Impact of age on outcomes after initial therapy with chemotherapy and different chemoimmunotherapy regimens in patients with chronic lymphocytic leukemia: results of sequential cancer and leukemia group B studies.
Chronic lymphocytic leukemia (CLL) is a disease of the elderly, yet few clinical trials include a significant number of older patients, and outcomes after specific therapies can be different depending on age. We examined patients enrolled onto successive first-line CALGB CLL trials to determine whether efficacy of regimens varied by age, focusing on ideal chemotherapy choice and benefit of immunotherapy addition to chemotherapy in older patients. Regimens included chlorambucil, fludarabine, fludarabine plus rituximab (FR), fludarabine with consolidation alemtuzumab, and FR with consolidation alemtuzumab. A total of 663 patients were evaluated for response, progression-free survival (PFS), and overall survival (OS) by age group. Interaction effects of fludarabine versus chlorambucil by age group (PFS, P = .046; OS, P = .006) showed that among patients younger than 70 years, PFS and OS was improved with fludarabine over chlorambucil (PFS: hazard ratio [HR] = 0.6, 95% CI, 0.5 to 0.8; OS: HR = 0.7, 95% CI, 0.5 to 0.9), but not in older adults (PFS, HR = 1.0, 95% CI, 0.6 to 1.7; OS: HR = 1.5, 95% CI, 0.9 to 2.3). In contrast, FR improved outcomes relative to fludarabine, irrespective of age (PFS: HR = 0.6, 95% CI, 0.4 to 0.7; OS: HR = 0.7, 95% CI, 0.5 to 0.9). Alemtuzumab consolidation did not provide benefit over similar regimens without alemtuzumab (P > .20), irrespective of age. These data support the use of chlorambucil as an acceptable treatment for many older patients with CLL and suggest rituximab is beneficial regardless of age. These findings bear relevance to both routine care of CLL patients 70 years and older and also future clinical trials in this population.


VH gene structure predicts a large potential anti-insulin repertoire.
The majority of insulin antibodies derived from immunization are IgG antibodies that cross-react extensively with the autologous hormone. To examine the relationship between VH genes expressed by such self-reactive antibodies and their germline (non-rearranged) counterparts, we used the polymerase chain reaction (PCR) to amplify and isolate the germline progenitors of anti-insulin VH genes derived from BALB/c mice immunized with beef or human insulin. Results indicate that two anti-insulin mAbs (123 and 124) express VH genes which arise from a small subset of the J558 gene family and are highly homologous to the VH gene used by the murine CD5 + B-cell tumor, BCL1. The anti-insulin IgG mAb 127 belongs to the VH-VIII (Vgam 3.2) family and the amplification and isolation of germline VH genes from this small family precisely identified only two somatic mutation events in the CDRH2 of mAb 127. Another anti-insulin mAb, 133, also shows two replacement substitutions in the CDRHs when compared to the germline encoded anti-dextran antibody 19.1.2. These findings indicate that the IgG response to this small self-protein uses multiple VH genes which are largely germline encoded with only a low level of somatic mutation in their CDRHs. Additionally, analysis of N-segment additions in CDRH3s indicates anti-insulin B cells may originate from both early (fetal) and adult repertoires. These data are consistent with the concept that the mechanisms of clonal anergy or deletion do not regulate anti-insulin B cells and indicate that there is a large potential VH gene repertoire for insulin.


Fried-Food Consumption Does Not Increase the Risk of Stroke in the Spanish Cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) Study.
The nutritional determinants of stroke and, more specifically, the association of frying with the risk of incident stroke have rarely been studied. Our aim was to evaluate prospectively the association between the consumption of fried food and the risk of incident stroke in the European Prospective Investigation into Cancer and Nutrition study using the Spanish cohort. Participants included 40,328 healthy adults (62% women) aged 29-69 y at study entry who were enrolled between 1992 and 1996. Participants were followed up until 31 December, 2017, at which time incident stroke (the main outcome) was measured. The main exposure measure was the percentage of energy obtained from fried-food consumption. Sex-specific quintiles were calculated. During a follow-up period of 23.5 y, 975 cases of stroke occurred (750 ischemic, 185 hemorrhagic, and 40 undetermined). Compared with those in the first (lowest) quintile of fried-food consumption, the multivariate HRs (95% CIs) of incident stroke in the consecutive quintiles were 1.05 (0.86, 1.30), 1.11 (0.90, 1.36), 1.05 (0.84, 1.31), and 0.91 (0.72, 1.15; P-trend = 0.45). There were no differences identified when subtypes of stroke were considered. In this Spanish cohort, whose participants mainly used olive oil or sunflower oil when frying, the consumption of fried food was not associated with an increased risk of incident stroke.


In vitro tolerance induction of neonatal murine B cells.
The susceptibility of neonatal and adult B lymphocytes to tolerance induction was analyzed by a modification of the in vitro splenic focus technique. This technique permits stimulation of individual hapten-specific clonal precursor cells from both neonatal and adult donors. Neonatal or adult BALB/c spleen cells were adoptively transferred into irradiated, syngeneic, adult recipients which had been carrier-primed to hemocyanin (Hy), thus maximizing stimulation to the hapten 2,4-dinitrophenyl coupled by Hy (DNP-Hy). Cultures were initially treated with DNP on several heterologous (non-Hy) carriers and subsequently stimulated with DNP-Hy. Whereas the responsiveness of adult B cells was not diminished by pretreatment with any DNP conjugate, the majority of the neonatal B-cell response was abolished by in vitro culture with all of the DNP-protein conjugates. During the 1st wk of life, the ability to tolerize neonatal splenic B cells progressively decreased. Thus, tolerance in this system is: (a) restricted to B cells early in development; (b) established by both tolerogens and immunogens; (c) achieved at low (10(-9) M determinant) antigen concentrations; and (d) highly specific, discriminating between DNP- and TNP-specific B cells. We conclude that: (a) B lymphocytes, during their development, mature through a stage in which they are extremely susceptible to tolerogenesis; (b) the specific interaction of B-cell antigen receptors with multivalent antigens, while irrelevant to mature B cells, is tolerogenic to neonatal (immature) B cells unless antigen is concomitantly recognized by primed T cells; and (c) differences in the susceptibility of immature and mature B lymphocytes to tolerance induction suggest intrinsic differences between neonatal and adult B cells and may provide a physiologically relevant model for the study of tolerance to self-antigens.


Control of Paratrichodorus allius and Corky Ringspot Disease of Potato in the Columbia Basin of Oregon.
Corky ringspot disease (CRS) of potato, caused by tobacco rattle virus that is vectored by stubby-root nematodes (Paratrichodorus spp.), is often controlled by aldicarb. When use of aldicarb on potato was suspended in 1989, an increase in crops rejected due to CRS in the Columbia Basin of the U.S. Pacific Northwest occurred. During 1992-94, several fumigant and nonfumigant nematicides were tested alone and in combination for control of P. allius and CRS. Aldicarb alone significantly reduced CRS but not to acceptable levels. Metam sodium or ethoprop alone did not control CRS, but metam sodium plus ethoprop provided adequate control under light disease pressure. Two or three postemergence applications of oxamyl, either with or without metham sodium, appeared to control CRS at low pressure. Fosthiazate reduced CRS incidence when used alone but not in combination with metam sodium. At low P. allius population densities, 1,3 dichloropropene (1,3-D) controlled CRS at 94 liters/ha, and rates of 140 liters/ha or greater were adequate at higher population densities. Treatment with 1,3-D plus chloropicrin was no better than 1,3-D alone and did not always control CRS. Combinations of 1,3-D at 94 liters/ha or greater plus metam sodium at 374 liters/ha or greater controlled CRS. Paratrichodorus allius numbers were higher and severity of CRS greater after wheat than after field corn, but P. allius declined rapidly after potato was planted and remained at low levels until harvest.


Change in area of geographic atrophy in the Age-Related Eye Disease Study: AREDS report number 26.
To characterize progression of geographic atrophy (GA) associated with age-related macular degeneration in AREDS as measured by digitized fundus photographs. Fundus photographs from 181 of 4757 AREDS participants with a GA area of at least 0.5 disc areas at baseline or from participants who developed bilateral GA during follow-up were scanned, digitized, and evaluated longitudinally. Geographic atrophy area was determined using planimetry. Rates of progression from noncentral to central GA and of vision loss following development of central GA included the entire AREDS cohort. Median initial lesion size was 4.3 mm(2). Average change in digital area of GA from baseline was 2.03 mm(2) (standard error of the mean, 0.24 mm(2)) at 1 year, 3.78 mm(2) (0.24 mm(2)) at 2 years, 5.93 mm(2) (0.34 mm(2)) at 3 years, and 1.78 mm(2) (0.086 mm(2)) per year overall. Median time to developing central GA after any GA diagnosis was 2.5 years (95% confidence interval, 2.0-3.0). Average visual acuity decreased by 3.7 letters at first documentation of central GA, and by 22 letters at year 5. Growth of GA area can be reliably measured using standard fundus photographs that are digitized and subsequently graded at a reading center. Development of GA is associated with subsequent further growth of GA, development of central GA, and loss in central vision.


The efficacy of the cyclin-dependent kinase 4/6 inhibitor in endometrial cancer.
PD-0332991, the selective cyclin-dependent kinase 4/6 inhibitor palbociclib, causes cell cycle arrest by inhibiting phosphorylation of retinoblastoma (Rb) protein. The aim of this study was to evaluate the therapeutic potential of PD-0332991 in endometrial cancer. Four human endometrial cancer cell lines, ECC, HEC1A, HEC108 and TEN, were treated with PD-0332991 and their function was evaluated. In vivo, the therapeutic efficacy was evaluated in a model of subcutaneous endometrial cancer. An immunohistochemical analysis was performed in 337 endometrial cancer specimens. A proliferation assay revealed that 2 of the 4 cell lines that expressed Rb were sensitive to PD-0332991 with an IC50 of 0.65 μM (HEC1A) and 0.58 μM (HEC108), respectively. Both cell lines had G0/G1 cell cycle arrest after treatment with PD-0332991 according to flow cytometry. In vivo, PD-0332991 had antitumoral efficacy with a reduction in the activity of Ki67 and phosphorylation of Rb. Immunohistochemical analyses revealed that the positive rate of Rb was 67.7%, however, there was no significant relationship between the expression levels of Rb and the tumor grade. PD-0332991 had therapeutic potential against endometrial cancer cell lines expressing Rb protein. Our immunohistochemical analysis revealed that approximately 70% of patients with endometrial cancer might have therapeutic indications for PD-0332991. Of note, the tumor grade had no impact on the indications for treatment.


Effect of vitamin D supplementation on endothelial dysfunction in hemodialysis patients.
Patients with chronic kidney disease (CKD) commonly experience 25-hydroxyvitamin D3 (25-OH-D3) deficiency, and these patients have a higher incidence of cardiovascular diseases (CVDs) due to endothelial dysfunction (ED). The aim of our study was to investigate the effect of 25-OH-D3 deficiency and its supplementation on ED in patients with CKD. Twenty-nine uremic patients on dialysis and 20 healthy controls were evaluated for ED by high-resolution Doppler ultrasonography of the brachial artery. In addition, 25-OH-D3-deficient patients (25-OH-D3 < 30 nmol/L) with CKD and healthy controls were evaluated for ED before and after 8 weeks of oral vitamin D (cholecalciferol, 50,000 units) treatment. All subjects were evaluated for percent flow-mediated dilatation (%FMD), percent endothelium-independent nitroglycerin-induced vasodilatation (%NID), and bilateral carotid intima-media thickness (CIMT). Patients on dialysis had lower %FMD and %NID 6.11 [2.27-12.74] and 10.96 [5.43-16.4], respectively, than controls 15.84 [8.19-22.49] and 21.74 [12.49-29.4], respectively (P < 0.05). Patients on dialysis had higher left and right CIMT (0.79 ± 0.15 and 0.78 ± 0.14, respectively) than controls (0.60 ± 0.09 and 0.59 ± 0.09, respectively; P < 0.05). In 25-OH-D3-deficient patients with CKD, after vitamin D treatment, %FMD was significantly increased in dialysis patients (10.25 [7.8-12.8]) compared to before supplementation (5.4 [2.77-6.15]; P < 0.001). These results indicated that dialysis patients had significantly lower blood 25-OH-D3 levels and higher CIMT than healthy subjects. In addition, vitamin D supplementation improved ED and increased %FMD in dialysis patients. Our findings suggest that vitamin D supplementation in dialysis patients might prevent CVD.


Review: Fluid biomarkers for frontotemporal dementias.
Frontotemporal dementias (FTDs) are clinically, genetically and pathologically heterogeneous neurodegenerative disorders that affect the frontal and anterior temporal lobes of the brain. They are relatively common causes of young-onset dementia and usually present with behavioural disturbance (behavioural variant FTD) or language impairment (primary progressive aphasia), but there is also overlap with motor neurone disease and the atypical parkinsonian disorders, corticobasal syndrome and progressive supranuclear palsy. At post mortem, neuronal inclusions containing tau, TDP-43 or infrequently FUS protein are seen in most cases. However, a poor correlation between clinical syndrome and underlying pathology means that it is difficult to diagnose the underlying molecular basis using clinical criteria. At this point, biomarkers for the underlying pathology come into play. This paper provides a brief update on fluid biomarkers for FTDs that may be useful to dissect the underlying molecular changes in patients presenting with signs of frontal and/or temporal lobe dysfunction. The hope is that such biomarkers, together with genetics and imaging, would be useful in clinical trials of novel drug candidates directed against specific pathologies and, in the long run, helpful in clinical practice to select the most appropriate treatment at the right dose for individual patients.


Trimetazidine prevents palmitate-induced mitochondrial fission and dysfunction in cultured cardiomyocytes.
Metabolic and cardiovascular disease patients have increased plasma levels of lipids and, specifically, of palmitate, which can be toxic for several tissues. Trimetazidine (TMZ), a partial inhibitor of lipid oxidation, has been proposed as a metabolic modulator for several cardiovascular pathologies. However, its mechanism of action is controversial. Given the fact that TMZ is able to alter mitochondrial metabolism, we evaluated the protective role of TMZ on mitochondrial morphology and function in an in vitro model of lipotoxicity induced by palmitate. We treated cultured rat cardiomyocytes with BSA-conjugated palmitate (25 nM free), TMZ (0.1-100 μM), or a combination of both. We evaluated mitochondrial morphology and lipid accumulation by confocal fluorescence microscopy, parameters of mitochondrial metabolism (mitochondrial membrane potential, oxygen consumption rate [OCR], and ATP levels), and ceramide production by mass spectrometry and indirect immunofluorescence. Palmitate promoted mitochondrial fission evidenced by a decrease in mitochondrial volume (50%) and an increase in the number of mitochondria per cell (80%), whereas TMZ increased mitochondrial volume (39%), and decreased mitochondrial number (56%), suggesting mitochondrial fusion. Palmitate also decreased mitochondrial metabolism (ATP levels and OCR), while TMZ potentiated all the metabolic parameters assessed. Moreover, pretreatment with TMZ protected the cardiomyocytes from palmitate-induced mitochondrial fission and dysfunction. TMZ also increased lipid accumulation in cardiomyocytes, and prevented palmitate-induced ceramide production. Our data show that TMZ protects cardiomyocytes by changing intracellular lipid management. Thus, the beneficial effects of TMZ on patients with different cardiovascular pathologies can be related to modulation of the mitochondrial morphology and function.


Prevalence, trajectories, and determinants of television viewing time in an ethnically diverse sample of young children from the UK.
Excessive screen viewing in early childhood is associated with poor physical and psycho-social health and poor cognitive development. This study aimed to understand the prevalence, trajectory and determinants of television viewing time in early childhood to inform intervention development. In this prospective longitudinal study, mothers of 1558 children (589 white British, 757 Pakistani heritage, 212 other ethnicities) completed questionnaires when their children were approximately 6, 12, 18, 24 and 36 months old. Mothers answered questions about their own and their child's TV-time. TV-time trajectories were estimated by linear longitudinal multilevel modeling, potential determinants were considered in models. The modelled trajectory estimated that 75% of children aged 12 months exceeded guidelines of zero screen-time. At 12 months of age an accelerated increase in TV-time was observed (<1 h/day at 14 months, >2 h/day by 30 months old). For every hour of mothers' TV-time and every hour the TV was on in the home, children's TV-time was 8 min and 1 min higher respectively at 6 months old (P < 0.05), and 15 min and 3 min higher respectively at 36 months old (P < 0.05). Children whose mothers did not agree that it was important their child did not watch too much TV, had 17 min more TV-time than their counterparts (P < 0.05). Children of first time mothers had 6 min more TV-time (P < 0.05). At 12 months of age, children of mothers experiencing stress watched 8 min more TV (P < 0.05). By 36 months, children of Pakistani heritage mothers had 22 min more TV-time than those of white British mothers (P < 0.05), and an additional 35 min of TV-time if their mother was not born in the UK (P < 0.05). High levels of TV-time were prevalent. Intervention developers should consider targeting interventions before 12 months of age. Modifiable determinants included mothers' own TV-time, the time the television is on in the home and mothers' attitude towards child TV-time. These behaviours may be key components to address in interventions for parents. Mothers experiencing stress, first time mothers, and Pakistani heritage mothers (particularly those born outside of the UK), may be priority groups for intervention.


Obsessive-compulsive disorder, impulse control disorders and drug addiction: common features and potential treatments.
The basic concepts underlying compulsive, impulsive and addictive behaviours overlap, which may help explain why laymen use these expressions interchangeably. Although there has been a large research effort to better characterize and disentangle these behaviours, clinicians and scientists are still unable to clearly differentiate them. Accordingly, obsessive-compulsive disorder (OCD), impulse control disorders (ICD) and substance-related disorders (SUD) overlap on different levels, including phenomenology, co-morbidity, neurocircuitry, neurocognition, neurochemistry and family history. In this review we summarize these issues with particular emphasis on the role of the opioid system in the pathophysiology and treatment of OCD, ICD and SUD. We postulate that with progression and chronicity of OCD, the proportion of the OCD-related behaviours (e.g. checking, washing, ordering and hoarding, among others) that are driven by impulsive 'rash' processes increase as involvement of more ventral striatal circuits becomes prominent. In contrast, as SUD and ICD progress, the proportion of the SUD- and ICD-related behaviours that are driven by compulsive 'habitual' processes increase as involvement of more dorsal striatal circuits become prominent. We are not arguing that, with time, ICD becomes OCD or vice versa. Instead, we are proposing that these disorders may acquire qualities of the other with time. In other words, while patients with ICD/SUD may develop 'compulsive impulsions', patients with OCD may exhibit 'impulsive compulsions'. There are many potential implications of our model. Theoretically, OCD patients exhibiting impulsive or addictive features could be managed with drugs that address the quality of the underlying drives and the involvement of neural systems. For example, agents for the reduction or prevention of relapse of addiction (e.g. heavy drinking), which modulate the cortico-mesolimbic dopamine system through the opioid (e.g. buprenorphine and naltrexone), glutamate (e.g. topiramate), serotonin (e.g. ondansetron) or γ-aminobutyric acid (e.g. baclofen and topiramate) systems, may prove to show some benefit in certain forms of OCD. Based on the available evidence, we suggest that the treatment of patients with these disorders must account for alterations in the underlying motivations and neurobiology of the condition. We provide an initial guide to the specific treatments that future clinical trials might consider in patients with OCD. For example, it might be wise to test naltrexone in patients with co-morbid SUD and ICD, topiramate in patients with co-morbid ICD and eating disorders, and baclofen in patients with co-morbid Tourette's syndrome. These trials could also include scales aimed at assessing underlying impulsivity (e.g. Barratt Impulsiveness Scale) to check whether this construct might predict response to drugs acting on the reward system.


Structural basis for bacterial lipoprotein relocation by the transporter LolCDE.
Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2-3.8 Å, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria.


Quality assessment of expert answers to lay questions about cystic fibrosis from various language zones in Europe: the ECORN-CF project.
The European Centres of Reference Network for Cystic Fibrosis (ECORN-CF) established an Internet forum which provides the opportunity for CF patients and other interested people to ask experts questions about CF in their mother language. The objectives of this study were to: 1) develop a detailed quality assessment tool to analyze quality of expert answers, 2) evaluate the intra- and inter-rater agreement of this tool, and 3) explore changes in the quality of expert answers over the time frame of the project. The quality assessment tool was developed by an expert panel. Five experts within the ECORN-CF project used the quality assessment tool to analyze the quality of 108 expert answers published on ECORN-CF from six language zones. 25 expert answers were scored at two time points, one year apart. Quality of answers was also assessed at an early and later period of the project. Individual rater scores and group mean scores were analyzed for each expert answer. A scoring system and training manual were developed analyzing two quality categories of answers: content and formal quality. For content quality, the grades based on group mean scores for all raters showed substantial agreement between two time points, however this was not the case for the grades based on individual rater scores. For formal quality the grades based on group mean scores showed only slight agreement between two time points and there was also poor agreement between time points for the individual grades. The inter-rater agreement for content quality was fair (mean kappa value 0.232 ± 0.036, p < 0.001) while only slight agreement was observed for the grades of the formal quality (mean kappa value 0.105 ± 0.024, p < 0.001). The quality of expert answers was rated high (four language zones) or satisfactory (two language zones) and did not change over time. The quality assessment tool described in this study was feasible and reliable when content quality was assessed by a group of raters. Within ECORN-CF, the tool will help ensure that CF patients all over Europe have equal possibility of access to high quality expert advice on their illness.


Nasopharyngeal muscle patch for the management of internal carotid artery injury in endoscopic endonasal surgery.
Injury to the internal carotid artery (ICA) is the most critical complication of endoscopic endonasal skull base surgery. Packing with a crushed muscle graft at the injury site has been an effective management technique to control bleeding without ICA sacrifice. Obtaining the muscle graft has typically required access to another surgical site, however. To address this concern, the authors investigated the application of an endonasally harvested longus capitis muscle patch for the management of ICA injury. One colored silicone-injected anatomical specimen was dissected to replicate the surgical access to the nasopharynx and the stepwise dissection of the longus capitis muscle in the nasopharynx. Two representative cases were selected to illustrate the application of the longus capitis muscle patch and the relevance of clinical considerations. A suitable muscle graft from the longus capitis muscle could be easily and quickly harvested during endoscopic endonasal skull base surgery. In the illustrative cases, the longus capitis muscle patch was successfully used for secondary prevention of pseudoaneurysm formation following primary bleeding control on the site of ICA injury. Nasopharyngeal harvest of a longus capitis muscle graft is a safe and practical method to manage ICA injury during endoscopic endonasal surgery.


Inhibition of oxidative metabolism by propionic acid and its reversal by carnitine in isolated rat hepatocytes.
The present study was designed to study the interaction of propionic acid and carnitine on oxidative metabolism by isolated rat hepatocytes. Propionic acid (10 mM) inhibited hepatocyte oxidation of [1-14C]-pyruvate (10 mM) by 60%. This inhibition was not the result of substrate competition, as butyric acid had minimal effects on pyruvate oxidation. Carnitine had a small inhibitory effect on pyruvate oxidation in the hepatocyte system (210 +/- 19 and 184 +/- 18 nmol of pyruvate/60 min per mg of protein in the absence and presence of 10 mM-carnitine respectively; means +/- S.E.M., n = 10). However, in the presence of propionic acid (10 mM), carnitine (10 mM) increased the rate of pyruvate oxidation by 19%. Under conditions where carnitine partially reversed the inhibitory effect of propionic acid on pyruvate oxidation, formation of propionylcarnitine was documented by using fast-atom-bombardment mass spectroscopy. Propionic acid also inhibited oxidation of [1-14C]palmitic acid (0.8 mM) by hepatocytes isolated from fed rats. The degree of inhibition caused by propionic acid was decreased in the presence of 10 mM-carnitine (41% inhibition in the absence of carnitine, 22% inhibition in the presence of carnitine). Propionic acid did not inhibit [1-14C]palmitic acid oxidation by hepatocytes isolated from 48 h-starved rats. These results demonstrate that propionic acid interferes with oxidative metabolism in intact hepatocytes. Carnitine partially reverses the inhibition of pyruvate and palmitic acid oxidation by propionic acid, and this reversal is associated with increased propionylcarnitine formation. The present study provides a metabolic basis for the efficacy of carnitine in patients with abnormal organic acid accumulation, and the observation that such patients appear to have increased carnitine requirements ('carnitine insufficiency').


Natural history and risk factors of early respiratory responses to exposure to cotton dust in newly exposed workers.
A prospective study of newly exposed cotton workers was performed to investigate the natural history of respiratory symptoms and lung function changes. A total of 157 workers naive to cotton dust exposure were investigated by questionnaire, spirometry, and skin tests. They were examined before employment (baseline) and at the end of the first week, and the first, third, sixth, and 12th month after starting work. Acute airway response was defined as either a cross-first-shift or a cross-week fall in forced expiratory volume in one second (FEV1). The longitudinal change of lung function over the year was also calculated. Five hundred seventy-two personal dust sampling and 191 endotoxin measurements were performed to assess the exposure. Forty percent of workers reported work-related symptoms in the first week of the study. Smoking, endotoxin, and dust concentrations were risk factors for all work-related symptoms. Acute airway responses were witnessed after immediate exposure. Female status was the only factor found to be predictive of acute airway response. The mean longitudinal fall in FEV1 at 1 year was 65.5 mL (standard error = 37.2). Age, early respiratory symptoms, and early fall in cross-week FEV1 were found to predict the 12-month fall in FEV1. Cross-first-shift and cross-week falls in FEV1 reduced in magnitude during the course of the study. This study of workers naive to cotton dust exposure has demonstrated that respiratory symptoms and acute airway responses develop early following first exposure, and a tolerance effect develops in those workers with the continued exposure. Current smoking and increasing exposure predicts the development of work-related lower respiratory tract symptoms, while early symptoms and acute airway changes across the working week predict the longitudinal loss of lung function at 1 year.


Ebola virus exploits a monocyte differentiation program to promote its entry.
Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo. However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection.


Comprehensive evaluation of peripheral nerve regeneration in the acute healing phase using tissue clearing and optical microscopy in a rodent model.
Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.


Notch-mediated post-translational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment.
Ngn3 is recognized as a regulator of pancreatic endocrine formation, and Notch signaling as an important negative regulator Ngn3 gene expression. By conditionally controlling expression of Ngn3 in the pancreas, we find that these two signaling components are dynamically linked. This connection involves transcriptional repression as previously shown, but also incorporates a novel post-translational mechanism. In addition to its ability to promote endocrine fate, we provide evidence of a competing ability of Ngn3 in the patterning of multipotent progenitor cells in turn controlling the formation of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1>Notch>Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated activation of the Notch>Hes1 pathway impacts formation of the trunk domain in the pancreas causing multipotent progenitors to lose acinar, while gaining endocrine and ductal, competence. The subsequent selection of fate from such bipotential progenitors is then governed by lateral inhibition, where Notch>Hes1-mediated Ngn3 protein destabilization serves to limit endocrine differentiation by reducing cellular levels of Ngn3. This system thus allows for rapid dynamic changes between opposing bHLH proteins in cells approaching a terminal differentiation event. Inhibition of Notch signaling leads to Ngn3 protein stabilization in the normal mouse pancreas explants. We conclude that the mutually exclusive expression pattern of Ngn3/Hes1 proteins in the mammalian pancreas is partially controlled through Notch-mediated post-translational regulation and we demonstrate that the formation of insulin-producing beta-cells can be significantly enhanced upon induction of a pro-endocrine drive combined with the inhibition of Notch processing.


Precise determination of water exchanges on a mineral surface.
Solvent exchanges on solid surfaces and dissolved ions are a fundamental property important for understanding chemical reactions, but the rates of fast exchanges are poorly constrained. We probed the diffusional motions of water adsorbed onto nanoparticles of the mineral barite (BaSO4) using quasi-elastic neutron scattering (QENS) and classical molecular dynamics (MD) to reveal the complex dynamics of water exchange along mineral surfaces. QENS data as a function of temperature and momentum transfer (Q) were fit using scattering functions derived from MD trajectories. The simulations reproduce the dynamics measured in the experiments at ambient temperatures, but as temperature is lowered the simulations overestimate slower motions. Decomposition of the MD-computed QENS intensity into contributions from adsorbed and unbound water shows that the majority of the signal arises from adsorbed species, although the dynamics of unbound water cannot be dismissed. The mean residence times of water on each of the four surface sites present on the barite {001} were calculated using MD: at room temperature the low barium site is 194 ps, whereas the high barium site contains two distributions of motions at 84 and 2.5 ps. These contrast to 13 ps residence time on both sulfate sites, with an additional surface diffusion exchange of 66 ps. Surface exchanges are similar to those of the aqueous ions calculated using the same force field: Baaq2+ is 208 ps and SO4aq2- is 5.8 ps. This work demonstrates how MD can be a reliable method to deconvolute solvent exchange reactions when quantitatively validated by QENS measurements.


Healthy Human Fecal Microbiota Transplantation into Mice Attenuates MPTP-Induced Neurotoxicity via AMPK/SOD2 Pathway.
Increasing evidence has shown that gut dysbacteriosis may play a crucial role in neuroinflammation in Parkinson's disease (PD). However, the specific mechanisms that link gut microbiota to PD remain unexplored. Given the critical roles of blood-brain barrier (BBB) dysfunction and mitochondrial dysfunction in the development of PD, we aimed to evaluate the interactions among the gut microbiota, BBB, and mitochondrial resistance to oxidation and inflammation in PD. We investigated the effects of fecal microbiota transplantation (FMT) on the physiopathology of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. The aim was to explore the role of fecal microbiota from PD patients and healthy human controls in neuroinflammation, BBB components, and mitochondrial antioxidative capacity via the AMPK/SOD2 pathway. Compared to control mice, MPTP-treated mice exhibited elevated levels of Desulfovibrio, whereas mice given FMT from PD patients exhibited enriched levels of Akkermansia and mice given FMT from healthy humans showed no significant alterations in gut microbiota. Strikingly, FMT from PD patients to MPTP-treated mice significantly aggravated motor impairments, dopaminergic neurodegeneration, nigrostriatal glial activation and colonic inflammation, and inhibited the AMPK/SOD2 signaling pathway. However, FMT from healthy human controls greatly improved the aforementioned MPTP-caused effects. Surprisingly, the MPTP-treated mice displayed a significant loss in nigrostriatal pericytes, which was restored by FMT from healthy human controls. Our findings demonstrate that FMT from healthy human controls can correct gut dysbacteriosis and ameliorate neurodegeneration in the MPTP-induced PD mouse model by suppressing microgliosis and astrogliosis, ameliorating mitochondrial impairments via the AMPK/SOD2 pathway, and restoring the loss of nigrostriatal pericytes and BBB integrity. These findings raise the possibility that the alteration in the human gut microbiota may be a risk factor for PD and provide evidence for potential application of FMT in PD preclinical treatment.


Population genomics of early events in the ecological differentiation of bacteria.
Genetic exchange is common among bacteria, but its effect on population diversity during ecological differentiation remains controversial. A fundamental question is whether advantageous mutations lead to selection of clonal genomes or, as in sexual eukaryotes, sweep through populations on their own. Here, we show that in two recently diverged populations of ocean bacteria, ecological differentiation has occurred akin to a sexual mechanism: A few genome regions have swept through subpopulations in a habitat-specific manner, accompanied by gradual separation of gene pools as evidenced by increased habitat specificity of the most recent recombinations. These findings reconcile previous, seemingly contradictory empirical observations of the genetic structure of bacterial populations and point to a more unified process of differentiation in bacteria and sexual eukaryotes than previously thought.


Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys.
Taxonomic classification of the thousands-millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases.


Genetic manipulation of centromere function.
A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL1 promoter from S. cerevisiae. By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucose-carbon source) from the GAL1 promoter, centromere function can be switched off or on, respectively. With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids. Moreover, it was possible to selectively induce the loss of a single chromosome from a mitotically dividing population of cells. We observed that the induction of chromosome III aneuploidy resulted in a dramatic change in cell morphology. The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element.


Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia.
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL.


Solution structure of the SGTA dimerisation domain and investigation of its interactions with the ubiquitin-like domains of BAG6 and UBL4A.
The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.


Genomic evolution of uveal melanoma arising in ocular melanocytosis.
Ocular melanocytosis is the most important predisposing condition for the eye cancer uveal melanoma (UM). Here, we present a patient who developed UM arising within ocular melanocytosis who was treated with enucleation (eye removal), which provided an invaluable opportunity to interrogate both the UM and adjacent uveal tissue containing the melanocytosis using whole-exome and deep-targeted sequencing. This analysis revealed a clonal PLCB4 mutation in the melanocytosis, confirming that this is indeed a neoplastic condition and explaining why it predisposes to UM. This mutation was present in 100% of analyzed UM cells, indicating that a PLCB4-mutant cell gave rise to the UM. The earliest aberrations specific to the tumor were loss of Chromosomes 1p, 3, and 9p, which were present in virtually all tumor cells. A mutation in BAP1 arose later on the other copy of Chromosome 3 in a tumor subclone, followed by a gain of Chromosome 8q. These findings provide a mechanistic explanation for the well-known clinical association between ocular melanocytosis and UM by showing that this predisposing condition introduces the first "hit" and thereby increases the stochastic likelihood of acquiring further aberrations leading to UM.


Effect of reciprocal complementation of two defective human immunodeficiency virus type 1 (HIV-1) molecular clones on HIV-1 cell tropism and virulence.
Human immunodeficiency virus type 1 (HIV-1) displays both interstrain and intrastrain genetic variability. Virus populations with extensive microheterogeneity have been defined as swarms or quasispecies. Many of the genomes within HIV-1 swarms appear to be defective in one or more genes required for viral replication. It is unclear to what extent defective viruses play a role in the process of HIV-1 infection or in the pathogenesis of AIDS. We have isolated two biologically active HIV-1 clones: LW 12.3, which contains defects in the vif and vpr genes, and MN ST.1, which has a defect in the vpu gene. LW 12.3 is unable to replicate in peripheral blood mononuclear cells (PBMC). The growth of MN-ST.1 in SupT1 cells is marked by a 3-week lag in extracellular virus production and by the presence of unusually abundant viral buds. We demonstrate here that coinfection of PBMC with these two partially defective HIV-1 clones extends the cellular host range of LW 12.3, significantly increases the replication rate of both viral genomes, and eliminates the delay in production observed with the vpu-defective MN ST.1. When the lesions in vpr and vif of LW 12.3 are repaired, the resultant virus grows normally in PBMC. This is also the case when only vif is repaired, indicating that complementation of LW 12.3 in PBMC by MN ST.1 is mediated by vif in trans. The reciprocal complementation results in a dramatic increase of HIV-1 virulence. This two-component model represents a simplified version of the in vivo situation and illustrates one way in which interaction of defective viruses could increase the spread of infection and progression of disease.


Inhibition of the expression and activity of cyclooxygenase-2 by chicory extract.
Chicory is a major source of fructans with reported prebiotic-bifidogenic properties. In the present study, the potential anti-inflammatory activities of chicory were investigated. Ethyl acetate chicory root extract produced a marked inhibition of prostaglandin E(2) (PGE(2)) production in human colon carcinoma HT29 cells treated with the pro-inflammatory agent TNF-alpha. Two independent mechanisms of action were identified: (1) a drastic inhibition of the induction by TNF-alpha of cyclooxygenase 2 (COX-2) protein expression and (2) a direct inhibition of COX enzyme activities with a significantly higher selectivity for COX-2 activity. The inhibition of TNF-alpha-dependent induction of COX-2 expression was mediated by an inhibition of NF-kappaB activation. A major sesquiterpene lactone of chicory root, the guaianolide 8-deoxylactucin, was identified as the key inhibitor of COX-2 protein expression present in chicory extract. Altogether, the data presented strongly support chicory root as a promising source of functional food ingredient, combining prebiotic and anti-inflammatory properties.


Alcohol Hangover Has Detrimental Impact Upon Both Executive Function and Prospective Memory.
The alcohol hangover (AH) is a state of general malaise following an evening of heavy episodic drinking when the blood alcohol concentration of the person reaches/approaches zero. The aim of the current study was to investigate what impact the AH has upon both executive function (EF) and prospective memory (PM). Previous research has shown that the AH has a detrimental effect upon cognitive abilities, including attention, working memory, and PM. The current study focused upon what impact AH might have upon both EF and related PM in the same cohort, both of which underpin everyday remembering. The current study compared an AH group (AHG) with a non-hangover group (NHG) on both EF and PM measures. Forty-one participants aged 18-29 years were tested; 19 comprised the AHG and 22 of whom made up the NHG (individuals who reported no heavy drinking the day before and did not report any significant hangover symptoms). A Verbal Fluency task measured EF and the Prospective Remembering Video Procedure measured PM. The Acute Hangover Rating Scale measured AH symptoms and severity, and a Digital Breath Analyzer Test measured their blood alcohol concentration (BAC). A Recreational Drug Use Questionnaire measured alcohol and other drug use. Anyone reporting having used an illicit substance across their lifetime (e.g., cannabis, ecstasy) or who smoked heavily were omitted from the study. Two univariate analyses of covariance compared the AHG and NHG groups on Verbal Fluency and Prospective Remembering Video Task scores (controlling for age, total alcohol units consumed per week, and the number of years spent drinking). The AHG recalled significantly fewer items on the Verbal Fluency task [F (1, 36) = 7.42, p < 0.01] and on the Prospective Remembering Video Task NHG [F (1, 36) = 14.9, p < 0.001] when compared with the NHG. Overall, it appeared that a state of AH significantly impaired both EF and PM. Given the importance of EF and PM to everyday remembering, these findings may have farther-reaching implications.


To be a nurse or a neighbour? A moral concern for psychiatric nurses living next door to individuals with a mental illness.
Several studies reveal that positive attitudes towards individuals with a mental illness are correlated with knowledge about mental illness. The aim of this study was to explore and describe psychiatric nurses' experiences of living next to people with mental health problems. In addition, it sought to identify and describe how they handle situations arising in a neighbourhood where people with a mental illness live. Two men and seven women participated in the study. The constant comparative method of grounded theory was used for data collection and analysis. The process of 'behaving as a nurse or not' was identified as a core category. Four subcategories were identified: 'receiving involuntary information', 'to take action or not', 'behaving as a mediator in the neighbourhood' and 'the freedom of choice'. The findings show that psychiatric nurses with professional knowledge about mental illness have moral concerns about their role as nurses during their leisure time. In conclusion, it is not obvious that psychiatric nurses want to live in the same neighbourhood as persons with a mental illness. However, this study shows that their knowledge about mental illness creates for them a moral dilemma consisting of a conflict between whether to care for these mentally ill persons or to preserve their own leisure time.


Monomeric and dimeric CXCL8 are both essential for in vivo neutrophil recruitment.
Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist in vivo, and if so, the relevance of each form for in vivo function is not known. In this study, using a 'trapped' non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease.


Optical coherence tomography evaluation of the corneal cap and stromal bed features after laser in situ keratomileusis for high myopia and astigmatism.
To study the corneal microstructure by optical coherence tomography (OCT) after laser in situ keratomileusis (LASIK) for high myopia with and without astigmatism. Nonrandomized self-controlled comparative trial. Sixty-three consecutive LASIK eyes with spherical equivalent refraction between -6.0 and -17.0 diopters (D) and astigmatism between 0.0 and -5.0 D were prospectively recruited for examination. LASIK was performed with the Chiron Hansatome microkeratome (160-microm fixed plate) and Summit Apex Plus excimer laser using a 5.5/6.0/6.5-mm multizone pattern. Proper preoperative calculations were performed to ensure stromal beds thicker than 250 microm. OCT imaging and measurement of corneal thickness was performed preoperatively. In addition, corneal cap and stromal bed thickness measurements were performed 1 day, 1 month, and 3 months postoperatively. The average central corneal pachymetry was 538.9 +/- 26.2 microm preoperatively. Mean corneal cap thickness measured 124.8 +/- 18.5 microm 1-day postoperatively. Mean stromal bed thickness was 295.2 +/- 37.1 microm on the first postoperative day. Compared with the 1-day postoperative examination, the average stromal bed thickness increased significantly by 5.9 microm (P = 0.001) and 7.2 microm (P = 0.001) at the 1-month and 3-month postoperative examinations, respectively. Mean difference between actual (118.7 +/- 27.8 microm) and predicted (104.1 +/- 20.8 microm) central ablation depths was 14.6 +/- 16.7 microm (P = 0.0001). A weak but statistically significant positive association was found between preoperative refraction and the difference between expected and real ablation depth values (R = 0.26; P = 0.042). Posterior stromal beds were more than 250-microm thick in 58 eyes (89.9%) 1 day postoperatively. This safety requirement improved at the 1-month postoperative examination, when the partial regression accounted for slightly thicker stromal beds and only two cases (3.2%) exhibited posterior stromal tissue thinner than 250 microm. These two cases were seen only for corrections exceeding 12 D (P = 0.04). OCT appears to be a useful tool for the evaluation of both the qualitative and quantitative anatomic outcome of LASIK. Corrections of higher degrees of ametropia run a higher risk of producing a thinner than expected central cornea. Particularly, corrections greater than 12 D may lead eventually to stromal beds thinner than 250 microm, despite proper preoperative calculations. Because corneal flaps are usually thinner than expected with the microkeratome used herein, adequate posterior corneal stroma is preserved in most instances.


The impact of informed consent on patient interest in prostate-specific antigen screening.
Because of the many uncertainties surrounding screening for prostate cancer, authorities recommend that patients be involved in the screening decision. To determine the impact of informed consent on patient interest in undergoing prostate-specific antigen (PSA) screening. Men 50 years or older with no prior PSA testing and no history of prostate cancer presenting to 1 of 4 university-affiliated primary care practices were eligible for enrollment. Patients were randomized to receive either a scripted informational intervention simulating an informed consent presentation (intervention group, n = 103) or a single sentence about the PSA (control group, n = 102). The main outcome measure was patient interest in undergoing PSA screening measured on a 5-point Likert scale. Patients who received the informational intervention were significantly less interested in undergoing PSA screening than controls (mean difference in interest, 0.8 on 5-point scale, P < .001). Informed patients were much less likely to indicate high interest in screening (odds ratio, 0.34; 95% confidence interval, 0.19-0.60; P < .001). In a multivariate model, family history of prostate cancer was associated with increased interest and advancing age with decreased interest in PSA screening, but the informational intervention remained the strongest predictor of interest. Among primary care patients of predominantly lower socioeconomic status, those who received informed consent were significantly less interested in PSA screening than those who did not. For physicians who offer the PSA as a screening test, this finding highlights the importance of apprising patients of the associated benefits, burdens, and uncertainties and allowing them to participate in the screening decision.


Examination of adduct formation in vivo in the mouse between benzo(a)pyrene and DNA of skin and hemoglobin of red blood cells.
We are interested in devising techniques which will allow us to measure and quantitate exposure to chemical carcinogens and which eventually can be used in risk analysis with humans. Our recent research with HPLC/fluorescence has demonstrated that we can detect, identify, and quantitate the binding of benzo(a)pyrene (BaP) with DNA of mouse skin. The technique not only allows femtomole amounts of BaPDE associated with DNA isolated from a single mouse skin to be detected using conventional instrumentation, but also establishes the stereochemical origin of the adduct, and has been employed in the investigation reported here to estimate the concomitant binding of BaP to hemoglobin in vivo. The temporal existence of BaPDE/DNA adducts in mouse skin over a 5-week period showed that at 35 days after treatment, approximately 15% of the initial adducts were still detectable even though DNA turnover would predict that they should have been deleted from the genome. The concentration of the major covalently bound adduct, anti-BaPDE/deoxyguanosine, relative to the total BaPDE/DNA adduct population remained essentially constant during the 5-week period. It is known that topically applied BaP is absorbed, metabolized, and excreted by the mouse. Examination of hemoglobin of mouse RBCs 24 hr after BaP treatment revealed covalent adduct formation exclusively via anti-BaPDE. The dose response of adduct binding to hemoglobin and DNA appeared to be similar.


Degradation pathways of pentachlorophenol and benzo(a)pyrene during heterogeneous photocatalysis.
Organic micropollutants, in particular those of anthropogenic origin, may have a toxic influence on water organisms. Photochemical oxidation processes are one of the most effective methods of decomposition of a wide range of those compounds. During the oxidation process a large number of different by-products are generated, which can still be biologically active. The development of analytical techniques, including the reduction of the detection limit to several fg/L, allows the identification of even trace concentrations of compounds. The paper presents the determination of pentachlorophenol and benzo(a)pyrene degradation pathways during the process of heterogeneous photocatalysis carried out in the presence of titanium dioxide. The gas chromatography-mass spectrometry (GC-MS) analysis of post-processing samples indicated the formation of different by-products of the parent micropollutants. Moreover, the toxicity assessment demonstrates for both tested micropollutants an increase in the toxicity within the whole time of the UV irradiation process run.


Comprehensive next-generation sequencing analyses of hypoparathyroidism: identification of novel GCM2 mutations.
In most patients with hypoparathyroidism (HP), the etiology is not defined clinically. Eight genes (AIRE, CASR, CLDN16, GATA3, GCM2, PTH, TBCE, and TRPM6) are known to be responsible genes associated with HP; however, no previous study has screened the eight responsible genes comprehensively in HP patients. This study was conducted to determine the genetic defect in HP patients. We also described clinical and molecular findings of two HP patients with novel GCM2 mutations. We enrolled 20 nonconsanguineous Japanese patients with child-onset permanent HP without 22q11 deletion. Mutations and genomic rearrangements involving the eight genes were screened by targeted next-generation sequencing (NGS). We also screened genetic rearrangements by array comparative genomic hybridization (aCGH) in the mutation-negative patients. A putative deletion, which was suspected by NGS, was additionally analyzed by droplet digital PCR (ddPCR) and junction PCR. Identified novel nucleotide-level GCM2 mutants were characterized in vitro. We identified seven patients with a single gene disorder, including a CASR mutation, GATA3 mutations, and novel GCM2 mutations (R367Tfs*15, T370M, and the deletion encompassing exon 1). This submicroscopic deletion, which had been suspected by NGS, could not be detected by aCGH and was confirmed by ddPCR and junction PCR. Functional studies of R367Tfs*- and T370M-GCM2 demonstrated a reduction of target gene transactivation in both. Using comprehensive NGS analyses, we identified the genetic defect in 35% of HP patients in our cohort and discovered novel GCM2 mutations including submicroscopic deletion that was undetectable by aCGH.


Relationship between periodontal inflammation and fetal growth in pregnant women: a cross-sectional study.
Periodontal disease in pregnant women, evaluated by probing pocket depth (PPD) or clinical attachment level (CAL), significantly increases the risk of subsequent preterm birth (PB) and/or low birth weight (LBW). However, PPD and CAL do not always reflect current periodontal tissue inflammation. Therefore, assessment of bleeding on probing index (BOP), an indicator of periodontal inflammation, may be appropriate. The objective of this cross-sectional study was to investigate the relationship between periodontal inflammation and fetal growth in pregnant women after periodontal treatment. We recruited 203 pregnant women (mean age 31.8 ± 4.5 years), including 20 subjects with periodontal inflammation and 183 periodontally healthy controls, after periodontal treatment. Fetal growth parameters as well as periodontal conditions were recorded. Perinatal gestational age was 39.4 ± 1.3 weeks; 8 subjects had a PB and 12 had an infant with a LBW. Results of multiple stepwise regression analysis indicated that birth length was negatively correlated with BOP% (β = -0.175; p = 0.002). Fetal femur length, and birth length and weight among mothers with low periodontal inflammation were significantly higher than those among mothers with high periodontal inflammation (p < 0.05). The LBW rate in the high periodontal inflammation group was significantly higher than that in the low periodontal inflammation group (p < 0.05). These results suggest that periodontal inflammation is correlated with fetal femur length, birth weight, and birth length.


Azathioprine in ulcerative colitis: final report on controlled therapeutic trial.
Eighty patients, all of whom were suffering from a frank clinical attack of ulcerative colitis, were admitted to the trial. The attack was treated with a standard course of corticosteroids and the patients were immediately placed on treatment with either azathioprine in a dose of 2.5 mg/kg body weight or dummy tablets. The trial tablets were continued for one year while the patients were maintained under regular clinical, sigmoidoscopic, histological, haematological, and biochemical surveillance. If a patient relapsed during such maintenance treatment he or she was treated with a further course of corticosteroids without interrupting maintenance treatment.In the treatment of an actual attack of ulcerative colitis the results in the attacks which brought the 80 patients into the trial show that no benefit came from the addition of azathioprine to a standard course of corticosteroid therapy.Patients admitted in their first attack of ulcerative colitis showed no benefit from the one-year maintenance treatment with azathioprine, the benefits of which were confined to patients admitted in a relapse of established disease. Even in these the difference between the treated group and the control group failed to reach statistical significance, but the difference was big enough to suggest that there is a prima facie case for regarding azathioprine as of some benefit in this group of patients.


Red blood cell populations and membrane levels of peroxiredoxin 2 as candidate biomarkers to reveal blood doping.
Blood doping represents one main trend in doping strategies. Blood doping refers to the practice of boosting the number of red blood cells (RBCs) in the bloodstream in order to enhance athletic performance, by means of blood transfusions, administration of erythropoiesis-stimulating substances, blood substitutes, natural or artificial altitude facilities, and innovative gene therapies. While detection of recombinant EPO and homologous transfusion is already feasible through electrophoretic, mass spectrometry or flow cytometry-based approaches, no method is currently available to tackle doping strategies relying on autologous transfusions. We exploited an in vitro model of autologous transfusion through a 1:10 dilution of concentrated RBCs after 30 days of storage upon appropriate dilution in freshly withdrawn RBCs from the same donor. Western blot towards membrane Prdx2 and Percoll density gradients were exploited to assess their suitability as biomarkers of transfusion. Membrane Prdx2 was visible in day 30 samples albeit not in day 0, while it was still visible in the 1:10 dilution of day 30 in day 0 RBCs. Cell gradients also highlighted changes in the profile of the RBC subpopulations upon dilution of stored RBCs in the fresh ones. From this preliminary in vitro investigation it emerges that Prdx2 and RBC populations might be further tested as candidate biomarkers of blood doping through autologous transfusion, though it is yet to be assessed whether the kinetics in vivo of Prdx2 exposure in the membrane of transfused RBCs will endow a sufficient time-window to allow reliable anti-doping testing.


Effects of Timely Control Intervention on the Spread of Middle East Respiratory Syndrome Coronavirus Infection.
The 2015 Middle East Respiratory Syndrome Coronavirus (MERS-CoV) outbreak in Korea caused major economic and social problems. The control intervention was conducted during the MERS-CoV outbreak in Korea immediately after the confirmation of the index case. This study investigates whether the early risk communication with the general public and mass media is an effective preventive strategy. The SEIR (Susceptible, Exposed, Infectious, Recovered) model with estimated parameters for the time series data of the daily MERS-CoV incidence in Korea was considered from May to December 2015. For 10,000 stochastic simulations, the SEIR model was computed using the Gillespie algorithm. Depending on the time of control intervention on the 20th, 40th, and 60th days after the identification of the index case, the box plots of MERS-CoV incidences in Korea were computed, and the results were analyzed via ANOVA. The box plots showed that there was a significant difference between the non-intervention and intervention groups (the 20th day, 40th day, and 60th day groups) and seemed to show no significant difference based on the time of intervention. However, the ANOVA revealed that early intervention was a good strategy to control the disease. Appropriate risk communication can secure the confidence of the general public in the public health authorities.


Hypoxia, not pulmonary vascular pressure, induces blood flow through intrapulmonary arteriovenous anastomoses.
Blood flow through intrapulmonary arteriovenous anastomoses (IPAVA) is increased by acute hypoxia during rest by unknown mechanisms. Oral administration of acetazolamide blunts the pulmonary vascular pressure response to acute hypoxia, thus permitting the observation of IPAVA blood flow with minimal pulmonary pressure change. Hypoxic pulmonary vasoconstriction was attenuated in humans following acetazolamide administration and partially restored with bicarbonate infusion, indicating that the effects of acetazolamide on hypoxic pulmonary vasoconstriction may involve an interaction between arterial pH and PCO2. We observed that IPAVA blood flow during hypoxia was similar before and after acetazolamide administration, even after acid-base status correction, indicating that pulmonary pressure, pH and PCO2 are unlikely regulators of IPAVA blood flow. Blood flow through intrapulmonary arteriovenous anastomoses (IPAVA) is increased with exposure to acute hypoxia and has been associated with pulmonary artery systolic pressure (PASP). We aimed to determine the direct relationship between blood flow through IPAVA and PASP in 10 participants with no detectable intracardiac shunt by comparing: (1) isocapnic hypoxia (control); (2) isocapnic hypoxia with oral administration of acetazolamide (AZ; 250 mg, three times a day for 48 h) to prevent increases in PASP; and (3) isocapnic hypoxia with AZ and 8.4% NaHCO3 infusion (AZ + HCO3 (-) ) to control for AZ-induced acidosis. Isocapnic hypoxia (20 min) was maintained by end-tidal forcing, blood flow through IPAVA was determined by agitated saline contrast echocardiography and PASP was estimated by Doppler ultrasound. Arterial blood samples were collected at rest before each isocapnic-hypoxia condition to determine pH, [HCO3(-)] and Pa,CO2. AZ decreased pH (-0.08 ± 0.01), [HCO3(-)] (-7.1 ± 0.7 mmol l(-1)) and Pa,CO2 (-4.5 ± 1.4 mmHg; P < 0.01), while intravenous NaHCO3 restored arterial blood gas parameters to control levels. Although PASP increased from baseline in all three hypoxic conditions (P < 0.05), a main effect of condition expressed an 11 ± 2% reduction in PASP from control (P < 0.001) following AZ administration while intravenous NaHCO3 partially restored the PASP response to isocapnic hypoxia. Blood flow through IPAVA increased during exposure to isocapnic hypoxia (P < 0.01) and was unrelated to PASP, cardiac output and pulmonary vascular resistance for all conditions. In conclusion, isocapnic hypoxia induces blood flow through IPAVA independent of changes in PASP and the influence of AZ on the PASP response to isocapnic hypoxia is dependent upon the H(+) concentration or Pa,CO2.


Improvement of mineral and bone metabolism markers is associated with better survival in haemodialysis patients: the COSMOS study.
Abnormalities in serum phosphorus, calcium and parathyroid hormone (PTH) have been associated with poor survival in haemodialysis patients. This COSMOS (Current management Of Secondary hyperparathyroidism: a Multicentre Observational Study) analysis assesses the association of high and low serum phosphorus, calcium and PTH with a relative risk of mortality. Furthermore, the impact of changes in these parameters on the relative risk of mortality throughout the 3-year follow-up has been investigated. COSMOS is a 3-year, multicentre, open-cohort, prospective study carried out in 6797 adult chronic haemodialysis patients randomly selected from 20 European countries. Using Cox proportional hazard regression models and penalized splines analysis, it was found that both high and low serum phosphorus, calcium and PTH were associated with a higher risk of mortality. The serum values associated with the minimum relative risk of mortality were 4.4 mg/dL for serum phosphorus, 8.8 mg/dL for serum calcium and 398 pg/mL for serum PTH. The lowest mortality risk ranges obtained using as base the previous values were 3.6-5.2 mg/dL for serum phosphorus, 7.9-9.5 mg/dL for serum calcium and 168-674 pg/mL for serum PTH. Decreases in serum phosphorus and calcium and increases in serum PTH in patients with baseline values of >5.2 mg/dL (phosphorus), >9.5 mg/dL (calcium) and <168 pg/mL (PTH), respectively, were associated with improved survival. COSMOS provides evidence of the association of serum phosphorus, calcium and PTH and mortality, and suggests survival benefits of controlling chronic kidney disease-mineral and bone disorder biochemical parameters in CKD5D patients.


High-Dose Chemotherapy With Hematopoietic Stem Cell Transplant in Patients With High-Risk Breast Cancer and 4 or More Involved Axillary Lymph Nodes: 20-Year Follow-up of a Phase 3 Randomized Clinical Trial.
Trials of adjuvant high-dose chemotherapy (HDCT) have failed to show a survival benefit in unselected patients with breast cancer, but long-term follow-up is lacking. To determine 20-year efficacy and safety outcomes of a large trial of adjuvant HDCT vs conventional-dose chemotherapy (CDCT) for patients with stage III breast cancer. This secondary analysis used data from a randomized phase 3 multicenter clinical trial of 885 women younger than 56 years with breast cancer and 4 or more involved axillary lymph nodes conducted from August 1, 1993, to July 31, 1999. Additional follow-up data were collected between June 1, 2016, and December 31, 2017, from medical records, general practitioners, the Dutch national statistical office, and nationwide cancer registries. Analysis was performed on an intention-to-treat basis. Statistical analysis was performed from February 1, 2018, to October 14, 2019. Participants were randomized 1:1 to receive 5 cycles of CDCT consisting of fluorouracil, 500 mg/m2, epirubicin, 90 mg/m2, and cyclophosphamide, 500 mg/m2, or HDCT in which the first 4 cycles were identical to CDCT and the fifth cycle was replaced by cyclophosphamide, 6000 mg/m2, thiotepa, 480 mg/m2, and carboplatin, 1600 mg/m2, followed by hematopoietic stem cell transplant. Main end points were overall survival and safety and cumulative incidence risk of a second malignant neoplasm or cardiovascular events. Of the 885 women in the study (mean [SD] age, 44.5 [6.6] years), 442 were randomized to receive HDCT, and 443 were randomized to receive CDCT. With 20.4 years median follow-up (interquartile range, 19.2-22.0 years), the 20-year overall survival was 45.3% with HDCT and 41.5% with CDCT (hazard ratio, 0.89; 95% CI, 0.75-1.06). The absolute improvement in 20-year overall survival was 14.6% (hazard ratio, 0.72; 95% CI, 0.54-0.95) for patients with 10 or more invoved axillary lymph nodes and 15.4% (hazard ratio, 0.67; 95% CI, 0.42-1.05) for patients with triple-negative breast cancer. The cumulative incidence risk of a second malignant neoplasm at 20 years or major cardiovascular events was similar in both treatment groups (20-year cumulative incidence risk for second malignant neoplasm was 12.1% in the HDCT group vs 16.2% in the CDCT group, P = .10), although patients in the HDCT group more often had hypertension (21.7% vs 14.3%, P = .02), hypercholesterolemia (15.7% vs 10.6%, P = .04), and dysrhythmias (8.6% vs 4.6%, P = .005). High-dose chemotherapy provided no long-term survival benefit in unselected patients with stage III breast cancer but did provide improved overall survival in very high-risk patients (ie, with ≥10 involved axillary lymph nodes). High-dose chemotherapy did not affect long-term risk of a second malignant neoplasm or major cardiovascular events. ClinicalTrials.gov Identifier: NCT03087409.


"Food Addiction" in Patients with Eating Disorders is Associated with Negative Urgency and Difficulties to Focus on Long-Term Goals.
The present study aimed to investigate if eating disorder patients differ in specific personality traits depending on a positive screening of food addiction (FA) and to find a model to predict FA in eating disorder patients using measures of personality and impulsivity. Two hundred seventy eight patients, having an eating disorder, self-reported on FA, impulsivity, personality, eating and general psychopathology. Patients were then split into two groups, depending on a positive or negative result on the FA screening. Analysis of variance was used to compare means between the two groups. Stepwise binary logistic regression was used to obtain a predictive model for the presence of FA. Patients with FA had lower self-directedness, and more negative urgency and lack of perseverance than patients not reporting addictive eating. The probability of FA can be predicted by high negative urgency, high reward dependence, and low lack of premeditation. Eating disorder patients who have more problems to pursue tasks to the end and to focus on long-term goals seem to be more likely to develop addictive eating patterns.


Common variants in HNF-1 alpha and risk of type 2 diabetes.
Mutations in the hepatocyte nuclear factor 1-alpha gene (HNF-1alpha, now known as the transcription factor 1 gene [TCF1]) cause the most common monogenic form of diabetes, MODY3, but it is not known if common variants in HNF-1a are associated with decreased transcriptional activity or phenotypes related to type 2 diabetes, or whether they predict future type 2 diabetes. We studied the effect of four common polymorphisms (rs1920792, I27L, A98V and S487N) in and upstream of the HNF-1alpha gene on transcriptional activity in vitro, and their possible association with type 2 diabetes and insulin secretion in vivo. Certain combinations of the I27L and A98V polymorphisms in the HNF-1alpha gene showed decreased transcriptional activity on the target promoters glucose transporter 2 (now known as solute carrier family 2 [facilitated glucose transporter], member 2) and albumin in both HeLa and INS-1 cells. In vivo, these polymorphisms were associated with a modest but significant impairment in insulin secretion in response to oral glucose. Insulin secretion deteriorated over time in individuals carrying the V allele of the A98V polymorphism (n = 2,293; p = 0.003). In a new case-control (n = 1,511 and n = 2,225 respectively) data set, the I27L polymorphism was associated with increased risk of type 2 diabetes, odds ratio (OR) = 1.5 (p = 0.002; multiple logistic regression), particularly in elderly (age > 60 years) and overweight (BMI > 25 kg/m(2)) patients (OR = 2.3, p = 0.002). This study provides in vitro and in vivo evidence that common variants in the MODY3 gene, HNF-1alpha, influence transcriptional activity and insulin secretion in vivo. These variants are associated with a modestly increased risk of late-onset type 2 diabetes in subsets of elderly overweight individuals.


Fisetin: a dietary antioxidant for health promotion.
Diet-derived antioxidants are now being increasingly investigated for their health-promoting effects, including their role in the chemoprevention of cancer. In general, botanical antioxidants have received much attention, as they can be consumed for longer periods of time without any adverse effects. Flavonoids are a broadly distributed class of plant pigments that are regularly consumed in the human diet due to their abundance. One such flavonoid, fisetin (3,3',4',7-tetrahydroxyflavone), is found in various fruits and vegetables, such as strawberry, apple, persimmon, grape, onion, and cucumber. Several studies have demonstrated the effects of fisetin against numerous diseases. It is reported to have neurotrophic, anticarcinogenic, anti-inflammatory, and other health beneficial effects. Although fisetin has been reported as an anticarcinogenic agent, further in-depth in vitro and in vivo studies are required to delineate the mechanistic basis of its observed effects. In this review article, we describe the multiple effects of fisetin with special emphasis on its anticancer activity as investigated in cell culture and animal models. Additional research focused toward the identification of molecular targets could lead to the development of fisetin as a chemopreventive/chemotherapeutic agent against cancer and other diseases.


Honey protects against wings posture error and molecular changes related to mitochondrial pathways induced by hypoxia/reoxygenation in adult Drosophila melanogaster.
We conducted an investigation to evaluate the effects of Brazilian Pampa biome honey and its major phenolic compounds on the development of an erected wings posture phenotype and related mitochondrial aspects induced by Hypoxia/Reoxygenation (H/R) in Drosophila melanogaster. Flies were pre-treated for 3 days with a 10% honey solution and different concentrations of caffeic acid and ρ-coumaric acid and then submitted to hypoxia for 3 h. We observed that after reoxygenation, some flies acquired an erected wings posture and that this feature may be related to mortality. In addition, H/R induced down-regulation of ewg mRNA expression, which could be associated to the observed complex phenotype. H/R also caused a dysregulation in opa1-like, ldh and diap genes expression and reduced O2 fluxes in flie's mitochondria. Honey mitigated opa1-like mRNA expression changes provoked by H/R. Differently from honey, caffeic and ρ-coumaric acids displayed no protective effects. In conclusion, we report for the first time the protective effects of honey against complex phenotypes and mitochondrial changes induced by H/R in adult flies.


Review of the sequential development of Loma salmonae (Microsporidia) based on experimental infections of rainbow trout (Oncorhynchus mykiss) and Chinook salmon (O. tshawytscha).
Loma salmonae is a common gill parasite of salmonids, and essentially all species in the genus Oncorhynchus are susceptible. Infections occur in both fresh and salt water. Loma salmonae is directly transmissible by ingestion of spores or infected tissue. The parasite infects the wall of blood vessels of various organs, but the gill is the primary site of infection. Initial infection occurs in the intestine, and xenomas are easily detected in the gills by standard histology at 4-6 wk post-exposure. A few presporogonic stages of the parasite are found in the heart endothelium prior to xenoma formation in the gills. Ultrastructure studies of early infections demonstrated that wandering blood cells transport the meronts to the gills, and that merogony occurs in pillar cells and other cells underlying the gill endothelium. Xenomas develop in these cells, resulting in hypertrophied host cells filled with spores. Xenomas ultimately rupture, and are associated with severe inflammation in which free spores are found in macrophages. The parasites are most pathogenic during this phase of the infection, resulting in severe vasculitis and clinical disease. Both rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus ishawytscha) recover from infections, but free spores persist in kidney and spleen phagocytes for many months after xenomas are absent in Chinook salmon. Fish that have recovered from the infection show strong immunity against the parasite, lasting up to 1 year. Fish are susceptible to infection by other routes of exposure by spores; co-habitation, anal gavage, and intramuscular, intraperitoneal and intravascular injection. Autoinfection probably occurs following release of spores in blood vessels after xenomas rupture. The optimal temperature for L. salmonae infections is 15-17 degrees C, with a permissive range of 11-20 degrees C.


Comparative isoschizomer profiling of cytosine methylation: the HELP assay.
The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.


Developmental regulation of mitochondrial biogenesis in Trypanosoma brucei.
The metabolism of Trypanosoma brucei undergoes a significant change as the parasite differentiates from the mammalian bloodstream form to the form found in the tse-tse fly vector. Because the mitochondria of bloodstream form cells lack cytochromes and several key citric acid cycle enzymes, the metabolism of these cells is mostly limited to glycolysis. The reducing equivalents generated by this process are passed to oxygen by a plant-like alternative oxidase. As cells differentiate to the insect form, they begin to oxidatively metabolize proline. The mitochondria of insect form cells contain functional, cytochrome-mediated electron transport chains and have complete complements of citric acid cycle enzymes. Although the characterization is far from complete, the nuclear and mitochondrial genes involved in the expression of these mitochondrial functions appear to be developmentally regulated at posttranscriptional and posttranslational levels. This review outlines some of the molecular processes that are associated with the developmental regulation of mitochondrial biogenesis and suggests some possible mechanisms of regulation.


Ischemic Strokes: Observations from a Hospital Based Stroke Registry in Bangladesh.
Background. Stroke is an important morbidity for low and middle income countries like Bangladesh. We established the first stroke registry in Bangladesh. Methods. Data was collected from stroke patients who were admitted in Department of Neurology of BIRDEM with first ever stroke, aged between 30 and 90 years. Patients with intracerebral hemorrhage, subarachnoid and subdural hemorrhage, and posttrauma features were excluded. Results. Data was gathered from 679 stroke patients. Mean age was 60.6 years. Almost 68% of patients were male. Small vessel strokes were the most common accounting for 45.4% of all the patients followed by large vessel getting affected in 32.5% of the cases. Only 16 (2.4%) died during treatment, and 436 (64.2%) patients had their mRS score of 3 to 5. Age greater than 70 years was associated with poor outcome on discharge [OR 1.79 (95% CI: 1.05 to 3.06)] adjusting for gender, duration of hospital stay, HDL, and pneumonia. Age, mRS, systolic blood pressure, urinary tract infection, pneumonia, and stroke severity explained the Barthel score. Conclusion. Mortality was low but most of patient had moderate to severe disability at discharge. Age, mRS, systolic blood pressure, urinary tract infection, pneumonia, and stroke severity influenced the Barthel score.


Modality-specific control processes in verbal versus spatial working memory.
Over the past decade, neuroimaging and electrophysiological studies of working memory (WM) have made progress in distinguishing the neural substrates of central executive (CE) functions from substrates of temporary storage subsystems. However, the degree to which CE-related processes and their substrates may be further fractionated is less clear. The present study measured event-related potentials (ERPs) in a running memory paradigm, to study modality-specific CE-related processes in verbal and spatial WM. Participants were asked to remember either verbal (digit identity) or spatial (digit location) information for the first or last three items in a variable length sequence of spatially distributed digit stimuli. Modality-specific WM demand-sensitive ERP amplitude effects were selectively observed over left prefrontal areas under verbal WM performance and over right prefrontal areas under spatial WM performance. In addition, distinct patterns of item-by-item sensitivity under high-CE-demand conditions suggested qualitatively different processing strategies for verbal versus spatial tasks. These results suggest that both modality-specific and task-general CE-related processes are likely operational in many WM situations and that careful dissociative methods will be needed to properly further fractionate and characterize these component CE-related processes and their neurological substrates.


Copy number loss of KDM5D may be a predictive biomarker for ATR inhibitor treatment in male patients with pulmonary squamous cell carcinoma.
A limited number of patients with lung squamous cell carcinoma (SCC) benefit clinically from molecular targeted drugs because of a lack of targetable driver alterations. We aimed to understand the prevalence and clinical significance of lysine-specific demethylase 5D (KDM5D) copy number loss in SCC and explore its potential as a predictive biomarker for ataxia-telangiectasia and Rad3-related (ATR) inhibitor treatment. We evaluated KDM5D copy number loss in 173 surgically resected SCCs from male patients using fluorescence in situ hybridization. KDM5D copy number loss was detected in 75 of the 173 patients (43%). Genome-wide expression profiles of the transcription start sites (TSSs) were obtained from 17 SCCs, for which the cap analysis of gene expression assay was performed, revealing that upregulated genes in tumors with the KDM5D copy number loss are associated with 'cell cycle', whereas downregulated genes in tumors with KDM5D copy number loss were associated with 'immune response'. Clinicopathologically, SCCs with KDM5D copy number loss were associated with late pathological stage (p = 0.0085) and high stromal content (p = 0.0254). Multiplexed fluorescent immunohistochemistry showed that the number of tumor-infiltrating CD8+ /T-bet+ T cells was lower in SCCs with KDM5D copy number loss than in wild-type tumors. In conclusion, approximately 40% of the male patients with SCC exhibited KDM5D copy number loss. Tumors in patients who show this distinct phenotype can be 'cold tumors', which are characterized by the paucity of tumor T-cell infiltration and usually do not respond to immunotherapy. Thus, they may be candidates for trials with ATR inhibitors.


First Report of Stolbur Phytoplasma Infection in Commercial Freesia hybrida Cultivars.
In January 2012, disease symptoms including chlorosis, leaf crinkle, leaf curving and stunting of whole plants, virescence, and curving and necrosis of flower stalks were observed in Freesia hybrida cvs. Evone, Honey Moon, Golden Gem, and Pallas in Icheon and Suwon (Gyeonggi Province in Korea). To determine a possible phytoplasma infection, the symptomatic freesia plants were examined for the presence of phytoplasma 16S rDNA fragment by PCR with the primer pair P1/P6 (2) and R16F1/R16R1 (in nested PCR), which amplifies phytoplasma 16S rDNA regions (4). An expected PCR product of ~1,096 bp was obtained from the symptomatic freesia plants, and they were designated as FreLN, Fre-phy-Ev4, Fre-phy-Ev6, Fre-phy-GG, Fre-phy-HM, and Fre-phy-Pal. The PCR products were sequenced and registered as GenkBank accessions AB695174 and AB709951-55. The sequence corresponding to symptomatic freesia had 98.8 to 99.4% identity with Stolbur phytoplasma strains in the 16S rDNA region, and it had only 95.7 to 96.3% identity with AY phytoplasma strains. In the ultra-thin sections of the leaf midribs, globous phytoplasmal bodies 54 to 214 nm in size were observed in sieve tube elements of phloem tissue. Fre-Phy-Ev6 and Fre-Phy-HM were doube-infected with Stolbur phytoplasma and Freesia mosaic virus (FreMV). Fre-Phy-Ev6 and Fre-Phy-HM revealed necrosis of flower stalks and flower color breaking besides curving of flower stalks. Therefore, flower color breaking and flower stalk necrosis were assumed to be caused by FreMV (1). Symptoms of chlorosis and stunting of whole plants shown in FreLN and virescence of Fre-phy-GG were typical symptoms of phytoplasmal diseases, while leaf crinkle, leaf curving, and curving of flower stalks appeared to be unique symptoms in F. hybrida. Stolbur phytoplasma was abundant in commercial freesia cultivation fields. Some of the cultivars, such as cv. Pallas, showed only curving of leaf and flower stalks without any typical symptom of phytoplasmal diseases. A phytoplasmal disease was reported in Poland in 2001 from F. hybrida exhibiting leaf chlorotic and necrotic spots, and classified as AY I-B based on RFLP analysis of PCR products (3). To our knowledge, this is the first report of Stolbur phytoplasma in F. hybrida. This result is significant because F. hybrida could be the infection source of Stolbur phytoplasma disease in floricultural crops. Interestingly, we found a prevalence of Stolbur phytoplasma in Petunia hybrida cultivars (GenBank Accession Nos. AB713757 to AB713758). High nucleotide sequence identity of 99.8% in the 16S rDNA region of Stolbur phytoplasma isolates from petunia and freesia support the inference that those Stolbur phytoplasma isolates could infect both floricultural crops. References: (1) A. A. Brunt. Freesia. Page 274 in: Virus and virus-like Diseases of Bulb and Flower Crops, John Wiley & Sons, Chichester, 1995. (2) S. Deng and C. Hiruki. J. Microbiol. Methods. 14:53, 1991. (3) M. Kamińska and H. Sliwa. Plant Dis. 85:336, 2001. (4) I. M. Lee et al. Phytopathology 84:559, 1994.