Effect of the (+)-CC-1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase.
(+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3-adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening [Lee, C.-S., Sun, D., Kizu, R., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 203-213]. In the studies described here, in which we have constructed site-directed DNA adducts on single-stranded DNA templates, we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead us to suspect that it is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)


The Intervention Probability Curve: Modeling the Practical Application of Threshold-Guided Decision-Making, Evaluated in Lung, Prostate, and Ovarian Cancers.
Diagnostic prediction models are useful guides when considering lesions suspicious for cancer, as they provide a quantitative estimate of the probability that a lesion is malignant. However, the decision to intervene ultimately rests on patient and physician preferences. The appropriate intervention in many clinical situations is typically defined by clinically relevant, actionable subgroups based upon the probability of malignancy. However, the "all-or-nothing" approach of threshold-based decisions is in practice incorrect. Here, we present a novel approach to understanding clinical decision-making, the intervention probability curve (IPC). The IPC models the likelihood that an intervention will be chosen as a continuous function of the probability of disease. We propose the cumulative distribution function as a suitable model. The IPC is explored using the National Lung Screening Trial and the Prostate Lung Colorectal and Ovarian Screening Trial datasets. Fitting the IPC results in a continuous curve as a function of pretest probability of cancer with high correlation (R2 > 0.97 for each) with fitted parameters closely aligned with professional society guidelines. The IPC allows analysis of intervention decisions in a continuous, rather than threshold-based, approach to further understand the role of biomarkers and risk models in clinical practice. We propose that consideration of IPCs will yield significant insights into the practical relevance of threshold-based management strategies and could provide a novel method to estimate the actual clinical utility of novel biomarkers.


Selective endothelin-A receptor blockade attenuates endotoxin-induced pulmonary hypertension and pulmonary vascular dysfunction.
Endothelin-1 is a potent mediator of sepsis-induced pulmonary hypertension (PH). The pulmonary vascular effects of selective blockade of endothelin receptor subtype A (ETAR) during endotoxemia remain unknown. We hypothesized that selective ETAR antagonism attenuates endotoxin-induced PH and improves pulmonary artery (PA) vasoreactivity. Adult male Sprague-Dawley rats (250-450 g) received lipopolysaccharide (LPS; Salmonella typhimurium; 20 mg/kg intraperitoneally) or vehicle 6 hours before hemodynamic assessment and tissue harvest. The selective ETAR antagonist sitaxsentan (10 or 20 mg/kg) or vehicle was injected intravenously 3 hours after receipt of LPS. Right ventricular systolic pressure, mean arterial pressure (MAP), cardiac output (CO), oxygenation (P/F ratio), and serum bicarbonate were measured. Bronchoalveolar lavage (BAL) cell differential and lung wet-to-dry ratios were obtained. Endothelium-dependent and endothelium-independent vasorelaxations were determined in isolated PA rings. PA interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) were measured. LPS caused PH, decreased MAP, CO, and serum bicarbonate, and increased PA IL-1β, IL-6, TNF-α, and iNOS mRNA. Sitaxsentan attenuated sepsis-induced PH and increased MAP. The P/F ratio, CO, serum bicarbonate, and BAL neutrophilia were not affected by sitaxsentan. In isolated PA rings, while not affecting phenylephrine-induced vasocontraction or endothelium-dependent relaxation, sitaxsentan dose-dependently attenuated LPS-induced alterations in endothelium-independent relaxation. PA cytokine mRNA levels were not significantly attenuated by ETAR blockade. We conclude that ETAR blockade attenuates endotoxin-induced alterations in systemic and PA pressures without negatively affecting oxygenation. This protective effect appears to be mediated not by attenuation of sepsis-induced cardiac dysfunction, acidosis, or alveolar inflammation but rather by improved endothelium-independent vasorelaxation.


Spontaneous Activation of Event Details in Episodic Future Simulation.
Episodic future simulation is supported by both the retrieval and recombination of episodic details. It remains unclear, however, how individuals retrieve episodic details from memory to construct possible future scenarios; for this people must use details related to the planned future events appropriately. A potentially relevant cognitive process is the spontaneous activation of intention observed in prospective memory (i.e., the intention superiority effect). Previous studies on prospective memory have shown that the approximation of retrieval opportunities for future intentions activate related information, suggesting that the intention superiority effect is context-sensitive. We hypothesized that the same cognitive process underlies future simulation-that is, details related to future events should spontaneously become activated at the appropriate moment of future simulation to make that simulation plausible. In Experiment 1, participants took part in future experiments and formed intentions to perform particular actions for the next experiments. Subsequently, they imagined events that could occur up until they arrived at the experimental room on the day of the next experiment. During this exercise, they did not imagine engaging in the required experimental task. We measured the conceptual activation of intention-related information via a recognition task using intended action words as targets. The results showed the intention superiority effect-concepts related to participants' future intentions became active when envisioning future events approaching the next experiment. In Experiments 2 and 3, we ensured that the intention superiority effect in future simulation was context-sensitive by adding a control condition that required participants to imagine events other than the approaching future experiments. These results indicated that concepts related to the intended actions were spontaneously activated when imagined future events became both temporally and spatially close to the future simulation. Our finding suggests that spontaneous activation of details approaching the context of a future simulation helps in constructing plausible future scenarios.


Basal Ganglia Neuromodulation Over Multiple Temporal and Structural Scales-Simulations of Direct Pathway MSNs Investigate the Fast Onset of Dopaminergic Effects and Predict the Role of Kv4.2.
The basal ganglia are involved in the motivational and habitual control of motor and cognitive behaviors. Striatum, the largest basal ganglia input stage, integrates cortical and thalamic inputs in functionally segregated cortico-basal ganglia-thalamic loops, and in addition the basal ganglia output nuclei control targets in the brainstem. Striatal function depends on the balance between the direct pathway medium spiny neurons (D1-MSNs) that express D1 dopamine receptors and the indirect pathway MSNs that express D2 dopamine receptors. The striatal microstructure is also divided into striosomes and matrix compartments, based on the differential expression of several proteins. Dopaminergic afferents from the midbrain and local cholinergic interneurons play crucial roles for basal ganglia function, and striatal signaling via the striosomes in turn regulates the midbrain dopaminergic system directly and via the lateral habenula. Consequently, abnormal functions of the basal ganglia neuromodulatory system underlie many neurological and psychiatric disorders. Neuromodulation acts on multiple structural levels, ranging from the subcellular level to behavior, both in health and disease. For example, neuromodulation affects membrane excitability and controls synaptic plasticity and thus learning in the basal ganglia. However, it is not clear on what time scales these different effects are implemented. Phosphorylation of ion channels and the resulting membrane effects are typically studied over minutes while it has been shown that neuromodulation can affect behavior within a few hundred milliseconds. So how do these seemingly contradictory effects fit together? Here we first briefly review neuromodulation of the basal ganglia, with a focus on dopamine. We furthermore use biophysically detailed multi-compartmental models to integrate experimental data regarding dopaminergic effects on individual membrane conductances with the aim to explain the resulting cellular level dopaminergic effects. In particular we predict dopaminergic effects on Kv4.2 in D1-MSNs. Finally, we also explore dynamical aspects of the onset of neuromodulation effects in multi-scale computational models combining biochemical signaling cascades and multi-compartmental neuron models.


Editing of transfer RNAs in Acanthamoeba castellanii mitochondria.
With the discovery of RNA editing, a process whereby the primary sequence of RNA is altered after transcription, traditional concepts of genetic information transfer had to be revised. The known RNA editing systems act mainly on messenger RNAs, introducing sequence changes that alter their coding properties. An editing system that acts on transfer RNAs is described here. In the mitochondria of Acanthamoeba castellanii, an amoeboid protozoan, certain transfer RNAs differ in sequence from the genes that encode them. The changes consist of single-nucleotide conversions (U to A, U to G, and A to G) that appear to arise posttranscriptionally, are localized in the acceptor stem, and have the effect of correcting mismatched base pairs. Editing thus restores the base pairing expected of a normal transfer RNA in this region.


Future of genetics of mood disorders research.
This report summarizes the deliberations of a panel with representation from diverse disciplines of relevance to the genetics of mood disorders. The major charge to the panel was to develop a strategic plan to employ the tools of genetics to advance the understanding, treatment, and outcomes for mood disorders. A comprehensive review of the evidence for the role of genetic factors in the etiology of mood disorders was conducted, and the chief impediments for progress in gene identification were identified. The National Institute of Mental Health (NIMH) portfolios in the Genetics Research Branch and the Division of Mental Disorders, Behavioral Sciences, AIDS, and all genetics training activities were reviewed. Despite some promising leads, there are still no confirmed linkage findings for mood disorders. Impediments to gene finding include the lack of phenotypic validity, variation in ascertainment sources and methodology across studies, and genetic complexity. With respect to linkage, the committee recommended that a large-scale, integrated effort be undertaken to examine existing data from linkage and association studies of bipolar disorders using identical phenotypes and statistical methods across studies to determine whether the suggestive linkage findings at some loci can be confirmed. Confirmation would justify more intensive approaches to gene finding. The committee recommended that the NIMH support continued efforts to identify the most heritable subtypes and endophenotypes of major depression using the tools of genetic epidemiology, neuroscience, and behavioral science. The field of genetic epidemiology was identified as an important future direction because population-based, epidemiologic studies of families and unrelated affected individuals assume increasing importance for common chronic diseases. To prepare for shifts to more complex genetic models, the committee recommended that the NIMH develop new interdisciplinary training strategies to prepare for the next generation of genetics research.


Silver and gold glyconanoparticles for colorimetric bioassays.
The color changes associated with the aggregation of metal nanoparticles has led to the development of colorimetric-based assays for a variety of target species. We have examined both silver- and gold-based nanoparticles in order to establish whether either metal exhibits optimal characteristics for bioassay development. These silver and gold nanoparticles have been stabilized with a self-assembled monolayer of a mannose derivative (2-mercaptoethyl alpha-d-mannopyranoside) with the aim of inducing aggregation by exploiting the well-known interaction between mannose and the lectin Concanavalin A (Con A). Both metal glyconanoparticles were determined to be ca. 16 nm in diameter (using TEM measurements). Aggregation was observed on addition of Con A to both silver and gold nanoparticles resulting in a shift in the surface plasmon absorption band and a consequent color change of the solution, which was monitored using UV-visible spectrophotometry. Mannose-stabilized silver nanoparticles at a concentration of 3 nM provide an assay for Con A with the largest linear range (between 0.08 and 0.26 microM). Additionally, the kinetic rate of aggregation of the silver-nanoparticle-based bioassay was significantly greater than that of the gold-nanoparticle system. However, in terms of sensitivity, the mannose-stabilized gold-nanoparticle-based assay was optimum with a limit of detection of 0.04 microM Con A, as compared with a value of 0.1 microM obtained for the mannose-stabilized silver nanoparticles. Additionally, a lactose derivative (11-mercapto-3,6,9-trioxaundecyl beta-D-lactoside) was used to stabilize gold nanoparticles to induce aggregation upon addition of the galactose specific lectin Ricinus communis agglutinin (RCA(120)). To examine the specificity of the bioassay, lactose-stabilized gold nanoparticles were mixed with a solution of mannose-stabilized silver nanoparticles to give an aggregation assay capable of detecting two different lectins. When either Con A or RCA(120) was added to the mixed glyconanoparticles, selective recognition of the respective natural ligand was shown by aggregation of a single metal nanoparticle. Centrifugation and removal of the aggregated species enabled further bioassay measurements using the second glyconanoparticle system.


Role of the central melanocortin system in cachexia.
Individuals affected with either acute or chronic diseases often show disorders of nutrient balance. In some cases, a devastating state of malnutrition known as cachexia arises, brought about by a synergistic combination of a dramatic decrease in appetite and an increase in metabolism of fat and lean body mass. Stimulation of the hypothalamic melanocortin 4 receptor (MC4-R) produces relative anorexia and increased metabolic rate, even in a relatively starved state. Here we demonstrate that cachexia induced by lipopolysaccharide administration and by tumor growth is ameliorated by central MC4-R blockade. MC4-R knock-out mice or mice administered the MC3-R/MC4-R antagonist, agouti-related peptide, resist tumor-induced loss of lean body mass, and maintain normal circadian activity patterns during tumor growth. The final tumor mass is not affected in these animals, providing further support for the potential role of MC4-R antagonism in the treatment of cachexia in disease states.


Intravaginal prostaglandin-E2 for cervical priming and induction of labour.
A prospective study examined the safety, efficacy and labour outcome in 436 women undergoing labour induction using intravaginal prostaglandin E2. Women with singleton pregnancies (235 nulliparas and 201 multiparas) were recruited if they had a clinically unfavourable cervix, and indications for induction. The mean (standard deviation) interval from initiation to delivery was statistically significantly shorter in multiparas than nulliparas: 13.5 hours (SD 1.8) versus 15.5 hours (SD 2.4). No more than 2 x 3 mg tablets were needed to achieve a clinically feasible cervix for amniotomy. The overall need for oxytocin augmentation of labour was 42%, significantly higher in nulliparas (47%) than multiparas (35%). Intrapartum complications, caesarean section and perinatal deaths showed no statistically significant differences between the groups.


EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract.
Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.


Exposure to hand-transmitted vibration and pain in the neck and upper limbs.
Workers exposed to hand-transmitted vibration (HTV) often experience aches and pains in the upper limbs, but there have been few studies of the pattern and severity of symptoms, or their relationship to the estimated dose of vibration. As part of a wider survey of vibration, we mailed a questionnaire about exposures to HTV and pain in the neck and upper limbs to a sample of men selected at random from the registration lists of 34 British general practices and the pay records of the armed services. Analysis was confined to the 1856 male respondents who had been employed in manual occupations for a year or more and who reported the last week as being representative of their job. Inquiry was made about pain in the neck, shoulder, elbow and wrist/hand over the past week and past year (including pains that limited normal activity). Subjects were classed according to their lifetime exposure to HTV and their estimated average daily vibration dose [A(8) r.m.s.] in the previous week. A total of 283 men had a minimum estimated A(8) greater than a proposed action level of 2.8 m/s(2), and in this group symptoms were about twice as prevalent as in manual workers who had never used vibratory tools. The excess risk was somewhat higher for distal sites than for proximal ones (e.g. the prevalence ratio for hand/wrist pain in the past week was 2.7 versus 1.8 for neck pain). This accords with the pattern of transmission of HTV to the upper limb, although a confounding effect from other ergonomic risk factors cannot be discounted.


RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin.
We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation.IMPORTANCE Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli, the Gram-negative bacterial paradigm, and V. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage.


Mid-term radiological and clinical results of incomplete triple pelvic osteotomy.
The aim of this study was to assess clinical and radiological results of incomplete triple pelvic osteotomy in acetabular dysplasia. Twenty-six hips of 24 patients (5 males, 19 females) treated with incomplete triple pelvic osteotomy by a single surgeon from February 1995 to October 2001 were retrospectively reviewed at an average follow-up time of 12 years. The mean age at the time of surgery was 21.6 years (range: 14-41). Radiological evaluation was based on the central edge angle, acetabular angle, acetabular index, acetabular head index and lateralisation. Clinical and radiological scoring was performed using the Harris scoring system, Ömeroğlu scoring system and the Tönnis criteria for osteoarthritis. There was significant improvements in all of the radiological parameters with 88.5% good radiological results, 96.2% excellent clinical results, no significant progression to osteoarthritis and no need for conversion to total hip arthroplasty. The rate of major complication was 11%. Retroversion was seen in 15.4% of the hips. Our results support the use of incomplete triple pelvic osteotomy as a safe choice in the treatment of acetabular dysplasia as it offers clinical and radiological benefits and contributes to the prevention of osteoarthritis. Level IV, therapeutic study.


Light-up probe based on AIEgens: dual signal turn-on for caspase cascade activation monitoring.
Direct monitoring of multiple enzyme activities in a given biological process is extremely important for disease diagnosis. Herein, we report a single fluorescent probe that targets two caspase activities in living cells. The probe consists of three parts that includes two AIE fluorogens with distinctive green and red emission colors excitable at a single wavelength, and a hydrophilic peptide as the substrate of the apoptosis initiator caspase-8 and the effector caspase-3. The probe is non-fluorescent in aqueous media. The green and red fluorescence can be sequentially turned on when the peptide substrate is cleaved by the cascade activation of caspase-8 and caspase-3 in early apoptotic HeLa cells induced by hydrogen peroxide. This sequential fluorescence turn-on allows real-time monitoring of the caspase cascade activation during the apoptotic process, which was further explored for evaluating the therapeutic efficiency of anticancer drugs. The probe design strategy developed in this study also proved to be general, which opens a new avenue for real-time, multiplexed imaging of cellular enzyme activity in a biological process.


Rupture of the extensor pollicis longus tendon.
Rupture of the extensor pollicis longus (EPL) tendon after nondisplaced fracture of the distal radius is a known complication, but can also occur in nonrheumatoid patients without a history of fracture. This study is a retrospective analysis of seven patients treated between 1985 and 1992. Five EPL ruptures occurred with nondisplaced Colles' fractures. Two involved a chronic tenosynovitis of the second dorsal extensor compartment of the wrist. The age of the patients ranged from 29 years to 68 years (mean, 42 years). Length of follow-up ranged from 6 weeks to 62 months (mean, 30 months). Rupture occurred at a median of 7 weeks (range, 2 weeks to 11 months) from the time of the distal radius fracture. Three extensor indicis proprius tendons and four free palmaris longus tendon grafts were performed. At final follow-up, six patients had an extensor lag < 10 degrees or normal thumb interphalangeal joint motion. One patient had a 25 degrees extensor lag. The causes of EPL rupture include mechanical irritation, attrition, and vascular impairment leading to delayed rupture. Synovitis of the extensor carpi radialis due to repetitive use may invade the EPL tendon and lead to rupture. Extensor indicis proprius transfer or free palmaris longus grafts yield good results after EPL rupture.


Women who fall by the roadside: gender, sexual risk and alcohol in rural Uganda.
To investigate community perceptions about the different relationship between alcohol consumption and sexual risk-taking for men and women in a high HIV prevalence African setting. Participatory learning and action (PLA) activities were conducted in five rural villages in south-western Uganda in 2002. For each village, discussions and visualization activities over the course of 5 days explored local concepts of fun, drinking alcohol and HIV-related behavioural risks. Twelve focus group discussions (FGDs) investigated emerging themes. Analysis is based on visual outputs, observation notes and focus group transcripts. Attendance at sex-segregated PLA sessions was open to all village residents. FGDs were purposively sampled from drinkers and general population groups. For men, drinking is conducted invariably outside the home, usually at night in bars, emphasizing independence, masculinity and freedom from domestic responsibilities. For women, drinking outside male supervision challenges feminine ideals of domesticity and signifies potential sexual vulnerability. Accepting drinks from men was viewed as signifying assent to sex and refusal could justify men resorting to sexual coercion. Even though drinking is seen to promote sexual risk, HIV prevention campaigns were considered unwelcome in bars. Communities preferred seminars involving drinkers and non-drinkers alike. Public drinking in this community serves as a marker for men willing to exercise privileges of independence (sexual and otherwise) and women willing to defy gender norms (and risk the sexual consequences). The social and symbolic context of drinking suggests why effective HIV prevention around alcohol should not be limited to drinking environments alone.


Gene Essentiality Analyzed by In Vivo Transposon Mutagenesis and Machine Learning in a Stable Haploid Isolate of Candida albicans.
Knowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungus Candida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicans essential genes, including 1,195 with high "essentiality confidence" scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential in C. albicans were 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogens C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Histoplasma capsulatum and highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies.IMPORTANCE Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.


TNK-tissue plasminogen activator in acute myocardial infarction. Results of the Thrombolysis in Myocardial Infarction (TIMI) 10A dose-ranging trial.
TNK-tissue plasminogen activator (TNK-TPA) is a genetically engineered variant of TPA, which in experimental models has a slower plasma clearance and greater fibrin specificity and is 80-fold more resistant to plasminogen activator inhibitor-1 than alteplase TPA. The thrombolysis in Myocardial Infarction (TIMI) 10A trial was a Phase 1, dose-ranging pilot trial designed to evaluate the pharmacokinetics, safety, and efficacy of TNK-TPA in patients with acute myocardial infarction. One hundred thirteen patients with acute ST-segment elevation myocardial infarction presenting within 12 hours and without contraindications to thrombolysis were enrolled and treated with a single bolus of TNK-TPA over 5 to 10 seconds with doses ranging from 5 to 50 mg. TNK-TPA demonstrated a plasma clearance of 151 +/- 55 mL/min and a half-life of 17 +/- 7 minutes. Comparable values for wild-type TPA are 572 +/- 132 mL/min and 3.5 +/- 1.4 minutes, respectively. Systemic fibrinogen and plasminogen levels fell by only 3% and 13%, respectively, at 1 hour after TNK-TPA administration. TIMI grade 3 flow at 90 minutes was achieved in 57% to 64% of patients at the 30- to 50-mg doses. Seven patients (6.2%) experienced a major hemorrhage, which occurred at a vascular access site in six patients. TNK-TPA has a prolonged half-life so it can be administered as a single bolus. TNK-TPA appears to be very fibrin specific, and the initial patency and safety profiles are encouraging. Further study of this new thrombolytic agent is ongoing.


Cytochrome P450 isozymes catalyzing the hepatic microsomal oxidation of 9-anthraldehyde to 9-anthracene carboxylic acid in adult male rats.
Microsomal aldehyde oxygenase (MALDO) activity for 9-anthraldehyde (9-AA) was significantly higher in the male than in the female adult rat liver. 9-AA MALDO activity was also significantly enhanced by pretreatment with dexamethasone and phenobarbital, whereas it was not significantly changed by 3-methylcholanthrene or acetone. Several cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyze the oxidation of 9-AA to 9-anthracene carboxylic acid (9-ACA) in the presence of NADPH, NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. Under the ordinary conditions of the reconstituted system, the catalytic activities (nmol/min/nmol P450) of cytochrome P450s, 2A1, 2B2, 2C6, 2C11 and 3A2 were 1.53 (1.37 in the presence of cytochrome b5), 1.20 (2.06), 4.87 (7.75), 18.0 (21.6) and 0.90 (1.17), respectively. Cytochrome P450 2C11 (CYP 2C11) showed the highest catalytic activity of the cytochromes examined. In the reconstituted system using the lipids extracted from microsomes, CYP 3A2 more effectively catalyzed the oxidation of 9-AA to 9-ACA, and its catalytic activity (nmol/min/nmol P450) was 3.33 or 6.61 in the absence or presence of cytochrome b5, respectively. The antibody against CYP 2C11 inhibited by 90% the hepatic microsomal oxidation of 9-AA MALDO activity in adult male rats, but the activity was not inhibited by antibody against CYP 3A2. These results show that the individual forms of cytochrome P450 have a catalytic activity for the oxidation of 9-AA to 9-ACA, and that CYP 2C11 is the major constitutive catalyst of 9-AA MALDO activity in untreated adult male rat liver.


Loss of 'complexity' and aging. Potential applications of fractals and chaos theory to senescence.
The concept of "complexity," derived from the field of nonlinear dynamics, can be adapted to measure the output of physiologic processes that generate highly variable fluctuations resembling "chaos." We review data suggesting that physiologic aging is associated with a generalized loss of such complexity in the dynamics of healthy organ system function and hypothesize that such loss of complexity leads to an impaired ability to adapt to physiologic stress. This hypothesis is supported by observations showing an age-related loss of complex variability in multiple physiologic processes including cardiovascular control, pulsatile hormone release, and electroencephalographic potentials. If further research supports this hypothesis, measures of complexity based on chaos theory and the related geometric concept of fractals may provide new ways to monitor senescence and test the efficacy of specific interventions to modify the age-related decline in adaptive capacity.


Performance of Dental Hygiene Students in Mass Fatality Training and Radiographic Imaging of Dental Remains.
Mass fatality incidents can overwhelm local, state and national resources quickly. Dental hygienists are widely distributed and have the potential to increase response teams' capacity. However, appropriate training is required. The literature is void of addressing this type of training for dental hygienists and scant in dentistry. Hence, the purpose of this study was to assess one facet of such training: Whether the use of multimedia is likely to enhance educational outcomes related to mass fatality training. A randomized, double-blind, pre- and post-test design was used to evaluate the effectiveness of comparable educational modules for 2 groups: a control group (n=19) that received low media training and a treatment group (n=20) that received multimedia training. Participants were second-year, baccalaureate dental hygiene students. Study instruments included a multiple-choice examination, a clinical competency-based radiology lab scored via a standardized rubric, and an assessment of interest in mass fatality education as a specialty. ANOVA was used to analyze results. Participants' pre- and post-test scores and clinical competency-based radiology lab scores increased following both educational approaches. Interest in mass fatality training also increased significantly for all participants (p=0.45). There was no significant difference in pre- and post-test multiple choice scores (p=0.6455), interest (p=0.9133) or overall competency-based radiology lab scores (p=0.997) between groups. Various educational technique may be effective for mass fatality training. However, mass fatality training that incorporates multimedia is an appropriate avenue for training instruction. Continued research about multimedia's role in this specialty area is encouraged.


[Sternal tuberculosis].
Isolated involvement of the sternum is rare, representing less than 1% of tuberculous osteomyelitis. In this report, a 51-years-old woman who was admitted to the hospital with a localized solid mass in the sternum has been presented. A soft, painful mass measuring 3 cm in diameter in the lower sternum was detected during physical examination. Radiological investigation revealed presternal soft tissue and bone expansion in the posterior side of lower sternum. The patient was treated with non-specific antibiotics for a suspected diagnosis of osteomyelitis but the lesion did not show any regression. Since the microbiological analysis of fine needle aspiration fluid demonstrated acid-fast bacilli in direct microscopy and Mycobacterium tuberculosis was isolated from the culture, anti-tuberculous therapy consisting of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and ethambutol (ETM) was started. Sputum and urine cultures of the patient yielded negative results in terms of tuberculosis. After the first month of the therapy, her skin lesion was completely healed. Since the strain was found to be resistant to isoniazid, the maintenance therapy has been applied as INH + RIF + PZA for nine months. The history of the patient indicated that one of her relatives had skin tuberculosis on the face. As a result the patient has been successfully treated with anti-tuberculosis combination therapy together with surgical debridement.


A study protocol for a randomised controlled trial evaluating clinical effects of platelet transfusion products: the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) trial.
Patients with chemotherapy-induced thrombocytopaenia frequently experience minor and sometimes severe bleeding complications. Unrestrictive availability of safe and effective blood products is presumed by treating physicians as well as patients. Pathogen reduction technology potentially offers the opportunity to enhance safety by reducing bacterial and viral contamination of platelet products along with a potential reduction of alloimmunisation in patients receiving multiple platelet transfusions. To test efficacy, a randomised, single-blinded, multicentre controlled trial was designed to evaluate clinical non-inferiority of pathogen-reduced platelet concentrates treated by the Mirasol system, compared with standard plasma-stored platelet concentrates using the percentage of patients with WHO grade ≥ 2 bleeding complications as the primary endpoint. The upper limit of the 95% CI of the non-inferiority margin was chosen to be a ≤ 12.5% increase in this percentage. Bleeding symptoms are actively monitored on a daily basis. The adjudication of the bleeding grade is performed by 3 adjudicators, blinded to the platelet product randomisation as well as by an automated computer algorithm. Interim analyses evaluating bleeding complications as well as serious adverse events are performed after each batch of 60 patients. The study started in 2010 and patients will be enrolled up to a maximum of 618 patients, depending on the results of consecutive interim analyses. A flexible stopping rule was designed allowing stopping for non-inferiority or futility. Besides analysing effects of pathogen reduction on clinical efficacy, the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) is designed to answer several other pending questions and translational issues related to bleeding and alloimmunisation, formulated as secondary and tertiary endpoints. Ethics approval was obtained in all 3 participating countries. Results of the main trial and each of the secondary endpoints will be submitted for publication in a peer-reviewed journal. NTR2106; Pre-results.


"You've got to settle down!": Mothers' perceptions of physical activity in their young children.
Mothers are important mediators of children's physical activity (PA) level and risk of obesity, however previous studies of maternal perceptions of child PA have been limited. Furthermore, it is unknown if maternal perceptions of child PA are predicted by family, mother and child characteristics. Therefore objectives of this study were to 1) evaluate maternal perceptions of PA in their children and 2) test associations of family, mother and child characteristics with these perceptions. 278 low-income mothers of children (mean age 70.9 months) participated in an audio-taped semi-structured interview. Transcripts were systematically analyzed using the constant comparative method and themes were generated. A coding scheme to classify the themes appearing in each transcript was developed and reliably applied. Anthropometrics were measured. Demographics and questionnaires (the Confusion, Hubbub and Order Scale, The Parenting Scale, and the Child Behavior Questionnaire (CBQ)) were collected. Logistic regression models were used to test the associations of family, mother and child characteristics with each theme. In this sample of low-income United States mothers, two themes emerged: 1) Mothers perceive their children as already very active (87.8%, n = 244), predicted by the child being younger, the child not being overweight, and higher child CBQ Activity Level; and 2) Mothers view their children's high activity level as problematic (27.0%, n = 75), predicted by lower Parenting Laxness, the child being male and lower child CBQ Inhibitory Control. Low-income United States mothers have unique perceptions of PA in their children; these beliefs are associated with characteristics of the child and mother but not characteristics of the family. Further understanding of contributors to maternal perceptions of child PA may inform future childhood obesity interventions. The influence of these perceptions on physical activity outcomes in low-income children should be pursued in future research.


Immune responses in children after vaccination with a typhoid Vi-tetanus toxoid conjugate vaccine in Bangladesh.
A cluster-randomized trial of Vi-TT was conducted in Dhaka, Bangladesh, using JE vaccine as the control. A subset of 1,500 children were randomly selected on 2:1 basis (Vi-TT vs JE) to assess immune response. Blood was collected before vaccination, and on days 28, 545 and 730 post-vaccination and plasma anti-Vi-IgG response was measured. A robust, persistent antibody response was induced after single dose of Vi-TT, even after 2 years of vaccination. While there is no accepted serological antibody threshold of protection, analyzing the antibodies of children who received Vi-TT provides evidence that may later be useful in predicting population protection.


Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents.
BioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria. To date, very few bioH genes have been characterized. In this study, we cloned and identified a novel bioH gene, bioHx, from an environmental metagenome by a functional metagenomic approach. The bioHx gene, encoding an enzyme that is capable of hydrolysis of p-nitrophenyl esters of fatty acids, was expressed in Escherichia coli BL21 using the pET expression system. The biochemical property of the purified BioHx protein was also investigated. Screening of an unamplified metagenomic library with a tributyrin-containing medium led to the isolation of a clone exhibiting lipolytic activity. This clone carried a 4,570-bp DNA fragment encoding for six genes, designated bioF, bioHx, fabG, bioC, orf5 and sdh, four of which were implicated in the de novo biotin biosynthesis. The bioHx gene encodes a protein of 259 aa with a calculated molecular mass of 28.60 kDa, displaying 24-39% amino acid sequence identity to a few characterized bacterial BioH enzymes. It contains a pentapeptide motif (Gly76-Trp77-Ser78-Met79-Gly80) and a catalytic triad (Ser78-His230-Asp202), both of which are characteristic for lipolytic enzymes. BioHx was expressed as a recombinant protein and characterized. The purified BioHx protein displayed carboxylesterase activity, and it was most active on p-nitrophenyl esters of fatty acids substrate with a short acyl chain (C4). Comparing BioHx with other known BioH proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and BioHx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known BioH enzymes. This ascribed BioHx as a novel carboxylesterase with a strong potential in industrial applications. This study constituted the first investigation of a novel bioHx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. The bioHx gene was successfully cloned, expressed and characterized. The results demonstrated that BioHx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. Our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioH genes from complex ecosystem.


Expression Analysis of p16, c-Myc, and mSin3A in Non-small Cell Lung Cancer by Computer Aided Scoring and Analysis (CASA).
Immunohistochemical analysis (IHC) of tissue microarray (TMA) slides enables large sets of tissue samples to be analyzed simultaneously on a single slide. However, manual evaluation of small cores on a TMA slide is time consuming and error prone. We describe a computer aided scoring and analysis (CASA) method to allow facile and reliable scoring of IHC staining using TMA containing 300 non-small cell lung cancer (NSCLC) cases. In the two previous published papers utilizing our TMA slides of lung cancer we examined 18 proteins involved in the chromatin machinery. We developed our study using more proteins of the chromatin complex and several transcription factors that facilitate the chromatin machinery. Then, a total of 78 antibodies were evaluated by CASA to derive a normalized intensity value that correlated with the overall staining status of the targeting protein. The intensity values for TMA cores were then examined for association to clinical variables and predictive significance individually and with other factors. RESULTs: Using our TMA, the intensity of several protein pairs were significantly correlated with an increased risk of death in NSCLC. These included c-Myc with p16, mSin3A with p16 and c-Myc with mSinA. Predictive values of these pairs remained significant when evaluated based on standard IHC scores. Our results demonstrate the usefulness of CASA as a valuable tool for systematic assessment of TMA slides to identify potential predictive biomarkers using a large set of primary human tissues.


Genome-wide detection of terpene synthase genes in holy basil (Ocimum sanctum L.).
Holy basil (Ocimum sanctum L.) and sweet basil (Ocimum basilicum L.) are the most commonly grown basil species in India for essential oil production and biosynthesis of potentially volatile and non-volatile phytomolecules with commercial significance. The aroma, flavor and pharmaceutical value of Ocimum species is a significance of its essential oil, which contains most of the monoterpenes and sesquiterpenes. A large number of plants have been studied for characterization and identification of terpene synthase genes, involved in terpenoids biosynthesis. The goal of this study is to discover and identify the putative functional terpene synthase genes in O. sanctum. HMMER search was performed by using a set of 13 well sequenced and annotated plant genomes including the newly sequenced genome of O. sanctum with Pfam-A database locally, using HMMER 3.0 hmmsearch for the two Pfam domains (PF01397 and PF03936). Using this search method 81 putative terpene synthases genes (OsaTPS) were identified in O. sanctum; the study further reveals 47 OsaTPS were putatively functional genes, 19 partial OsaTPS, and 15 OsaTPS as probably pseudogenes. All these identified OsaTPS genes were compared with other plant species, and phylogenetic analysis reveals the subfamily classification of OsaTPS in TPS-a, -b, -c, -e, -f and TPS-g subfamilies clusters. This genome-wide identification of OsaTPS genes, their phylogenetic analysis and secondary metabolite pathway mapping predictions together provide a comprehensive understanding of the TPS gene family in Ocimum sanctum and offer opportunities for the characterization and functional validation of numbers of terpene synthase genes.


The Corneal Basement Membranes and Stromal Fibrosis.
The purpose of this review was to provide detailed insights into the pathophysiology of myofibroblast-mediated fibrosis (scarring or late haze) after corneal injury, surgery, or infection. Literature review. The epithelium and epithelial basement membrane (EBM) and/or endothelium and Descemet's basement membrane (BM) are commonly disrupted after corneal injuries, surgeries, and infections. Regeneration of these critical regulatory structures relies on the coordinated production of BM components, including laminins, nidogens, perlecan, and collagen type IV by epithelial, endothelial, and keratocyte cells. Whether a cornea, or an area in the cornea, heals with transparency or fibrosis may be determined by whether there is injury to one or both corneal basement membranes (EBM and/or Descemet's BM) and delayed or defective regeneration or replacement of the BM. These opaque myofibroblasts, and the disordered extracellular matrix these cells produce, persist in the stroma until the EBM and/or Descemet's BM is regenerated or replaced. Corneal stromal fibrosis (also termed "stromal scarring" or "late haze") occurs as a consequence of BM injury and defective regeneration in both the anterior (EBM) and posterior (Descemet's BM) cornea. The resolution of fibrosis and return of stromal transparency depends on reestablished BM structure and function. It is hypothesized that defective regeneration of the EBM or Descemet's BM allows key profibrotic growth factors, including transforming growth factor beta-1 (TGF-β1) and TGF-β2, to penetrate the stroma at sustained levels necessary to drive the development and maintenance of mature opacity-producing myofibroblasts from myofibroblast precursors cells, and studies suggest that perlecan and collagen type IV are the critical components in EBM and Descemet's BM that bind TGF-β1, TGF-β2, platelet-derived growth factor, and possibly other growth factors, and regulate their bioavailability and function during homeostasis and corneal wound healing.


Exacerbation of thrombotic events by diesel exhaust particle in mouse model of hypertension.
Several epidemiological studies have shown that acute exposure to particulate air pollution is associated with increases in cardiovascular morbidity and mortality, and that these effects are especially exacerbated among individuals with pre-existing compromised cardiovascular function such as hypertension. This study was undertaken to determine the cardiovascular effect of diesel exhaust on TO mice made hypertensive by implanting osmotic minipump infusing angiotensin II or vehicle (control). On day 13, the animals were intratracheally instilled with either DEP (15 μg/mouse) or saline. 24 h later, pulmonary exposure to DEP had significantly decreased the systolic blood pressure (SBP) in hypertensive (HT) mice (P<0.01), but not in normotensive (NT) mice. The number of leukocytes and red blood cells, and the plasma interleukin 6 concentration in plasma, however, were not affected in any of the animals. The PaO₂ was decreased, and PaCO₂ increased in DEP-treated HT mice compared to NT mice treated with DEP (P<0.05). The number of circulating platelets was significantly increased in DEP-treated HT versus saline-treated HT and DEP-treated NT mice. Moreover, in NT mice, DEP exposure induced a prothrombotic effect in pial arterioles compared with saline-treated NT mice (P<0.05). Interestingly, in DEP-treated HT mice, the prothrombotic events were significantly aggravated compared with saline-treated HT and DEP-treated NT mice. The direct addition of DEP (0.1-1 μg/ml) to untreated mouse blood significantly induced in vitro platelet aggregation in a dose-dependent fashion, and these effects were more pronounced in blood of HT mice. In vitro exposure to DEP (0.25-1 μg/ml) led to activated intravascular coagulation, an effect that was confirmed by a shortening of both the activated partial thromboplastin time (aPTT) and the prothrombin time (PT). The effect of DEP on aPTT was potentiated in the plasma of HT mice. It can be concluded that the thrombotic events caused by DEP are exacerbated by hypertension in mice. Our findings, therefore, provide a possible plausible explanation for the cardiovascular morbidity and mortality accompanying urban air pollution.


Determination of antibody responses to meningococcal antigens by ELISA.
Enzyme-linked immunosorbent assay (ELISA, EIA) is a highly versatile and sensitive technique that can be used for quantitative as well as qualitative determination of almost any antigen or antibody. Reagents are stable, non-radioactive and, in most cases, commercially available. Owing to the simplicity and versatility of the method, ELISA represents probably one of the most used methods for studying antibody responses and antibody levels. Since Engvall and Perlman's first paper describing the ELISA in 1971 (1), almost all laboratories working in serology or immunology have designed their own assays with different protocols for coating with antigens, incubation conditions, detecting systems, and ways of reporting of the results. In most cases, there is no need for strict interlaboratory standardization of ELISAs and each laboratory will develop a system that suits their needs. However, for some ELISAs, e.g., used in diagnostic laboratories and in vaccine trials, standardization is important, and this is considered in "Meningococcal Disease" Edited by AJ Pollard and MCJ Maiden, (1a).


Crocus sativus restores dopaminergic and noradrenergic damages induced by lead in Meriones shawi: A possible link with Parkinson's disease.
Lead (Pb) is a metal element released into the atmosphere and a major source of environmental contamination. The accumulation and concentration of this metal in a food web may lead to the intoxication of the body, more precisely, the nervous system (NS). In addition, Pb-exposure can cause structural and functional disruption of the NS. Studies have shown that Pb-exposure could be a risk factor in the development of Parkinson's disease (PD). The latter is related to dopaminergic deficiency that may be triggered by genetic and environmental factors such as Pb intoxication. In this study, we have evaluated, in one hand, the neurotoxic effect of Pb (25 mg / kg B.W i.p) for three consecutive days on dopaminergic system and locomotor performance in Merione shawi. In the other hand, the possible restorative potential of C. sativus (CS) (50 mg / kg BW) by oral gavage. The immunohistochemical approach has revealed that Pb-intoxicated Meriones show a significant increase of Tyrosine Hydroxylase (TH) levels within the Substantia Nigra compacta (SNc), Ventral Tegmental Area (VTA), Locus Coeruleus (LC), Dorsal Striatum (DS) and Medial Forebrain Bundle (MFB), unlike the control meriones, a group intoxicated and treated with Crocus sativus hydroethanolic extract (CSHEE) and treated group by CSHEE. Treatment with CSHEE, has shown a real potential to prevent all Pb-induced damages. In fact, restores the TH levels by 92%, 90%, 88%, 90% and 93% in SNc, VTA, LC, DS and MFB respectively, similarly, locomotor activity dysfunction in Pb-intoxicaed meriones was reinstated by 90%. In this study, we have revealed a new pharmacological potential of Crocus sativus that can be used as a neuroprotective product for neurodegenerative disorders, especially, which implying dopaminergic and noradrenergic injuries, like PD, trigged by heavy metals.


Combined Detection of Urinary Micro Albumin, α1-microglobulin and N-acetyl-β-D-glucosaminidase in the Early Diagnosis of Diabetic Nephropathy.
To analyze the values of combined detection of urinary micro albumin (mAlb), α1-microglobulin (α1-MG) and N-acetyl-β-D-glucosaminidase (NAG) in the early diagnosis of diabetic nephropathy (DN). Ninety-four patients with early DN who were admitted to the hospital between April 2015 and April 2016 were selected and set as a DN group. Moreover, seventy-six patients with diabetes who were admitted to the hospital in the same period were selected and set as a diabetes group, and sixty-four healthy people were selected as set as a control group. The urinary mAlb, α1-MG and NAG of the three groups were detected. Moreover, the patients were divided into a favorable blood glucose control group and a poor blood glucose control group according to the blood glucose control condition of the patients. The detection results of the three groups were compared and statistically analyzed. The urinary mAlb, α1-MG and NAG levels of the DN group were significantly higher than those of the diabetes group and control group, and the differences had statistical significance (P<0.05). The detection indicator values of the favorable blood glucose control group were much lower than those of the poor blood glucose control group, and the difference was statistically significant (P<0.05). The positive rate of the combined detection of mAlb, α1-MG and NAG levels was 90.2%, which was much higher than that of single indicator (P<0.05). Combined detection of urinary mAlb, α1-MG and NAG is sensitive in diagnosing early renal damages in DN patients.


Enhanced myogenic responsiveness of renal interlobular arteries in spontaneously hypertensive rats.
We recently demonstrated that the interlobular artery (ILA) constricts in response to elevating renal arterial pressure (RAP), suggesting that the ILA contributes to renal autoregulation. In the present study, we examined the segmental myogenic responsiveness of the ILA in kidneys from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The tapered nature of the ILA allowed us to characterize the regional responsiveness, using the basal diameter to define segments as either proximal (greater than 60 microns), intermediate (40-60 microns), or distal (less than 40 microns). At 80 mm Hg, segmental diameters were similar in WKY and SHR arteries (proximal, 76.0 +/- 3.1 versus 71.6 +/- 3.5 microns; intermediate, 48.2 +/- 1.4 versus 48.1 +/- 1.7 microns; distal, 30.7 +/- 0.9 versus 27.9 +/- 1.3 microns for WKY and SHR, respectively). In both strains, intermediate and distal segments exhibited graded reductions in diameter as RAP was elevated, whereas proximal segments did not. Pressure-induced decrements in the diameters of distal ILA segments were similar in WKY (-24 +/- 2%) and SHR (-20 +/- 2%; p greater than 0.1). The intermediate ILA of SHR exhibited an augmented myogenic responsiveness, constricting at lower RAP levels and exhibiting greater maximal decrements in diameter at 180 mm Hg (i.e., -19 +/- 2% and -12 +/- 2% for SHR and WKY, respectively; p less than 0.05). Nifedipine (1.0 microM) reduced pressure-induced vasoconstriction of intermediate and distal ILA segments by 56 +/- 11% and 79 +/- 7%, respectively, in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)


Gamma oscillatory firing reveals distinct populations of pyramidal cells in the CA1 region of the hippocampus.
Hippocampal place cells that fire together within the same cycle of theta oscillations represent the sequence of positions (movement trajectory) that a rat traverses on a linear track. Furthermore, it has been suggested that the encoding of these and other types of temporal memory sequences is organized by gamma oscillations nested within theta oscillations. Here, we examined whether gamma-related firing of place cells permits such discrete temporal coding. We found that gamma-modulated CA1 pyramidal cells separated into two classes on the basis of gamma firing phases during waking theta periods. These groups also differed in terms of their spike waveforms, firing rates, and burst firing tendency. During gamma oscillations one group's firing became restricted to theta phases associated with the highest gamma power. Consequently, on the linear track, cells in this group often failed to fire early in theta-phase precession (as the rat entered the place field) if gamma oscillations were present. The second group fired throughout the theta cycle during gamma oscillations, and maintained gamma-modulated firing at different stages of theta-phase precession. Our results suggest that the two different pyramidal cell classes may support different types of population codes within a theta cycle: one in which spike sequences representing movement trajectories occur across subsequent gamma cycles nested within each theta cycle, and another in which firing in synchronized gamma discharges without temporal sequences encode a representation of location. We propose that gamma oscillations during theta-phase precession organize the mnemonic recall of population patterns representing places and movement paths.


The genome of Cardinium cBtQ1 provides insights into genome reduction, symbiont motility, and its settlement in Bemisia tabaci.
Many insects harbor inherited bacterial endosymbionts. Although some of them are not strictly essential and are considered facultative, they can be a key to host survival under specific environmental conditions, such as parasitoid attacks, climate changes, or insecticide pressures. The whitefly Bemisia tabaci is at the top of the list of organisms inflicting agricultural damage and outbreaks, and changes in its distribution may be associated to global warming. In this work, we have sequenced and analyzed the genome of Cardinium cBtQ1, a facultative bacterial endosymbiont of B. tabaci and propose that it belongs to a new taxonomic family, which also includes Candidatus Amoebophilus asiaticus and Cardinium cEper1, endosymbionts of amoeba and wasps, respectively. Reconstruction of their last common ancestors' gene contents revealed an initial massive gene loss from the free-living ancestor. This was followed in Cardinium by smaller losses, associated with settlement in arthropods. Some of these losses, affecting cofactor and amino acid biosynthetic encoding genes, took place in Cardinium cBtQ1 after its divergence from the Cardinium cEper1 lineage and were related to its settlement in the whitefly and its endosymbionts. Furthermore, the Cardinium cBtQ1 genome displays a large proportion of transposable elements, which have recently inactivated genes and produced chromosomal rearrangements. The genome also contains a chromosomal duplication and a multicopy plasmid, which harbors several genes putatively associated with gliding motility, as well as two other genes encoding proteins with potential insecticidal activity. As gene amplification is very rare in endosymbionts, an important function of these genes cannot be ruled out.


[Effect of a single-locus mutation of the silver-blue color allele in mink on the dopaminergic system of the brain].
The peculiarities of dopamine metabolism and dopamine receptors in the brain of homozygous silver-blue (pp) minks were examined in comparison with wild-type (PP) minks. A pp mutation was found to affect the dopamine turnover in corpus striatum, which is one of the major dopaminergic brain structures. In silver-blue minks, increased dopamine catabolic enzyme activity monoamine oxidase type B (MAO B), together with the decreased level of dopamine and the tendency for the level of the metabolite 3,4-dihydrophenyl acetic acid to increase, were demonstrated in this brain region. No significant alterations in the level of another dopamine metabolite, homovanillic acid, or in D1 and D2 receptor density were shown. In the midbrain and hippocampus, an elevated level of MAO B activity was detected. We found no modifications in receptor properties, nor in dopamine turnover in a mesolimbic dopamine structure, the nucleus accumbens. We propose that the primary effect of pp mutation is an increase in MAO B activity resulting in dopamine turnover alterations and probably in dopamine-dependent behavior (pleiotropic cascade).


Protocol for a randomised controlled trial of an outreach support program for family carers of older people discharged from hospital.
Presentations to hospital of older people receiving family care at home incur substantial costs for patients, families, and the health care system, yet there can be positive carer outcomes when systematically assessing/addressing their support needs, and reductions in older people's returns to hospital attributed to appropriate discharge planning. This study will trial the Further Enabling Care at Home program, a 2-week telephone outreach initiative for family carers of older people returning home from hospital. Hypotheses are that the program will (a) better prepare families to sustain their caregiving role and (b) reduce patients' re-presentations/readmissions to hospital, and/or their length of stay; also that reduced health system costs attributable to the program will outweigh costs of its implementation. In this randomised controlled trial, family carers of older patients aged 70+ discharged from a Medical Assessment Unit in a Western Australian tertiary hospital, plus the patients themselves, will be recruited at discharge (N = 180 dyads). Carers will be randomly assigned (block allocation, assessors blinded) to receive usual care (control) or the new program (intervention). The primary outcome is the carer's self-reported preparedness for caregiving (Preparedness for Caregiving Scale administered within 4 days of discharge, 2-3 weeks post-discharge, 6 weeks post-discharge). To detect a clinically meaningful change of two points with 80 % power, 126 carers need to complete the study. Patients' returns to hospital and subsequent length of stay will be ascertained for a minimum of 3 months after the index admission. Regression analyses will be used to determine differences in carer and patient outcomes over time associated with the group (intervention or control). Data will be analysed using an Intention to Treat approach. A qualitative exploration will examine patients' and their family carers' experiences of the new program (interviews) and explore the hospital staff's perceptions (focus groups). Process evaluation will identify barriers to, and facilitators of, program implementation. A comprehensive economic evaluation will determine cost consequences. This study investigates a novel approach to identifying and addressing family carers' needs following discharge from hospital of the older person receiving care. If successful, the program has potential to be incorporated into routine post-discharge support. Australian and New Zealand Clinical Trial Registry: ACTRN12614001174673 .


Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning.
Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34(+) HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34(+) cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2, 603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5' ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.


The primary cilium is a self-adaptable, integrating nexus for mechanical stimuli and cellular signaling.
Mechanosensation is crucial for cells to sense and respond to mechanical signals within their local environment. While adaptation allows a sensor to be conditioned by stimuli within the environment and enables its operation in a wide range of stimuli intensities, the mechanisms behind adaptation remain controversial in even the most extensively studied mechanosensor, bacterial mechanosensitive channels. Primary cilia are ubiquitous sensory organelles. They have emerged as mechanosensors across diverse tissues, including kidney, liver and the embryonic node, and deflect with mechanical stimuli. Here, we show that both mechanical and chemical stimuli can alter cilium stiffness. We found that exposure to flow stiffens the cilium, which deflects less in response to subsequent exposures to flow. We also found that through a process involving acetylation, the cell can biochemically regulate cilium stiffness. Finally, we show that this altered stiffness directly affects the responsiveness of the cell to mechanical signals. These results demonstrate a potential mechanism through which the cell can regulate its mechanosensing apparatus.


The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila.
The protein phosphatase 2A (PP2A) regulatory subunit Tap42 is essential for target of rapamycin (TOR)-mediated signaling in yeast, but its role in higher eukaryotes has not been established. Here we show that Tap42 does not contribute significantly to TOR signaling in Drosophila, as disruption of the Tap42 gene does not cause defects in cell growth, metabolism, or S6-kinase activity characteristic of TOR inactivation. In addition, Tap42 is not required for increased cell growth in response to activation of TOR signaling. Instead, we find that Tap42 mutations cause disorganization of spindle microtubules in larval neuroblasts, leading to a preanaphase mitotic arrest in these cells. Loss of Tap42 ultimately results in increased JNK signaling, caspase activation, and cell death. These phenotypes are associated with increased accumulation and nuclear localization of PP2A in Tap42 mutant cells. Our results demonstrate that the role of Tap42 in TOR signaling has not been conserved in higher eukaryotes, indicating fundamental differences in the mechanisms of TOR signaling between yeast and higher eukaryotes.


Cellular and molecular changes associated with inhibitory effect of pioglitazone on neointimal growth in patients with type 2 diabetes after zotarolimus-eluting stent implantation.
To investigate the mechanistic basis underlying antirestenosis and the antiatherogenic effect of pioglitazone in patients with type 2 diabetes mellitus who were undergoing zotarolimus-eluting stent implantation. Recent studies highlight the beneficial effect of pioglitazone in attenuating neointimal growth after stent implantation. Patients with coronary artery diseases were randomly assigned to pioglitazone (n=47) or placebo (n=47) after stent implantation. Pioglitazone significantly reduced neointimal hyperplasia within the stented lesion and attenuated total plaque burden in the in-segment regions of the stent, as assessed by intravascular ultrasonography at the 8-month follow-up. These changes were preceded by reduced circulating natural killer (NK) cells, diminished interleukin 6 and monocyte chemoattractant protein-1 levels, and downregulation of chemokine receptor 2 at 2 days after stent implantation; and an elevated interleukin 10 level at 10 days after implantation. Furthermore, the proliferation and migration of vascular smooth muscle cells were inhibited in the presence of pioglitazone-treated patient serum, demonstrating that the antiproliferative effects of pioglitazone occurred concurrently with its antiinflammatory action. Our data present early cellular and immunologic changes by pioglitazone that might have been associated with antirestenotic and antiatherogenic effects in diabetic patients. Inhibiting proinflammatory responses while promoting antiinflammatory circuits, together with an antiproliferative action, may, in part, account for the antirestenotic effect of pioglitazone by altering vascular remodeling processes in the early phase.


Epigenetic alterations following early postnatal stress: a review on novel aetiological mechanisms of common psychiatric disorders.
Stressor exposure during early life has the potential to increase an individual's susceptibility to a number of neuropsychiatric conditions such as mood and anxiety disorders and schizophrenia in adulthood. This occurs in part due to the dysfunctional stress axis that persists following early adversity impairing stress responsivity across life. The mechanisms underlying the prolonged nature of this vulnerability remain to be established. Alterations in the epigenetic signature of genes involved in stress responsivity may represent one of the neurobiological mechanisms. The overall aim of this review is to provide current evidence demonstrating changes in the epigenetic signature of candidate gene(s) in response to early environmental adversity. More specifically, this review analyses the epigenetic signatures of postnatal adversity such as childhood abuse or maltreatment and later-life psychopathology in human and animal models of early life stress. The results of this review shows that focus to date has been on genes involved in the regulation of hypothalamic-pituitary-adrenal (HPA) axis and its correlation to subsequent neurobiology, for example, the role of glucocorticoid receptor gene. However, epigenetic changes in other candidate genes such as brain-derived neurotrophic factor (BDNF) and serotonin transporter are also implicated in early life stress (ELS) and susceptibility to adult psychiatric disorders. DNA methylation is the predominantly studied epigenetic mark followed by histone modifications specifically acetylation and methylation. Further, these epigenetic changes are cell/tissue-specific in regulating expression of genes, providing potential biomarkers for understanding the trajectory of early stress-induced susceptibility to adult psychiatric disorders.


Porcine reproductive and respiratory syndrome antibody detection on filter discs.
Two enzyme-linked immunosorbent assay (ELISA) kits were evaluated for detection of porcine reproductive and respiratory syndrome (PRRS) antibodies in pooled sera and filter discs (FDs). Elution and incubation procedures and positive thresholds for both ELISAs were determined using FDs collected from sixty non-infected pigs and five pigs with low PRRS-antibody titres. Eighty paired samples (serum/FD) from infected pigs were titrated using both ELISAs. The authors thus showed that five sera or five FDs could be pooled in one test without significant loss of sensitivity. Compared to individual sera, method sensitivity was found to be 79% and specificity 97.5%, based on data from 200 pools of FDs collected on 15 PRRS-infected farms and 120 pools collected on 71 non-infected farms. To balance loss of sensitivity, two pools of five samples from sows and one pool of five samples from finishing pigs can be tested as an alternative to seven and five single sera, respectively.


Assessing the value of the NHIS for studying changes in state coverage policies: the case of New York.
(1) To assess the effects of New York's Health Care Reform Act of 2000 on the insurance coverage of eligible adults and (2) to explore the feasibility of using the National Health Interview Survey (NHIS) as opposed to the Current Population Survey (CPS) to conduct evaluations of state health reform initiatives. We take advantage of the natural experiment that occurred in New York to compare health insurance coverage for adults before and after the state implemented its coverage initiative using a difference-in-differences framework. We estimate the effects of New York's initiative on insurance coverage using the NHIS, comparing the results to estimates based on the CPS, the most widely used data source for studies of state coverage policy changes. Although the sample sizes are smaller in the NHIS, the NHIS addresses a key limitation of the CPS for such evaluations by providing a better measure of health insurance status. Given the complexity of the timing of the expansion efforts in New York (which encompassed the September 11, 2001 terrorist attacks), we allow for difference in the effects of the state's policy changes over time. In particular, we allow for differences between the period of Disaster Relief Medicaid (DRM), which was a temporary program implemented immediately after September 11th, and the original components of the state's reform efforts-Family Health Plus (FHP), an expansion of direct Medicaid coverage, and Healthy New York (HNY), an effort to make private coverage more affordable. 2000-2004 CPS; 1999-2004 NHIS. We find evidence of a significant reduction in uninsurance for parents in New York, particularly in the period following DRM. For childless adults, for whom the coverage expansion was more circumscribed, the program effects are less promising, as we find no evidence of a significant decline in uninsurance. The success of New York at reducing uninsurance for parents through expansions of both public and private coverage offers hope for new strategies to expand coverage. The NHIS is a strong data source for evaluations of many state health reform initiatives, providing a better measure of insurance status and supporting a more comprehensive study of state innovations than is possible with the CPS.


Soluble or insoluble fibre in irritable bowel syndrome in primary care? Randomised placebo controlled trial.
To determine the effectiveness of increasing the dietary content of soluble fibre (psyllium) or insoluble fibre (bran) in patients with irritable bowel syndrome. Randomised controlled trial. General practice. 275 patients aged 18-65 years with irritable bowel syndrome. 12 weeks of treatment with 10 g psyllium (n=85), 10 g bran (n=97), or 10 g placebo (rice flour) (n=93). The primary end point was adequate symptom relief during at least two weeks in the previous month, analysed after one, two, and three months of treatment to assess both short term and sustained effectiveness. Secondary end points included irritable bowel syndrome symptom severity score, severity of abdominal pain, and irritable bowel syndrome quality of life scale. The proportion of responders was significantly greater in the psyllium group than in the placebo group during the first month (57% v 35%; relative risk 1.60, 95% confidence interval 1.13 to 2.26) and the second month of treatment (59% v 41%; 1.44, 1.02 to 2.06). Bran was more effective than placebo during the third month of treatment only (57% v 32%; 1.70, 1.12 to 2.57), but this was not statistically significant in the worst case analysis (1.45, 0.97 to 2.16). After three months of treatment, symptom severity in the psyllium group was reduced by 90 points, compared with 49 points in the placebo group (P=0.03) and 58 points in the bran group (P=0.61 versus placebo). No differences were found with respect to quality of life. Fifty four (64%) of the patients allocated to psyllium, 54 (56%) in the bran group, and 56 (60%) in the placebo group completed the three month treatment period. Early dropout was most common in the bran group; the main reason was that the symptoms of irritable bowel syndrome worsened. Psyllium offers benefits in patients with irritable bowel syndrome in primary care. Clinical trials NCT00189033.


Mycorrhiza-induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity.
Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.


Influence of the electrolyte's pH on the properties of electrochemically deposited hydroxyapatite coating on additively manufactured Ti64 alloy.
Properties of the hydroxyapatite obtained by electrochemical assisted deposition (ED) are dependent on several factors including deposition temperature, electrolyte pH and concentrations, applied potential. All of these factors directly influence the morphology, stoichiometry, crystallinity, electrochemical behaviour, and particularly the coating thickness. Coating structure together with surface micro- and nano-scale topography significantly influence early stages of the implant bio-integration. The aim of this study is to analyse the effect of pH modification on the morphology, corrosion behaviour and in vitro bioactivity and in vivo biocompatibility of hydroxyapatite prepared by ED on the additively manufactured Ti64 samples. The coatings prepared in the electrolytes with pH = 6 have predominantly needle like morphology with the dimensions in the nanometric scale (~30 nm). Samples coated at pH = 6 demonstrated higher protection efficiency against the corrosive attack as compared to the ones coated at pH = 5 (~93% against 89%). The in vitro bioactivity results indicated that both coatings have a greater capacity of biomineralization, compared to the uncoated Ti64. Somehow, the coating deposited at pH = 6 exhibited good corrosion behaviour and high biomineralization ability. In vivo subcutaneous implantation of the coated samples into the white rats for up to 21 days with following histological studies showed no serious inflammatory process.


Analysis of HSP90-related folds with MED-SuMo classification approach.
Three-dimensional structural information is critical for understanding functional protein properties and the precise mechanisms of protein functions implicated in physiological and pathological processes. Comparison and detection of protein binding sites are key steps for annotating structures with functional predictions and are extremely valuable steps in a drug design process. In this research area, MED-SuMo is a powerful technology to detect and characterize similar local regions on protein surfaces. Each amino acid residue's potential chemical interactions are represented by specific surface chemical features (SCFs). The MED-SuMo heuristic is based on the representation of binding sites by a graph structure suitable for exploration by an efficient comparison algorithm. We use this approach to analyze one particular SCOP superfamily which includes HSP90 chaperone, MutL/DNA topoisomerase, histidine kinases, and alpha-ketoacid dehydrogenase kinase C (BCK). They share a common fold and a common region for ATP-binding. To analyze both similar and differing features of this fold, we use a novel classification method, the MED-SuMo multi approach (MED-SMA). We highlight common and distinct features of these proteins. The different clusters created by MED-SMA yield interesting observations. For instance, one cluster gathers three types of proteins (HSP90, topoisomerase VI, and BCK) which all bind the drug radicicol.


Integrated Analysis of Exosomal Protein Biomarkers on Alternating Current Electrokinetic Chips Enables Rapid Detection of Pancreatic Cancer in Patient Blood.
Pancreatic ductal adenocarcinoma (PDAC) typically has nonspecific symptoms and is often found too late to treat. Because diagnosis of PDAC involves complex, invasive, and expensive procedures, screening populations at increased risk will depend on developing rapid, sensitive, specific, and cost-effective tests. Exosomes, which are nanoscale vesicles shed into blood from tumors, have come into focus as valuable entities for noninvasive liquid biopsy diagnostics. However, rapid capture and analysis of exosomes with their protein and other biomarkers have proven difficult. Here, we present a simple method integrating capture and analysis of exosomes and other extracellular vesicles directly from whole blood, plasma, or serum onto an AC electrokinetic microarray chip. In this process, no pretreatment or dilution of sample is required, nor is it necessary to use capture antibodies or other affinity techniques. Subsequent on-chip immunofluorescence analysis permits specific identification and quantification of target biomarkers within as little as 30 min total time. In this initial validation study, the biomarkers glypican-1 and CD63 were found to reflect the presence of PDAC and thus were used to develop a bivariate model for detecting PDAC. Twenty PDAC patient samples could be distinguished from 11 healthy subjects with 99% sensitivity and 82% specificity. In a smaller group of colon cancer patient samples, elevated glypican-1 was observed for metastatic but not for nonmetastatic disease. The speed and simplicity of ACE exosome capture and on-chip biomarker detection, combined with the ability to use whole blood, will enable seamless "sample-to-answer" liquid biopsy screening and improve early stage cancer diagnostics.


Update on treatment options for spinal brucellosis.
We evaluated the efficacy and tolerability of antibiotic regimens and optimal duration of therapy in complicated and uncomplicated forms of spinal brucellosis. This is a multicentre, retrospective and comparative study involving a total of 293 patients with spinal brucellosis from 19 health institutions. Comparison of complicated and uncomplicated spinal brucellosis was statistically analysed. Complicated spinal brucellosis was diagnosed in 78 (26.6%) of our patients. Clinical presentation was found to be significantly more acute, with fever and weight loss, in patients in the complicated group. They had significantly higher leukocyte and platelet counts, erythrocyte sedimentation rates and C-reactive protein levels, and lower haemoglobulin levels. The involvement of the thoracic spine was significantly more frequent in complicated cases. Spondylodiscitis was complicated, with paravertebral abscess in 38 (13.0%), prevertebral abscess in 13 (4.4%), epidural abscess in 30 (10.2%), psoas abscess in 10 (3.4%) and radiculitis in 8 (2.7%) patients. The five major combination regimens were: doxycycline 200 mg/day, rifampicin 600 mg/day and streptomycin 1 g/day; doxycycline 200 mg/day, rifampicin 600 mg/day and gentamicin 5 mg/kg; doxycycline 200 mg/day and rifampicin 600 mg/day; doxycycline 200 mg/day and streptomycin 1 g/day; and doxycycline 200 mg/day, rifampicin 600 mg/day and ciprofloxacin 1 g/day. There were no significant therapeutic differences between these antibiotic groups; the results were similar regarding the complicated and uncomplicated groups. Patients were mostly treated with doxycycline and rifampicin with or without an aminoglycoside. In the former subgroup, complicated cases received antibiotics for a longer duration than uncomplicated cases. Early recognition of complicated cases is critical in preventing devastating complications. Antimicrobial treatment should be prolonged in complicated spinal brucellosis in particular.


Lower Functional Connectivity in Vestibular-Limbic Networks in Individuals With Subclinical Agoraphobia.
Background: Agoraphobia was described in 1871 as a condition of fear-related alterations in spatial orientation and locomotor control triggered by places or situations that might cause a patient to panic and feel trapped. In contemporary nosology, however, this original concept of agoraphobia was split into two diagnostic entities, i.e., the modern anxiety disorder of agoraphobia, consisting solely of phobic/avoidant symptoms in public spaces, and the recently defined vestibular disorder of persistent postural perceptual dizziness (PPPD), characterized by dizziness, and unsteadiness exacerbated by visual motion stimuli. Previous neuroimaging studies found altered brain activity and connectivity in visual-vestibular networks of patients with PPPD vs. healthy controls. Neuroticism and introversion, which pre-dispose to both agoraphobia and PPPD, influenced brain responses to vestibular and visual motion stimuli in patients with PPPD. Similar neuroimaging studies have not been undertaken in patients with agoraphobia in its current definition. Given their shared history and pre-disposing factors, we sought to test the hypotheses that individuals with agoraphobic symptoms have alterations in visual-vestibular networks similar to those of patients with PPPD, and that these alterations are influenced by neuroticism and introversion. Methods: Drawing from the Human Connectome Project (HCP) database, we matched 52 participants with sub-clinical agoraphobia and 52 control subjects without agoraphobic symptoms on 19 demographic and psychological/psychiatric variables. We then employed a graph-theoretical framework to compare resting-state functional magnetic resonance images between groups and evaluated the interactive effects of neuroticism and introversion on the brain signatures of agoraphobia. Results: Individuals with subclinical agoraphobia had lower global clustering, efficiency and transitivity relative to controls. They also had lower connectivity metrics in two brain networks, one positioned to process incoming visual space-motion information, assess threat, and initiate/inhibit behavioral responses (visuospatial-emotional network) and one positioned to control and monitor locomotion (vestibular-navigational network). Introversion interacted with agoraphobic symptoms to lower the connectivity of the visuospatial-emotional network. This contrasted with previous findings describing neuroticism-associated higher connectivity in a narrower visual-spatial-frontal network in patients with PPPD. Conclusion: Functional connectivity was lower in two brain networks in subclinical agoraphobia as compared to healthy controls. These networks integrate visual vestibular and emotional response to guide movement in space.


N-acetyl-L-tryptophan, a substance-P receptor antagonist attenuates aluminum-induced spatial memory deficit in rats.
Neuroinflammation plays an important role in the pathophysiology of Alzheimer's disease. Neurokinin substance P is a key mediator which modulates neuroinflammation through neurokinin receptor. Involvement of substance P in Alzheimer's disease is still plausible and various controversies exist in this hypothesis. Preventing the deleterious effects of substance P using N-acetyl-L-tryptophan, a substance P antagonist could be a promising therapeutic strategy. This study was aimed to evaluate the effect of N-acetyl-L-tryptophan on aluminum induced spatial memory alterations in rats. Memory impairment was induced using aluminum chloride (AlCl3) at a dose of 10 mg/kg for 42 d. After induction of dementia, rats were exposed to 30 and 50 mg/kg of N-acetyl-L-tryptophan for 28 d. Spatial memory alterations were measured using Morris water maze. Acetylcholinesterase activity and antioxidant enzyme glutathione level were assessed in hippocampus, frontal cortex and striatum. The higher dose of N-acetyl-L-tryptophan (50 mg/kg) significantly improved the aluminum induced memory alterations. N-acetyl-L-tryptophan exposure resulted in significant increase in acetylcholinesterase activity and glutathione level in hippocampus. The neuroprotective effect of N-acetyl-L-tryptophan could be due to its ability to block substance P mediated neuroinflammation, reduction in oxidative stress and anti-apoptotic properties. To conclude, N-acetyl-L-tryptophan may be considered as a novel neuroprotective therapy in Alzheimer's disease.


Theoretical insight into the regioselective ring-expansions of bicyclic aziridinium ions.
Transient bicyclic aziridinium ions are known to undergo ring-expansion reactions, paving the way to functionalized nitrogen-containing heterocycles. In this study, the regioselectivity observed in the ring-expansion reactions of 1-azoniabicyclo[n.1.0]alkanes was investigated from a computational viewpoint to study the ring-expansion pathways of two bicyclic systems with different ring sizes. Moreover, several nucleophiles leading to different experimental results were investigated. The effect of solvation was taken into account using both explicit and implicit solvent models. This theoretical rationalization provides valuable insight into the observed regioselectivity and may be used as a predictive tool in future studies.


Are forest disturbances amplifying or canceling out climate change-induced productivity changes in European forests?
Recent studies projecting future climate change impacts on forests mainly consider either the effects of climate change on productivity or on disturbances. However, productivity and disturbances are intrinsically linked because 1) disturbances directly affect forest productivity (e.g. via a reduction in leaf area, growing stock or resource-use efficiency), and 2) disturbance susceptibility is often coupled to a certain development phase of the forest with productivity determining the time a forest is in this specific phase of susceptibility. The objective of this paper is to provide an overview of forest productivity changes in different forest regions in Europe under climate change, and partition these changes into effects induced by climate change alone and by climate change and disturbances. We present projections of climate change impacts on forest productivity from state-of-the-art forest models that dynamically simulate forest productivity and the effects of the main European disturbance agents (fire, storm, insects), driven by the same climate scenario in seven forest case studies along a large climatic gradient throughout Europe. Our study shows that, in most cases, including disturbances in the simulations exaggerate ongoing productivity declines or cancel out productivity gains in response to climate change. In fewer cases, disturbances also increase productivity or buffer climate-change induced productivity losses, e.g. because low severity fires can alleviate resource competition and increase fertilization. Even though our results cannot simply be extrapolated to other types of forests and disturbances, we argue that it is necessary to interpret climate change-induced productivity and disturbance changes jointly to capture the full range of climate change impacts on forests and to plan adaptation measures.


Drosophila miR-277 controls branched-chain amino acid catabolism and affects lifespan.
Development, growth and adult survival are coordinated with available metabolic resources, ascertaining that the organism responds appropriately to environmental conditions. MicroRNAs are short (21-23 nt) regulatory RNAs that confer specificity on the RNA-induced silencing complex (RISC) to inhibit a given set of mRNA targets. We profiled changes in miRNA expression during adult life in Drosophila melanogaster and determined that miR-277 is downregulated during adult life. Molecular analysis revealed that this miRNA controls branched-chain amino acid (BCAA) catabolism and as a result it can modulate the activity of the TOR kinase, a central growth regulator, in cultured cells. Metabolite analysis in cultured cells as well as flies suggests that the mechanistic basis may be an accumulation of branched-chain α-keto-acids (BCKA), rather than BCAAs, thus avoiding potentially detrimental consequences of increased branched chain amino acid levels on e.g., translational fidelity. Constitutive miR-277 expression shortens lifespan and is synthetically lethal with reduced insulin signaling, indicating that metabolic control underlies this phenotype. Transgenic inhibition with a miRNA sponge construct also shortens lifespan, in particular on protein-rich food. Thus, optimal metabolic adaptation appears to require tuning of cellular BCAA catabolism by miR-277.