Has the prevalence of health care services use increased over the last decade (2001-2009) in elderly people?

Abstract:
OBJECTIVES (1) To describe the prevalence of general practitioner visits and hospitalization according to sex and age groups; (2) to identify which factors are independently associated with a higher use of health care services among elderly Spanish; and (3) to study the time trends in the prevalence of use of health care services 2001-2009.
STUDY DESIGN Observational study. We analyzed data from the Spanish National Health Surveys conducted in 2001 (n=21,058), 2003 (n=21,650), 2006 (n=29,478) and 2009 (n=22,188). We included responses from adults aged 65 years and older.
OUTCOME MEASURES The main variables were the number of general practitioner visits in the last 4 weeks and hospitalization in the past year. We stratified the adjusted models by the main variables. We analyzed socio-demographic characteristics, health related variables, using multivariate logistic regression models.
RESULTS The total number of subjects was 24,349 (15,041 woman, 9309 men). Women were significantly older than men (P<0.001). Women had higher prevalence of general practitioner visits than men in all surveys. Men had significantly higher prevalence of hospitalizations than women in the years 2001, 2006 and 2009. When we adjusted the hospitalization by possible confounders using logistic regressions, men had a higher probability of being hospitalized than women (OR 1.53, 1.39-1.69). The variables that were significantly associated with a higher use of health care services were lower educational level, worse self-rated health, chronic conditions, polypharmacy, and the level of disability. The number of general practitioner visits among women and men significantly increased from 2001 to 2009 (women: OR 1.43, 1.27-1.61; men: OR 1.71, 1.49-1.97).
CONCLUSION The current study revealed an increase in health care services utilization from 2001 to 2009 in the older Spanish population.


Is endothelin-1 an aggravating factor in the development of acute pancreatitis?

Abstract:
OBJECTIVE We have reported previously that cerulein-induced edematous pancreatitis would transform into hemorrhagic pancreatitis by administration of endothelin-1 in rats. In the present study, we tried to protect rat model from developing into hemorrhagic pancreatitis with BQ123 (an ETA receptor antagonist).
METHODS The rat model was made by 5-hour restraint water-immersion stress and two intraperitoneal injections of cerulein (40 micrograms/kg) at hourly interval. BQ123 (3 or 6 mg/kg) was administered intravenously 30 minutes before and 2 hours after the first cerulein injection.
RESULTS Acute hemorrhagic pancreatitis was induced in all rats treated with cerulin + stress. The score for pancreatic hemorrhage was 2.4 +/- 0.2 in this group. In the rats pretreated with BQ123, the score was reduced to 1.0 +/- 0.0, pancreas wet weight and serum amylase activity were significantly reduced, and histologic alterations in the pancreas lightened, also the local pancreatic blood flow improved without affecting the systemic blood pressure.
CONCLUSION These results suggest that endothelin-1 should play a role in aggravating the development of acute hemorrhagic pancreatitis, through its action on the pancreatic microcirculation.


Malnutrition, a new inducer for arterial calcification in hemodialysis patients?

Abstract:
BACKGROUND Arterial calcification is a significant cardiovascular risk factor in hemodialysis patients. A series of factors are involved in the process of arterial calcification; however, the relationship between malnutrition and arterial calcification is still unclear.
METHODS 68 hemodialysis patients were enrolled in this study. Nutrition status was evaluated using modified quantitative subjective global assessment (MQSGA). Related serum biochemical parameters were measured. And the radial artery samples were collected during the arteriovenous fistula surgeries. Hematoxylin/eosin stain was used to observe the arterial structures while Alizarin red stain to observe calcified depositions and classify calcified degree. The expressions of bone morphogenetic protein 2 (BMP2) and matrix Gla protein (MGP) were detected by immunohistochemistry and western blot methods.
RESULTS 66.18% hemodialysis patients were malnutrition. In hemodialysis patients, the calcified depositions were mainly located in the medial layer of the radial arteries and the expressions of BMP2 and MGP were both increased in the calcified areas. The levels of serum albumin were negatively associated with calcification score and the expressions of BMP2 and MGP. While MQSGA score, serum phosphorus and calcium × phosphorus product showed positive relationships with calcification score and the expressions of BMP2 and MGP.
CONCLUSION Malnutrition is prevalent in hemodialysis patients and is associated with arterial calcification and the expressions of BMP2 and MGP in calcified radial arteries. Malnutrition may be a new inducer candidate for arterial calcification in hemodialysis patients.


Does glomerular hyperfiltration in pregnancy damage the kidney in women with more parities?

Abstract:
OBJECTIVES We aimed to investigate the glomerular hyperfiltration due to pregnancy in women with more parities.
METHODS Five hundred women aged 52.57 +/- 8.08 years, without a history of hypertension, diabetes mellitus or complicated pregnancy were involved in the study. They were divided into three groups. Group 1: women with no or one parity (n = 76); group 2: women with two or three parities (n = 333); group 3: women with four or more parities (n = 91). Laboratory parameters and demographical data were compared between the three groups.
RESULTS Mean age, serum urea and serum creatinine were similar between three groups. Patients in group 3 had significantly higher GFR values compared to groups 1 and 2 (109.44 +/- 30.99, 110.76 +/- 30.22 and 121.92 +/- 34.73 mL/min/1.73 m(2) for groups 1, 2 and 3, respectively; P = 0.008 for group 1 vs group 3; P = 0.002 for group 2 vs group 3).
CONCLUSION In our study, we suggest that glomerular hyperfiltration due to pregnancy does not have adverse effects on kidney in women with more parities. Pregnancy may have possible protective mechanisms for kidney against adverse effects of glomerular hyperfiltration.


Subclavian steal syndrome: can the blood pressure difference between arms predict the severity of steal?

Abstract:
BACKGROUND AND PURPOSE A side-to-side difference in systolic brachial arterial blood pressure is a common finding in subclavian artery stenosis and is frequently used as a screening tool for subclavian steal syndrome (SSS). It was the goal of this retrospective study to investigate the relationship between different vertebral artery waveform types and the side-to-side difference in systolic blood pressure in patients with sonographically proven SSS.
METHODS The records of 1860 patients from the Neuroultrasound Laboratory between January 2000 and December 2000 were screened for the diagnosis of SSS in the final ultrasound report. In all patients, bilateral brachial arterial blood pressure was measured in a sitting position prior to the ultrasound examination. Vertebral artery waveforms were classified as (1) systolic deceleration, (2) alternating flow, and (3) complete reversal at rest. Blood pressure difference as calculated by normal-side blood pressure minus lesion-side blood pressure was compared with the 3 Doppler waveform types.
RESULTS SSS was found in 51 of 1860 (2.7%) ultrasonography studies of 49 patients (17 men, 32 women; mean age 65.3 +/- 10.5 years). Two patients (4%) had bilateral SSS. In 3 patients (6%), SSS was related to an innominate artery stenosis. Waveform analysis showed a completely reversed flow in 16 (31%), an alternating flow in 24 (47%), and a systolic deceleration in 11 (22%) cases. Systolic blood pressure difference was significantly higher in the complete reversal and alternating groups than in the systolic deceleration group (P<.001).
CONCLUSION Brachial systolic blood pressure difference is related to the severity of SSS and can be used as a screening tool for SSS. However, it performed better in severe steal than milder steal phenomena.


Is eligibility for a chemotherapy protocol a good prognostic factor for invasive bladder cancer after radical cystectomy?

Abstract:
PURPOSE To assess whether eligibility to an adjuvant chemotherapy protocol in itself represents a good prognostic factor after radical cystectomy for bladder cancer.
PATIENTS AND METHODS Between April 1984 and May 1989, our institution entered 35 patients with invasive bladder cancer into the Swiss Group for Clinical and Epidemiological Cancer Research (SAKK) study 09/84. They were randomly assigned to either observation or three postoperative courses of cisplatin monotherapy after cystectomy. This study had a negative result. The outcome of these 35 patients (protocol group) was compared with an age- and tumor-stage-matched cohort (matched group; n = 35) who also underwent cystectomy during the same period, but were not entered into the SAKK study, as well as the remaining 57 patients treated during the study period for the same indication (remaining group).
RESULTS Median overall survival decreased from 76.3 months in the protocol group to 52.1 months in the matched group and to 20.3 months in the remaining group. The respective times of median recurrence-free survival were 67.2, 16.0, and 9.4 months. Tumor progression occurred in 46% of the protocol group compared with 69% in the matched group and 65% in the remaining group (P<.05). Cancer-related death was noted in 40% of the protocol group, 57% in the matched group, and 56% in the remaining group.
CONCLUSION These data suggest that being willing and fit enough for a chemotherapy protocol is a good prognostic factor for invasive bladder cancer. This eligibility bias emphasizes the need for prospective, randomized trials, and indicates that single-group studies using historical or matched controls have to be interpreted with caution.


Does a geriatric oncology consultation modify the cancer treatment plan for elderly patients?

Abstract:
BACKGROUND This study was performed to describe the treatment plan modifications after a geriatric oncology clinic. Assessment of health and functional status and cancer assessment was performed in older cancer patients referred to a cancer center.
PATIENTS AND METHODS Between June 2004 and May 2005, 105 patients 70 years old or older referred to a geriatric oncology consultation at the Institut Curie cancer center were included. Functional status, nutritional status, mood, mobility, comorbidity, medication, social support, and place of residence were assessed. Oncology data and treatment decisions were recorded before and after this consultation. Data were analyzed for a possible correlation between one domain of the assessment and modification of the treatment plan.
RESULTS Patient characteristics included a median age of 79 years and a predominance of women with breast cancer. About one half of patients had an independent functional status. Nearly 15% presented severe undernourishment. Depression was suspected in 53.1% of cases. One third of these patients had>2 chronic diseases, and 74% of patients took>or =3 medications. Of the 93 patients with an initial treatment decision, the treatment plan was modified for 38.7% of cases after this assessment. Only body mass index and the absence of depressive symptoms were associated with a modification of the treatment plan.
CONCLUSION The geriatric oncology consultation led to a modification of the cancer treatment plan in more than one third of cases. Further studies are needed to determine whether these modifications improve the outcome of these older patients.


Does left atrial appendage (LAA) occlusion device alter the echocardiography and electrocardiogram parameters in patients with atrial fibrillation?

Abstract:
BACKGROUND The alterations of echocardiography and electrocardiogram (ECG) in patients received left atrial appendage LAA occlusion therapy are still unclear. The present study was to evaluate the influence of LAA occlusion device on echocardiography and ECG changes in patients with atrial fibrillation (AF).
METHODS Seventy-three patients who had undergone Watchman, LAmbre and Lefort were enrolled in this study. Echocardiography and ECG results at pre- and post-operation were collected. Besides, echocardiography was also performed during follow-up visits at 1, 6 and 12months after discharge.
RESULTS After LAA occlusion, a slight and measureable movement of QRS electric axis was observed in most patients. The significant differences were also observed in heart rate (HR) and the mean-mean QT interval between pre- and post-operation for all patients. There existed no significant difference in echocardiographic parameters between before and after device implantation. However, a larger left atrial (LA) diameter was detected by echocardiography during follow-up visit at 6months when compared with pre-operation parameters. Similarly, aortic root diameter (ARD) was also larger during follow-up at 12months than the baseline dimension in pre-operation.
CONCLUSION LAA occlusion device resulted in a slightly movement in QRS axis, reduced HR and increased the mean-mean QT interval duration. In addition, LA diameter and ARD seemed to be larger after device implantation.


Can ki-67 play a role in prediction of breast cancer patients' response to neoadjuvant chemotherapy?

Abstract:
BACKGROUND Currently the choice of breast cancer therapy is based on prognostic factors. The proliferation marker Ki-67 is used increasingly to determine the method of therapy. The current study analyses the predictive value of Ki-67 in foreseeing breast cancer patients' responses to neoadjuvant chemotherapy.
METHODS This study includes patients with invasive breast cancer treated between 2008 and 2013. The clinical response was assessed by correlating Ki-67 to histological examination, mammography, and ultrasonography findings.
RESULTS The average Ki-67 value in our patients collectively (n = 77) is 34.9 ± 24.6%. The average Ki-67 value is the highest with 37.4 ± 24.0% in patients with a pCR. The Ki-67 values do not differ significantly among the 3 groups: pCR versus partial pathological response versus stable disease/progress (P = 0.896). However, Ki-67 values of patients with luminal, Her2 enriched, and basal-like cancers differed significantly from each other. Furthermore, within the group of luminal tumors Ki-67 values of patients with versus without pCR also differed significantly.
CONCLUSION Our data shows that the Ki-67 value predicts the response to neoadjuvant chemotherapy as a function of the molecular subtype, reflecting the daily routine concerning Ki-67 and its impressing potential and limitation as a predictive marker for neoadjuvant chemotherapy response.


Intravenous administration of metoclopramide by 2 min bolus vs 15 min infusion: does it affect the improvement of headache while reducing the side effects?

Abstract:
OBJECTIVE To determine the therapeutic effect (alleviation of vascular type headache) and side effects of a slow intravenous metoclopramide infusion over 15 min compared with those effects of a bolus intravenous metoclopramide infusion over 2 min in the treatment of patients with recent onset vascular type headache.
MATERIAL AND METHODS All adults treated with metoclopramide for vascular type headache were eligible for entry into this clinical randomised double blinded trial. This study compared the effects of two different rates of intravenous infusion of metoclopramide over a period of 13 months at a university hospital emergency department. During the trial, side effects and headache scores were recorded at baseline (0 min), and then at 5, 15, 30 and 60 min. Repeated measures analysis of variance was used to compare the medication's efficacy and side effects.
RESULTS A total of 120 patients presenting to the emergency department met the inclusion criteria. Of these, 62 patients (51.7%) were given 10 mg metoclopramide as a slow intravenous infusion over 15 min (SIG group) and 58 patients (48.3%) were given 10 mg metoclopramide intravenous bolus infusion over 2 min (BIG group). 17 of the 58 patients in the BIG group (29.3%) and 4 of the 62 patients (6.5%) in the SIG group had akathisia (p = 0.001). There were no significant differences between the BIG and SIG groups in terms of mean headache scores (p = 0.34) and no adverse reactions in the study period. Metoclopramide successfully relieved the headache symptom(s) of patients in both the BIG and SIG groups.
CONCLUSION Slowing the infusion rate of metoclopramide is an effective strategy for the improvement of headache and reducing the incidence of akathisia in patients with vascular type headache.


Is the breast best for children with a family history of atopy?

Abstract:
BACKGROUND Previous studies reported that breast-feeding protects children against a variety of diseases, but these studies were generally conducted on "high-risk" or hospitalized children. This paper describes the results of our study on the effects of breast-feeding on rate of illness in normal children with a family history of atopy.
METHODS A historic cohort approach of 794 children with a family history of atopy was used to assess the effects of breast-feeding on illness rates. Family history of atopy was based on allergic diseases in family members as registered by the family physician. Illness data from birth onwards were available from the Continuous Morbidity Registration of the Department of Family Medicine. Information on breast-feeding was collected by postal questionnaire. We then compared rates of illness between children with a family history of atopy who were and who were not breast-fed.
RESULTS Breast-feeding was related to lower levels of childhood illness both in the first and the first three years of life. In the first year of life they had fewer episodes of gastroenteritis, lower respiratory tract infections, and digestive tract disorders. Over the next three years of life they had fewer respiratory tract infections and skin infections.
CONCLUSION Our results suggest a protective effect of breast-feeding among children with a family history of atopy that is not confined to the period of breast-feeding but continues during the first three years of life. Breast-feeding should be promoted in children with a family history of atopy.


Can patient coaching reduce racial/ethnic disparities in cancer pain control?

Abstract:
PURPOSE Minority patients with cancer experience worse control of their pain than do their white counterparts. This disparity may, in part, reflect more miscommunication between minority patients and their physicians. Therefore, we examined whether patient coaching could reduce disparities in pain control in a secondary analysis of a randomized controlled trial.
METHODS Sixty-seven English-speaking adult cancer outpatients, including 15 minorities, with moderate pain over the prior 2 weeks were randomly assigned to the experimental (N = 34) or control group (N = 33). Experimental patients received a 20-minute individualized education and coaching session to increase knowledge of pain self-management, to redress personal misconceptions about pain treatment, and to rehearse an individually scripted patient-physician dialog about pain control. The control group received standardized information on controlling pain. Data on average pain (0-10 scale) were collected at enrollment and 2-week follow-up.
RESULTS At enrollment, minority patients had significantly more pain than their white counterparts (6.0 vs 5.0, P = 0.05). At follow-up, minorities in the control group continued to have more pain (6.4 vs 4.7, P = 0.01), whereas in the experimental group, disparities were eliminated (4.0 vs 4.3, P = 0.71). The effect of the intervention on reducing disparities was significant (P = 0.04).
CONCLUSION Patient coaching offers promise as a means of reducing racial/ethnic disparities in pain control. Larger studies are needed to validate these findings and to explore possible mechanisms.


Does prior benign prostate biopsy predict outcome for patients treated with radical perineal prostatectomy?

Abstract:
OBJECTIVES To determine the effect of prior benign prostate biopsies on the surgical and clinical outcomes of patients treated with radical perineal prostatectomy for prostate cancer.
METHODS A total of 1369 patients with clinically localized prostate cancer underwent radical prostatectomy by a single surgeon between 1991 and 2001. A subset of 203 patients (14.9%), who had undergone at least one prior benign prostate biopsy for a rising prostate-specific antigen and/or abnormal digital rectal examination, constituted our study population. A total of 1115 patients with no prior biopsy represented our control group. After prostatectomy, patients were evaluated at 6-month intervals for biochemical evidence of recurrence, defined as a prostate-specific antigen level of 0.5 ng/mL or greater.
RESULTS Patients with a prior benign biopsy had more favorable pathologic features with more organ-confined (74% versus 64%; P<0.001) and less margin-positive (9.8% versus 18%) disease. Only 24 patients (12%) in the study group (versus 20% in control group; P = 0.01) had eventual evidence of biochemical failure. Kaplan-Meier analyses suggested that patients with prior benign biopsies have improved biochemical disease-free survival, especially for those with more aggressive disease (Gleason sum 7 or greater; P<0.01). Overall, patients in the study group had lower probability (odds ratio 0.57, P<0.001) of biochemical failure compared with those in the control group.
CONCLUSION A prior benign prostate biopsy may be independently associated with more favorable surgical and biochemical outcomes after prostatectomy. Additional studies are needed to confirm these findings.


Does patient position during liver surgery influence the risk of venous air embolism?

Abstract:
BACKGROUND It is generally believed that positioning of the patient in a head-down tilt (Trendelenberg position) decreases the likelihood of a venous air embolism during liver resection.
METHODS The physiological effect of variation in horizontal attitude on central and hepatic venous pressure was measured in 10 patients during liver surgery. Hemodynamic indices were recorded with the operating table in the horizontal, 20 degrees head-up and 20 degrees head-down positions.
RESULTS There was no demonstrable pressure gradient between the hepatic and central venous levels in any of the positions. The absolute pressures did, however, vary in a predictable way, being highest in the head-down and lowest during head-up tilt. However, on no occasion was a negative intraluminal pressure recorded.
CONCLUSION The effect on venous pressures caused by the change in patient positioning alone during liver surgery does not affect the risk of venous air embolism.


Do Indigenous Australians age prematurely?

Abstract:
OBJECTIVE To assess whether Indigenous Australians age prematurely compared with other Australians, as implied by Australian Government aged care policy, which uses age 50 years and over for population-based planning for Indigenous people compared with 70 years for non-indigenous people.
METHODS Cross-sectional analysis of aged care assessment, hospital and health survey data comparing Indigenous and non-indigenous age-specific prevalence of health conditions. Analysis of life tables for Indigenous and non-indigenous populations comparing life expectancy at different ages.
RESULTS At age 63 for women and age 65 for men, Indigenous people had the same life expectancy as non-indigenous people at age 70. There is no consistent pattern of a 20-year lead in age-specific prevalence of age-associated conditions for Indigenous compared with other Australians. There is high prevalence from middle-age onwards of some conditions, particularly diabetes (type unspecified), but there is little or no lead for others.
CONCLUSION The idea that Indigenous people age prematurely is not well supported by this study of a series of discrete conditions. The current focus and type of services provided by the aged care sector may not be the best way to respond to the excessive burden of chronic disease and disability of middle-aged Indigenous people.


Do oblique views add value in the diagnosis of spondylolysis in adolescents?

Abstract:
BACKGROUND Anteroposterior, lateral, and right and left oblique lumbar spine radiographs are often a standard part of the evaluation of children who are clinically suspected of having spondylolysis. Recent concerns regarding radiation exposure and costs have brought the value of oblique radiographs into question. The purpose of the present study was to determine the diagnostic value of oblique views in the diagnosis of spondylolysis.
METHODS Radiographs of fifty adolescents with L5 spondylolysis without spondylolisthesis and fifty controls were retrospectively reviewed. All controls were confirmed not to have spondylolysis on the basis of computed tomographic scanning, magnetic resonance imaging, or bone scanning. Anteroposterior, lateral, and right and left oblique radiographs of the lumbar spine were arranged into two sets of slides: one showing four views (anteroposterior, lateral, right oblique, and left oblique) and one showing two views (anteroposterior and lateral only). The slides were randomly presented to four pediatric spine surgeons for diagnosis, with four-view slides being presented first, followed by two-view slides. The slides for twenty random patients were later reanalyzed in order to calculate of intra-rater agreement. A power analysis demonstrated that this study was adequately powered. Inter-rater and intra-rater agreement were assessed on the basis of the percentage of overall agreement and intraclass correlation coefficients (ICCs). PCXMC software was used to generate effective radiation doses. Study charges were determined from radiology billing data.
RESULTS There was no significant difference in sensitivity and specificity between four-view and two-view radiographs in the diagnosis of spondylolysis. The sensitivity was 0.59 for two-view studies and 0.53 for four-view studies (p = 0.33). The specificity was 0.96 for two-view studies and 0.94 for four-view studies (p = 0.60). Inter-rater agreement, intra-rater agreement, and agreement with gold-standard ICC values were in the moderate range and also demonstrated no significant differences. Percent overall agreement was 78% for four-view studies and 82% for two-view studies. The radiation effective dose was 1.26 mSv for four-view studies and 0.72 mSv for two-view studies (difference, 0.54 mSv). The charge for four-view studies was $145 more than that for two-view studies.
CONCLUSION There is no difference in sensitivity and specificity between four-view and two-view studies. Although oblique views have long been considered standard practice by some, our data could not identify a diagnostic benefit that might outweigh the additional cost and radiation exposure.


Are many colorectal cancers due to missed adenomas?

Abstract:
BACKGROUND An unknown number of colorectal cancers could be due to missed adenomas during previous endoscopy. Data in the literature are sparse. A large cross-sectional study was done in a prospective database of all patients diagnosed with colorectal cancer.
METHODS All consecutive endoscopies over a period of 15 years, in which colorectal cancer was diagnosed were included. All patients who underwent more than one endoscopy and in whom ultimately cancer was diagnosed were studied separately.
RESULTS Colorectal cancer was diagnosed in 835 patients. Twenty-five patients underwent a previous endoscopy without a cancer diagnosis. These 25 patients were divided into three groups according to the time between the endoscopy in which the cancer was detected and the previous endoscopy. Five out of these 25 patients underwent regular surveillance. Only 11 patients had no argument for regular follow-up. Assuming that these cancers developed from an adenoma than only 11 out of 835 (1.3%) cancers were missed in the adenoma phase. There was no difference in the size of the tumour between the three groups of patients.
CONCLUSION In normal daily practice, only a small number of clinically important adenomas are missed. The problem of missed adenomas probably is being exaggerated.


Is there any interest to perform ultrasonography in boys with undescended testis?

Abstract:
OBJECTIVE To evaluate the accuracy of ultrasonographic examination in boys with an undescended testis.
MATERIAL AND METHODS All patients who were referred to the paediatric surgeon after detection of an undescended testis were evaluated prospectively between November 2001 and November 2004. Among these 377 patients, 87 were referred with an ultrasonogram previously prescribed by the referring primary physician. The results of the ultrasonogram were compared to the results of the clinical examination of the paediatric surgeon and, in cases of no palpable testis, to the surgical findings.
RESULTS Ultrasonography did not detect the retractile testes. Ultrasonography detected 67% of the palpable undescended testes. In cases of no palpable testis, the ultrasonographic examination missed the abdominal testes and sometimes other structures were falsely interpreted as a testis.
CONCLUSION Sonography has no place in the diagnosis of undescended testis.


Are sugars-free medicines more erosive than sugars-containing medicines?

Abstract:
OBJECTIVE The reduced use of sugars-containing (SC) liquid medicines has increased the use of other dose forms, potentially resulting in more widespread dental effects, including tooth wear. The aim of this study was to assess the erosive potential of 97 paediatric medicines in vitro.
METHODS The study took the form of in vitro measurement of endogenous pH and titratable acidity (mmol). Endogenous pH was measured using a pH meter, followed by titration to pH 7.0 with 0.1-M NaOH.
RESULTS Overall, 55 (57%) formulations had an endogenous pH of<5.5. The mean (+/- SD) endogenous pH and titratable acidity for 41 SC formulations were 5.26 +/- 1.30 and 0.139 +/- 0.133 mmol, respectively; for 56 sugars-free (SF) formulations, these figures were 5.73 +/- 1.53 and 0.413 +/- 1.50 mmol (P>0.05). Compared with their SC bioequivalents, eight SF medicines showed no significant differences for pH or titratable acidity, while 15 higher-strength medicines showed lower pH (P = 0.035) and greater titratable acidity (P = 0.016) than their lower-strength equivalents. Chewable and dispersible tablets (P<0.001), gastrointestinal medicines (P = 0.002) and antibiotics (P = 0.007) were significant predictors of higher pH. In contrast, effervescent tablets (P<0.001), and nutrition and blood preparations (P = 0.021) were significant predictors of higher titratable acidity.
CONCLUSION Paediatric SF medicines were not more erosive than SC medicines in vitro; a more significant predictor of their erosive potential was dose form.


Microstructural development of human newborn cerebral white matter assessed in vivo by diffusion tensor magnetic resonance imaging.

Abstract: Alterations of the architecture of cerebral white matter in the developing human brain can affect cortical development and result in functional disabilities. A line scan diffusion-weighted magnetic resonance imaging (MRI) sequence with diffusion tensor analysis was applied to measure the apparent diffusion coefficient, to calculate relative anisotropy, and to delineate three-dimensional fiber architecture in cerebral white matter in preterm (n = 17) and full-term infants (n = 7). To assess effects of prematurity on cerebral white matter development, early gestation preterm infants (n = 10) were studied a second time at term. In the central white matter the mean apparent diffusion coefficient at 28 wk was high, 1.8 microm2/ms, and decreased toward term to 1.2 microm2/ms. In the posterior limb of the internal capsule, the mean apparent diffusion coefficients at both times were similar (1.2 versus 1.1 microm2/ms). Relative anisotropy was higher the closer birth was to term with greater absolute values in the internal capsule than in the central white matter. Preterm infants at term showed higher mean diffusion coefficients in the central white matter (1.4 +/- 0.24 versus 1.15 +/- 0.09 microm2/ms, p = 0.016) and lower relative anisotropy in both areas compared with full-term infants (white matter, 10.9 +/- 0.6 versus 22.9 +/- 3.0%, p = 0.001; internal capsule, 24.0 +/- 4.44 versus 33.1 +/- 0.6% p = 0.006). Nonmyelinated fibers in the corpus callosum were visible by diffusion tensor MRI as early as 28 wk; full-term and preterm infants at term showed marked differences in white matter fiber organization. The data indicate that quantitative assessment of water diffusion by diffusion tensor MRI provides insight into microstructural development in cerebral white matter in living infants.


Induction of myelodysplasia by myeloid-derived suppressor cells.

Abstract: Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.


BC1 RNA, the transcript from a master gene for ID element amplification, is able to prime its own reverse transcription.

Abstract: ID elements are short interspersed elements (SINEs) found in high copy number in many rodent genomes. BC1 RNA, an ID-related transcript, is derived from the single copy BC1 RNA gene. The BC1 RNA gene has been shown to be a master gene for ID element amplification in rodent genomes. ID elements are dispersed through a process termed retroposition. The retroposition process involves a number of potential regulatory steps. These regulatory steps may include transcription in the appropriate tissue, transcript stability, priming of the RNA transcript for reverse transcription and integration. This study focuses on priming of the RNA transcript for reverse transcription. BC1 RNA gene transcripts are shown to be able to prime their own reverse transcription in an efficient intramolecular and site-specific fashion. This self-priming ability is a consequence of the secondary structure of the 3'-unique region. The observation that a gene actively amplified throughout rodent evolution makes a RNA capable of efficient self-primed reverse transcription strongly suggests that self-priming is at least one feature establishing the BC1 RNA gene as a master gene for amplification of ID elements.


The DNA Methylome of Human Peripheral Blood Mononuclear Cells

Abstract: DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.


The human myelin basic protein gene is included within a 179-kilobase transcription unit: expression in the immune and central nervous systems.

Abstract: Two human Golli (for gene expressed in the oligodendrocyte lineage)-MBP (for myelin basic protein) cDNAs have been isolated from a human oligodendroglioma cell line. Analysis of these cDNAs has enabled us to determine the entire structure of the human Golli-MBP gene. The Golli-MBP gene, which encompasses the MBP transcription unit, is approximately 179 kb in length and consists of 10 exons, seven of which constitute the MBP gene. The human Golli-MBP gene contains two transcription start sites, each of which gives rise to a family of alternatively spliced transcripts. At least two Golli-MBP transcripts, containing the first three exons of the gene and one or more MBP exons, are produced from the first transcription start site. The second family of transcripts contains only MBP exons and produces the well-known MBPs. In humans, RNA blot analysis revealed that Golli-MBP transcripts were expressed in fetal thymus, spleen, and human B-cell and macrophage cell lines, as well as in fetal spinal cord. These findings clearly link the expression of exons encoding the autoimmunogen/encephalitogen MBP in the central nervous system to cells and tissues of the immune system through normal expression of the Golli-MBP gene. They also establish that this genetic locus, which includes the MBP gene, is conserved among species, providing further evidence that the MBP transcription unit is an integral part of the Golli transcription unit and suggest that this structural arrangement is important for the genetic function and/or regulation of these genes.


Targeting A20 Decreases Glioma Stem Cell Survival and Tumor Growth

Abstract: Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.


Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases

Abstract: Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type–specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)–mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.


Empirical Bayesian models for analysing molecular serotyping microarrays

Abstract:
BACKGROUND Microarrays offer great potential as a platform for molecular diagnostics, testing clinical samples for the presence of numerous biomarkers in highly multiplexed assays. In this study applied to infectious diseases, data from a microarray designed for molecular serotyping of Streptococcus pneumoniae was used, identifying the presence of any one of 91 known pneumococcal serotypes from DNA extracts. This microarray incorporated oligonucleotide probes for all known capsular polysaccharide synthesis genes and required a statistical analysis of the microarray intensity data to determine which serotype, or combination of serotypes, were present within a sample based on the combination of genes detected.
 RESULTS We propose an empirical Bayesian model for calculating the probabilities of combinations of serotypes from the microarray data. The model takes into consideration the dependencies between serotypes, induced by genes they have in common, and by homologous genes which, although not identical, are similar to each other in sequence. For serotypes which are very similar in capsular gene composition, extra probes are included on the microarray, providing additional information which is integrated into the Bayesian model. For each serotype combination with high probability, a second model, a Bayesian random effects model is applied to determine the relative abundance of each serotype.
 CONCLUSIONS To assess the accuracy of the proposed analysis we applied our methods to experimental data from samples containing individual serotypes and samples containing combinations of serotypes with known levels of abundance. All but two of the known serotypes of S. pneumoniae that were tested as individual samples could be uniquely determined by the Bayesian model. The model also enabled the presence of combinations of serotypes within samples to be determined. Serotypes with very low abundance within a combination of serotypes can be detected (down to 2% abundance in this study). As well as detecting the presence of serotype combinations, an approximate measure of the percentage abundance of the serotypes within the combination can be obtained.


Loss of immune escape mutations during persistent HCV infection in pregnancy enhances replication of vertically transmitted viruses

Abstract: Globally, about 1% of pregnant women are persistently infected with the hepatitis C virus (HCV). Mother-to-child transmission of HCV occurs in 3-5% of pregnancies and accounts for most new childhood infections. HCV-specific CD8(+) cytotoxic T lymphocytes (CTLs) are vital in the clearance of acute HCV infections, but in the 60-80% of infections that persist, these cells become functionally exhausted or select for mutant viruses that escape T cell recognition. Increased HCV replication during pregnancy suggests that maternofetal immune tolerance mechanisms may further impair HCV-specific CTLs, limiting their selective pressure on persistent viruses. To assess this possibility, we characterized circulating viral quasispecies during and after consecutive pregnancies in two women. This revealed a loss of some escape mutations in HLA class I epitopes during pregnancy that was associated with emergence of more fit viruses. CTL selective pressure was reimposed after childbirth, at which point escape mutations in these epitopes again predominated in the quasispecies and viral load dropped sharply. Importantly, the viruses transmitted perinatally were those with enhanced fitness due to reversion of escape mutations. Our findings indicate that the immunoregulatory changes of pregnancy reduce CTL selective pressure on HCV class I epitopes, thereby facilitating vertical transmission of viruses with optimized replicative fitness.


Acute administration of recombinant Angiopoietin-1 ameliorates multiple-organ dysfunction syndrome and improves survival in murine sepsis.

Abstract:
INTRODUCTION Endothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis.
 METHODS Polymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1 μg of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8h thereafter. Sham-operated animals served as time-matched controls.
 RESULTS Compared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model.
 CONCLUSIONS In a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept.


CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2.

Abstract: It is thought that the H19 imprinting control region (ICR) directs the silencing of the maternally inherited Igf2 allele through a CTCF-dependent chromatin insulator. The ICR has been shown to interact physically with a silencer region in Igf2, differentially methylated region (DMR)1, but the role of CTCF in this chromatin loop and whether it restricts the physical access of distal enhancers to Igf2 is not known. We performed systematic chromosome conformation capture analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF binding sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF target site within Igf2 DMR1, showing that CTCF can coordinate regional epigenetic marks. This systematic chromosome conformation capture analysis of an imprinting cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome.


De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation.

Abstract: Telomerase-negative tumor cells use an alternative lengthening of telomeres (ALT) pathway that involves DNA recombination and repair to maintain their proliferative potential. The cytological hallmark of this process is the accumulation of promyelocytic leukemia (PML) nuclear protein at telomeric DNA to form ALT-associated PML bodies (APBs). Here, the de novo formation of a telomeric PML nuclear subcompartment was investigated by recruiting APB protein components. We show that functionally distinct proteins were able to initiate the formation of bona fide APBs with high efficiency in a self-organizing and self-propagating manner. These included: (1) PML and Sp100 as the constituting components of PML nuclear bodies, (2) telomere repeat binding factors 1 and 2 (TRF1 and TRF2, respectively), (3) the DNA repair protein NBS1 and (4) the SUMO E3 ligase MMS21, as well as the isolated SUMO1 domain, through an interacting domain of another protein factor. By contrast, the repair factors Rad9, Rad17 and Rad51 were less efficient in APB nucleation but were recruited to preassembled APBs. The artificially created APBs induced telomeric extension through a DNA repair mechanism, as inferred from their colocalization with sites of non-replicative DNA synthesis and histone H2A.X phosphorylation, and an increase of the telomere repeat length. These activities were absent after recruitment of the APB factors to a pericentric locus and establish APBs as functional intermediates of the ALT pathway.


Effects of an opal termination codon preceding the nsP4 gene sequence in the O'Nyong-Nyong virus genome on Anopheles gambiae infectivity.

Abstract: The genomic RNA of an alphavirus encodes four different nonstructural proteins, nsP1, nsP2, nsP3, and nsP4. The polyprotein P123 is produced when translation terminates at an opal termination codon between nsP3 and nsP4. The polyprotein P1234 is produced when translational readthrough occurs or when the opal termination codon has been replaced by a sense codon in the alphavirus genome. Evolutionary pressures appear to have maintained genomic sequences encoding both a stop codon (opal) and an open reading frame (arginine) as a general feature of the O'nyong-nyong virus (ONNV) genome, indicating that both are required at some point. Alternate replication of ONNVs in both vertebrate and invertebrate hosts may determine predominance of a particular codon at this locus in the viral quasispecies. However, no systematic study has previously tested this hypothesis in whole animals. We report here the results of the first study to investigate in a natural mosquito host the functional significance of the opal stop codon in an alphavirus genome. We used a full-length cDNA clone of ONNV to construct a series of mutants in which the arginine between nsP3 and nsP4 was replaced with an opal, ochre, or amber stop codon. The presence of an opal stop codon upstream of nsP4 nearly doubled (75.5%) the infectivity of ONNV over that of virus possessing a codon for the amino acid arginine at the corresponding position (39.8%). Although the frequency with which the opal virus disseminated from the mosquito midgut did not differ significantly from that of the arginine virus on days 8 and 10, dissemination did began earlier in mosquitoes infected with the opal virus. Although a clear fitness advantage is provided to ONNV by the presence of an opal codon between nsP3 and nsP4 in Anopheles gambiae, sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated codon usage at this position corresponded with that of the virus administered in the blood meal. These results suggest that while selection of ONNV variants is occurring, de novo mutation at the position between nsP3 and nsP4 does not readily occur in the mosquito. Taken together, these results suggest that the primary fitness advantage provided to ONNV by the presence of an opal codon between nsP3 and nsP4 is related to mosquito infectivity.


Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

Abstract: Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.


Fra-1 protooncogene regulates IL-6 expression in macrophages and promotes the generation of M2d macrophages

Abstract: The tumor microenvironment (TME) plays a prominent role in the growth of tumor cells. As the major inflammatory component of the TME, M2d macrophages are educated by the TME such that they adopt an immunosuppressive role that promotes tumor metastasis and progression. Fra-1 forms activator protein-1 heterodimers with Jun partners and drives gene transcription. Fra-1 is thought to drastically induce tumorigenesis and progression. However, the functional role of Fra-1 in the generation of M2d macrophages is poorly understood to date. Here, we demonstrate that 4T1 mammary carcinoma cells, when co-cultured with RAW264.7 macrophage cells, skew the RAW264.7 macrophage cell differentiation into M2d macrophages. The 4T1 cells stimulate de novo overexpression of Fra-1 in RAW264.7 cells, and then Fra-1 binds to the interleukin 6 (IL-6) promoter to increase the production of the cytokine IL-6 in RAW264.7 cells. IL-6 acts in an autocrine fashion to skew RAW264.7 macrophage cell differentiation into M2d macrophages. These findings open new insights into how to reverse M2d macrophage-induced immune tolerance to improve the efficacy of immunotherapeutic approaches.


Analysis of SiO2 nanoparticles binding proteins in rat blood and brain homogenate

Abstract: A multitude of nanoparticles, such as titanium oxide (TiO2), zinc oxide, aluminum oxide, gold oxide, silver oxide, iron oxide, and silica oxide, are found in many chemical, cosmetic, pharmaceutical, and electronic products. Recently, SiO2 nanoparticles were shown to have an inert toxicity profile and no association with an irreversible toxicological change in animal models. Hence, exposure to SiO2 nanoparticles is on the increase. SiO2 nanoparticles are routinely used in numerous materials, from strengthening filler for concrete and other construction composites, to nontoxic platforms for biomedical application, such as drug delivery and theragnostics. On the other hand, recent in vitro experiments indicated that SiO2 nanoparticles were cytotoxic. Therefore, we investigated these nanoparticles to identify potentially toxic pathways by analyzing the adsorbed protein corona on the surface of SiO2 nanoparticles in the blood and brain of the rat. Four types of SiO2 nanoparticles were chosen for investigation, and the protein corona of each type was analyzed using liquid chromatography-tandem mass spectrometry technology. In total, 115 and 48 plasma proteins from the rat were identified as being bound to negatively charged 20 nm and 100 nm SiO2 nanoparticles, respectively, and 50 and 36 proteins were found for 20 nm and 100 nm arginine-coated SiO2 nanoparticles, respectively. Higher numbers of proteins were adsorbed onto the 20 nm sized SiO2 nanoparticles than onto the 100 nm sized nanoparticles regardless of charge. When proteins were compared between the two charges, higher numbers of proteins were found for arginine-coated positively charged SiO2 nanoparticles than for the negatively charged nanoparticles. The proteins identified as bound in the corona from SiO2 nanoparticles were further analyzed with ClueGO, a Cytoscape plugin used in protein ontology and for identifying biological interaction pathways. Proteins bound on the surface of nanoparticles may affect functional and conformational properties and distributions in complicated biological processes.


Inferring nucleosome positions with their histone mark annotation from ChIP data

Abstract:
MOTIVATION The nucleosome is the basic repeating unit of chromatin. It contains two copies each of the four core histones H2A, H2B, H3 and H4 and about 147 bp of DNA. The residues of the histone proteins are subject to numerous post-translational modifications, such as methylation or acetylation. Chromatin immunoprecipitiation followed by sequencing (ChIP-seq) is a technique that provides genome-wide occupancy data of these modified histone proteins, and it requires appropriate computational methods.
 RESULTS We present NucHunter, an algorithm that uses the data from ChIP-seq experiments directed against many histone modifications to infer positioned nucleosomes. NucHunter annotates each of these nucleosomes with the intensities of the histone modifications. We demonstrate that these annotations can be used to infer nucleosomal states with distinct correlations to underlying genomic features and chromatin-related processes, such as transcriptional start sites, enhancers, elongation by RNA polymerase II and chromatin-mediated repression. Thus, NucHunter is a versatile tool that can be used to predict positioned nucleosomes from a panel of histone modification ChIP-seq experiments and infer distinct histone modification patterns associated to different chromatin states.    AVAILABILITY The software is available at http://epigen.molgen.mpg.de/nuchunter/.


Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains.

Abstract: Early region 3 (E3) of group C human adenoviruses (Ad) encodes several inhibitors of tumor necrosis factor alpha (TNF-alpha) cytolysis, including an E3 14.7-kDa protein (E3-14.7K) and a heterodimer containing two polypeptides of 10.4 and 14.5 kDa. To understand the mechanism by which the viral proteins inhibit TNF-alpha functions, the E3-14.7K protein was used to screen a HeLa cell cDNA library to search for interacting proteins in the yeast two-hybrid system. A novel protein containing multiple leucine zipper domains without any significant homology with any known protein was identified and has been named FIP-2 (for 14.7K-interacting protein). FIP-2 interacted with E3-14.7K both in vitro and in vivo. It colocalized with Ad E3-14.7K in the cytoplasm, especially near the nuclear membrane, and caused redistribution of the viral protein. FIP-2 by itself does not cause cell death; however, it can reverse the protective effect of E3-14.7K on cell killing induced by overexpression of the intracellular domain of the 55-kDa TNF receptor or by RIP, a death protein involved in the TNF-alpha and Fas apoptosis pathways. Deletion analysis indicates that the reversal effect of FIP-2 depends on its interaction with E3-14.7K. Three major mRNA forms of FIP-2 have been detected in multiple human tissues, and expression of the transcripts was induced by TNF-alpha treatment in a time-dependent manner in two different cell lines. FIP-2 has consensus sequences for several potential posttranslational modifications. These data suggest that FIP-2 is one of the cellular targets for Ad E3-14.7K and that its mechanism of affecting cell death involves the TNF receptor, RIP, or a downstream molecule affected by either of these two molecules.


PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening.

Abstract: The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) at telomeres. Here, we dissected the role of APBs in a human ALT cell line by performing an RNA interference screen using an automated 3D fluorescence microscopy platform and advanced 3D image analysis. We identified 29 proteins that affected APB formation, which included proteins involved in telomere and chromatin organization, protein sumoylation and DNA repair. By integrating and extending these findings, we found that APB formation induced clustering of telomere repeats, telomere compaction and concomitant depletion of the shelterin protein TRF2 (also known as TERF2). These APB-dependent changes correlated with the induction of a DNA damage response at telomeres in APBs as evident by a strong enrichment of the phosphorylated form of the ataxia telangiectasia mutated (ATM) kinase. Accordingly, we propose that APBs promote telomere maintenance by inducing a DNA damage response in ALT-positive tumor cells through changing the telomeric chromatin state to trigger ATM phosphorylation.


Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung.

Abstract: The mechanisms by which a primary tumor affects a selected distant organ before tumor cell arrival remain to be elucidated. This report shows that Gr-1+CD11b+ cells are significantly increased in lungs of mice bearing mammary adenocarcinomas before tumor cell arrival. In the premetastatic lungs, these immature myeloid cells significantly decrease IFN-gamma production and increase proinflammatory cytokines. In addition, they produce large quantities of matrix metalloproteinase 9 (MMP9) and promote vascular remodeling. Deletion of MMP9 normalizes aberrant vasculature in the premetastatic lung and diminishes lung metastasis. The production and activity of MMP9 is selectively restricted to lungs and organs with a large number of Gr-1+CD11b+ cells. Our work reveals a novel protumor mechanism for Gr-1+CD11b+ cells that changes the premetastatic lung into an inflammatory and proliferative environment, diminishes immune protection, and promotes metastasis through aberrant vasculature formation. Thus, inhibition of Gr-1+CD11b+ cells could normalize the premetastatic lung environment, improve host immunosurveillance, and inhibit tumor metastasis.


Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

Abstract: In many eukaryotes, RNA-dependent RNA polymerases (RdRPs) play key roles in the RNAi pathway. They have been implicated in the recognition and processing of aberrant transcripts triggering the process, and in amplification of the silencing response. We have tested the functions of RdRP genes from the ciliate Paramecium tetraurelia in experimentally induced and endogenous mechanisms of gene silencing. In this organism, RNAi can be triggered either by high-copy, truncated transgenes or by directly feeding cells with double-stranded RNA (dsRNA). Surprisingly, dsRNA-induced silencing depends on the putatively functional RDR1 and RDR2 genes, which are required for the accumulation of both primary siRNAs and a distinct class of small RNAs suggestive of secondary siRNAs. In contrast, a third gene with a highly divergent catalytic domain, RDR3, is required for siRNA accumulation when RNAi is triggered by truncated transgenes. Our data further implicate RDR3 in the accumulation of previously described endogenous siRNAs and in the regulation of the surface antigen gene family. While only one of these genes is normally expressed in any clonal cell line, the knockdown of RDR3 leads to co-expression of multiple antigens. These results provide evidence for a functional specialization of Paramecium RdRP genes in distinct RNAi pathways operating during vegetative growth.


Blood stem cells emerge from aortic endothelium by a novel type of cell transition

Abstract: The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.


Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation

Abstract:
OBJECTIVE To determine whether the novel avian influenza H7N9 virus can transmit from person to person and its efficiency.
 DESIGN Epidemiological investigations conducted after a family cluster of two patients with avian H7N9 in March 2013.
 SETTING Wuxi, Eastern China.
 PARTICIPANTS Two patients, their close contacts, and relevant environments. Samples from the patients and environments were collected and tested by real time reverse transcriptase-polymerase chain reaction (rRT-PCR), viral culture, and haemagglutination inhibition assay. Any contacts who became ill had samples tested for avian H7N9 by rRT-PCR. Paired serum samples were obtained from contacts for serological testing by haemagglutination inhibition assays.
 MAIN OUTCOMES MEASURES Clinical data, history of exposure before the onset of illnesses, and results of laboratory testing of pathogens and further analysis of sequences and phylogenetic tree to isolated strains.
 RESULTS The index patient became ill five to six days after his last exposure to poultry. The second patient, his daughter aged 32, who provided unprotected bedside care in the hospital, had no known exposure to poultry. She developed symptoms six days after her last contact with her father. Two strains were isolated successfully from the two patients. Genome sequence and analyses of phylogenetic trees showed that both viruses were almost genetically identical. Forty three close contacts of both patients were identified. One had mild illness but had negative results for avian H7N9 by rRT-PCR. All 43 close contacts tested negative for haemagglutination inhibition antibodies specific for avian H7N9.
 CONCLUSIONS The infection of the daughter probably resulted from contact with her father (the index patient) during unprotected exposure, suggesting that in this cluster the virus was able to transmit from person to person. The transmissibility was limited and non-sustainable.


Genetic Variation in the Interleukin-28B Gene Is Associated with Spontaneous Clearance and Progression of Hepatitis C Virus in Moroccan Patients

Abstract:
BACKGROUND Genetic variation in the IL28B gene has been strongly associated with treatment outcomes, spontaneous clearance and progression of the hepatitis C virus infection (HCV). The aim of the present study was to investigate the role of polymorphisms at this locus with progression and outcome of HCV infection in a Moroccan population.
 METHODS We analyzed a cohort of 438 individuals among them 232 patients with persistent HCV infection, of whom 115 patients had mild chronic hepatitis and 117 had advanced liver disease (cirrhosis and hepatocellular carcinoma), 68 individuals who had naturally cleared HCV and 138 healthy subjects. The IL28B SNPs rs12979860 and rs8099917 were genotyped using a TaqMan 5' allelic discrimination assay.
 RESULTS The protective rs12979860-C and rs8099917-T alleles were more common in subjects with spontaneous clearance (77.9% vs 55.2%; p = 0.00001 and 95.6% vs 83.2%; p = 0.0025, respectively). Individuals with clearance were 4.69 (95% CI, 1.99-11.07) times more likely to have the C/C genotype for rs12979860 polymorphism (p = 0.0017) and 3.55 (95% CI, 0.19-66.89) times more likely to have the T/T genotype at rs8099917. Patients with advanced liver disease carried the rs12979860-T/T genotype more frequently than patients with mild chronic hepatitis C (OR = 1.89; 95% CI, 0.99-3.61; p = 0.0532) and this risk was even more pronounced when we compared them with healthy controls (OR = 4.27; 95% CI, 2.08-8.76; p = 0.0005). The rs8099917-G allele was also associated with advanced liver disease (OR = 2.34; 95% CI, 1.40-3.93; p = 0.0100).
 CONCLUSIONS In the Moroccan population, polymorphisms near the IL28B gene play a role both in spontaneous clearance and progression of HCV infection.


Quantitative Detection of Hepatitis C Virus RNA by Light Cycler PCR and Comparison with Two Different PCR Assays

Abstract: The new Light Cycler technology was adapted to the detection of hepatitis C virus (HCV) RNA in clinical samples. Sera from 81 patients were tested by Light Cycler PCR, AMPLICOR HCV Monitor assay, and in-house PCR. Our data demonstrate that Light Cycler is a fast and reliable method for the detection and quantitation of HCV RNA.


Targeting arginine metabolism pathway to treat arginine-dependent cancers.

Abstract: The significant disparities in metabolism between tumor and normal cells have inspired the development of metabolism-based anti-tumor therapeutics. Arginine is a semi-essential amino acid because normal cells can not only synthesize arginine de novo but also take up extracellular arginine. Several types of tumors have abnormalities in arginine metabolism enzymes and completely rely on extracellular arginine to support necessary biological processes. This property is referred to as arginine auxotrophy. Taking advantage of characteristic arginine auxotrophy in tumors, arginine deprivation, which is generally induced by the use of arginine deiminase (ADI) and arginase I, has been investigated as a novel strategy for cancer therapy. Arginine deprivation demonstrated promising efficacy against arginine-auxotrophic tumors. By integrating perspectives from both clinical oncologists and laboratory scientists, this article reviews the important aspects of arginine deprivation as a promising anticancer therapy.


Long-term immune deficiency after allogeneic stem cell transplantation: B-cell deficiency is associated with late infections.

Abstract: Immune reconstitution was analyzed in 140 consecutive patients who were 2-year disease-free and who underwent myeloablative allogeneic transplantation. A CD4 and CD8 defect was observed involving naive, terminally differentiated, memory and competent cells and above limits values for activated subsets. Natural killer cells normalize at six months while we observed expansion of CD19(+)/CD5(+) B cells after three months and a persisting defect of memory B cells. Chronic graft-versus-host disease did not influence significantly those parameters for CD8 subsets while the naïve and competent CD4 subsets were strongly affected. But the most profound impact of chronic graft-versus-host disease was on B-cell subsets, especially on the memory B population. The cumulative incidence of late severe infections was low (14% at four years). Using Cox's models, only low B-cell counts at 12 (P=0.02) and 24 (P=0.001) months were associated with the hazard of developing late infection, in particular if patients did not develop chronic graft-versus-host disease.


Control of glutamate clearance and synaptic efficacy by glial coverage of neurons.

Abstract: Analysis of excitatory synaptic transmission in the rat hypothalamic supraoptic nucleus revealed that glutamate clearance and, as a consequence, glutamate concentration and diffusion in the extracellular space, is associated with the degree of astrocytic coverage of its neurons. Reduction in glutamate clearance, whether induced pharmacologically or associated with a relative decrease of glial coverage in the vicinity of synapses, affected transmitter release through modulation of presynaptic metabotropic glutamate receptors. Astrocytic wrapping of neurons, therefore, contributes to the regulation of synaptic efficacy in the central nervous system.


A Functional Neuroimaging Study of Sound Localization: Visual Cortex Activity Predicts Performance in Early-Blind Individuals

Abstract: Blind individuals often demonstrate enhanced nonvisual perceptual abilities. However, the neural substrate that underlies this improved performance remains to be fully understood. An earlier behavioral study demonstrated that some early-blind people localize sounds more accurately than sighted controls using monaural cues. In order to investigate the neural basis of these behavioral differences in humans, we carried out functional imaging studies using positron emission tomography and a speaker array that permitted pseudo-free-field presentations within the scanner. During binaural sound localization, a sighted control group showed decreased cerebral blood flow in the occipital lobe, which was not seen in early-blind individuals. During monaural sound localization (one ear plugged), the subgroup of early-blind subjects who were behaviorally superior at sound localization displayed two activation foci in the occipital cortex. This effect was not seen in blind persons who did not have superior monaural sound localization abilities, nor in sighted individuals. The degree of activation of one of these foci was strongly correlated with sound localization accuracy across the entire group of blind subjects. The results show that those blind persons who perform better than sighted persons recruit occipital areas to carry out auditory localization under monaural conditions. We therefore conclude that computations carried out in the occipital cortex specifically underlie the enhanced capacity to use monaural cues. Our findings shed light not only on intermodal compensatory mechanisms, but also on individual differences in these mechanisms and on inhibitory patterns that differ between sighted individuals and those deprived of vision early in life.


High-Infiltration of Tumor-Associated Macrophages Predicts Unfavorable Clinical Outcome for Node-Negative Breast Cancer

Abstract: The tumor microenvironment is composed of tumor cells, fibroblasts, endothelial cells and infiltrating immune cells, which may inhibit or promote tumor growth and progression. The objectives of this retrospective study were to characterize the density of tumor-associated macrophages (TAMs) in breast cancer, and to correlate the density of TAMs with clinicopathological parameters. Paraffin-embedded specimens and clinicopathological data, including up to 5 years follow-up information, were obtained from 172 breast cancer patients. Immunohistochemical staining for CD68 (marker for macrophages) was performed and evaluated in a blinded fashion. We found that TAMs were significantly frequent in high histopathological grade breast cancer patients. Breast cancer patients with a high density of TAMs had significantly lower rates of disease-free survival and 5-year overall survival than patients with low density of TAMs. Furthermore, high-infiltration of TAMs indicated worse survival rate for patients with node-negative breast cancer. In conclusion, the number of TAMs in the tumor stroma is an independent predictor of survival time for breast cancer patients. High-infiltration of TAMs is a significant unfavorable prognostic factor for patients with invasive breast cancer and, as such, is a potentially useful prognostic marker for breast cancer.


Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract, Cochlea, and Neural Tube Development

Abstract: Dishevelled (Dvl) proteins are important signaling components of both the canonical beta-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3(-/-) mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3(-/-) and LtapLp/+ mutants, Dvl3(+/-);LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.


Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast

Abstract: The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.


Efficacy of the 6-month thrice-weekly regimen in the treatment of new sputum smear-positive pulmonary tuberculosis under clinical trial conditions.

Abstract:
BACKGROUND Under the Revised National Tuberculosis Control Programme of India, patients with new smear-positive pulmonary tuberculosis are treated with a thrice-weekly regimen of antitubercular drugs (2H(3)R(3)Z(3)E(3)/4H(3)R(3) [H isoniazid, R rifampicin, Z pyrazinamide and E ethambutol]) for 6 months. We conducted a retrospective analysis of the efficacy andtolerability of this regimen under clinical trial conditions in HIV-negative patients with newly diagnosed smear-positive pulmonary tuberculosis.
 METHODS We retrospectively analysed the data on patients assigned to the control regimen (2H (3)R(3)Z(3)E(3)/4H(3)R(3)) in two clinical trials during 2001-06 at the National Institute for Research in Tuberculosis, Chennai, India.
 RESULTS Of the 268 patients treated with this regimen, data for efficacy analysis were available for 249. At the end of treatment, of 249 patients, 238 (96%) had a favourable status. Treatment failure occurred in the remaining 11: 7 in whom the organisms were initially drug-susceptible and 4 with initial drug resistance. Of the 238 patients who had a favourable status at the end of treatment, 14 (6%) had recurrence of tuberculosis during the following 24 months. In the intention-to-treat analysis, 245 (94%) of 262 patients had a favourable status at the end of treatment. Of the 28 patients with initial drug resistance, 24 (86%) had a favourable outcome. Only 4 of these 24 patients were found to have recurrence of tuberculosis in 2 years of follow-up. Among the 221 patients initially infected with drug-susceptible organisms, drug resistance did not develop in any of the 7 patients in whom the treatment failed or the 10 who had recurrence of tuberculosis. Further, 5 of the 7 patients in whom the treatment failed continued to excrete drug-susceptible bacilli at 6 months. Adverse drug reactions were observed in 38 (14%) of the 262 patients. Only 3 (1.1%) needed a modification in the treatment.
 CONCLUSION This thrice-weekly 6-month regimen of antitubercular drugs, when administered under full supervision, is associated with a high rate of favourable treatment outcomes in HIV-negative patients with newly diagnosed sputum smearpositive pulmonary tuberculosis. There are few adverse drug reactions in these patients.


Curcumin attenuates inflammatory response in IL-1beta-induced human synovial fibroblasts and collagen-induced arthritis in mouse model.

Abstract: Curcumin, a major component of turmeric, has been shown to exhibit anti-oxidant and anti-inflammatory activities. The present study was performed to determine whether curcumin is efficacious against both collagen-induced arthritis (CIA) in mice and IL-1beta-induced activation in fibroblast-like synoviocytes (FLSs). DBA/1 mice were immunized with bovine type II collagen (CII) and treated with curcumin every other day for 2weeks after the initial immunization. For arthritis, we evaluated the incidence of disease and used an arthritis index based on paw thickness. In vitro proliferation of CII- or concanavalin A-induced splenic T cells was examined using IFN-gamma production. Pro-inflammatory cytokines TNF-alpha and IL-1beta were examined in the mouse ankle joint and serum IgG1 and IgG2a isotypes were analyzed. The expression levels of prostaglandin E(2) (PGE(2)), cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs) in human FLSs were also determined. The results showed that compared with untreated CIA mice, curcumin-treated mice downregulated clinical arthritis score, the proliferation of splenic T cells, expression levels of TNF-alpha and IL-1beta in the ankle joint, and expression levels of IgG2a in serum. Additionally, by altering nuclear factor (NF)-kappaB transcription activity in FLSs, curcumin inhibited PGE(2) production, COX-2 expression, and MMP secretion. These results suggest that curcumin can effectively suppress inflammatory response by inhibiting pro-inflammatory mediators and regulating humoral and cellular immune responses.


Antimicrobial peptide pleurocidin synergizes with antibiotics through hydroxyl radical formation and membrane damage, and exerts antibiofilm activity.

Abstract:
BACKGROUND Pleurocidin, a 25-mer antimicrobial peptide (AMP), is known to exert bactericidal activity. However, the synergistic activity and mechanism(s) of pleurocidin in combination with conventional antibiotics, and the antibiofilm effect of the peptide are poorly understood.
 METHODS The interaction between pleurocidin and antibiotics was evaluated using checkerboard assay. To study the mechanism(s) involved in their synergism, we detected hydroxyl radical formation using 3'-(p-hydroxyphenyl) fluorescein, measured the NAD(+)/NADH ratio by NAD(+) cycling assay, observed change in bacterial viability with the hydroxyl radical scavenger thiourea, and investigated cytoplasmic membrane damage using propidium iodide. Also, the antibiofilm effect of pleurocidin was examined with the tissue culture plate method.
 RESULTS All combinations of pleurocidin and antibiotics showed synergistic interaction against bacterial strains (fractional inhibitory concentration index (FICI)≤0.5) except for Enterococcus faecium treated with a combination of the peptide and ampicillin (FICI=0.75). We identified that pleurocidin alone and in combinations with antibiotics induced formation of hydroxyl radicals. The oxidative stress was caused by a transient NADH depletion and the addition of thiourea prevented bacterial death, especially in the case of the combined treatment of pleurocidin and ampicillin showing synergisms. The combination of pleurocidin and erythromycin increased permeability of bacterial cytoplasmic membrane. Additionally, pleurocidin exhibited a potent inhibitory effect on preformed biofilm of bacterial organisms. In conclusion, pleurocidin synergized with antibiotics through hydroxyl radical formation and membrane-active mechanism, and exerted antibiofilm activity.    GENERAL SIGNIFICANCE The synergistic effect between pleurocidin and antibiotics suggests the AMP is a potential therapeutic agent and adjuvant for antimicrobial chemotherapy.


High-mobility group box-1 protein induces osteogenic phenotype changes in aortic valve interstitial cells.

Abstract:
OBJECTIVES Calcific aortic valve (AV) disease is known to be an inflammation-related process. High-mobility group box-1 (HMGB1) protein and Toll-like receptor 4 (TLR4) have been reported to participate in several inflammatory diseases. The purpose of the present study was to determine whether the HMGB1-TLR4 axis is involved in calcific AV disease, and to evaluate the effect of HMGB1, and its potential mechanisms, on the pro-osteogenic phenotype change of valvular interstitial cells (VICs).
 METHODS Expression of HMGB1 and TLR4 in human calcific AVs was evaluated using immunohistochemical staining and immunoblotting. Cultured VICs were used as an in vitro model. The VICs were stimulated with HMGB1 for analysis, with versus without TLR4 small interfering ribonucleic acid (siRNA), c-Jun N-terminal kinase mitogen-activated protein kinase (JNK MAPK), and nuclear factor kappa-B (NF-κB) inhibitors.
 RESULTS Enhanced accumulation of HMGB1 and TLR4 was observed in calcific valves. Moreover, we found that HMGB1 induced high levels of pro-inflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. In addition, HMGB1 induced phosphorylation of JNK MAPK and NF-κB. However, these effects were markedly suppressed by siRNA silencing of TLR4. In addition, blockade of JNK MAPK and NF-κB phosphorylation prohibited HMGB1-induced production of pro-osteogenic factors, and mineralization of VICs.
 CONCLUSIONS The HMGB1 protein may promote osteoblastic differentiation and calcification of VICs, through the TLR4-JNK-NF-κB signaling pathway.


RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae.

Abstract: We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.


Generation of mice with a conditional allele for the p75(NTR) neurotrophin receptor gene.

Abstract: The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.


Universal voluntary HIV testing with immediate antiretroviral therapy as a strategy for elimination of HIV transmission: a mathematical model.

Abstract:
BACKGROUND Roughly 3 million people worldwide were receiving antiretroviral therapy (ART) at the end of 2007, but an estimated 6.7 million were still in need of treatment and a further 2.7 million became infected with HIV in 2007. Prevention efforts might reduce HIV incidence but are unlikely to eliminate this disease. We investigated a theoretical strategy of universal voluntary HIV testing and immediate treatment with ART, and examined the conditions under which the HIV epidemic could be driven towards elimination.
 METHODS We used mathematical models to explore the effect on the case reproduction number (stochastic model) and long-term dynamics of the HIV epidemic (deterministic transmission model) of testing all people in our test-case community (aged 15 years and older) for HIV every year and starting people on ART immediately after they are diagnosed HIV positive. We used data from South Africa as the test case for a generalised epidemic, and assumed that all HIV transmission was heterosexual.
 FINDINGS The studied strategy could greatly accelerate the transition from the present endemic phase, in which most adults living with HIV are not receiving ART, to an elimination phase, in which most are on ART, within 5 years. It could reduce HIV incidence and mortality to less than one case per 1000 people per year by 2016, or within 10 years of full implementation of the strategy, and reduce the prevalence of HIV to less than 1% within 50 years. We estimate that in 2032, the yearly cost of the present strategy and the theoretical strategy would both be US$1.7 billion; however, after this time, the cost of the present strategy would continue to increase whereas that of the theoretical strategy would decrease.
 INTERPRETATION Universal voluntary HIV testing and immediate ART, combined with present prevention approaches, could have a major effect on severe generalised HIV/AIDS epidemics. This approach merits further mathematical modelling, research, and broad consultation.


Common variants in DGKK are strongly associated with risk of hypospadias

Abstract: Hypospadias is a common congenital malformation of the male external genitalia. We performed a genome-wide association study using pooled DNA from 436 individuals with hypospadias (cases) and 494 controls of European descent and selected the highest ranked SNPs for individual genotyping in the discovery sample, an additional Dutch sample of 133 cases and their parents, and a Swedish series of 266 cases and 402 controls. Individual genotyping of two SNPs (rs1934179 and rs7063116) in DGKK, encoding diacylglycerol kinase κ, produced compelling evidence for association with hypospadias in the discovery sample (allele-specific odds ratio (OR) = 2.5, P = 2.5 × 10−11 and OR = 2.3, P = 2.9 × 10−9, respectively) and in the Dutch (OR = 3.9, P = 2.4 × 10−5 and OR = 3.8, P = 3.4 × 10−5) and Swedish (OR = 2.5, P = 2.6 × 10−8 and OR = 2.2, P = 2.7 × 10−6) replication samples. Expression studies showed expression of DGKK in preputial tissue of cases and controls, which was lower in carriers of the risk allele of rs1934179 (P = 0.047). We propose DGKK as a major risk gene for hypospadias.


Neutrophils Suppress Intraluminal NK Cell-Mediated Tumor Cell Clearance and Enhance Extravasation of Disseminated Carcinoma Cells.

Abstract: UNLABELLED Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion-metastasis cascade remain poorly understood. Here, we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b(+)/Ly6G(+) neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL1β and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their cross-talk with host cells and disseminating carcinoma cells.    SIGNIFICANCE This study provides important insights into the systemic contributions of neutrophils to cancer metastasis by identifying how neutrophils facilitate intermediate steps of the invasion-metastasis cascade. We demonstrate that neutrophils suppress natural killer cell activity and increase extravasation of tumor cells. Cancer Discov; 6(6); 630-49. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.


Histones are incorporated in trans during reassembly of the yeast PHO5 promoter.

Abstract: In yeast, remodeling of PHO5 promoter chromatin upon activation is accompanied by transient hyperacetylation and subsequent eviction of histones from the promoter in trans. In the course of rerepression, nucleosomes have to be reassembled on the promoter. We have analyzed where the histones for reassembly of the inactive promoter chromatin come from. The use of a strain with two differently tagged and differently regulated versions of histone H3 allowed us to discriminate between histones originating from the chromatin fraction and histones arising from the soluble histone pool. In this way, we show that the incorporated histones originate from a source in trans. Promoter closure occurs very rapidly, and the histone chaperones Asf1 and Hir1 as well as the SWI/SNF nucleosome remodeling complex appear to be important for rapid reassembly of nucleosomes at the PHO5 promoter.