Catabolic repression in early-diverging anaerobic fungi is partially mediated by natural antisense transcripts.
Early-diverging anaerobic fungi (order: Neocallimastigomycota), lignocelluolytic chytrid-like fungi central to fiber degradation in the digestive tracts of large herbivores, are attractive sources of cellulases and hemicellulases for biotechnology. Enzyme expression is tightly regulated and coordinated through mechanisms that remain unelucidated to optimize hydrolytic efficiency. Our analysis of anaerobic fungal transcriptomes reveals hundreds of cis-natural antisense transcripts (cis-NATs), which we hypothesize play an integral role in this regulation. Through integrated genomic and transcriptomic sequencing on a range of catabolic substrates, we validate these NATs in three species (Anaeromyces robustus, Neocallimasix californiae, and Piromyces finnis), and analyze their expression patterns and prevalence to gain insight into their function. NAT function was diverse and conserved across the three fungal genomes studied, with 10% of all metabolic process NATs associated with lignocellulose hydrolysis. Despite these similarities, however, only eleven gene targets were conserved orthologs. Several NATs were dynamically regulated by lignocellulosic substrates while their gene targets were unregulated. This observation is consistent with a hypothesized, but untested, regulatory mechanism where selected genes are exclusively regulated at the transcriptional/post-transcriptional level by NATs. However, only genes with high NAT relative expression levels displayed this phenomenon, suggesting a selection mechanism that favors larger dynamic ranges for more precise control of gene expression. In addition to this mode, we observed two other possible regulatory fates: canonical transcriptional regulation with no NAT response, and positive co-regulation of target mRNA and cognate NAT, which we hypothesize is a fine-tuning strategy to locally negate control outputs from global regulators. Our work reveals the complex contributions of antisense RNA to the catabolic response in anaerobic fungi, highlighting its importance in understanding lignocellulolytic activity for bioenergy applications. More importantly, the relative expression of NAT to target may form a critical determinant of transcriptional vs post-transcriptional (NAT) control of gene expression in primitive anaerobic fungi.


The effect of lanthanum, quercetin and dinitrophenol on calcium-evoked electrical responses in hamster eggs.
At room temperature micro-injections of calcium or strontium produced transient hyperpolarizations with an associated rise in input conductance. By contrast micro-injections of potassium, barium, magnesium, cobalt or lanthanum did not produce hyperpolarizations. The reversal potential for the hyperpolarizing response was about -80 mV. Some responses to calcium injections appeared to suffer from an additional transient leak conductance generated by the injected current. In these cases the recovery of the potential and the conductance to normal values was prolonged. The reversal potential of this additional leak pathway was about -10 mV. Experiments designed to investigate the role of active calcium extrusion from the cells showed that extracellular lanthanum or quercetin caused a pronounced slowing of the recovery phase of the potential and conductance response to calcium injection. The metabolic uncoupler dinitrophenol also prolonged the calcium-evoked responses. The replacement of extracellular sodium by lithium or choline produced no alteration in the time course of the calcium-evoked responses, thus suggesting that sodium-calcium exchange exerts no rate control on the recovery phase of those responses.


Colloidal stability of self-assembled monolayer-coated gold nanoparticles: the effects of surface compositional and structural heterogeneity.
Surface heterogeneity plays an important role in controlling colloidal phenomena. This study investigated the self-aggregation and bacterial adsorption of self-assembled monolayer coated gold nanoparticles (AuNPs) with different surface compositional and structural heterogeneity. Evaluation was performed on AuNPs coated with (1) one ligand with charged terminals (MUS), (2) two homogeneously distributed ligands with respectively charged and nonpolar terminals (brOT) and (3) two ligands with respectively charged and nonpolar terminals with stripe-like distribution (OT). The brOT particles have less negative electrophoretic mobility (EPM) values, smaller critical coagulation concentration (CCC) and larger adsorption rate on Escherichia coli than that of AuNPs with homogeneously charged groups, in good agreement with DLVO predictions. Although the ligand composition on the surface of AuNPs is the same, OT particles have less negative EPM values and faster rate of bacterial adsorption, but much larger CCC compared to brOT. The deviation of OT particles from brOT and MUS in their self-aggregation behavior reflects the effects of surface heterogeneity on electrical double layer structures at the interface. Results from the present study demonstrated that, besides chemical composition, organization of ligands on particle surface is important in determining their colloidal stability.


Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation.
Stat1 and Stat3 are latent transcriptional factors activated initially through phosphorylation on single tyrosine residues induced by cytokine and growth factor occupation of cell surface receptors. Here we show that phosphorylation on a single serine (residue 727) in each protein is also required for maximal transcriptional activity. Both cytokines and growth factors are capable of inducing the serine phosphorylation of Stat1 and Stat3. These experiments show that gene activation by Stat1 and Stat3, which obligatorily require tyrosine phosphorylation to become active, also depends for maximal activation on one or more of the many serine kinases.


Introduction to Section II: RNA methods to approach CFTR expression.
In this section, we review methods for the analysis of the CFTR gene and its transcript. First, we discuss techniques to accurately measure levels of CFTR mRNA in primary human cells; next, protocols for measuring CFTR transcripts that contain premature termination codons and for evaluating the role of nonsense-mediated decay which targets these transcripts; a further chapter considers methodology to investigate pre-mRNA splicing. The penultimate chapter concentrates on methods for evaluating microRNA regulation of gene expression in the context of airway disease. The final chapter considers methods to evaluate the chromatin structure of the active CFTR locus and to analyse the cis-acting regulatory elements that control it.


Toxoplasma infection and milk consumption: Meta-analysis of assumptions and evidences.
Toxoplasmosis is the most widespread infection worldwide. It occurs within congenital contamination, organ transplant or immune system depression. Primary infection is mainly foodborne with the ingestion of raw or undercooked meat, unwashed fruit-vegetables, unhygienic water or contaminated milk. Gaps in current knowledge about the risk assessment of Toxoplasma gondii by milk consumption are noted. Contradictory data are observed within risk assessment of milk consumption and toxoplasmosis occurrence. While some papers reported positive correlations between drinking milk and infection transmission to human, other studies stated nonsignificant influence of milk or milk products consumption. New debate about the detection of the parasite in the milk matrix from different hosts raised interrogations. To figure out the real contribution and the potential correlations of milkborne way in toxoplasmic infection, meta-analysis approach was investigated. Overall analysis showed heterogeneous responses and led to state that statistically dairy matrix (other than milk), Bovidae products, agricultural population and countries in Africa, Europe and Southeast Asia are not linked to milkborne toxoplasmosis. The most involved factors are Capridae products, immune-depressed population and North America, Middle East, and Latin territories. The current work advanced those parameters that could affect the public health and should be envisioned in further epidemiological analysis.


Physiological determinants of hyperreactivity to stress in borderline hypertension.
Blood pressure hyperreactivity during stress is characteristic of borderline hypertension in white men. The present study evaluated the hemodynamic basis of this hyperreactivity and assessed its physiological basis in terms of sympathetic nervous system function. Cardiovascular adjustments to an aversive reaction time test were compared with those of the forehead cold pressor test, representing stressors that elicit active behavioral responses in contrast to passive tolerance of aversive stimulation. As anticipated, blood pressure increases were greater in 12 borderline hypertensive men compared with 21 age-matched normotensive men during the active reaction time stressor but not during the passive cold pressor test. The pressor hyperreactivity in borderline hypertensives was associated with excessive rises in plasma epinephrine and norepinephrine, leading to greater increases in cardiac output, despite evidence that the cardiac beta-adrenergic receptors in these subjects were downregulated compared with those of normotensive subjects. During the cold pressor test, borderline hypertension was associated with greater increases in systemic vascular resistance, which, in the presence of normal baroreceptor reflex function, led to an attenuation of cardiac output, thus producing no greater net effect on blood pressure than seen in normotensive subjects. Evidence of vascular hypertrophy in the borderline hypertensive subjects was considered to account for their vascular hyperreactivity to cold pressor stimulation. Collectively, the observations in this study further support the view that the early stages of hypertension in white men are characterized by sympathetic nervous system hyperreactivity, but only in association with tasks that elicit active behavioral coping responses.(ABSTRACT TRUNCATED AT 250 WORDS)


The role of social-cognitive and emotional factors on testicular self-examination.
This study determined the role of social-cognitive and affective factors in promoting testicular self-examination. Male participants (N = 115) rated their perceptions of testicular cancer, social-cognitive variables (attitude, subjective norm, and perceived control), and their emotions towards testicular cancer (anxiety and shame) and testicular self-examination (anticipated regret and relief). Participants also stated whether or not they had performed a testicular self-examination within the last month. Perceived control and anticipated relief positively predicted testicular self-examination within the last month. Both these factors also positively predicted the intention to self-examine in the future. Intention was also positively predicted by attitude and negatively predicted by shame. These results highlight the importance of social-cognitive and emotional factors in promoting health screening. Targeting these factors might improve the effectiveness of testicular self-examination interventions. Copyright © 2016 John Wiley & Sons, Ltd.


Measuring the Performance of Neural Models.
Good metrics of the performance of a statistical or computational model are essential for model comparison and selection. Here, we address the design of performance metrics for models that aim to predict neural responses to sensory inputs. This is particularly difficult because the responses of sensory neurons are inherently variable, even in response to repeated presentations of identical stimuli. In this situation, standard metrics (such as the correlation coefficient) fail because they do not distinguish between explainable variance (the part of the neural response that is systematically dependent on the stimulus) and response variability (the part of the neural response that is not systematically dependent on the stimulus, and cannot be explained by modeling the stimulus-response relationship). As a result, models which perfectly describe the systematic stimulus-response relationship may appear to perform poorly. Two metrics have previously been proposed which account for this inherent variability: Signal Power Explained (SPE, Sahani and Linden, 2003), and the normalized correlation coefficient (CC norm , Hsu et al., 2004). Here, we analyze these metrics, and show that they are intimately related. However, SPE has no lower bound, and we show that, even for good models, SPE can yield negative values that are difficult to interpret. CC norm is better behaved in that it is effectively bounded between -1 and 1, and values below zero are very rare in practice and easy to interpret. However, it was hitherto not possible to calculate CC norm directly; instead, it was estimated using imprecise and laborious resampling techniques. Here, we identify a new approach that can calculate CC norm quickly and accurately. As a result, we argue that it is now a better choice of metric than SPE to accurately evaluate the performance of neural models.


Antiviral effect of oryzacystatin, a proteinase inhibitor in rice, against herpes simplex virus type 1 in vitro and in vivo.
Oryzacystatin (OC) is the first-described cystatin originating from rice seed; it consists of two molecular species, OC-I and OC-II, which have antiviral action against poliovirus in vitro (H. Kondo, S. Ijiri, K. Abe, H. Maeda, and S. Arai, FEBS Lett. 299:48-50, 1992). In the experiments reported here, we investigated the effects of OC-I and OC-II on the replication of herpes simplex virus type 1 (HSV-1) in vitro and in vivo. HSV-1 was inoculated onto monolayers of monkey kidney epithelial cells (CV-1 cells) at a multiplicity of infection of 0.1 PFU per cell. After adsorption of the virus onto cells, the cultures were incubated in the presence of either OC-I or OC-II in the concentration range of 1.0 to 300 microM, and the supernatant virus yield was quantitated at 24 h. The effective concentration for 90% inhibition of HSV-1 was 14.8 microM, while a cytotoxic effect on CV-1 cells without infection of HSV-1 was not observed below 500 microM OC-I. Therefore, the apparent in vitro chemotherapeutic index was estimated to be more than 33. In the mouse model of HSV-1-induced keratitis and encephalopathy, topical administration of OC-I to the mouse cornea produced a significant decrease in virus production in the cornea (mean virus yields: 3.11 log10 PFU in the treated group and 4.37 log10 PFU in the control group) and significant improvement in survival rates (P = 0.01). The in vivo antiherpetic effect of OC-I was comparable to that of acyclovir, indicating that topical treatment of HSV-1 infection in humans with OC-I might be possible. Our data also suggest the importance of some thiol proteinases, which may be derived from either the host's cells or HSV-1, during the replication process of HSV-1.


Mitochondrial carbonic anhydrase.
We have assayed carbonic anhydrase activity (carbonate dehydratase, carbonate hydro-lyase, EC 18.104.22.168) and bicarbonate permeability in suspensions of broken and intact guinea pig mitochondria by monitoring the disappearance of C16O18O. We found significant activity in preparations from liver and skeletal muscle, but not in preparations from heart muscle, brain, and kidney. Intact mitochondria containing carbonic anhydrase produce a two-phase acceleration of the disappearance of the labeled CO2, which indicates that the enzyme is located in a region more accessible to CO2 than to HCO3-. Acetazolamide inhibits the enzyme activity instantly in broken mitochondria but only after a delay in intact mitochondria, indicating that the enzyme is in a region not immediately accessible to the inhibitor. Sonication of mitochondria containing carbonic anhydrase activity releases the enzyme, which remains in the supernatant after sedimentation of the submitochondrial particles. This shows that mitochondrial carbonic anhydrase is in the matrix compartment and not in, or bound to, the inner membrane. The activity of the enzyme increases markedly with increasing pH. The enzyme activity of intact mitochondria is greater than that of the broken mitochondria at the same pH of the suspending fluid, corresponding to an intramitochondrial pH that is 0.2-0.5 unit more alkaline.


A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases.
The 3'----5' exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.


EpiDex® Swiss field trial 2004-2008.
Approximately 20% of leg ulcers remain unresponsive to the best conservative standard of care. So far, these patients could either receive conventional skin grafts or had to accept their intractable wound. Skin substitutes from cell culture may represent a promising alternative to heal a major part of these patients on a non-surgical, potentially more cost-effective basis. To systematically evaluate the first 68 patients treated in Switzerland (Swiss EpiDex® field trial 2004-2008). Retrospective study on EpiDex treatment of a complete consecutive series of 68 patients with chronic wounds (66 chronic leg ulcers, 2 sores) unresponsive to best conservative standard of care. The primary end point was complete wound closure within 9 months after transplantation, the secondary end points change of wound surface area, pain reduction and overall judgement by the patient. Adverse effects were infection, dermatitis and others. Calculation of treatment costs was made. By the end of the study, 50/68 (74%) of patients had their wound completely healed [venous 29/37 (78%); mixed 7/9 (78%); others 14/22 (64%)]; 10/68 (15%) had the wound surface area reduced by >50%, and 8/68 (12%) did not respond to the EpiDex treatment. Wound pain disappeared completely in 78% and partially in 13%. Fifteen patients (22%) received antibiotics for wound infection, and 2 (3%) developed dermatitis (not related to the local therapy). Average treatment costs for venous ulcers amounted to EUR 5,357, compared to EUR 5,722-8,622 reimbursed according to the German DRG system (2010) for an in-patient skin graft. EpiDex may effectively heal up to three quarters of recalcitrant chronic leg ulcers. Thus, it represents an intermediate step to avoid costly in-patient split-skin mesh graft treatments. Patients remain mobilized, and a donor site is avoided. Large wound size or a necrotic wound bed limit the use of EpiDex. Otherwise, it offers the opportunity to avoid conventional skin grafts in a significant number of chronic leg ulcer patients.


The inositol polyphosphate 5-phosphatases: traffic controllers, waistline watchers and tumour suppressors?
Phosphoinositide signals regulate cell proliferation, differentiation, cytoskeletal rearrangement and intracellular trafficking. Hydrolysis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3, by inositol polyphosphate 5-phosphatases regulates synaptic vesicle recycling (synaptojanin-1), hematopoietic cell function [SHIP1(SH2-containing inositol polyphosphate 5-phosphatase-1)], renal cell function [OCRL (oculocerebrorenal syndrome of Lowe)] and insulin signalling (SHIP2). We present here a detailed review of the characteristics of the ten mammalian 5-phosphatases. Knockout mouse phenotypes and underexpression studies are associated with significant phenotypic changes, indicating non-redundant roles, despite, in many cases, overlapping substrate specificity and tissue expression. The extraordinary complexity in the control of phosphoinositide signalling continues to be revealed.


Preference-Sensitive Management of Post-Mammography Decisions in Breast Cancer Diagnosis.
Decision models representing the clinical situations where treatment options entail a significant risk of morbidity or mortality should consider the variations in risk preferences of individuals. In this study, we develop a stochastic modeling framework that optimizes risk-sensitive diagnostic decisions after a mammography exam. For a given patient, our objective is to find the utility maximizing diagnostic decisions where we define the utility over quality-adjusted survival duration. We use real data from a private mammography database to numerically solve our model for various utility functions. Our choice of utility functions for the numerical analysis is driven by actual patient behavior encountered in clinical practice. We find that invasive diagnostic procedures such as biopsies are more aggressively used than what the optimal risk-neutral policy would suggest, implying a far-sighted (or equivalently risk-seeking) behavior. When risk preferences are incorporated into the clinical practice, policy makers should bear in mind that a welfare loss in terms of survival duration is inevitable as evidenced by our structural and empirical results.


Modelling the impact of prior immunity, case misclassification and bias on case-control studies in the investigation of outbreaks of cryptosporidiosis.
Cryptosporidiosis is the most common cause of outbreaks of disease linked to mains water supply in the United Kingdom and the second commonest in the United States. Recent evidence has suggested that prior population immunity may have an impact on the epidemiology of waterborne outbreaks and in particular prior immunity may reduce the power of case-control studies for demonstrating association between disease and water consumption behaviour. However, the degree of impact of prior immunity on the power of epidemiological studies is not yet clear. This paper reports the results of some simple mathematical models of outbreaks of waterborne disease in populations with varying levels of immunity due to prior water and non-water exposure. The basic outbreak model was run on a spreadsheet. To determine the impact of prior immunity on case-control studies, further analysis was done using a Monte Carlo method to simulate sampling from cases and controls. It was found that moderate degrees of prior immunity due to water associated disease could markedly reduce the relative risk of water consumption on illness in waterborne outbreaks. In turn this would reduce the power of case-control studies. In addition, this model was used to demonstrate the impact of case misclassification and recall bias on case-control studies. Again it was found that within the model, the results of case-control studies could be significantly affected by both these sources of error. Anyone conducting epidemiological investigations of potentially waterborne outbreaks of disease should be aware of the epidemiological problems. Mistakes from case-control studies will be minimized if the outbreak team pays considerable attention to the descriptive phase of the investigation and if case-control studies are conducted as soon as possible after an outbreak is detected.