EC(50)s) increased 7.3- and 12.2-fold were isolated after 13 and 14 passages, respectively. Cloning and DNA sequencing of the RT region from the first resistant isolate identified a K65R mutation (AAA to AGA) in 10 of 10 clones. The role of this mutation in DXG resistance was confirmed by site-specific mutagenesis of HIV-1(LAI). The K65R mutation also conferred greater than threefold cross-resistance to 2',3'-dideoxycytidine, 2', 3'-dideoxyinosine, 2',3'-dideoxy-3'-thiacytidine, 9-(2-phosphonylmethoxyethyl)adenine, 2-amino-6-chloropurine dioxolane, dioxolanyl-5-fluorocytosine, and diaminopurine dioxolane but had only marginal effects on 3'-azido-3'-deoxthymidine (AZT) susceptibility. However, when introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q), the K65R mutation reversed the AZT resistance. DNA sequencing of RT clones derived from the second resistant isolate identified the L74V mutation, previously reported to cause ddI resistance. The L74V mutation also decreased the AZT resistance when the mutation was introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q) but to a lesser degree than the K65R mutation did. These findings indicate that DXG and certain 2',3'-dideoxy compounds (e.g., ddI) can select for the same resistance mutations and thus may not be optimal for use in combination. However, the combination of AZT with DXG or its orally bioavailable prodrug (-)-beta-D-2, 6-diaminopurine-dioxolane should be explored because of the suppressive effects of the K65R and L74V mutations on AZT resistance.


Arterial infusion of cisplatin plus S-1 against unresectable intrahepatic cholangiocarcinoma.
Conventional regimens for unresectable intrahepatic cholangiocarcinoma are considered of limited effectiveness. To evaluate the efficacy and toxicity of combination chemotherapy with hepatic arterial infusion of IA-call (a fine-powder formulation of cisplatin) plus oral S-1 in patients with unresectable intrahepatic cholangiocarcinoma. The clinicopathological data and long-term outcome of 12 patients who were received with IA-call plus S-1 were compared with those of 16 patients who were received other treatments, such as radiation therapy, trans-arterial chemoembolization, and systemic chemotherapy. The IA-call plus S-1 regimen consisted of IA-call (65 mg/m2, administered into the hepatic artery) on day 1 and oral S-1 (60 mg/m2/day) on days 1-28, every 42 days, repeated cycle. Prognostic factors of these patients were evaluated by uni- and multivariate analysis. There was no significant difference between the two groups in the disease status, such as the number of tumor and the tumor size. The overall survival was significantly longer in the patients receiving the arterial IA-call and S-1 regimen (median survival time = 10.1; range, 3.6-24.2 months) than in the receiving other treatments (median survival time = 4.0; range; 0.3-24.2 months, p = 0.01). The multivariate analysis revealed that chemotherapy regimen was significantly related to survival, with a hazard ratio of 3.97 (p = 0.02). In the IA-call plus S-1 group, the overall response rate was 33.3%. The major toxic effect was grade 3 anemia, occurring in 1 patient (4.5%). Combination chemotherapy with arterial IA-call plus oral S-1 is an effective regimen that may improve survival in patients with unresectable intrahepatic cholangiocarcinoma.


Human genetic evidence for involvement of CD137 in atherosclerosis.
Atherosclerosis is an inflammatory disease and the main cause of cardiovascular disease. Inflammation promotes plaque instability and clinical disease, such as myocardial infarction, stroke and peripheral vascular disease. Subclinical atherosclerosis begins with thickening of the arterial intimal layer, and increased intima-media thickness (IMT) in the carotid artery is a widely used measurement of subclinical atherosclerosis. Activation of CD137 (tumor necrosis factor receptor super family 9) promotes inflammation and disease development in murine atherosclerosis. CD137 is expressed in human atherosclerosis, but its role is largely unknown. This study uses a genetic approach to investigate CD137 in human atherosclerotic disease. In publicly available data on genotype and gene expression from the HapMap project, the minor T allele of rs2453021, a single nucleotide polymorphism in CD137, was significantly associated with CD137 gene expression. In the PROCARDIS and Wellcome Trust Case Control Consortium (WTCCC) cohorts of 13,029 cases and controls, no significant association was detected between the minor T allele of rs2453021 and risk for coronary artery disease or myocardial infarction. However, in the IMPROVE multicenter study of 3,418 individuals, the minor T allele of rs2453021 was associated with increased IMT of the common carotid artery (CCA), as measured by ultrasonography, with presence of plaque in CCA and with increased incidence of adverse noncardiac vascular events. Taken together, this study shows that the minor T allele of rs2453021 is associated with increased IMT in the CCA and increased risk of incident noncardiac vascular events, thus providing the first human genetic evidence for involvement of CD137 in atherosclerosis.