Experience of laparoscopic cholecystectomy under spinal anesthesia with low-pressure pneumoperitoneum--prospective study of 300 cases.
Having long experience of open upper abdominal surgery under spinal anesthesia and laparoscopic cholecystectomy under general anesthesia, we performed this study of laparoscopic cholecystectomy with low-pressure pneumoperitoneum under spinal anesthesia to assess its safety and feasibility. In a private rural health set-up, 300 patients were selected prospectively for laparoscopic cholecystectomy under low-pressure (8 mm) pneumoperitoneum under spinal anesthesia in a span of three years. Only 3.5 ml of 0.5% bupivacaine was used for spinal anesthesia. Fourth port positioned at lower than usual at the level of umbilicus, change of position of the table with different stages of operation, massaging of right shoulder in cases of shoulder pain, removal of smoke if formed during dissection to diminish shoulder pain and holding the body of the gallbladder by the fourth port grasper at the level of lower margin of the liver in cases of long gallbladder were some modifications of standard laparoscopic cholecystectomy made in this study. We successfully performed the operations in 291 patients without major complications. Four patients denied operation under spinal anesthesia. Spinal anesthesia was converted to general anesthesia in two patients due to severe shoulder pain. The operation was converted to open cholecystectomy in three patients. Mean age was 34.6 years (range 21-82 years). Mean BMI was -23.1 (range 20.8-28.3). Mean duration of operation was 39.6 min (range 18-78 min). Mean O 2 saturation was 97.6%. Mean peak respiratory rate was 23.4 (range 16-38). 90.08% patients complained of right shoulder pain--most of them managed by shoulder massage alone. All patients were satisfied on follow up. Laparoscopic cholecystectomy under spinal anesthesia with low-pressure pneumoperitoneum can be performed safely and satisfactorily without major complications by experienced surgeons.


Overactivation of intestinal SREBP2 in mice increases serum cholesterol.
Sterol Response Element Binding Protein 2 (SREBP2) transcription factor is a master regulator of cholesterol homeostasis. Treatment with statins, inhibitors of cholesterol synthesis, activates intestinal SREBP2, which may hinder their cholesterol-lowering effects. Overactivation of SREBP2 in mouse liver was shown to have no effect on plasma cholesterol. However, the influence of activating intestinal SREBP2 on plasma cholesterol is not known. We have generated a novel transgenic mouse model with intestine specific overexpression of active SREBP2 (ISR2) driven by villin promoter. ISR2 mice showed overexpression of active SREBP2 specifically in the intestine. Microarray analysis of jejunal RNA from ISR2 mice showed a significant increase in genes involved in fatty acid and cholesterol synthesis. Cholesterol and triglyceride (TG) in jejunum and liver (mg/g protein) were significantly increased in ISR2 vs wild type mice. Serum Cholesterol was significantly increased in VLDL and LDL fractions whereas the level of serum triglycerides was decreased in ISR2 vs wild type mice. In conclusion, activation of intestinal SREBP2 alone seems to be sufficient to increase plasma cholesterol, highlighting the essential role of intestine in maintaining cholesterol homeostasis in the body.


Urinary incontinence: an unexpected large problem among young females. Results from a population-based study.
The International Continence Society has defined urinary incontinence as a condition in which involuntary loss of urine is objectively demonstrable and is a social or hygiene problem. Urinary incontinence is presumably a common health problem among women even in younger ages. The primary aim was to investigate the prevalence of urinary incontinence (UI) in a female population with a special focus on younger women (18-30 years old). The secondary aim was to investigate the association between UI and number of deliveries, use of contraceptives or oestrogen substitutions, and urinary tract infections (UTIs). A population-based study with a self-administered questionnaire was set in the community of Surahammar, Sweden. Subjects were all women (3493) aged 18-70 years living in Surahammar during 1995. The main outcome measures were the prevalence of UI and variables such as number of deliveries, use of contraceptives or oestrogen substitutions, and UTIs. Twenty-six per cent of the women reported problems of UI. The prevalence of UI in younger women was 12%. The number of reported complaints of UTIs was significantly higher in the women with UI compared with women without urinary incontinence (wUI). In the younger women UTI, nulliparous or having given birth to one or two children were most frequent in those with UI. The use of contraceptives was more common in younger women without UI (P < 0.05). However, the use of oestrogen was more common in older women in the age group 51-70 years with UI (P < 0.01). Our findings have shown that 26% of the women who took part in the survey reported problems of UI. Among women below 30 years of age, 12% reported complaints of UI. We found a high prevalence of UI in younger women with a UTI, not taking oestrogen, nulliparous or having given birth to one or two children. There are needs for further investigations with a special focus on younger women.


IMRT QA using machine learning: A multi-institutional validation.
To validate a machine learning approach to Virtual intensity-modulated radiation therapy (IMRT) quality assurance (QA) for accurately predicting gamma passing rates using different measurement approaches at different institutions. A Virtual IMRT QA framework was previously developed using a machine learning algorithm based on 498 IMRT plans, in which QA measurements were performed using diode-array detectors and a 3%local/3 mm with 10% threshold at Institution 1. An independent set of 139 IMRT measurements from a different institution, Institution 2, with QA data based on portal dosimetry using the same gamma index, was used to test the mathematical framework. Only pixels with ≥10% of the maximum calibrated units (CU) or dose were included in the comparison. Plans were characterized by 90 different complexity metrics. A weighted poison regression with Lasso regularization was trained to predict passing rates using the complexity metrics as input. The methodology predicted passing rates within 3% accuracy for all composite plans measured using diode-array detectors at Institution 1, and within 3.5% for 120 of 139 plans using portal dosimetry measurements performed on a per-beam basis at Institution 2. The remaining measurements (19) had large areas of low CU, where portal dosimetry has a larger disagreement with the calculated dose and as such, the failure was expected. These beams need further modeling in the treatment planning system to correct the under-response in low-dose regions. Important features selected by Lasso to predict gamma passing rates were as follows: complete irradiated area outline (CIAO), jaw position, fraction of MLC leafs with gaps smaller than 20 or 5 mm, fraction of area receiving less than 50% of the total CU, fraction of the area receiving dose from penumbra, weighted average irregularity factor, and duty cycle. We have demonstrated that Virtual IMRT QA can predict passing rates using different measurement techniques and across multiple institutions. Prediction of QA passing rates can have profound implications on the current IMRT process.


Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells.
Estrogens and antiestrogens influence the G(1) phase of the cell cycle. In MCF-7 breast cancer cells, estrogen stimulated cell cycle progression through loss of the kinase inhibitor proteins (KIPs) p27 and p21 and through G(1) cyclin-dependent kinase (cdk) activation. Treatment with antiestrogen drugs, Tamoxifen or ICI 182780, caused cell cycle arrest, with up-regulation of both p21 and p27 levels, an increase in their binding to cyclin E-cdk2, and kinase inhibition. The requirement for these KIPs in the arrests induced by estradiol depletion or by antiestrogens was investigated with antisense. Antisense inhibition of p21 or p27 expression in estradiol-depleted or antiestrogenarrested MCF-7 led to abrogation of cell cycle arrest, with loss of cyclin E-associated KIPs, activation of cyclin E-cdk2, and S phase entrance. These data demonstrate that depletion of either p21 or p27 can mimic estrogen-stimulated cell cycle activation and indicate that both of these KIPs are critical mediators of the therapeutic effects of antiestrogens in breast cancer.


Does tagging transparent fish increase predation risk? A laboratory study with glass eel (Anguilla anguilla) and sea bass (Dicentrarchus labrax).
Barriers in the estuaries of the rivers prevent the immigration of glass eels (Anguilla anguilla) arriving on the European coast every spring. This leads to an unnatural accumulation of migrating glass eels below the barriers, and this may lead to additional losses in glass eels by piscivorous fish. The proportion of predation losses can be estimated using mark-recapture techniques and abundance estimates in combination with stomach content analysis of piscivorous fish. Nonetheless, whether tagging transparent glass eels increases predation risk and what the digestion rate of glass eel is in piscivorous fish are unknown. This study aimed to determine whether there is an increased predation risk for tagged glass eel; it also studies glass eel digestion status in piscivorous fish after appointed time frames. A laboratory experiment with 48 trials was conducted. Tagged (visible implanted elastomer, VIE) and untagged glass eels were exposed to small (19.1-24.4 cm) and large (31.9-43.5 cm) sea bass (Dicentrarchus labrax) during a 2 h trial. In 48% of the trials, successful predation was present and 13% showed clear predation attempts in which bass did not capture glass eels. No significant difference was found in predation rate between tagged and untagged glass eels and between red and blue tagged glass eels. Large sea bass predated more, but all sizes consumed glass eel under laboratory conditions. Stomach content analysis showed intact glass eel bodies 4-6 h after ending the 2 h trial and parts of glass eel bodies up to 16-18 h. This study showed that tagging does not increase predation in mark-recapture studies using VIE-tags in transparent glass eel. It also shows that the proportion of predation in relation to local glass eel abundance can be estimated if stomach content analysis is conducted within 4-6 h after predation.


Distinct visual resolution supports aperture shaping in natural and pantomime-grasping.
Pantomime-grasping is a "simulated" motor response wherein an individual grasps to an area dissociated from a physical target. The task has been used in the apraxia literature as a proxy for natural grasping (i.e., physically grasping a target); however, it is important to recognize that the task's decoupled spatial relations between stimulus and response renders the top-down processing of target features (e.g., size) that accumulating evidence has shown to be mediated by visual information functionally distinct from natural grasping. Here, we examined whether the visual information supporting pantomime-grasps exhibits a visual resolution power commensurate with natural grasps. Participants were presented with a target and nontarget that differed in size below the perceptual threshold (i.e., 0.5 mm or ∼1.3%) and were asked to make a perceptual judgment about the target (i.e., "smaller" or "larger" than the nontarget) before and after completing natural and pantomime-grasps. Results showed that perceptual judgments "before" and "after" natural and pantomime-grasps did not reliably distinguish between target and nontarget. Natural grasp peak grip apertures (PGAs) scaled to target size and were comparable for "before" and "after" perceptual judgment trials-a result indicating that haptic feedback from physically grasping the target did not "boost" perceptual accuracy. Most notably, pantomime-grasp PGAs were insensitive to target size; that is, responses elicited a visual resolution power less than natural grasps. These results provide convergent evidence that pantomime-grasps are mediated by the same visual information as obligatory perceptions and do not provide a proxy for natural grasps. (PsycInfo Database Record (c) 2022 APA, all rights reserved).


Structural basis of human pregnane X receptor activation by the hops constituent colupulone.
Hops extracts are used to alleviate menopausal symptoms and as an alternative to hormone replacement therapy, but they can produce potentially harmful drug-drug interactions. The nuclear xenobiotic receptor pregnane X receptor (PXR) is promiscuously activated by a range of structurally distinct chemicals. It has a key role in the transcriptional regulation of genes that encode xenobiotic metabolism enzymes. In this study, hops extracts are shown to induce the expression of numerous drug metabolism and excretion proteins. The beta-bitter acid colupulone is demonstrated to be a bioactive component and direct activator of human PXR. The 2.8-A resolution crystal structure of the ligand binding domain of human PXR in complex with colupulone was elucidated, and colupulone was observed to bind in a single orientation stabilized by both van der Waals and hydrogen bonding contacts. The crystal structure also indicates that related alpha- and beta-bitter acids have the capacity to serve as PXR agonists as well. Taken together, these results reveal the structural basis for drug-drug interactions mediated by colupulone and related constituents of hops extracts.


Glutathione production is regulated via distinct pathways in stressed and non-stressed cortical neurons.
Peroxynitrite-mediated damage has been linked to numerous neurological and neurodegenerative diseases, including stroke, Alzheimer's and Parkinson's Diseases, amyotrophic lateral sclerosis and multiple sclerosis. Studies on the toxic effects of peroxynitrite in neurons have focused primarily on adverse effects resulting from the nitration of cellular proteins as the principal mode of toxicity while the consequences of the modulation of kinase pathways by peroxynitrite have received relatively less attention. Our results show that treatment of primary rat neurons with the peroxynitrite donor, SIN-1, leads to decreases in glutathione (GSH) levels and cell viability via a novel extracellular-signal-related kinase (ERK)/c-Myc phosphorylation pathway and a reduction in the nuclear expression of NF-E2-related factor-2 (Nrf2) that down-regulate the expression of glutamate cysteine ligase, the rate limiting enzyme for GSH synthesis. The flavonoid fisetin protects against the SIN-1-mediated alterations in ERK/c-Myc phosphorylation, nuclear Nrf2 levels, glutamate cysteine ligase levels, GSH concentration and cell viability. We also show that inhibition of mitogen-activated protein kinase kinase or Raf kinase can increase GSH levels in unstressed primary rat neurons through the same ERK/c-Myc phosphorylation pathway. Together, these results demonstrate that distinct signaling pathways modulate GSH metabolism in unstressed and stressed cortical neurons.


Purification of plant-derived antibodies through direct immobilization of affinity ligands on cellulose.
Plants possess enormous potential as factories for the large scale production of therapeutic reagents such as recombinant proteins and antibodies. A major factor limiting commercial advances of plant-derived pharmaceuticals is the cost and inefficiency of purification. As a model system, we have developed a simple yet robust method for immobilizing affinity capture ligands onto solid supports by interfacing the secreted expression and coupling of a chimeric fusion protein in Pichia pastoris to microcrystalline cellulose in a single step. The fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD), was tethered directly onto cellulose resins added to P. pastoris cultures and subsequently used for antibody purification. Both the antibody-binding protein L and protein G domains were functional, as demonstrated by the ability of cellulose-immobilized LG-CBD to purify both a scFv antibody fragment from yeast and a human IgG1 monoclonal antibody from transgenic tobacco. Furthermore, combining two P. pastoris strains expressing LG-CBD and scFv with CP-102 cellulose in a single culture allowed for easy recovery of biologically active scFv. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, single-use antibody purification reagents. Straightforward preparation of purification reagents will help make antibody purification from genetically modified crop plants feasible and address one of the major bottlenecks facing commercialization of plant-derived pharmaceuticals.


Impact of daily temperature fluctuations on dengue virus transmission by Aedes aegypti.
Most studies on the ability of insect populations to transmit pathogens consider only constant temperatures and do not account for realistic daily temperature fluctuations that can impact vector-pathogen interactions. Here, we show that diurnal temperature range (DTR) affects two important parameters underlying dengue virus (DENV) transmission by Aedes aegypti. In two independent experiments using different DENV serotypes, mosquitoes were less susceptible to virus infection and died faster under larger DTR around the same mean temperature. Large DTR (20 °C) decreased the probability of midgut infection, but not duration of the virus extrinsic incubation period (EIP), compared with moderate DTR (10 °C) or constant temperature. A thermodynamic model predicted that at mean temperatures <18 °C, DENV transmission increases as DTR increases, whereas at mean temperatures >18 °C, larger DTR reduces DENV transmission. The negative impact of DTR on Ae. aegypti survival indicates that large temperature fluctuations will reduce the probability of vector survival through EIP and expectation of infectious life. Seasonal variation in the amplitude of daily temperature fluctuations helps to explain seasonal forcing of DENV transmission at locations where average temperature does not vary seasonally and mosquito abundance is not associated with dengue incidence. Mosquitoes lived longer and were more likely to become infected under moderate temperature fluctuations, which is typical of the high DENV transmission season than under large temperature fluctuations, which is typical of the low DENV transmission season. Our findings reveal the importance of considering short-term temperature variations when studying DENV transmission dynamics.


Specific PGF(2alpha) receptor (FP) antagonism and human uterine contractility in vitro.
PGF(2alpha) acts through its receptor, FP, as an important smooth muscle contractile agent. The aim of this study was to investigate the effects of specific FP antagonism, using the novel-specific FP non-competitive antagonist THG113.31, on spontaneous and agonist-elicited contractions in pregnant and non-pregnant human myometrium in vitro. Scientific study. University hospital and laboratories. Population Women undergoing caesarean section or hysterectomy. Biopsies of human myometrium were obtained at elective caesarean section (n= 22) and from hysterectomy specimens from premenopausal women (n= 8). Dissected strips were mounted in tissue baths under physiological conditions. The effects of THG113.31 on spontaneous and oxytocin-induced contractions, in pregnant myometrium, and on phenylephrine-induced contractions, in non-pregnant myometrium, were measured. The effects of PGF(2alpha) on spontaneous contractions, in pregnant tissue, in the presence and absence of THG113.31, were investigated. The integrals of contractile activity measured were compared with those from simultaneously run control experiments. The pD(2) and mean maximal effect observed for THG113.31, and for PGF(2alpha) in the presence and absence of THG113.31, were calculated. Changes in contractility. THG113.31 exerted a potent relaxant effect in both spontaneous and oxytocin-induced contractility in pregnant tissue (P < 0.001), and phenylephrine-induced contractility in non-pregnant tissue (P < 0.001), compared with control experiments. PGF(2alpha) exerted a significant contractile effect on spontaneous contractions in pregnant tissue and this effect was not significantly attenuated by THG113.31 (P > 0.05). THG113.31 exerted a significant relaxant effect on human spontaneous and oxytocin-induced contractility but did not alter PGF(2alpha)-elicited contractility. These data raise questions about the exact mechanism of effect of THG113.31 and its interaction with FP. The uterorelaxant potency of THG113.31 in human myometrium in vitro indicates that it may be of limited use as a tocolytic compound.


A model for chronic, intrahypothalamic thyroid hormone administration in rats.
In addition to the direct effects of thyroid hormone (TH) on peripheral organs, recent work showed metabolic effects of TH on the liver and brown adipose tissue via neural pathways originating in the hypothalamic paraventricular and ventromedial nucleus (PVN and VMH). So far, these experiments focused on short-term administration of TH. The aim of this study is to develop a technique for chronic and nucleus-specific intrahypothalamic administration of the biologically active TH tri-iodothyronine (T3). We used beeswax pellets loaded with an amount of T3 based on in vitro experiments showing stable T3 release (∼5 nmol l(-1)) for 32 days. Upon stereotactic bilateral implantation, T3 concentrations were increased 90-fold in the PVN region and 50-fold in the VMH region after placing T3-containing pellets in the rat PVN or VMH for 28 days respectively. Increased local T3 concentrations were reflected by selectively increased mRNA expression of the T3-responsive genes Dio3 and Hr in the PVN or in the VMH. After placement of T3-containing pellets in the PVN, Tshb mRNA was significantly decreased in the pituitary, without altered Trh mRNA in the PVN region. Plasma T3 and T4 concentrations decreased without altered plasma TSH. We observed no changes in pituitary Tshb mRNA, plasma TSH, or plasma TH in rats after placement of T3-containing pellets in the VMH. We developed a method to selectively and chronically deliver T3 to specific hypothalamic nuclei. This will enable future studies on the chronic effects of intrahypothalamic T3 on energy metabolism via the PVN or VMH.


Skin microbiota and allergic symptoms associate with exposure to environmental microbes.
A rural environment and farming lifestyle are known to provide protection against allergic diseases. This protective effect is expected to be mediated via exposure to environmental microbes that are needed to support a normal immune tolerance. However, the triangle of interactions between environmental microbes, host microbiota, and immune system remains poorly understood. Here, we have studied these interactions using a canine model (two breeds, n = 169), providing an intermediate approach between complex human studies and artificial mouse model studies. We show that the skin microbiota reflects both the living environment and the lifestyle of a dog. Remarkably, the prevalence of spontaneous allergies is also associated with residential environment and lifestyle, such that allergies are most common among urban dogs living in single-person families without other animal contacts, and least common among rural dogs having opposite lifestyle features. Thus, we show that living environment and lifestyle concurrently associate with skin microbiota and allergies, suggesting that these factors might be causally related. Moreover, microbes commonly found on human skin tend to dominate the urban canine skin microbiota, while environmental microbes are rich in the rural canine skin microbiota. This in turn suggests that skin microbiota is a feasible indicator of exposure to environmental microbes. As short-term exposure to environmental microbes via exercise is not associated with allergies, we conclude that prominent and sustained exposure to environmental microbiotas should be promoted by urban planning and lifestyle changes to support health of urban populations.


INTEROCC case-control study: lack of association between glioma tumors and occupational exposure to selected combustion products, dusts and other chemical agents.
The aim was to investigate possible associations between glioma (an aggressive type of brain cancer) and occupational exposure to selected agents: combustion products (diesel and gasoline exhaust emissions, benzo(a)pyrene), dusts (animal dust, asbestos, crystalline silica, wood dust) and some other chemical agents (formaldehyde, oil mist, sulphur dioxide). The INTEROCC study included cases diagnosed with glioma during 2000-2004 in sub-regions of seven countries. Population controls, selected from various sampling frames in different centers, were frequency or individually matched to cases by sex, age and center. Face-to-face interviews with the subject or a proxy respondent were conducted by trained interviewers. Detailed information was collected on socio-economic and lifestyle characteristics, medical history and work history. Occupational exposure to the 10 selected agents was assessed by a job exposure matrix (JEM) which provides estimates of the probability and level of exposure for different occupations. Using a 25% probability of exposure in a given occupation in the JEM as the threshold for considering a worker exposed, the lifetime prevalence of exposure varied from about 1% to about 15% for the different agents. Associations between glioma and each of the 10 agents were estimated by conditional logistic regression, and using three separate exposure indices: i) ever vs. never; ii) lifetime cumulative exposure; iii) total duration of exposure. The study sample consisted of 1,800 glioma cases and 5,160 controls. Most odds ratio estimates were close to the null value. None of the ten agents displayed a significantly increased odds ratio nor any indication of dose-response relationships with cumulative exposure or with duration of exposure. Thus, there was no evidence that these exposures influence risk of glioma.


[Incidence of type 2 diabetes and its diagnosis process in the decade 1991-2000 in a primary health care centre].
To determine the annual incidence and reasons for diagnosing type 2 diabetes mellitus (T2DM) from 1991 to 2000. We performed a retrospective, longitudinal study. The clinical records of all new cases of T2DM registered in an urban primary care center over a 10-year period were reviewed. The annual incidence and prevalence in the population aged more than 14 years old were calculated. Statistical analyses of temporal trends and periodicity (monthly, seasonal and annual) were performed with ARIMA models (Box-Jenkins), Poisson parametrical regression and semiparametrical (GAM) models. There were 598 true new cases of T2DM (mean age [SD]: 59.7 [11.4] years; 51.3% women). The most frequent associated cardiovascular risk factors were obesity (58.5%) and hypertension (68.2%). The main reasons for diagnosing T2DM were the presence of previous hyperglycemia (64.7%) and the application of protocols for other cardiovascular risk factors (12.4%).The annual incidence was 37.9 per 10,000 persons (95% CI, 34.9-40.9) with no differences between sexes. The prevalence of T2DM at the beginning and end of the study period was 4.4 (95% CI, 2.0-4.8) and 5.5% (95% CI, 5.2-5.9) (25% relative increase). There was no significant temporal trend in the incidence of T2DM over the years. The observed incidence of T2DM is high compared with that reported in other studies. The increase in prevalence was not related to a progressive increase in the incidence. The most frequent reasons for diagnosing T2DM were previous hyperglycemia and the application of protocols for other cardiovascular risk factors. This finding seems to be related to an early diagnosis and could benefit these patients.


Virulent Treponema pallidum 47 kDa antigen regulates the expression of cell adhesion molecules and binding of T-lymphocytes to cultured human dermal microvascular endothelial cells.
Perivasculitis and endothelial cell abnormalities are prominent histopathologic features of syphilis. Various cutaneous lesions are the main clinical features of syphilis. We examined whether Treponema pallidum 47 kDa antigen regulates the expression of cell adhesion molecules on human dermal microvascular endothelial cells (HDMEC) and the regulation of T-lymphocytes binding to HDMEC. Using immunofluorescence flow cytometry and enzyme-linked immunosorbent assay (ELISA), we demonstrated that T. pallidum upregulated the expression of adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. The 47 kDa antigen of T. pallidum also activated endothelium as measured by the upregulation of the expression of adhesion molecules on HDMEC, and it also promoted an increased adherence of T-lymphocytes to HDMEC. The expressions of ICAM-1 and VCAM-1 on HDMEC and the adherence of T-lymphocytes to HDMEC were inhibited by treatment with anti-TNF-alpha antibody or anti-IL-1alpha antibody. These results show that T. pallidum or T. pallidum-specific 47 kDa antigen are capable of stimulating HDMEC to increase the expression of ICAM-1, VCAM-1 and E-selectin and thereby, promote the adherence of T-lymphocytes. The whole process may play an important role in the immunopathogenesis of syphilis and it is likely that TNF-alpha and IL-1alpha are involved.


Preoperative 3-Dimensional Angiography Data and Intraoperative Real-Time Vascular Data Integrated in Microscope-Based Navigation by Automatic Patient Registration Applying Intraoperative Computed Tomography.
To establish a workflow integrating preoperative 3-dimensional (3D) angiography data and intraoperative real-time vascular information in microscope-based navigation for aneurysm and arteriovenous malformation (AVM) surgery. In 7 patients (3 with AVMs and 4 with aneurysms), preoperative 3D rotational angiography or computed tomography (CT) or magnetic resonance angiography data were navigated applying a 32-slice movable CT scanner for low-dose registration scanning. The 3D vasculature was segmented and visualized by microscope-based navigation along with navigated intraoperative real-time imaging data from indocyanin green angiography and duplex ultrasonography. Automatic registration applying intraoperative CT resulted in high accuracy (registration error, 0.80 ± 0.79 mm). The effective radiation dose of the registration CT scans (0.28-0.42 mSv) was only approximately one-sixth of a standard diagnostic head CT scan. The 3D vessel architecture could be visualized accurately in the operating microscope heads-up display and on the navigation screens in the same projection as the view angle of the surgeon, both facilitating orientation in 3D space, providing a better understanding of anatomy. In addition, intraoperative real-time modalities could be coregistered with high precision, providing further information during the course of the vascular procedure. Registration CT imaging facilitates integrating preoperative and intraoperative vascular image data with a low registration error and low radiation exposure for the patient, improving the understanding of 3D vascular anatomy during surgery with easier identification of feeding vessels in AVMs, and of the projection and configuration of aneurysms.


Racial/ethnic differences in alcohol use trajectories among adolescents involved in child welfare.
This study examined racial/ethnic differences in growth trajectories of alcohol use for a nationally representative sample of U.S. adolescents in the child welfare system (CWS), and how individual, family, and child welfare (i.e., proximal) factors predicted alcohol use trajectories for these adolescents. The study included 1,080 Hispanic, African American, and White adolescents aged 11 to 14 years old (at baseline) from the National Survey of Child and Adolescent Well-Being dataset, which is a nationally representative sample of U.S. children in the CWS. Latent growth modeling was used to determine alcohol use growth over 36 months, and multigroup analyses were conducted to examine racial/ethnic differences on alcohol use trajectories and the proximal factors predicting these trajectories. Findings indicated that CWS adolescents demonstrated similar trends in alcohol use growth and initiation compared to adolescents in the general population, especially the Hispanic CWS adolescents. This may reflect an overall shift in nativity status of Hispanic youth in the overall U.S. population. African American adolescents had the fewest significant predictors; this may suggest that factors more relevant for these adolescents and their alcohol use are missing from CWS research. Family-level factors were found to differentially affect use for CWS adolescents compared to adolescents in the general population. Overall, these findings point to a need for improvements in the assessment of CWS adolescents of color in research and practice settings to fully capture the complexity of experiences for these youth and their families. (PsycINFO Database Record (c) 2019 APA, all rights reserved).


Assessing type I collagen expression and quality in cellular models of osteogenesis imperfecta.
Osteogenesis imperfecta (OI) is a group of genetic disorders of bone formation characterized by soft and shorter brittle bones in affected individuals. OI is generally considered a collagenopathy resulting from abnormal expression of type I collagen. As assay system to detect the cellular level and quality of type I collagen would help in rapid and correct detection of OI from the diagnostic perspectives. Here, we report an immunofluorescence assay for detection of type I collagen in fibroblast models of OI and represented them into two broad categories based on the expression level and aggregation characteristics of pro-α1(I). Cell phenotypic assays of pro-α1(I) in OI-related gene knocked down fibroblasts revealed aggregates of pro-α1(I) in conditions with knockdown of SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, TMEM38B, MESD, and KDELR2, whereas pro-α1(I) expression was very low in fibroblasts which had knockdown of IFITM5, SP7, BMP1, WNT1, CREB3L1, MBTPS2, and CCDC134. The expression of pro-α1(I) showed abundant and non-aggregated distribution in the fibroblasts with knockdown of non-OI skeletal disorder-related genes (RAB33B and IFT52). The in vitro assay accurately detected abnormally expressed pro-α1(I) levels in cellular models of various types of OI. Thus, this procedure represents a promising point-of-detection assay for potential diagnosis and therapeutic decisions in OI.


Past and present patterns of connectivity among populations of four cryptic species of freshwater mussels Velesunio spp. (Hyriidae) in central Australia.
We examined genetic structure and levels of connectivity among subpopulations within each of four cryptic species belonging to the freshwater mussel genus Velesunio. We used allozymes and a fragment of the mitochondrial cytochrome c oxidase I gene to examine genetic variation in populations from isolated waterholes, belonging to four major inland drainages in eastern Australia. Based on evidence from other invertebrates in the region we predicted that, in each species, we would find evidence of historical connectivity among populations from different drainages. This was clearly not the case, as for the two species that occurred in more than one drainage there was evidence of both current and past restriction to gene flow. Moreover, given the potential for extensive dispersal of these mussels through the river systems during flood times via their fish hosts, we predicted low levels of genetic variation among populations from waterholes in the same drainage. Contrary to our expectations, all four species showed some evidence of restricted gene flow among waterholes within drainages. This suggests that either (a) mussel larvae are not produced during flood times, when their fish hosts would be free to move between waterholes, or (b) mussel larvae are attached to their hosts at these times, but the fish movement is limited between waterholes.


The light field camera: extended depth of field, aliasing, and superresolution.
Portable light field (LF) cameras have demonstrated capabilities beyond conventional cameras. In a single snapshot, they enable digital image refocusing and 3D reconstruction. We show that they obtain a larger depth of field but maintain the ability to reconstruct detail at high resolution. In fact, all depths are approximately focused, except for a thin slab where blur size is bounded, i.e., their depth of field is essentially inverted compared to regular cameras. Crucial to their success is the way they sample the LF, trading off spatial versus angular resolution, and how aliasing affects the LF. We show that applying traditional multiview stereo methods to the extracted low-resolution views can result in reconstruction errors due to aliasing. We address these challenges using an explicit image formation model, and incorporate Lambertian and texture preserving priors to reconstruct both scene depth and its superresolved texture in a variational Bayesian framework, eliminating aliasing by fusing multiview information. We demonstrate the method on synthetic and real images captured with our LF camera, and show that it can outperform other computational camera systems.


Killer cell immunoglobulin-like receptor 3DL1 variation modifies HLA-B*57 protection against HIV-1.
HLA-B*57 control of HIV involves enhanced CD8+ T cell responses against infected cells, but extensive heterogeneity exists in the level of HIV control among B*57+ individuals. Using whole-genome sequencing of untreated B*57+ HIV-1-infected controllers and noncontrollers, we identified a single variant (rs643347A/G) encoding an isoleucine-to-valine substitution at position 47 (I47V) of the inhibitory killer cell immunoglobulin-like receptor KIR3DL1 as the only significant modifier of B*57 protection. The association was replicated in an independent cohort and across multiple outcomes. The modifying effect of I47V was confined to B*57:01 and was not observed for the closely related B*57:03. Positions 2, 47, and 54 tracked one another nearly perfectly, and 2 KIR3DL1 allotypes differing only at these 3 positions showed significant differences in binding B*57:01 tetramers, whereas the protective allotype showed lower binding. Thus, variation in an immune NK cell receptor that binds B*57:01 modifies its protection. These data highlight the exquisite specificity of KIR-HLA interactions in human health and disease.


PrP-deficient mice are resistant to scrapie.
Prusiner proposed that the infectious agent of scrapie, the prion, is PrPSc, a modified form of the normal host protein PrPC. Prn-p0/0 mice devoid of PrPC showed normal development and behavior. When inoculated with mouse scrapie prions they remained free of scrapie symptoms for at least 16 months while wild type controls all died within 6 months. Propagation of infectivity in the PrP null mice, if any, was less than 10(-5) that in wild type animals. Surprisingly, heterozygous Prn-p0/+ mice also showed enhanced resistance to scrapie. After introduction of Syrian hamster PrP transgenes, Prn-p0/0 mice became highly susceptible to hamster but not to mouse prions. These experiments show that PrPC, possibly at close to normal levels, is required for the usual susceptibility to scrapie and that lack of homology between incoming prions and the host's PrP genes retards disease.


A Simple Method for Optimization of Reference Gene Identification and Normalization in DNA Microarray Analysis.
Comparative DNA microarray analyses typically yield very large gene expression data sets that reflect complex patterns of change. Despite the wealth of information that is obtained, the identification of stable reference genes is required for normalization of disease- or drug-induced changes across tested groups. This is a prerequisite in quantitative real-time reverse transcription-PCR (qRT-PCR) and relative RT-PCR but rare in gene microarray analysis. The goal of the present study was to outline a simple method for identification of reliable reference genes derived from DNA microarray data sets by comparative statistical analysis of software-generated and manually calculated candidate genes. DNA microarray data sets derived from whole-blood samples obtained from 14 Zucker diabetic fatty (ZDF) rats (7 lean and 7 diabetic obese) were used for the method development. This involved the use of software-generated filtering parameters to accomplish the desired signal-to-noise ratios, 75th percentile signal manual normalizations, and the selection of reference genes as endogenous controls for target gene expression normalization. The combination of software-generated and manual normalization methods yielded a group of 5 stably expressed, suitable endogenous control genes which can be used in further target gene expression determinations in whole blood of ZDF rats. This method can be used to correct for potentially false results and aid in the selection of suitable endogenous control genes. It is especially useful when aimed to aid the software in cases of borderline results, where the expression and/or the fold change values are just beyond the pre-established set of acceptable parameters.


Efficacy and safety of stress ulcer prophylaxis in critically ill patients: a network meta-analysis of randomized trials.
Stress ulcer prophylaxis (SUP) is commonly prescribed in the intensive care unit. However, data from systematic reviews and conventional meta-analyses are limited by imprecision and restricted to direct comparisons. We conducted a network meta-analysis of randomized clinical trials (RCTs) to examine the safety and efficacy of drugs available for SUP in critically ill patients. We searched MEDLINE, EMBASE, and the Cochrane Library Central Register of Controlled Trials through April 2017 for randomized controlled trials that examined the efficacy and safety of proton pump inhibitors (PPIs), histamine-2 receptor antagonists (H2RAs), and sucralfate for SUP in critically ill patients. No date or language restrictions were applied. Data on study characteristics, methods, outcomes, and risk of bias were abstracted by two reviewers. Of 96 potentially eligible studies, we included 57 trials enrolling 7293 patients. The results showed that PPIs are probably more effective for preventing clinically important gastrointestinal bleeding (CIB) than H2RAs [odds ratio (OR) 0.38; 95% confidence interval (95% CI) 0.20, 0.73], sucralfate (OR 0.30; 95% CI 0.13, 0.69), and placebo (OR 0.24; 95% CI 0.10, 0.60) (all moderate quality evidence). There were no convincing differences among H2RA, sucralfate, and placebo. PPIs probably increase the risk of developing pneumonia compared with H2RAs (OR 1.27; 95% CI 0.96, 1.68), sucralfate (OR 1.65; 95% CI 1.20, 2.27), and placebo (OR 1.52; 95% CI 0.95, 2.42) (all moderate quality). Mortality is probably similar across interventions (moderate quality). Estimates of baseline risks of bleeding varied significantly across studies, and only one study reported on Clostridium difficile infection. Definitions of pneumonia varied considerably. Most studies on sucralfate predate pneumonia prevention strategies. Our results provide moderate quality evidence that PPIs are the most effective agents in preventing CIB, but they may increase the risk of pneumonia. The balance of benefits and harms leaves the routine use of SUP open to question.


Overview of toxicological aspects of polybrominated diphenyl ethers: a flame-retardant additive in several consumer products.
Polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants due to their long half-life and widespread use as flame retardants in several consumer products, including plastics. In addition to other actions, these compounds are characterized as thyroid hormone disruptors. Thyroid hormones affect the function of nearly all tissues via their effects on cellular metabolism and the essential roles they play in differentiation and growth. Interference with thyroid hormone homeostasis by these environmental compounds, therefore, has the potential to impact development and every system in the body. Their presence in human breast milk is particularly troubling due to exposure of nursing children. The last trimester of pregnancy up to 2 years of age corresponds to a time of rapid neurodevelopment and represents a period of vulnerability to environmental insults. Rodent studies indicate that PBDEs may act as developmental neurotoxicants and effects on the reproductive system have been reported as well. Concerns exist regarding possible impacts of exposure, in particular ones which occur during development, on human health. This paper is part of a series of articles regarding contaminants in plastic and provides an overview regarding PBDEs, a class of flame-retardant additives to plastic. PBDEs possess a similar structure to the polychlorinated biphenyls (PCBs) previously used as lubricants in electrical generators and transformers until production was prohibited approximately 25 years ago. Parallels between the two compounds will be briefly made and in particular, as more epidemiological studies on PCBs are available than on PBDEs, a few examples concerning thyroid homeostasis, cognitive function and sexually dimorphic behavior in humans will be mentioned.


Benzyl isothiocyanate-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species and regulated by Bax and Bak.
Epidemiologic studies have revealed an inverse correlation between dietary intake of cruciferous vegetables and the risk of breast cancer. We now show that cruciferous vegetable constituent benzyl isothiocyanate (BITC) effectively suppresses growth of cultured human breast cancer cells (MDA-MB-231 and MCF-7) by causing G(2)-M phase cell cycle arrest and apoptosis induction. On the other hand, a normal mammary epithelial cell line (MCF-10A) is significantly more resistant to growth arrest and apoptosis by BITC compared with breast cancer cells. The BITC-mediated cell cycle arrest was associated with a decrease in levels of proteins involved in regulation of G(2)-M transition, including cyclin B1, cyclin-dependent kinase 1, and cell division cycle 25C. The BITC-induced apoptosis correlated with induction of proapoptotic proteins Bax (MCF-7) and Bak (MDA-MB-231 and MCF-7) and down-regulation of antiapoptotic proteins Bcl-2 and Bcl-xL (MDA-MB-231). The SV40-immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice were significantly more resistant to BITC-induced DNA fragmentation compared with wild-type mouse embryonic fibroblasts. The BITC treatment caused rapid disruption of the mitochondrial membrane potential, leading to cytosolic release of apoptogenic molecules, which was accompanied by formation of autophagosome-like structures as revealed by transmission electron microscopy. The BITC-mediated apoptosis was associated with generation of reactive oxygen species and cleavage of caspase-9, caspase-8, and caspase-3. Apoptosis induction by BITC was significantly attenuated in the presence of a combined superoxide dismutase and catalase mimetic EUK134 as well as caspase inhibitors. In conclusion, the present study reveals a complex signaling leading to growth arrest and apoptosis induction by BITC.


Muscarinic agonist-induced burst firing in immature rat olfactory cortex neurons In vitro.
Age-related changes in pre-/postsynaptic muscarinic (mAChR) and metabotropic-glutamate (mGluR) responsiveness were studied in slices of olfactory cortex from both immature [postnatal day 16-22 (P16-P22)] and adult (>/=P40) rats, using a conventional intracellular recording technique. In adult neurons, bath application of the mAChR agonist oxotremorine-M (OXO-M; 10 microM), or the selective mGluR agonist 1-aminocyclopentane-1S-3R-dicarboxylic acid (1S,3R-ACPD; 10 microM) evoked sustained membrane depolarizations, increases in input resistance, intense repetitive firing, and the appearance of a slow poststimulus afterdepolarizing potential (sADP). Excitatory postsynaptic potentials (EPSPs) evoked by local electrical stimulation of association fiber terminals were also depressed. In contrast, in neurons from immature slices, the 10 microM OXO-M-induced membrane depolarization was followed by the appearance of spontaneous rhythmic epileptiform activity, which was voltage independent and reversible on drug wash out. Epileptiform bursts were abolished or reduced by coapplication of tetrodotoxin (1 microM), atropine (1 microM), pirenzepine (100-200 nM), the N-methyl-D-aspartate (NMDA) receptor antagonist -amino-5-phosphonovaleric acid (-APV; 100 microM), the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5-20 microM), the anesthetic-sedative barbiturate pentobarbitone (100 microM), or by raising the extracellular Mg2+ concentration, whereas a clear facilitatory effect was exhibited by the selective gamma-aminobutyric acid-A (GABAA) receptor blocker (-)-bicuculline methiodide (10 microM). The epileptogenic effects induced by OXO-M were indistinguishable from those produced by 4-aminopyridine (4-AP; 100-200 microM), although these latter actions were unaffected by atropine. In slices from immature animals, electrical stimulation of layer III association fibers in the presence of 10 microM OXO-M was accompanied by a dramatic prolongation of evoked depolarizing postsynaptic potentials (PSPs), with the appearance of recurrent superimposed spike discharges. This effect was readily reversed on wash out of OXO-M. No comparable age-dependent differences were observed in the nature or time course of 1S,3R-ACPD-evoked pre- (or post)synaptic responses, even in immature cells where muscarinic epileptiform activity had previously been demonstrated. We suggest that the overall susceptibility toward muscarinic-induced epileptiform discharge in immature olfactory cortical neurons may depend on the functional integrity of presynaptic inhibitory mAChRs; additional contributing mechanisms were also considered.


Cloning of CYP2J2 gene and identification of functional polymorphisms.
CYP2J2 is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that possess potent anti-inflammatory, vasodilatory, and fibrinolytic properties. We cloned and sequenced the entire CYP2J2 gene (approximately 40.3 kb), which contains nine exons and eight introns. We then sequenced the CYP2J2 exons and intron-exon boundaries in 72 healthy persons representing African, Asian, and European/white populations as part of the National Institutes of Health/National Institute of Environmental Health Sciences Environmental Genome Single Nucleotide Polymorphism Program. A variety of polymorphisms were found, four of which resulted in coding changes (Arg158Cys, Ile192Asn, Asp342Asn, and Asn404Tyr). A fifth variant (Thr143Ala) was identified by screening a human heart cDNA library. All five variant cDNAs of CYP2J2 were generated by site-directed mutagenesis and expressed in Sf9 insect cells by using a baculovirus system. The recombinant wild-type and variant CYP2J2 proteins immunoreacted with peptide-based antibodies to CYP2J2 and displayed typical cytochrome P450 (P450) CO-difference spectra; however, the Asn404Tyr and Ile192Asn variants also had prominent spectral peaks at 420 nm. The ability of these variants to metabolize arachidonic acid and linoleic acid was compared with that of wild-type CYP2J2. Three variants (Asn404Tyr, Arg158Cys, and Thr143Ala) showed significantly reduced metabolism of both arachidonic acid and linoleic acid. The Ile192Asn variant showed significantly reduced activity toward arachidonic acid only. The Asp342Asn variant showed similar metabolism to wild-type CYP2J2 for both endogenous substrates. Based on these data, we conclude that allelic variants of the human CYP2J2 gene exist and that some of these variants result in a P450 protein that has reduced catalytic function. Insofar as CYP2J2 products have effects in the cardiovascular system, we speculate that these variants may be functionally relevant.


Radiofrequency neurotomy in chronic lumbar and sacroiliac joint pain: A meta-analysis.
Effective treatment of low back pain (LBP) originating in the lumbar and sacroiliac joints is difficult to achieve. The objective of the current study was to compare the clinical effectiveness of radiofrequency (RF) neurotomy versus conservative nonsurgical approaches for the management of chronic lumbar and sacroiliac joint pain. The PICOS framework was adhered to (P [population]: patients with a history of chronic function-limiting lumbar and sacroiliac joint pain lasting at least 6 months; I [intervention]: RF neurotomy; C [comparator]: other nonsurgical treatments; O [outcomes]: the Oswestry Disability Index (ODI), measurement for pain, and a quality of life (QoL) questionnaire; S [study design]: meta-analysis). Two trained investigators systematically searched Medline, Cochrane, EMBASE, and ISI Web of Knowledge databases for relevant studies published in English through March 2019. Patients treated with RF neurotomy (n = 528) had significantly greater improvement in ODI scores, pain scores and QoL measured by EQ-5D compared with controls (n = 457); however, significant heterogeneity was observed when data were pooled from eligible studies. In subgroup analyses, patients who received RF neurotomy had a significantly greater improvement in ODI scores compared with those with sham treatment. Patients treated with RF achieved significantly greater improvement in pain scores compared with controls who received sham treatment or medical treatment. In a subgroup analysis of pain in the sacroiliac joint and in lumbar facet joints, the RF neurotomy group achieved a significantly greater improvement in ODI score and pain scores compared with the control group. The ODI score and pain score were improved after 2 months of follow up in the analyses stratified by follow-up duration. Use of RF neurotomy as an intervention for chronic lumbar and sacroiliac joint pain led to improved function; however, larger, more directly comparable studies are needed to confirm this study's findings.


Occurrence and significance of epithelial-mesenchymal transition in breast cancer.
By contrast with developmental epithelial-mesenchymal transition (EMT), where epithelial characteristics undergo transformation to a mesenchymal-like phenotype in a coordinated fashion, oncogenic EMT occurs in the context of unpredictable genetic changes present in the tumour cells, as well as an abnormal tumour microenvironment. Therefore, a partial form of EMT has been proposed as variably participating in the establishment of invasive phenotype in different types of breast carcinoma, in keeping with their morphological and phenotypical diversity. A complex network of signalling pathways and transcription factors appears responding to various growth factors and cytokines released by stromal and neoplastic elements, endowing the system with abundant regulatory opportunities. The process of EMT is largely elusive in histopathological preparations, prompting doubts regarding its significance in tumour progression. This might be related to the presumed focal occurrence of EMT in the majority of tumours. Detailed topological studies might facilitate understanding of the orchestration of events taking place in vivo. Even more importantly, clinical correlations can be endeavoured and, in parallel with advancement in molecular pathology, a contribution to taxonomy refinement can be envisaged.


Race-dependent association of sulfidogenic bacteria with colorectal cancer.
Colorectal cancer (CRC) incidence is higher in African Americans (AAs) compared with non-Hispanic whites (NHWs). A diet high in animal protein and fat is an environmental risk factor for CRC development. The intestinal microbiota is postulated to modulate the effects of diet in promoting or preventing CRC. Hydrogen sulfide, produced by autochthonous sulfidogenic bacteria, triggers proinflammatory pathways and hyperproliferation, and is genotoxic. We hypothesised that sulfidogenic bacterial abundance in colonic mucosa may be an environmental CRC risk factor that distinguishes AA and NHW. Colonic biopsies from uninvolved or healthy mucosa from CRC cases and tumour-free controls were collected prospectively from five medical centres in Chicago for association studies. Sulfidogenic bacterial abundance in uninvolved colonic mucosa of AA and NHW CRC cases was compared with normal mucosa of AA and NHW controls. In addition, 16S rDNA sequencing was performed in AA cases and controls. Correlations were examined among bacterial targets, race, disease status and dietary intake. AAs harboured a greater abundance of sulfidogenic bacteria compared with NHWs regardless of disease status. Bilophila wadsworthia-specific dsrA was more abundant in AA cases than controls. Linear discriminant analysis of 16S rRNA gene sequences revealed five sulfidogenic genera that were more abundant in AA cases. Fat and protein intake and daily servings of meat were significantly higher in AAs compared with NHWs, and multiple dietary components correlated with a higher abundance of sulfidogenic bacteria. These results implicate sulfidogenic bacteria as a potential environmental risk factor contributing to CRC development in AAs.


Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization.
A mouse DNA polymerase accompanied by a novel RNA polymerase activity and its specific protein factor (stimulating factor) were purified from Ehrlich ascites tumor cells and partially characterized. The DNA polymerase was thought to be a subspecies of DNA polymerase alpha, and to be accompanied by or copurified with RNA polymerase activity capable of synthesizing RNA, which was probably utilized as a primer for subsequent DNA polymerization on a template of poly(dT) or poly(dC). This coupled reaction by RNA and DNA polymerase activities required the stimulating factor in addition to ribo- and deoxyribonucleotide substrates, although the degree of requirement depended on the kind of template and ribonucleotide substrate: the activity to incorporate dATP with poly(dT) plus ATP depended greatly on the stimulating factor, while the activity to incorporate dGTP with poly(dC) did not when GTP was added at high concentrations. GDP could be substituted for GTP, but the activity with poly(dC) plus GDP depended largely on the stimulating factor. Involvement of known RNA polymerases in the activity with poly(dT) was excluded, because addition of purified mouse RNA polymerases I and II had no effect on the incorporation of dATP, and alpha-amanitin (100 micrograms/ml) did not inhibit the incorporations of dATP and ATP. Analysis of the inhibition by the nucleotide analog 2',3'-dideoxynucleoside 5'-triphosphate (ddNTP) further supported the involvement of new RNA polymerase; ddNTPs inhibited the activities with poly(dT) and poly(dC) significantly more than RNA polymerases I and II or DNA polymerase alpha activity with poly(dT) . oligo(rA) and poly(dC) . oligo(dG) as template. Lineweaver-Burk analysis of the inhibitions showed that ddATP inhibited competitively with respect to ATP, and ddGTP inhibited competitively with respect to GDP but noncompetitively with respect to GTP.


Oncostatin M promotes excitotoxicity by inhibiting glutamate uptake in astrocytes: implications in HIV-associated neurotoxicity.
Elevated levels of oncostatin M (OSM), an interleukin-6 cytokine family member, have been observed in HIV-1-associated neurocognitive disorders (HAND) and Alzheimer's disease. However, the function of OSM in these disease conditions is unclear. Since deficient glutamate uptake by astrocytes is instrumental in HAND-associated neurotoxicity, we hypothesized that OSM impairs glutamate uptake in astrocytes and thereby promotes neuronal excitotoxicity. Primary cultures of mouse cortical astrocytes, neurons, microglia, and BV2 cell line were used. The expression of glutamate transporters (GLAST/EAAT1 and GLT-1/EAAT2) was investigated using real-time PCR and Western blot, and their activity was assessed by measuring (3)H-D-aspartate uptake. Neuronal toxicity was measured using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay and immunocytochemistry. A chimeric HIV-1 that infects murine cells (EcoHIV/NL4-3-GFP virus (EcoHIV)) was used to investigate whether the virus induces OSM, OSM receptor (OSMR)-β, glycoprotein 130 (gp130), GLT-1, GLAST (mRNA and protein), and OSM release (ELISA) in cultured BV2 cells, primary microglia, or astrocytes. Statistical analyses of the data were performed using one-way ANOVA (to allow multiple comparisons) and two-tailed Student's t test. OSM treatment (10 ng/mL) time-dependently reduced GLAST and GLT-1 expression and inhibited (3)H-D-aspartate uptake in cultured astrocytes in a concentration-dependent manner, an effect prevented by the Janus kinase (JAK)/signal transducers and activators of transcription (STAT)3 inhibitor AG490. Down-regulation of astrocytic glutamate transport by OSM resulted in NMDA receptor-dependent excitotoxicity in cortical neurons. Infection with EcoHIV induced OSM gene expression and protein release in BV2 cells and microglia, but not in astrocytes. Conversely, EcoHIV caused a fivefold increase in OSMR-β mRNA (but not gp130) and protein in astrocytes, but not in microglia, which did not express OSMR-β protein. Finally, astrocytic expression of GLAST gene was unaffected by EcoHIV, whereas GLT-1 mRNA was increased by twofold. We provide first evidence that activation of JAK/STAT3 signaling by OSM inhibits glutamate uptake in astrocytes, which results in neuronal excitotoxicity. Our findings with EcoHIV suggest that targeting OSMR-β signaling in astrocytes might alleviate HIV-1-associated excitotoxicity.


High-dose B-vitamin supplements and risk for age-related cataract: a population-based prospective study of men and women.
Previous studies that have investigated the association between B-vitamin supplement use and risk for cataract yield conflicting results. The aim of this study was to examine the association between use of high-dose B-vitamin supplements (approximately 10 times recommended daily intake) and risk for age-related cataract in a population-based prospective study of 13 757 women from the Swedish Mammography Cohort and 22 823 men from the Cohort of Swedish Men. Dietary supplement use and potential confounders were assessed using a questionnaire at baseline. Information on cataract diagnosis and extraction was obtained through linkage to registers. During the follow-up period between January 1998 and December 2011, we identified 8395 cataract cases (3851 for women and 4544 for men). The use of B vitamins plus other supplements and B vitamins only was associated with 9 % (95 % CI 2, 17) and 27 % (95 % CI 12, 43) increased risk for cataract, respectively. The hazard ratios for use of B vitamins only and risk for cataract stratified by different age groups were as follows: <60 years: 1·88 (95 % CI 1·47, 2·39); 60-69 years: 1·21 (95 % CI 0·96, 1·53); and ≥70 years: 1·09 (95 % CI 0·91, 1·31) (P interaction=0·002). Our results suggest that the use of high-dose B-vitamin supplements was associated with an increased risk for cataract. This association might be confined to younger participants.


Hepatitis B virus antigen: validation and immunologic characterization of low-titer serums with 125 I-antibody.
Radioimmunnoassay was used to determine the serologic subspecificities of 85 blood donor serums positive for hepatitis B virus-associated antigen. There was complete agreement with results obtained by immunoprecipitatiotn of 43 serums. The remaining 42 serums were negative by immunoprecipitation but followed type-specific immunology by radioimmunoassay, and this served as a validation for authentic hepatitis B virus antigen.


Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells.
Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9 Å and space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.


Anti-TNF drugs in patients with hepatitis B or C virus infection: safety and clinical management.
Drugs targeting TNF-α biological activity are increasingly used for the treatment of immune-mediated diseases, like rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Since TNF-α is a mediator of the immune response against viral infections, use of TNF-α inhibitors in patients with concurrent HBV or HCV infection can promote viral reactivation and potentially fatal liver failure. This paper reviews TNF mechanisms of action in viral hepatitis B and C, recommendations for managing HBV and HCV-infected patients receiving treatment with anti-TNF drugs, safety and anti-TNF hepatotoxicity. In hepatitis B surface antigen (HBsAg) carriers undergoing anti-TNF therapy, either anti-HBV treatment or prophylaxis is mandatory to prevent hepatitis reactivation, whereas HBsAg-negative antibody to hepatitis B core antigen (anti-HBc) seropositive patients require watchful monitoring, only. Conversely, in HCV-infected patients, TNF-α inhibition by specific drugs is safe and could be even beneficial, as TNF-α pathways are involved in perpetuating liver inflammation and fibrosis progression in HCV. HBV- or HCV-infected patients should be referred to a hepatologist for expert clinical management whenever antiviral therapy is deemed necessary or hepatitis reactivation occurs.


Mechanisms of apoptosis in Crustacea: What conditions induce versus suppress cell death?
Arthropoda is the largest of all animal phyla and includes about 90% of extant species. Our knowledge about regulation of apoptosis in this phylum is largely based on findings for the fruit fly Drosophila melanogaster. Recent work with crustaceans shows that apoptotic proteins, and presumably mechanisms of cell death regulation, are more diverse in arthropods than appreciated based solely on the excellent work with fruit flies. Crustacean homologs exist for many major proteins in the apoptotic networks of mammals and D. melanogaster, but integration of these proteins into the physiology and pathophysiology of crustaceans is far from complete. Whether apoptosis in crustaceans is mainly transcriptionally regulated as in D. melanogaster (e.g., RHG 'killer' proteins), or rather is controlled by pro- and anti-apoptotic Bcl-2 family proteins as in vertebrates needs to be clarified. Some phenomena like the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) are apparently lacking in crustaceans and may represent a vertebrate invention. We speculate that differences in regulation of the intrinsic pathway of crustacean apoptosis might represent a prerequisite for some species to survive harsh environmental insults. Pro-apoptotic stimuli described for crustaceans include UV radiation, environmental toxins, and a diatom-produced chemical that promotes apoptosis in offspring of a copepod. Mechanisms that serve to depress apoptosis include the inhibition of caspase activity by high potassium in energetically healthy cells, alterations in nucleotide abundance during energy-limited states like diapause and anoxia, resistance to opening of the calcium-induced MPTP, and viral accommodation during persistent viral infection. Characterization of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell death.


Internalization of carcinogenic lead chromate particles by cultured normal human lung epithelial cells: formation of intracellular lead-inclusion bodies and induction of apoptosis.
Occupational exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate, has been associated with lung cancer and respiratory tract toxicity. We have previously shown that apoptosis is a major mode of death in cultured rodent cells treated with soluble sodium chromate and particulate lead chromate. Here we report the cellular and molecular effects of lead chromate and sodium chromate in normal human lung small airway epithelial (HSAE) cells, which may be one of the targets for Cr(VI)-induced lung cancer and respiratory tract toxicity. Phagocytosed lead chromate particles and intracellular lead-inclusion bodies (LIB) were observed by transmission electron microscopy and confirmed by X-ray analysis. HSAE cells exposed to lead chromate and sodium chromate underwent dose-dependent apoptosis. The cellular uptake and genomic interactions of both Cr and lead (Pb) were examined by inductively coupled plasma mass spectrometry (ICPMS) coupled with a novel, direct-injection high-efficiency nebulizer (DIHEN). Using this approach, we have quantitated a dose-dependent formation of Cr-DNA adducts and DNA-associated Pb in lead chromate-treated HSAE cells. The formation of LIB in normal human lung cells exposed to lead chromate indicates that ionic Pb is released from the particles and thus might contribute to the cell toxicity caused by lead chromate. Internalization and dissolution of lead chromate particles and the interaction of ionic Cr and Pb with DNA, may be components of the mechanism of lead chromate carcinogenesis. Lead chromate-induced apoptosis may be a mechanism to eliminate cells with chromium- and/or lead-damaged DNA.


Keratin overexpression levels correlate with the extent of spontaneous pancreatic injury.
Mutation of the adult hepatocyte keratins K8 and K18 predisposes to liver disease. In contrast, exocrine pancreas K8 and K18 are dispensable and are co-expressed with limited levels of membrane-proximal K19 and K20. Overexpression of mutant K18 or genetic ablation of K8 in mouse pancreas is well tolerated whereas overexpression of K8 causes spontaneous chronic pancreatitis. To better understand the effect of exocrine pancreatic keratin overexpression, we compared transgenic mice that overexpress K18, K8, or K8/K18, associated with minimal, modest, or large increases in keratin expression, respectively, with nontransgenic wild-type (WT) mice. Overexpression of the type-II keratin K8 up-regulated type-I keratins K18, K19, and K20 and generated K19/K20-containing neocytoplasmic typical or short filaments; however, overexpression of K18 had no effect on K8 levels. K8- and K18-overexpressing pancreata were histologically similar to WT, whereas K8/K18 pancreata displayed age-enhanced vacuolization and atrophy of the exocrine pancreas and exhibited keratin hyperphosphorylation. Zymogen granules in K8/K18 pancreata were 50% smaller and more dispersed than their normal apical concentration but were twice as numerous as in WT controls. Therefore, modest keratin overexpression has minor effects on the exocrine pancreas whereas significant keratin overexpression alters zymogen granule organization and causes aging-associated exocrine atrophy. Keratin absence or mutation is well tolerated after pancreatic but not liver injury, whereas excessive overexpression is toxic to the pancreas but not the liver when induced under basal conditions.


Oxygen diffusion into multiwalled carbon nanotube doped polystrene latex films using fluorescence technique.
This study examines the oxygen diffusion into polystyrene (PS) latex/multiwalled carbon nanotube (MWNT) nanocomposite films (PS/MWNT) consisting of various amounts of MWNT via steady state fluorescence technique (SSF). PS/MWNT films were prepared from the mixture of MWNT and pyrene (P)-labeled PS latexes at various compositions at room temperature. These films were then annealed at 170 °C above glass transition (Tg) temperature of PS. Fluorescence quenching measurements were performed for each film separately to evaluate the effect of MWNT content on oxygen diffusion. The Stern-Volmer equation for fluorescence quenching is combined with Fick's law for diffusion to derive the mathematical expressions. Diffusion coefficients (D) were produced and found to be increased from 1.1 × 10(-12) to 41 × 10(-12) cm(2)s(-1) with increasing MWNT content. This increase was explained via the existence of large amounts of pores in composite films which facilitate oxygen penetration into the structure.


Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes.
Synthetic triterpenoids have been developed, which are potent inducers of cytoprotective enzymes and inhibitors of inflammation, greatly improving on the weak activity of naturally occurring triterpenoids. An imidazolide triterpenoid derivative, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im or TP235), has been previously shown to potently protect against hepatic tumorigenesis, acting in part by inducing cytoprotective genes through Keap1-Nrf2-antioxidant response element (ARE) signaling. In these studies, the pharmacodynamic activity of CDDO-Im is characterized in two distinct lines of ARE reporter mice and by measuring increases in Nqo1 transcript levels as a marker of cytoprotective gene induction. Oral administration of CDDO-Im induces ARE-regulated cytoprotective genes in many tissues in the mouse, including liver, lung, kidney, intestines, brain, heart, thymus, and salivary gland. CDDO-Im induces Nqo1 RNA transcripts in some organs at doses as low as 0.3 mumol/kg body weight (orally). A structure activity evaluation of 15 additional triterpenoids (a) confirmed the importance of Michael acceptor groups on both the A and C rings, (b) showed the requirement for a nitrile group at C-2 of the A ring, and (c) indicated that substituents at C-17 dramatically affected pharmacodynamic action in vivo. In addition to CDDO-Im, other triterpenoids, particularly the methyl ester CDDO-Me (TP155) and the dinitrile TP225, are extremely potent inducers of cytoprotective genes in mouse liver, lung, small intestine mucosa, and cerebral cortex. This pharmacodynamic characterization highlights the chemopreventive promise of several synthetic triterpenoids in multiple target organs.


Mycophenolate mofetil after methotrexate failure or intolerance in the treatment of scleritis and uveitis.
To evaluate the outcomes of treatment with mycophenolate mofetil in patients with scleritis and uveitis refractory to or intolerant of methotrexate. Retrospective noncomparative case series. Eighty-five patients with scleritis and/or uveitis who failed with or did not tolerate methotrexate and were subsequently treated with mycophenolate mofetil between 1998 and 2006. We reviewed medical records of patients who were treated with mycophenolate mofetil after methotrexate intolerance or failure at one tertiary uveitis referral practice. We recorded dose and duration of methotrexate and mycophenolate mofetil therapy, inflammation grade, Snellen visual acuity (VA), use of other immunomodulatory therapy, and adverse events. Multivariate logistic regression was used to identify factors associated with inflammation control. Control of inflammation, steroid-sparing effect, VA, and adverse effects were assessed. Inflammation was controlled with mycophenolate mofetil in 47 patients (55%), with 5 achieving durable remission off all medication. In multivariate logistic regression analysis that adjusted for gender and age, the odds of inflammation control were lower for patients with scleritis (odds ratio [OR], 0.19; 95% confidence interval [CI], 0.04-0.93; P = 0.04) than for patients without scleritis. Among patients without scleritis, the odds of inflammation control were lower for patients with juvenile idiopathic arthritis (JIA)-associated uveitis (OR, 0.14; CI, 0.02-0.81, P = 0.03) compared to patients without JIA-associated uveitis. Eight of the 11 patients (73%) who were taking concomitant prednisone were able to taper their dose to <10 mg daily. Visual acuity declined in a greater percentage of patients who were unresponsive to mycophenolate mofetil (29%) compared with that of patients who responded to mycophenolate mofetil (9%). Side effects requiring discontinuation of mycophenolate mofetil occurred in 18 patients (21%). Mycophenolate mofetil was effective in controlling inflammation in approximately half of the patients who had previously failed with or did not tolerate methotrexate. The odds of inflammation control were less in patients with the diagnoses of scleritis and JIA.


Use of preoperative embolization prior to Transplant nephrectomy.
After a failed transplant, management of a non-functional graft with pain or recurrent infections can be challenging. Transplant nephrectomy (TN) can be a morbid procedure with the potential for significant blood loss. Embolization of the renal artery alone has been proposed as a method of reducing complications from an in vivo failed kidney transplant. While this does yield less morbidity, it may not address an infected graft or refractory hematuria or rejection. We elected to begin preoperative embolization to assess if this would help decrease the blood loss and transfusion rate associated with TN. We performed a retrospective analysis of all patients who underwent non-emergent TN at our institution. Patients who had functioning grafts that later failed were included in analysis. TN was performed for recurrent infections, pain or hematuria. We evaluated for blood loss (EBL) during TN, transfusion rate and length of hospital stay. A total of 16 patients were identified. Nine had preoperative embolization or no blood flow to the graft prior to TN. The remaining 7 did not have preoperative embolization. The shortest time from transplant to TN was 8 months and the longest 18 years with an average of 6.3 years. Average EBL for the embolized patients (ETN) was 143.9cc compared to 621.4cc in the non-embolized (NETN) group (p=0.041). Average number of units of blood transfused was 0.44 in the ETN with only 3/9 patients requiring transfusion. The NETN patients had average of 1.29 units transfused with 5/7 requiring transfusion. The length of stay was longer for the ETN (5.4 days) compared to 3.9 in the NETN. No intraoperative complications were seen in either group and only one patient had a postoperative ileus in the NETN. Embolization prior to TN significantly decreases the EBL but does not significantly decrease transfusion rate. However, patients do require a significantly longer hospitalization with embolization due to the time needed for embolization. Larger studies are needed to determine if embolization before transplant nephrectomy reduces the transfusion rates and overall complications.


A differential display strategy identifies Cryptic, a novel EGF-related gene expressed in the axial and lateral mesoderm during mouse gastrulation.
We have developed a differential display screening approach to identify mesoderm-specific genes, relying upon the differentiation of embryonic stem (ES) cells in vitro. Using this strategy, we have isolated a novel murine gene that encodes a secreted molecule containing a variant epidermal growth factor-like (EGF) motif. We named this gene Cryptic, based on its predicted protein sequence similarity with Cripto, which encodes an EGF-related growth factor. Based on their strong sequence similarities, we propose that Cryptic, Cripto, and the Xenopus FRL-1 gene define a new family of growth factor-like molecules, which we name the 'CFC' (Cripto, Frl-1, and Cryptic) family. Analysis of Cryptic expression by in situ hybridization shows that it is expressed during gastrulation in two spatial domains that correspond to the axial and lateral mesoderm. In the first domain of expression, Cryptic expression is progressively localized to the anterior primitive streak, the head process, and the node and notochordal plate. In the second domain, Cryptic expression is initially concentrated in the lateral region of the egg cylinder, and is later found circumferentially in the intermediate and lateral plate mesoderm. Furthermore, Cryptic expression can also be detected at the early head-fold stage in the midline neuroectoderm, and consequently is an early marker for the prospective floor plate of the neural tube. Expression of Cryptic ceases at the end of gastrulation, and has not been observed in later embryonic stages or in adult tissues. Thus, Cryptic encodes a putative signaling molecule whose expression suggests potential roles in mesoderm and/or neural patterning during gastrulation.


Biofeedback in medicine: who, when, why and how?
Biofeedback is a mind-body technique in which individuals learn how to modify their physiology for the purpose of improving physical, mental, emotional and spiritual health. Much like physical therapy, biofeedback training requires active participation on the part of patients and often regular practice between training sessions. Clinical biofeedback may be used to manage disease symptoms as well as to improve overall health and wellness through stress management training. Research has shown that biofeedback interventions are efficacious in treating a variety of medical conditions, and many Americans are turning to biofeedback and other less traditional therapies for their routine healthcare.Clinical biofeedback training is growing increasingly popular in the USA, as many people are seeking out relatively new approaches to healthcare. This article provides an overview of clinical biofeedback training, outlines two models of training, details research which has established how effective biofeedback is in patients with a given disease, and describes who should be referred for biofeedback training.


Activation and inactivation of taurine efflux in hyposmotic and isosmotic swelling in cortical astrocytes: role of ionic strength and cell volume decrease.
A decrease in intracellular ionic strength appears involved in the activation of swelling-elicited 3H-taurine efflux in cortical cultured astrocytes. Hyposmotic (50%) or isosmotic urea-induced swelling leading to a decrease of intracellular ionic strength, activated 3H-taurine efflux from a rate constant of about 0.008 min(-1) to 0.33 min(-1) (hyposmotic) and 0.59 min(-1) (urea). This efflux rate was markedly lower (maximal 0.03 min(-1)) in isosmotic swelling caused by K+ accumulation, where there is no decrease in ionic strength, or in cold (10 degrees C) hyposmotic medium (maximal 0.18 min(-1)), where swelling is reduced and consequently intracellular ionic strength is less affected. Also, astrocytes pretreated with hyperosmotic medium, which recover cell volume by ion accumulation, did not release 3H-taurine when they swelled by switching to isosmotic medium, but when volume was recovered by accumulation of urea, taurine release was restored. These results point to a key role of ionic strength in the activation of osmosensitive 3H-taurine efflux. In contrast, its inactivation was independent of the change in ionic strength but appears related to the reduction in cell volume after swelling, since despite the extent or direction of the change in ionic strength, the 3H-taurine efflux did not inactivate in isosmotic KCl-elicited swelling when cell volume did not recover nor in hyposmotic swelling when RVD was impaired by replacing NaCl in the medium by permeant osmolytes.


Pay-for-performance schemes that use patient and provider categories would reduce payment disparities.
Providers that care for disproportionate numbers of disadvantaged patients tend to perform less well than other providers on quality measures commonly used in pay-for-performance programs. This can lead to the undesired effect of redistributing resources away from providers that most need them to improve care. We present a new pay-for-performance scheme that retains the motivational aspects of standard incentive designs while avoiding undesired effects. We tested an alternative incentive payment approach that started with a standard incentive payment allocation but then "post-adjusted" provider payments using predefined patient or provider characteristics. We evaluated whether such an approach would mitigate the negative effects of redistributions of payments across provider organizations in California with disparate patient populations. The post-adjustment approach nearly doubled payments to disadvantaged provider organizations and greatly reduced payment differentials across provider organizations according to patients' income, race/ethnicity, and region. The post-adjustment of payments could be a useful supplement to paying for improvement, aligning the goals of disparity reduction and quality improvement.


Vaccination with a DNA vaccine encoding herpes simplex virus type 1 VP22 linked to antigen generates long-term antigen-specific CD8-positive memory T cells and protective immunity.
Intradermal vaccination with DNA encoding herpes simplex virus type 1 (HSV-1) VP22 linked to antigen leads to spread of antigen within the epithelium and results in enhanced antigen-specific CD8+ T cell immune responses in vaccinated mice. In this study, we characterized the number of antigen-expressing dendritic cells (DCs) in the draining lymph nodes of vaccinated mice and determined whether the linkage of VP22 to antigen would influence the ability of antigen-expressing DCs to activate antigen-specific CD8+ T cells in vivo. Vaccination with DNA encoding HSV-1 VP22 linked to human papillomavirus type 16 E7 antigen generated more antigen-expressing DCs in the draining lymph nodes of vaccinated mice than E7 alone. In addition, the linkage of VP22 to E7 improved the MHC class I presentation of E7 in transfected DCs and led to enhanced activation of E7-specific CD8+ T cells. We also observed that vaccination with DNA encoding VP22 linked to E7 generated more E7-specific CD8+ memory T cells, and enhanced long-term protective antitumor immunity against an E7-expressing tumor in vaccinated mice compared with vaccination with DNA encoding E7 alone. Thus, administration of DNA encoding VP22 linked to antigen represents a plausible approach for the development of protective DNA vaccines.


Sustaining sleep spindles through enhanced SK2-channel activity consolidates sleep and elevates arousal threshold.
Sleep spindles are synchronized 11-15 Hz electroencephalographic (EEG) oscillations predominant during nonrapid-eye-movement sleep (NREMS). Rhythmic bursting in the reticular thalamic nucleus (nRt), arising from interplay between Ca(v)3.3-type Ca(2+) channels and Ca(2+)-dependent small-conductance-type 2 (SK2) K(+) channels, underlies spindle generation. Correlative evidence indicates that spindles contribute to memory consolidation and protection against environmental noise in human NREMS. Here, we describe a molecular mechanism through which spindle power is selectively extended and we probed the actions of intensified spindling in the naturally sleeping mouse. Using electrophysiological recordings in acute brain slices from SK2 channel-overexpressing (SK2-OE) mice, we found that nRt bursting was potentiated and thalamic circuit oscillations were prolonged. Moreover, nRt cells showed greater resilience to transit from burst to tonic discharge in response to gradual depolarization, mimicking transitions out of NREMS. Compared with wild-type littermates, chronic EEG recordings of SK2-OE mice contained less fragmented NREMS, while the NREMS EEG power spectrum was conserved. Furthermore, EEG spindle activity was prolonged at NREMS exit. Finally, when exposed to white noise, SK2-OE mice needed stronger stimuli to arouse. Increased nRt bursting thus strengthens spindles and improves sleep quality through mechanisms independent of EEG slow waves (<4 Hz), suggesting SK2 signaling as a new potential therapeutic target for sleep disorders and for neuropsychiatric diseases accompanied by weakened sleep spindles.


Newly identified and diverse plastid-bearing branch on the eukaryotic tree of life.
The use of molecular methods is altering our understanding of the microbial biosphere and the complexity of the tree of life. Here, we report a newly discovered uncultured plastid-bearing eukaryotic lineage named the rappemonads. Phylogenies using near-complete plastid ribosomal DNA (rDNA) operons demonstrate that this group represents an evolutionarily distinct lineage branching with haptophyte and cryptophyte algae. Environmental DNA sequencing revealed extensive diversity at North Atlantic, North Pacific, and European freshwater sites, suggesting a broad ecophysiology and wide habitat distribution. Quantitative PCR analyses demonstrate that the rappemonads are often rare but can form transient blooms in the Sargasso Sea, where high 16S rRNA gene copies mL(-1) were detected in late winter. This pattern is consistent with these microbes being a member of the rare biosphere, whose constituents have been proposed to play important roles under ecosystem change. Fluorescence in situ hybridization revealed that cells from this unique lineage were 6.6 ± 1.2 × 5.7 ± 1.0 μm, larger than numerically dominant open-ocean phytoplankton, and appear to contain two to four plastids. The rappemonads are unique, widespread, putatively photosynthetic algae that are absent from present-day ecosystem models and current versions of the tree of life.


Focus on Kir6.2: a key component of the ATP-sensitive potassium channel.
ATP-sensitive potassium (K(ATP)) channels are found in a wide variety of cell types where they couple cell metabolism to electrical activity. In glucose-sensing tissues, these channels respond to fluctuating changes in blood glucose concentration, but in other tissues they are activated only under ischemic conditions or in response to hormonal stimulation. Although K(ATP) channels in different tissues have different regulatory subunits, in almost all cases (except vascular smooth muscle) the pore-forming subunit is the inwardly rectifying K(+) channel Kir6.2. This article reviews recent studies of Kir6.2, focussing on the relation between channel structure and function, and on naturally occurring mutations in Kir6.2 that lead to human disease. New insights into the location of the ATP-binding site, the permeation pathway for K(+), and the gating of the pore provided by homology modelling are discussed in relation to functional studies. Gain-of-function mutations in Kir6.2 cause permanent neonatal diabetes mellitus (PNDM) by reducing the ATP sensitivity of the K(ATP) channel and increasing the K(ATP) current, which is predicted to inhibit beta-cell electrical activity and insulin secretion. Mutations at specific residues, that cause a greater decrease in ATP sensitivity, are associated with additional neurological symptoms. The molecular mechanism underlying the differences in ATP sensitivity produced by these two classes of mutations is discussed. We speculate on how some mutations lead to neurological disease and why no obvious cardiac symptoms are observed. We also consider the implications of these studies for type-2 diabetes.


Proteomic analysis of chemonaive pediatric osteosarcomas and corresponding normal bone reveals multiple altered molecular targets.
With a view to identify the proteins involved in transformation, metastasis or chemoresistance in pediatric osteosarcoma, we carried out a new experimental approach based on comparison of the proteomic profile of paired samples of osteosarcoma and normal bone tissues from the same patient. The proteomic profiles of five pairs of cell lines (normal vs tumoral) were obtained by two-dimensional difference gel electrophoresis. We detected 56 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify some of these proteins, 16 of which were chosen on the basis of the change of their relative abundance between osteosarcomas and paired normal bones and also because their involvement was supported by the genomic analysis. Two of the 16 proteins, Alpha-crystallin B chain (CRYAB) and ezrin (EZR1), were selected for further studies: an immunohistochemical analysis of a TMA (tissue microarray) and real-time PCR for a set of 14 osteosarcoma/normal-bone pairs. The results of this second tier of studies confirmed that there were significant increases in the amounts of CRYAB and ezrin, especially in advanced stages of the disease. Our overall conclusion is that proteomic profiling of paired samples of osteosarcoma and normal bone tissues from the same patient is a practicable and potentially powerful way of initiating and proceeding with a search for proteins and genes involved in pediatric osteosarcoma.


Antibiotics increase gut metabolism and antioxidant proteins and decrease acute phase response and necrotizing enterocolitis in preterm neonates.
The appropriate use of antibiotics for preterm infants, which are highly susceptible to develop necrotizing enterocolitis (NEC), is not clear. While antibiotic therapy is commonly used in neonates with NEC symptoms and sepsis, it remains unknown how antibiotics may affect the intestine and NEC sensitivity. We hypothesized that broad-spectrum antibiotics, given immediately after preterm birth, would reduce NEC sensitivity and support intestinal protective mechanisms. Preterm pigs were treated with antibiotics for 5 d (oral and systemic doses of gentamycin, ampicillin and metrodinazole; AB group) and compared with untreated pigs. Only the untreated pigs showed evidence of NEC lesions and reduced digestive function, as indicated by lowered villus height and activity of brush border enzymes. In addition, 53 intestinal and 22 plasma proteins differed in expression between AB and untreated pigs. AB treatment increased the abundance of intestinal proteins related to carbohydrate and protein metabolism, actin filaments, iron homeostasis and antioxidants. Further, heat shock proteins and the complement system were affected suggesting that all these proteins were involved in the colonization-dependent early onset of NEC. In plasma, acute phase proteins (haptoglobin, complement proteins) decreased, while albumin, cleaved C3, ficolin and transferrin increased. Depressed bacterial colonization following AB treatment increases mucosal integrity and reduces bacteria-associated inflammatory responses in preterm neonates. The plasma proteins C3, ficolin, and transferrin are potential biomarkers of the colonization-dependent NEC progression in preterm neonates.


Delayed visual deficits and monocular blindness after endovascular treatment of large and giant paraophthalmic aneurysms.
Paraophthalmic aneurysms may exert mass effect on the optic apparatus. Although surgical clipping and endovascular coiling of these aneurysms can be complicated by immediate postoperative visual deterioration, endovascular coil embolization has the unique risk of visual complications later (>24 h) in the perioperative period. Six patients with a delayed onset of vision loss after technically successful coil embolization of paraophthalmic region aneurysms were identified. All available clinical, angiographic, and cross sectional imaging for these patients, in addition to histopathological data, were reviewed. Six patients who underwent endovascular treatment of paraclinoid aneurysms at our institutions developed delayed postoperative visual decline. Four were treated with combination hydrogel-coated and bare platinum coils, one with hydrogel-coated coils, and one with bare platinum coils. Three patients presented with some degree of visual impairment caused by their aneurysms. Catheter angiography performed after the visual decline revealed no etiology in any of the cases. Magnetic resonance imaging was performed in all patients and was unremarkable in two. At follow-up, two had improved, three remained unchanged, and one patient died before any follow-up assessment of her vision. Both acute and delayed visual disturbances can present after the endovascular treatment of carotid artery paraophthalmic aneurysms. Delayed visual deterioration can be observed up to 35 days after embolization. Although the cause is still undefined, it is likely that the more delayed visual deterioration can be attributed to progression of mass effect and/or perianeurysmal inflammatory change. Our case series raises the possibility that this phenomenon may be more likely with HydroCoil (HydroCoil Embolic System; MicroVention, Aliso Viejo, CA). This possibility should be taken into account by neurointerventionists when selecting a coil type to treat large paraophthalmic aneurysms.