Exocytosis in chromaffin cells: evidence for a MgATP-independent step that requires a pertussis toxin-sensitive GTP-binding protein.
We have previously described that mastoparan, an amphiphilic tetradecapeptide that activates heterotrimeric G-proteins, inhibits Ca(2+)-induced MgATP-dependent secretion from streptolysin-O-permeabilized chromaffin cells [Vitale, Mukai, Rouot, Thiersé, Aunis and Bader (1993) J. Biol. Chem. 268, 14715-14723]. Our observations suggest the involvement of an inhibitory G(o)-protein, possibly located on the membrane of secretory granules, in the final stages of the exocytotic pathway in chromaffin cells. Here, we demonstrate that mastoparan is also able to stimulate the Ca(2+)-dependent secretion of catecholamines in the absence of MgATP in the medium. This MgATP-independent secretion is totally blocked by tetanus toxin, a potent inhibitor of exocytosis in all neurosecretory cells so far investigated, suggesting that the mastoparan target is a component of the exocytotic machinery. Mas17, a mastoparan analogue inactive on G-proteins, had no effect on catecholamine secretion whereas both Mas7, a highly active analogue of mastoparan, and AlF4-, which selectively activates trimeric G-proteins, triggered MgATP-independent secretion. Non-hydrolysable GTP analogues (GTP[S] and p[NH]ppG) mimicked the dual effects of mastoparan on secretion: they inhibited exocytosis in the presence of MgATP and stimulated MgATP-independent secretion. The different potencies displayed by these two analogues suggest the involvement of two distinct G-proteins. Accordingly, the mastoparan-induced MgATP-independent secretion is highly sensitive to pertussis toxin (PTX) whereas the inhibition by mastoparan of secretion in the presence of MgATP is resistant to PTX treatment. When permeabilized cells were incubated with mastoparan, the release of arachidonic acid increased in a PTX-sensitive manner. 7,7-Dimethyl-5,8-eicosadienoic acid, a potent inhibitor of intracellular phospholipase A2, inhibited both the arachidonate release and the MgATP-independent catecholamine secretion evoked by mastoparan. In contrast, neomycin, an inhibitor of phospholipase C, had no significant effect on either the release of arachidonic acid or the secretion of catecholamines provoked by mastoparan. We conclude that two distinct heterotrimeric G-proteins act in series in the exocytotic pathway in chromaffin cells: one controls an ATP-dependent priming step through an effector pathway that remains to be determined, and the second is involved in a late Ca(2+)-dependent step which does not require MgATP but possibly involves the generation of arachidonic acid.


Endothelial F-actin changes in the alkali burned rabbit cornea.
The healing mechanism of corneal endothelium after alkali burn was not completely understood. Rabbit cornea was burned with 1N sodium hydxoside for 1 minute. Endothelial F-actin was stained with NBD-phallacidin in regular sequence to find out the details of endothelial healing after alkali burn. F-actin was completely destroyed leaving a sharp margin against the unaffected area 1 hour after the burn. In the 3, 5 and 7 day specimens, highly active F-actin reactions were noted at the wound margin. New multiple F-actin layers, arising from the intact endothelium near the wound margin, were noted in the 9 day specimen. In the 8 1/2 month specimen, the endothelial defected area was covered by large primitive cells, each of which showed F-actin fiber bundles in the cytoplasm with a large nuclear shadow. Nearly all of the large primitive cells showed F-actin fibers arranged in shapes of cell junctions. Twelve months after the burn, endothelial defects were not found. Nearly all of the endothelial cells were normal in size and shape except for some mushroom-like projections toward the anterior chamber in some areas. Nineteen months after the burn, the endothelial cells were normal. Endothelial wound healing process can be continued even 1 year after the alkali burn in rabbit cornea.


Observational tools for assessment of procedural skills: a systematic review.
Assessment by direct observation of procedural skills is an important source of constructive feedback. The aim of this study was to identify observational tools for technical skill assessment, to assess characteristics of these tools, and to assess their usefulness for assessment. Included studies reported tools for observational assessment of technical skills. A total of 106 articles were included. Three main categories included global assessment scales evaluating generic skills (n = 29), task-specific methods assessing procedure-specific skills (n = 30), and combinations of tools evaluating both generic and task-specific skills (n = 47). In most studies, content validity was not evaluated using an accepted scientific method. All tools were assessed for inter-rater reliability and construct validity. Data on feasibility, acceptability, and educational impact were sparse. There is evidence of validity and reliability for observational assessment tools at the trainee level. In most studies a comprehensive analysis of the tools was not achieved. Evaluation of technical skill using current observational assessment tools is not reliable and valid at the specialist level. Future research needs to focus on further systematic tool development and analysis, especially at the specialist level.


Effect of short- and long-term beta-adrenergic blockade on lipolysis during fasting in humans.
Stable isotope tracers and indirect calorimetry were used to evaluate the importance of beta-adrenergic stimulation of lipolysis and triglyceride-fatty acid cycling during fasting in healthy human volunteers. Each subject was studied after 12 and 84 h of fasting both with and without propranolol infusion (protocol 1) and when oral propranolol treatment was given throughout fasting (protocol 2). In protocol 1, the rates of appearance of glycerol and palmitic acid increased from 3.04 +/- 0.19 and 1.78 +/- 0.17 mumol.kg lean body mass-1.min-1, respectively, after 12 h of fasting to 5.28 +/- 0.31 and 3.47 +/- 0.15 mumol.kg lean body mass-1.min-1, respectively, after 84 h of fasting (P less than 0.005). The rate of triglyceride-fatty acid cycling increased from 97 +/- 8 to 169 +/- 5 mumol/min (P less than 0.005). Intravenous propranolol infusion decreased the rate of lipolysis after both 12 and 84 h of fasting, but the magnitude of the antilipolytic effect was much greater after 84 h (P less than 0.005). In protocol 2, the rate of lipolysis and triglyceride-fatty acid cycling was still increased by fasting despite beta-adrenergic blockade with oral propranolol. This study demonstrates that beta-adrenergic stimulation contributes to the mobilization of fat during fasting. However, other mechanism(s) can increase lipolysis and triglyceride-fatty acid cycling when beta-adrenergic receptors are continuously blocked.


[Observer variation in diagnostic procedures related to cardiothoracic surgery].
The results reliability is a predominant condition in the validity of clinical research. The observer agreement determination is a useful procedure in the confirmation of the obtained results in clinical research. The present work is a presentation of the basis about observer agreement and a review of national publications in cardiology, pneumology and cardiothoracic surgery between january 1980 and july 1993, where we found just two publications with the application of observer agreement determination in echocardiography, electrocardiography, microscopy and hemodynamics. It was concluded that is necessary the increase in determination of observer agreement and inform this results in our publications, excluding the variability attributed to patients, diagnostics equipment or the circumstances.


Systematic review of UV-based therapy for psoriasis.
UV-based therapies, which include narrow-band (NB) UVB, broad-band (BB) UVB, and psoralen and UVA (PUVA), are well known treatment options for moderate to severe plaque psoriasis. However, there are limited evidence-based reviews on their efficacy, short-term safety, and tolerability. The aim of the study was to evaluate the efficacy, short-term safety, and tolerability of UV-based therapy in the treatment of adults with moderate to severe plaque psoriasis. We performed a systematic review and meta-analysis of randomized controlled trials (RCTs) evaluating NB-UVB, BB-UVB, and PUVA in adults with moderate to severe plaque-type psoriasis. Our efficacy outcomes were ≥ Psoriasis Area and Severity Index (PASI)-75 and clearance. We evaluated the short-term safety and tolerability from the percentage of adverse effects and withdrawal due to adverse effects, respectively. Forty-one RCTs, with a total of 2,416 patients, met the eligibility criteria and were included in the analysis. In regard to PASI-75 in monotherapy trials, PUVA (mean: 73 %, 95 % CI 56-88) was the most effective modality. Trials with BB-UVB also showed a high PASI-75 (73 %) but with a wide CI (18-98) due to heterogeneity of the total available three studies. This was followed by NB-UVB (mean: 62 %, 95 % CI 45-79) then bath PUVA (mean: 47 %, 95 % CI 30-65). In regard to clearance in the monotherapy trials, PUVA (mean: 79 %, 95 % CI 69-88) was superior to NB-UVB (mean: 68 %, 95 % CI 57-78), BB-UVB (mean: 59 %, 95 % CI 44-72), and bath PUVA (mean: 58 %, 95 % CI 44-72). The percentages of asymptomatic erythema development in monotherapy trials were 64 % for BB-UVB, 57 % for NB-UVB, 45 % for PUVA, and 34 % for bath PUVA. Symptomatic erythema or blistering percentages for the monotherapy trials were as follows: 7.8 % for NB-UVB, 2 % for BB-UVB, 17 % for PUVA, and 21 % for bath PUVA. The percentages of withdrawal due to adverse effects were 2 % for NB-UVB, 4.6 % for BB-UVB, 5 % for PUVA, and 0.7 % for bath PUVA monotherapy trials. As a monotherapy, PUVA was more effective than NB-UVB, and NB-UVB was more effective than BB-UVB and bath PUVA in the treatment of adults with moderate to severe plaque-type psoriasis, based on clearance as an end point. Based on PASI-75, the results were similar except for BB-UVB, which showed a high mean PASI-75 (73 %) that was similar to PUVA, but with a wide CI (18-98). The short-term adverse effects were mild as shown by the low rate of withdrawal due to adverse effects.


Homocysteine induces heme oxygenase-1 expression via transcription factor Nrf2 activation in HepG2 cell.
Elevated level of plasma homocysteine has been related to various diseases. Patients with hyperhomocysteinemia can develop hepatic steatosis and fibrosis. We hypothesized that oxidative stress induced by homocysteine might play an important role in pathogenesis of liver injury. Also, the cellular response designed to combat oxidative stress is primarily controlled by the transcription factor Nrf2, a principal inducer of anti-oxidant and phase II-related genes. HepG2 cells were treated with homocysteine in different time periods. Glutathione content was measured by flowcytometry. Using electrophoretic mobility shift assay (EMSA) and Western-blotting, anti-oxidant response element (ARE)-binding activity of Nrf2 for heme ocygenase-1 (HO-1) was demonstrated. Real time RT-PCR and Western-blotting were performed to evaluate whether homocysteine was able to induce mRNA and protein expression of HO-1. The role of Nrf2 in cellular response to homocysteine is substantiated by the following observations in HepG2 cells exposed to homocysteine (i) Western-blotting revealed that Nrf2 is strongly stabilized and became detectable in nuclear extracts. (ii) EMSA demonstrated increased binding of Nrf2 to oligomers containing HO-1 promoter-specific ARE-binding site. (iii) Real time RT-PCR and Western-blotting revealed increased mRNA and protein expression of inducible gene HO-1 after treatment with homocysteine. Data presented in the current study provide direct evidence that the immediate cellular response to oxidative stress provoked by homocysteine is orchestrated mainly by the Nrf2-ARE pathway. Therefore, induction of Nrf2-ARE-dependent expression of HO-1 could be a therapeutic option for hepatic cells damage induced by homocysteine.


Hallucinations on demand: the utility of experimentally induced phenomena in hallucination research.
Despite the desire to delve deeper into hallucinations of all types, methodological obstacles have frustrated development of more rigorous quantitative experimental techniques, thereby hampering research progress. Here, we discuss these obstacles and, with reference to visual phenomena, argue that experimentally induced phenomena (e.g. hallucinations induced by flickering light and classical conditioning) can bring hallucinations within reach of more objective behavioural and neural measurement. Expanding the scope of hallucination research raises questions about which phenomena qualify as hallucinations, and how to identify phenomena suitable for use as laboratory models of hallucination. Due to the ambiguity inherent in current hallucination definitions, we suggest that the utility of phenomena for use as laboratory hallucination models should be represented on a continuous spectrum, where suitability varies with the degree to which external sensory information constrains conscious experience. We suggest that existing strategies that group pathological hallucinations into meaningful subtypes based on hallucination characteristics (including phenomenology, disorder and neural activity) can guide extrapolation from hallucination models to other hallucinatory phenomena. Using a spectrum of phenomena to guide scientific hallucination research should help unite the historically separate fields of psychophysics, cognitive neuroscience and clinical research to better understand and treat hallucinations, and inform models of consciousness. This article is part of the theme issue 'Offline perception: voluntary and spontaneous perceptual experiences without matching external stimulation'.


Ultrathin nickel hydroxide on carbon coated 3D-porous copper structures for high performance supercapacitors.
An ultrathin nickel hydroxide layer electrodeposited on a carbon-coated three-dimensional porous copper structure (3D-C/Cu) is suggested as an additive and binder-free conductive electrode with short electron path distances, large electrochemical active sites, and improved structural stability, for high performance supercapacitors. The 3D-porous copper structure (3D-Cu) provides high electrical conductivity and facilitates electron transport between the Ni(OH)2 active materials and the current collector of the Ni-plate. A carbon coating was applied to the 3D-Cu to prevent the oxidation of Cu, without degrading the electron transport behavior of the 3D-Cu. The 3D-Ni(OH)2/C/Cu exhibited a high specific capacitance of 1860 F g-1 at 1 A g-1, and good cycling performance, with an 86.5% capacitance retention after 10 000 cycles. When tested in a two-electrode system, an asymmetric supercapacitor exhibited an energy density of 147.9 W h kg-1 and a power density of 37.0 kW kg-1. These results open a new area of ultrahigh-performance supercapacitors, supported by 3D-Cu electrodes.


Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.
We studied recovery of poly(3-hydroxybutyric acid) (PHB) from Alcaligenes eutrophus and a recombinant Escherichia coli strain harboring the A. eutrophus poly(3-hydroxyalkanoic acid) biosynthesis genes. The amount of PHB degraded to a lower-molecular-weight compound in A. eutrophus during the recovery process was significant when sodium hypochlorite was used, but the amount degraded in the recombinant E. coli strain was negligible. However, there was no difference between the two microorganisms in the patterns of molecular weight change when PHB was recovered by using dispersions of a sodium hypochlorite solution and chloroform. To understand these findings, we examined purified PHB and lyophilized cells containing PHB by using a differential scanning calorimeter, a thermogravimetric analyzer, and nuclear magnetic resonance. The results of our analysis of lyophilized whole cells containing PHB with the differential scanning calorimeter suggested that the PHB granules in the recombinant E. coli strain were crystalline, while most of the PHB in A. eutrophus was in a mobile amorphous state. The stability of the native PHB in the recombinant E. coli strain during sodium hypochlorite treatment seemed to be due to its crystalline morphology. In addition, as determined by the thermogravimetric analyzer study, lyophilized cell powder of the recombinant E. coli strain containing PHB exhibited greater thermal stability than purified PHB obtained by chloroform extraction. The PHB preparations extracted from the two microorganisms had identical polymer properties.


A study of the mechanism of the chaperone-like function of an scFv of human creatine kinase by computer simulation.
A new application of antibodies is to use them as macromolecular chaperones. Protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. However, not all antibodies that bind to one antigen could act as a chaperone. Experiments show that some screened anti-human creatine kinase single chain antibodies (scFV) could assist in the folding and stabilizing of the enzyme, while others could not. We built the model of the single chain antibody (scFv-A4) that increased the stability of human creatine kinase (HCK) by the homology modeling method. Epitopes of human creatine kinase were predicted by computer and then the binding of scFv-A4 and HCK was modeled with computer. The calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scFv-A4-HCK complex. Based on the above study we gave an explanation about how scFv-A4 could act as a macromolecular chaperone assisting the folding of HCK. This study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies' chaperone-like function.


Smoking, drinking and incident tuberculosis in rural India: population-based case-control study.
To investigate the extent to which smoking and/or drinking can increase the incidence of pulmonary tuberculosis (TB), a population-based case-control study was conducted in rural south India. A total of 1839 males and 870 females treated in 2000-03 by state TB clinics were interviewed at home in 2004-05 about their education, smoking and drinking habits before disease onset. As controls, 2134 men and 2119 women without TB were randomly chosen from case villages and interviewed. Incidence rate ratios (RRs) are from logistic regression, adjusted for age and education. No women smoked or drank. The main analyses are of men aged 35-64 years, 949 cases treated for new pulmonary TB and 1963 controls. In the study, 81.5% of the cases and 55.2% of the controls had ever smoked, yielding a standardized ever- vs never-smoker TB incidence RR of 2.7 [95% confidence interval (CI) 2.2-3.3, P < 0.00001). Among control ever-smokers 96% still smoked, 71% used only bidis (mean 17 per day) and 28% used only cigarettes (mean 7 per day). After additional adjustment for alcohol, this RR was 2.2 (95% CI 1.7-2.7, P < 0.00001), but even among those who had never drunk alcohol the standardized ever- vs never-smoker RR was 2.6 (95% CI 2.0-3.6, P < 0.00001). The corresponding RRs for ever- vs never-drinking were somewhat less extreme: 2.2 (95% CI 1.8-2.6, P < 0.00001) without adjustment for smoking, 1.5 (95% CI 1.2-1.9, P = 0.00004) with adjustment for smoking and 2.1 (95% CI 1.4-3.0, 2P = 0.0001) among those who had never smoked. Among control ever-drinkers, 96% still drank and 99% used only spirits (mean 0.3 l/week). This study of reliably confirmed disease (by the criteria of state TB clinics) demonstrates an increased incidence of pulmonary TB among those who smoke and among those who drink. The effects of smoking after adjustment for drinking were more definite than those of drinking after adjustment for smoking.


Clinical significance of ALDH1 combined with DAPI expression in patients with esophageal carcinoma.
Esophageal carcinoma is the most common type of tumor, with the incidence in China accounting for 50% of cases worldwide and the majority of patients not surviving due to tumor recurrence. According to the cancer stem cell theory, tumor development and recurrence is due to the excitation of a cancer stem cell. Aldehyde dehydrogenase 1 (ALDH1) is an appropriate marker for cancer stem cells and the present study aimed at determining the function of ALDH1 in human esophageal carcinoma. Indirect fluorescence antibody staining was used to investigate the association between the level of ALDH1 protein expression and clinicopathological parameters, including sex, age, vein invasion, degree of tumor cell differentiation and clinical stage. DAPI was used to stain the nuclei of tumor cells and exclude non-tumor cells. The results of the present study revealed that ALDH1 expression was associated with the level of tumor cell differentiation, tumor-node-metastasis stage and lymphatic invasion. In addition, increased expression of ALDH1 was identified in esophageal carcinoma tissues compared with in healthy esophageal tissues. Therefore, ALDH1 may be used as a parameter for the pathology of esophageal carcinoma.


Provider Perspectives on Integrating Sensor-Captured Patient-Generated Data in Mental Health Care.
The increasing ubiquity of health sensing technology holds promise to enable patients and health care providers to make more informed decisions based on continuously-captured data. The use of sensor-captured patient-generated data (sPGD) has been gaining greater prominence in the assessment of physical health, but we have little understanding of the role that sPGD can play in mental health. To better understand the use of sPGD in mental health, we interviewed care providers in an intensive treatment program (ITP) for veterans with post-traumatic stress disorder. In this program, patients were given Fitbits for their own voluntary use. Providers identified a number of potential benefits from patients' Fitbit use, such as patient empowerment and opportunities to reinforce therapeutic progress through collaborative data review and interpretation. However, despite the promise of sensor data as offering an "objective" view into patients' health behavior and symptoms, the relationships between sPGD and therapeutic progress are often ambiguous. Given substantial subjectivity involved in interpreting data from commercial wearables in the context of mental health treatment, providers emphasized potential risks to their patients and were uncertain how to adjust their practice to effectively guide collaborative use of the FitBit and its sPGD. We discuss the implications of these findings for designing systems to leverage sPGD in mental health care.


Can intranasal drug use reduce HCV infection among injecting drug users?
Preventing HCV infection among people who inject drugs is a difficult public health challenge. We examined the potential role of intranasal drug use in reducing HCV acquisition. Subjects were recruited from IDUs entering the Beth Israel drug detoxification program from 2005 to 2010. A structured interview was administered and serum samples were collected for HCV testing. 726 active injecting drug users were recruited from 2005 to 2010. HCV prevalence was 71%, 90% reported recent heroin injection and 44% reported recent intranasal heroin use. In a multiple logistic regression analysis, being HCV seropositive was associated with more years injecting, Latino ethnicity, previous testing for HCV, and recent injection of speedball, and negatively associated with recent intranasal use of heroin (AOR=0.52, 95% CI 0.33-0.82) and intranasal use of speedball (AOR=0.41, 95% CI 0.31-0.80). The association between intranasal heroin use and lower HCV seroprevalance was observed among both new injectors and persons with long injecting histories (16+ years since first injection). Encouraging intranasal use as an alternative to injection among persons currently injecting drugs may be a viable strategy for reducing HCV transmission.


Localization of amiloride-sensitive Na+ channels in intestinal epithelia.
Polyclonal antibodies raised against purified bovine renal papillary amiloride-sensitive Na+ channels were used to localize Na(+)-channel proteins in mouse and piglet small intestine. Immunostaining using the avidin-biotin-peroxidase technique revealed epithelial Na(+)-channel epitopes localized to apical regions of villus enterocytes in jejunal tissues of both species. Anti-Na(+)-channel antibodies also stained apical borders of villus enterocytes in piglet ileum and apical borders of surface cells in the piglet distal colon. On immunoblots of jejunal, colonic, and renal tissues the anti-Na(+)-channel antibodies recognized one to three polypeptides of apparent molecular masses similar to those found in bovine renal epithelial Na(+)-channel protein (the 55-65, 110-116, and 150-kDa subunits). The antibodies also recognized a polypeptide in the 40- to 45-kDa range in mouse intestine, which is comparable to the 35- to 40-kDa subunit of a renal Na+ channel. The results demonstrate that epitopes comparable to those present in renal amiloride-sensitive Na+ channels are found in apical regions of absorptive epithelial cells in the mammalian small and large intestine.


Design and Calibration of a Low-Cost SDI-12 Soil Moisture Sensor.
Water is the main limiting factor in agricultural production as well as a scarce resource that needs to be optimized. The measurement of soil water with sensors is an efficient way for optimal irrigation management. However, commercial sensors are still too expensive for most farmers. This paper presents the design, development and calibration of a new capacitive low-cost soil moisture sensor that incorporates SDI-12 communication, allowing one to select the calibration equation for different soils. The sensor was calibrated in three different soils and its variability and accuracy were evaluated. Lower but cost-compensated accuracy was observed in comparing it with commercial sensors. Field tests have demonstrated the temperature influence on the sensor and its capability to efficiently detect irrigation and rainfall events.


DNA Computing: NOT Logic Gates See the Light.
DNA-based Boolean logic gates (for example, AND, OR, and NOT) can be assembled into complex computational circuits that generate an output signal in response to specific patterns of oligonucleotide inputs. However, the fundamental nature of NOT gates, which convert the absence of an input into an output, makes their implementation within DNA-based circuits difficult. Premature execution of a NOT gate before completion of its upstream computation introduces an irreversible error into the circuit. By utilizing photocaging groups, we developed a novel DNA gate design that prevents gate function until irradiation at a certain time point. Optical activation provides temporal control over circuit performance by preventing premature computation and is orthogonal to all other components of DNA computation devices. Using this approach, we designed NAND and NOR logic gates that respond to synthetic microRNA sequences. We further demonstrate the utility of the NOT gate within multilayer circuits in response to a specific pattern of four microRNAs.


Tone-sequence analysis in the auditory cortex of awake macaque monkeys.
The present study analyzed neuronal responses to two-tone sequences in the auditory cortex of three awake macaque monkeys. The monkeys were passively exposed to 430 different two-tone sequences, in which the frequency of the first tone and the interval between the first and the second tone in the sequence were systematically varied. The frequency of the second tone remained constant and was matched to the single-tone frequency sensitivity of the neurons. Multiunit activity was recorded from 109 sites in the primary auditory cortex and posterior auditory belt. We found that the first tone in the sequence could inhibit or facilitate the response to the second tone. Type and magnitude of poststimulatory effects depended on the sequence parameters and were related to the single-tone frequency sensitivity of neurons, similar to previous observations in the auditory cortex of anesthetized animals. This suggests that some anesthetics produce, at the most, moderate changes of poststimulatory inhibition and facilitation in the auditory cortex. Hence many properties of the sequence-sensitivity of neurons in the auditory cortex measured in anesthetized preparations can be applied to neurons in the auditory cortex of awake subjects.


Endoplasmic reticulum stress-induced apoptosis in intestinal epithelial cells: a feed-back regulation by mechanistic target of rapamycin complex 1 (mTORC1).
Endoplasmic reticulum (ER) stress is associated with multiple pathological processes of intestinal diseases. Despite a critical role of mechanistic target of rapamycin complex 1 (mTORC1) in regulating cellular stress response, the crosstalk between mTORC1 and ER stress signaling and its contribution to the intestinal barrier function is unknown. In the present study, we showed that intestinal epithelial cells (IEC-6) incubated with tunicamycin led to caspase-3-dependent apoptotic cell death. The induction of cell death was accompanied by activation of unfolded protein response as evidenced by increased protein levels for BiP, p-IRE1α, p-eIF2α, p-JNK, and CHOP. Further study demonstrated that tunicamycin-induced cell death was enhanced by rapamycin, a specific inhibitor of mTORC1. Consistently, tunicamycin decreased transepithelial electrical resistance (TEER) and increased permeability of the cells. These effects of tunicamycin were exacerbated by mTORC1 inhibitor. Taken together, the data presented here identified a previously unknown crosstalk between an unfold protein response and mTORC1 signaling in the intestinal epithelium. This feed-back loop regulation on ER stress signaling by mTORC1 is critical for cell survival and intestinal permeability in epithelial cells.


Thiolation and Carboxylation of Glutathione Synergistically Enhance Its Lead-Detoxification Capabilities.
The natural tripeptide glutathione (GSH) is a ubiquitous compound harboring various biological tasks, among them interacting with essential and toxic metal ions. Yet, although weakly binding the poisonous metal lead (Pb), GSH poorly detoxifies it. β-Mercaptoaspartic acid is a new-to-nature novel amino acid that was found to enhance the Pb-detoxification capability of a synthetic cyclic tetrapeptide. Aiming to explore the advantages of noncanonical amino acids (ncAAs) of this nature, we studied the detoxification capabilities of GSH and three analogue peptides, each of which contains at least one ncAA that harbors both free carboxylate and thiolate groups. A thorough investigation that includes in vitro detoxification and mechanistic evaluations, metal-binding affinity, metal selectivity, and computational studies shows that these ncAAs are highly beneficial in additively enhancing Pb binding and reveals the importance of both high affinity and metal selectivity in synergistically reducing Pb toxicity in cells. Hence, such ncAAs join the chemical toolbox against Pb poisoning and pollution, enabling peptides to strongly and selectively bind the toxic metal ion.


Problems in measuring the effectiveness of cognitive therapy for pathological gambling.
Cognitive therapy is a relatively new approach to the treatment of pathological gambling. Theoretically, there are strong grounds for believing that cognitive treatments will be effective in helping individuals cut back and stop excessive levels of gambling. However, there is evidence that cognitive therapy for pathological gambling is being confused with cognitive-behaviour therapy. In this paper, the distinction between treatments that are cognitive and those that are cognitive-behavioural is highlighted. Such a distinction has strong implications for the manualisation of therapy. Additionally, a range of problems that confront the evaluation of all therapies for pathological gambling is considered. Spontaneous recovery without therapeutic intervention has been documented in both field studies of both problem and non-problem players and controlled trials of cognitive therapy compared to a waiting list control group. The implications of the phenomenon of spontaneous recovery for the evaluation of cognitive therapy are described. Other problems common to all evaluations of psychotherapies are considered in relation to gambling and recommendations made for outcome study designs.


Studies on the composition of egg-white ovomucin.
1. Purified ovomucin was isolated as an insoluble glycoprotein complex from thick egg white. 2. A homogeneous glycoprotein, designated alpha-ovomucin, of molecular weight 210000 and containing N-acetylglucosamine (6.7%, w/w), N-acetylgalactosamine (0.6%, w/w), galactose (1.8%, w/w), mannose (4.6%, w/w), N-acetylneuraminic acid (1.0%, w/w) and sulphate (0.7%, w/w), was isolated from preparations of reduced ovomucin by sedimentation equilibrium in a density gradient of caesium chloride formed in the presence of 4m-guanidine hydrochloride. 3. A carbohydrate-rich fraction, designated beta-ovomucin (which is homogeneous by sedimentation-velocity analysis in 5m-guanidine hydrochloride but which is heterogeneous by analytical sedimentation equilibrium in a density gradient of caesium chloride in the presence of 4m-guanidine hydrochloride), containing N-acetylglucosamine (11.0%, w/w), N-acetylgalactosamine (8.7%, w/w), galactose (19.2%, w/w), mannose (4.1%, w/w), N-acetylneuraminic acid (13.8%, w/w) and sulphate (2.7%, w/w), was also obtained from preparations of reduced ovomucin by the density-gradient method. 4. Mild acid hydrolysis of the unfractionated ovomucin complex showed that N-acetylneuraminic acid occupied a terminal position of the oligosaccharide chains. 5. Alkaline beta-elimination reactions with the unfractionated ovomucin complex indicated that N-acetylgalactosamine was linked by alkali-labile bonds to hydroxy amino acids.


Challenging the Sanctity of Donorism: Patient Tissue Providers as Payment-Worthy Contributors.
Many research projects rely on human biological materials and some of these projects generate revenue. Recently, it has been argued that investigators have a moral claim to share in the revenue generated by these projects, whereas persons who provide the biological material have no such claim (Truog, Kesselheim, and Joffe 2012). In this paper, we critically analyze this view and offer a positive proposal for why tissue providers have a moral claim to benefit. Focusing on payment as a form of benefit, we argue that research is a joint project and propose a contribution principle for paying participants in those joint projects. We distinguish between contributions that shape a project's revenue generating properties, grounding a claim to payment, and contributions that fail to ground such a claim. We conclude, contrary to existing arguments and practices, that some tissue providers have a moral claim to payment beyond compensation for risk and burden. This conclusion suggests that investigators, institutions, and sponsors should reconsider the fairness of their current practices.


Predictors of next-generation sequencing panel selection using a shared decision-making approach.
The introduction of next-generation sequencing panels has transformed the approach for genetic testing in cancer patients, however, established guidelines for their use are lacking. A shared decision-making approach has been adopted by our service, where patients play an active role in panel selection and we sought to identify factors associated with panel selection and report testing outcomes. Demographic and clinical data were gathered for female breast and/or ovarian cancer patients aged 21 and over who underwent panel testing. Panel type was classified as 'breast cancer panel' (BCP) or 'multi-cancer panel' (MCP). Stepwise multiple logistic regression analysis was used to identify clinical factors most predictive of panel selection. Of the 265 included subjects, the vast majority selected a broader MCP (81.5%). Subjects who chose MCPs were significantly more likely to be ≥50 years of age (49 vs. 31%; p < 0.05), Chinese (76 vs. 47%; p < 0.001) and have a personal history of ovarian cancer (41 vs. 8%; p < 0.001) with the latter two identified as the best predictors of panel selection. Family history of cancer was not significantly associated with panel selection. There were no statistically significant differences in result outcomes between the two groups. In summary, our findings demonstrate that the majority of patients have a preference for interrogating a larger number of genes beyond those with established testing guidelines, despite the additional likelihood of uncertainty. Individual factors, including cancer history and ethnicity, are the best predictors of panel selection.


Relationship between didanosine exposure and surrogate marker response in human immunodeficiency virus-infected outpatients.
We used information available from routine clinic visits to characterize the pharmacokinetics of didanosine in 82 human immunodeficiency virus-infected patients. A total of 271 blood samples were collected for the measurement of didanosine concentrations in plasma (mean +/- standard deviation [SD], 3.30 +/- 2.21 samples/patient). Bayesian estimates of didanosine oral clearance (CL[oral]) were obtained for these patients by the POSTHOC option within the NONMEM software package. Population priors from a previous NONMEM analysis of didanosine pharmacokinetics were used. The mean +/- SD CL(oral) was 132 +/- 27.7 liters/h, which agrees reasonably well with estimates obtained from previous pharmacokinetic studies of didanosine. Estimates of individual didanosine exposure were then used to consider potential relationships between drug exposure and surrogate marker response over a 6-month period. No correlations were found between the didanosine area under the concentration-time curve from 0 to 6 months and the absolute CD4 cell count (r = 0.305; 0.1 < P < 0.2), weight response (r = 0.0857; P > 0.4), or percentage of CD4 lymphocytes (r = 0.0559; P > 0.4). Future efforts to characterize didanosine exposure in outpatients by random sampling methods should involve more directed efforts to limit residual variability in the data.


The incidence and outcome of severe brain trauma - Design and first results of an epidemiological study in an urban area.
Epidemiological data on the incidence, the prehospital and hospital care and the outcome of traumatic brain injury in Germany are scarce. It is therefore difficult to estimate the importance of this injury with respect to magnitude as well as effectiveness and efficiency of therapeutic concepts. We therefore planned a study that was supposed to provide population based epidemiological data in the field of severe brain trauma from the site of the accident until discharge from hospital.All 90.000 prehospital emergencies that were cared for by emergency physicians in Cologne from January 1990 until December 1996 were screened for identification of severe brain trauma. Their clinical course was reviewed using standard records and patients were included if they had their accident within the city of Cologne and fullfilled the final inclusion criteria of GCS ≤ 8 or AISHead ≥ 3. 650 eligible patients were identified of whom 530 had complete datasets (follow-up 80 %). Univariate statistical analysis was performed for all relevant variables. The main study endpoints were incidence and outcome of severe brain trauma.The annual incidence of severe brain trauma in Cologne (1 mio. inhabitants) was 93. The average age was 39 years and 71 % of the patients were male. The average prehospital GCS was 6.8, the average prehospital Trauma Score was 8.3 points. 49 % of the study population suffered from multiple injuries. The overall mortality rate was 46,6 %. 60 % of deaths occurred within the prehospital setting.The incidence of severe brain trauma in Cologne in this study was significantly lower compared to what could be expected from the literature. The overall mortality was high, especially the high prehospital death rate is striking.


The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4.
The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.


Developing physician pay arrangements: the cash and care equation.
Developing physician compensation packages that help a healthcare organization meet its business objectives while satisfying physician pay expectations requires new ways of linking pay to physician performance. Such compensation arrangements specifically should include pay tied to defined performance standards, compensation linked to group performance, performance incentives based on realistic, achievable goals, work performance measured by common criteria, and similar pay ensured for similar work. Final pay arrangements also should include items that are sometimes overlooked, such as fully delineated job responsibilities, performance measures aligned correctly with performance areas, and the value of benefits considered in the cash compensation levels.


Evidence for enhancing childhood obesity treatment from a dual-process perspective: A systematic literature review.
Impulsivity contributes to poor outcomes of existing childhood obesity treatments. Conceptualised within dual-process models, this self-regulation failure reflects the operation of strong automatic processing (heightened food responsivity) and/or weak regulatory processing (poor self-control). This systematic literature review examined the evidence for the self-regulation failure hypothesis from a dual-process models perspective to evaluate its potential for enhancing childhood obesity treatment. Searches were conducted from six databases. Eligibility criteria included: (1) recruited a child or adolescent sample; (2) measured or manipulated one or more automatic and/or regulatory processes (attentional bias, approach bias, working memory, inhibitory control, executive function); (3) used a cross-sectional, longitudinal or experimental design; and (4) included a primary outcome measure that was eating/weight related and/or pertained to the underlying process(es). The search identified 147 eligible studies. Despite methodological variations and inconsistent findings across studies, evidence points to a stronger impact of automatic processes and in particular a reduced capacity for regulatory processing in youngsters with overweight/obesity. Emerging evidence suggests that these processes can be altered by targeted training to curtail food intake and associated weight gain. An intervention protocol based on the dual-process framework has the potential to enhance current childhood obesity treatments. Recommendations for future research are provided.


S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and ameliorates COVID-19 severity in hamsters.
In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (Mpro; also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to submicromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern, including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), has remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.


Lopinavir/ritonavir for the treatment of SARS, MERS and COVID-19: a systematic review.
Lopinavir/ritonavir has been used for the treatment of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) coronavirus infections. It has been suggested that, based on this experience, this drug should also be studied in SARS-CoV2 infection. We performed a systematic review of the literature regarding the use of lopinavir/ritonavir for the treatment of these three infections. We systematically searched the PubMed database from inception to April 30th, 2020, to identify in-vitro and animal studies and any reports of human use of lopinavir/ritonavir for the treatment of SARS, MERS and COVID-19. We also searched the Clinicatrial.gov to identify ongoing trials. Five in-vitro studies evaluated the effect of lopinavir/ritonavir in SARS. Three additional in-vitro studies reported the EC50 of the antiviral activity of lopinavir/ritonavir in MERS. We identified no in vitro studies evaluating the effect of lopinavir/ritonavir on the novel coronavirus. Two retrospective matched-cohort studies reported the use of lopinavir/ritonavir in combination with ribavirin for SARS patients. Three case reports and one retrospective study described the use of lopinavir/ritonavir in MERS. Twenty-two papers describe the use of lopinavir/ritonavir in adult patients with COVID-19. The existing literature does not suffice for assessing whether Lopinavir/ritonavir has any benefit in SARS, MERS or COVID-19.


Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor Cav 1.1 subunit share common binding sites on the skeletal ryanodine receptor.
Maurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Cav1.1) subunit. This domain belongs to the II-III loop of Cav1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021-1631 and 3201-3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351-3507 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. Moreover, when expressed in COS-7 cells, RyR1 carrying a deletion of fragment 7 shows a loss of interaction with both peptide A and maurocalcine. At the functional level, this deletion abolishes the maurocalcine induced stimulation of [3H]ryanodine binding onto microsomes of transfected COS-7 cells without affecting the caffeine and ATP responses.


IL-23 dampens the allergic response to Cryptococcus neoformans through IL-17-independent and -dependent mechanisms.
The cytokines IL-23 and IL-17 have been implicated in resistance to cryptococcal disease, but it is not clear whether IL-23-mediated production of IL-17 promotes fungal containment following pulmonary challenge with Cryptococcus neoformans. We used mice lacking IL-23 (IL-23p19(-/-)) or IL-17RA (IL-17RA(-/-)), and wild type (WT) C57BL/6 mice to examine the IL-23/IL-17 axis after intranasal infection with the C. neoformans strain 52D. The absence of IL-23 or IL-17RA had no effect on pulmonary or brain fungal burden at 1 or 6 weeks after infection. However, survival of IL-23p19(-/-) mice was reduced compared to IL-17RA(-/-) mice. IL-I7 production by CD4 T cells and natural killer T (NKT) cells was impaired in IL-23p19(-/-) lungs, but was not completely abolished. Both IL-23p19(-/-) and IL-17RA(-/-) mice exhibited impaired neutrophil recruitment, increased serum levels of IgE and IgG2b, and increased deposition of YM1/YM2 crystals in the lung, but only IL-23p19(-/-) mice developed persistent lung eosinophilia. Although survival of IL-17RA(-/-) and WT mice was similar after 17 weeks of infection, only surviving IL-17RA(-/-) mice exhibited cryptococcal dissemination to the blood. These data demonstrate that IL-23 dampens the allergic response to cryptococcal infection through IL-17-independent suppression of eosinophil recruitment and IL-17-dependent regulation of antibody production and crystal deposition. Furthermore, IL-23, and to a lesser extent IL-17, contribute to disease resistance.


Evaluation of interventions to prevent vasovagal reactions among whole blood donors: rationale and design of a large cluster randomised trial.
Vasovagal reactions (VVRs) are the most common acute complications of blood donation. Responsible for substantial morbidity, they also reduce the likelihood of repeated donations and are disruptive and costly for blood services. Although blood establishments worldwide have adopted different strategies to prevent VVRs (including water loading and applied muscle tension [AMT]), robust evidence is limited. The Strategies to Improve Donor Experiences (STRIDES) trial aims to reliably assess the impact of four different interventions to prevent VVRs among blood donors. STRIDES is a cluster-randomised cross-over/stepped-wedge factorial trial of four interventions to reduce VVRs involving about 1.4 million whole blood donors enrolled from all 73 blood donation sites (mobile teams and donor centres) of National Health Service Blood and Transplant (NHSBT) in England. Each site ("cluster") has been randomly allocated to receive one or more interventions during a 36-month period, using principles of cross-over, stepped-wedge and factorial trial design to assign the sequence of interventions. Each of the four interventions is compared to NHSBT's current practices: (i) 500-ml isotonic drink before donation (vs current 500-ml plain water); (ii) 3-min rest on donation chair after donation (vs current 2 min); (iii) new modified AMT (vs current practice of AMT); and (iv) psychosocial intervention using preparatory materials (vs current practice of nothing). The primary outcome is the number of in-session VVRs with loss of consciousness (i.e. episodes involving loss of consciousness of any duration, with or without additional complications). Secondary outcomes include all in-session VVRs (i.e. with and without loss of consciousness), all delayed VVRs (i.e. those occurring after leaving the venue) and any in-session non-VVR adverse events or reactions. The STRIDES trial should yield novel information about interventions, singly and in combination, for the prevention of VVRs, with the aim of generating policy-shaping evidence to help inform blood services to improve donor health, donor experience, and service efficiency. ISRCTN: 10412338. Registration date: October 24, 2019.


Phytotoxic Activity and Structure-Activity Relationships of Radicinin Derivatives against the Invasive Weed Buffelgrass (Cenchrus ciliaris).
Radicinin (1), is a fungal dihydropyranopyran-4,5-dione isolated together with some analogues, namely 3-epi-radicinin, radicinol, 3-epi-radicinol, and cochliotoxin (2-5), from the culture filtrates of the fungus Cochliobolus australiensis, a foliar pathogen of buffelgrass (Cenchrus ciliaris), an invasive weed in North America. Among the different metabolites 1 showed target-specific activity against the host plant and no toxicity on zebrafish embryos, promoting its potential use to develop a natural bioherbicide formulation to manage buffelgrass. These data and the peculiar structural feature of 1 suggested to carry out a structure-activity relationship study, preparing some key hemisynthetic derivatives and to test their phytotoxicity. In particular, p-bromobenzoyl, 5-azidopentanoyl, stearoyl, mesyl and acetyl esters of radicinin were semisynthesized as well as the monoacetyl ester of 3-epi-radicinin, the diacetyl esters of radicinol and its 3 epimer, and two hexa-hydro derivatives of radicinin. The spectroscopic characterization and the activity by leaf puncture bioassay against buffelgrass of all the derivatives is reported. Most of the compounds showed phytotoxicity but none of them had comparable or higher activity than radicinin. Thus, the presence of an α,β unsaturated carbonyl group at C-4, as well as, the presence of a free secondary hydroxyl group at C-3 and the stereochemistry of the same carbon proved to be the essential feature for activity.


Identification of Differentially Expressed miRNAs between a Wheat K-type Cytoplasmic Male Sterility Line and Its Near-Isogenic Restorer Line.
K-type cytoplasmic male sterility (KCMS) lines were ideal material for three-line hybrid wheat system due to the major role in hybrid wheat production. In this study, the morphology of developing microspore and mature pollen was compared between a KCMS line and its near-isogenic restorer line (KCMS-NIL). The most striking difference is that the microspore was unable to develop into tricellular pollen in the KCMS line. MicroRNA plays vital roles in flowering and gametophyte development. Small RNA sequencing identified a total of 274 known and 401 novel miRNAs differentially expressed between two lines or two developmental stages. Most of miRNAs with high abundance were differentially expressed at the uninucleate stage, and their expression level recovered or remained at the binucleate stage. Further degradome sequencing identified target genes which were mainly enriched in transcription regulation, phytohormone signaling and RNA degradation pathways. Combining with the transcriptome data, a correlation was found between the abnormal anther development, such as postmeiotic mitosis cessation, deformative pollen wall and the chromosome condensation of the vegetative cell, and the alterations in the related miRNA and their targets expression profiles. According to the correlation and pathway analysis, we propose a hypothetic miRNA-mediated network for the control of KCMS restoration.


A neural basis of speech-in-noise perception in older adults.
We investigated a neural basis of speech-in-noise perception in older adults. Hearing loss, the third most common chronic condition in older adults, is most often manifested by difficulty understanding speech in background noise. This trouble with understanding speech in noise, which occurs even in individuals who have normal-hearing thresholds, may arise, in part, from age-related declines in central auditory processing of the temporal and spectral components of speech. We hypothesized that older adults with poorer speech-in-noise (SIN) perception demonstrate impairments in the subcortical representation of speech. In all participants (28 adults, age 60-73 yr), average hearing thresholds calculated from 500 to 4000 Hz were ≤ 25 dB HL. The participants were evaluated behaviorally with the Hearing in Noise Test (HINT) and neurophysiologically using speech-evoked auditory brainstem responses recorded in quiet and in background noise. The participants were divided based on their HINT scores into top and bottom performing groups that were matched for audiometric thresholds and intelligent quotient. We compared brainstem responses in the two groups, specifically, the average spectral magnitudes of the neural response and the degree to which background noise affected response morphology. In the quiet condition, the bottom SIN group had reduced neural representation of the fundamental frequency of the speech stimulus and an overall reduction in response magnitude. In the noise condition, the bottom SIN group demonstrated greater disruption in noise, reflecting reduction in neural synchrony. The role of brainstem timing is particularly evident in the strong relationship between SIN perception and quiet-to-noise response correlations. All physiologic measures correlated with SIN perception. Adults in the bottom SIN group differed from the audiometrically matched top SIN group in how speech was neurally encoded. The strength of subcortical encoding of the fundamental frequency appears to be a factor in successful speech-in-noise perception in older adults. Given the limitations of amplification, our results suggest the need for inclusion of auditory training to strengthen central auditory processing in older adults with SIN perception difficulties.


Transcriptional activation of the integrated chromatin-associated human immunodeficiency virus type 1 promoter.
The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression involves a complex interplay between cellular transcription factors, chromatin-associated proviral DNA, and the virus-encoded transactivator protein, Tat. Here we show that Tat transactivates the integrated HIV-1 long terminal repeat (LTR), even in the absence of detectable basal promoter activity, and this transcriptional activation is accompanied by chromatin remodeling downstream of the transcription initiation site, as monitored by increased accessibility to restriction endonucleases. However, with an integrated promoter lacking both Sp1 and NF-kappaB sites, Tat was unable to either activate transcription or induce changes in chromatin structure even when it was tethered to the HIV-1 core promoter upstream of the TATA box. Tat responsiveness was observed only when Sp1 or NF-kappaB was bound to the promoter, implying that Tat functions subsequent to the formation of a specific transcription initiation complex. Unlike Tat, NF-kappaB failed to stimulate the integrated transcriptionally silent HIV-1 promoter. Histone acetylation renders the inactive HIV-1 LTR responsive to NF-kappaB, indicating that a suppressive chromatin structure must be remodeled prior to transcriptional activation by NF-kappaB. Taken together, these results suggest that Sp1 and NF-kappaB are required for the assembly of transcriptional complexes on the integrated viral promoter exhibiting a continuum of basal activities, all of which are fully responsive to Tat.


Lightweight deep neural networks for cholelithiasis and cholecystitis detection by point-of-care ultrasound.
Emergency physicians (EPs) frequently deal with abdominal pain, including that is caused by either gallstones or acute cholecystitis. Easy access and low cost justify point-of-care ultrasound (POCUS) use as a first-line test to detect these diseases; yet, the detection performance of POCUS by EPs is unreliable, causing misdiagnoses with serious impacts. This study aimed to develop a machine learning system to detect and localize gallstones and to detect acute cholecystitis by ultrasound (US) still images taken by physicians or technicians for preliminary diagnoses. Abdominal US images (> 89,000) were collected from 2386 patients in a hospital database. We constructed training sets for gallstones with or without cholecystitis (N = 10,971) and cholecystitis with or without gallstones (N = 7348) as positives. Validation sets were also constructed for gallstones (N = 2664) and cholecystitis (N = 1919). We applied a single-shot multibox detector (SSD) and a feature pyramid network (FPN) to classify and localize objects using image features extracted by ResNet-50 for gallstones, and MobileNet V2 to classify cholecystitis. The deep learning models were pretrained using the COCO-2017 and ILSVRC-2012 datasets. Using the validation sets, the SSD-FPN-ResNet-50 and MobileNet V2 achieved areas under the receiver operating characteristics curve of 0.92 and 0.94, respectively. The inference speeds were 21 (47.6 frames per second, fps) and 7 ms (142.9 fps). A machine learning system was developed to detect and localize gallstones, and to detect cholecystitis, with acceptable discrimination and speed. This is the first study to develop this system for either gallstone or cholecystitis detection with absence or presence of each one. After clinical trials, this system may be used to assist EPs, including those in remote areas, for detecting these diseases.


Insulin stimulates Na+, Cl-, Ca2+, and Mg2+ transports in TAL of mouse nephron: cross-potentiation with AVP.
Insulin (Ins) decreases Na+ delivery in the final urine. To determine whether the loop of Henle participates in this reduction, the effects of Ins were tested on cortical (CTAL) and medullary thick ascending limbs (MTAL) of the mouse nephron, microperfused in vitro. In the MTAL, Ins increased the transepithelial potential difference (Vt) and the Na+ and Cl- net reabsorption fluxes (JNa and JCl, respectively) in a dose-dependent manner, the threshold being below 10(-9) M. At 10(-7) M, Ins reversibly increased JNa and JCl, leaving Mg2+ and Ca2+ fluxes (JMg and JCa, respectively) close to zero. In the CTAL, 10(-7) M Ins reversibly increased Vt, JNa, JCl, JMg, and JCa. In CTAL segments perfused under asymmetrical conditions, with a bath-to-lumen-directed NaCl gradient (lumen 50 mM NaCl, bath 150 mM NaCl), addition of 10(-7) M Ins to the bath resulted in a large increase in JMg and JCa. Thus the responses of CTAL and MTAL to Ins are in all ways similar to those already reported for the adenosine 3',5'-cyclic monophosphate (cAMP)-generating hormones acting on these nephron segments. When 10(-10) M arginine vasopressin (AVP) and 10(-7) M Ins were used in combination, previous addition of one hormone to the bath potentiated the response to the second hormone. In cAMP accumulation experiments, performed in the presence of a phosphodiesterase inhibitor, the amounts of cAMP formed with 10(-7) M Ins and 10(-10) M AVP (which elicit maximal physiological responses in these segments) were in the same range.(ABSTRACT TRUNCATED AT 250 WORDS)


Microbially-enhanced composting of olive mill solid waste (wet husk): bacterial and fungal community dynamics at industrial pilot and farm level.
Bacterial and fungal community dynamics during microbially-enhanced composting of olive mill solid waste (wet husk), used as a sole raw material, were analysed in a process carried out at industrial pilot and at farm level by the PCR-DGGE profiling of the 16 and 26S rRNA genes. The use of microbial starters enhanced the biotransformation process leading to an earlier and increased level of bacterial diversity. The bacterial community showed a change within 15 days during the first phases of composting. Without microbial starters bacterial biodiversity increased within 60 days. Moreover, the thermophilic phase was characterized by the highest bacterial biodiversity. By contrast, the biodiversity of fungal communities in the piles composted with the starters decreased during the thermophilic phase. The biodiversity of the microbial populations, along with physico-chemical traits, evolved similarly at industrial pilot and farm level, showing different maturation times.


GNINA 1.0: molecular docking with deep learning.
Molecular docking computationally predicts the conformation of a small molecule when binding to a receptor. Scoring functions are a vital piece of any molecular docking pipeline as they determine the fitness of sampled poses. Here we describe and evaluate the 1.0 release of the Gnina docking software, which utilizes an ensemble of convolutional neural networks (CNNs) as a scoring function. We also explore an array of parameter values for Gnina 1.0 to optimize docking performance and computational cost. Docking performance, as evaluated by the percentage of targets where the top pose is better than 2Å root mean square deviation (Top1), is compared to AutoDock Vina scoring when utilizing explicitly defined binding pockets or whole protein docking. GNINA, utilizing a CNN scoring function to rescore the output poses, outperforms AutoDock Vina scoring on redocking and cross-docking tasks when the binding pocket is defined (Top1 increases from 58% to 73% and from 27% to 37%, respectively) and when the whole protein defines the binding pocket (Top1 increases from 31% to 38% and from 12% to 16%, respectively). The derived ensemble of CNNs generalizes to unseen proteins and ligands and produces scores that correlate well with the root mean square deviation to the known binding pose. We provide the 1.0 version of GNINA under an open source license for use as a molecular docking tool at https://github.com/gnina/gnina .


Polarization independent two-way variable optical attenuator based on polymer-stabilized cholesteric liquid crystal.
We have investigated the optical properties of the polymer-stabilized cholesteric liquid crystal (CLC). It was observed that the reflectance was decreased and the transmittance was increased with the increase of the applied voltage. Based on this property, a polarization independent two-way variable optical attenuator (VOA) has been demonstrated by sandwiching a lambda/2 film between two left handed polymer-stabilized CLC films. Different from a conventional VOA, the VOA based on our developed polymer-stabilized CLC can continuously change the optical intensity in both the reflection and transmission directions by applying voltage on it. This unique property will allow it to be widely used in many applications, such as optical communications.


The effect of increased FIO(2) before tracheal extubation on postoperative atelectasis.
General anesthesia promotes pulmonary atelectasis, which can be eliminated by a vital capacity (VC) maneuver (inflation of the lungs to 40 cm H(2)O for 15 s). High-inspired oxygen concentration favors recurrence of atelectasis. Therefore, 100% oxygen before tracheal extubation may contribute to atelectasis. To evaluate whether the use of 100% oxygen before extubation increases the amount of postoperative atelectasis, we studied 30 adults scheduled for elective surgery of the extremities. Ten minutes before the presumed end of surgery, patients were randomly assigned to (a) a fraction of inspired oxygen (FIO(2)) = 1.0 (n = 10), (b) VC maneuver + FIO(2) = 1.0 (n = 10), or (c) VC maneuver + FIO(2) = 0.4 (n = 10). The amount of atelectasis was measured by computed tomography scan, and oxygenation was studied by arterial blood gas analysis. Data were analyzed by one-way analysis of variance with Bonferroni correction. Results are presented as mean +/- SD; P < 0.05 was considered significant. In the VC maneuver + FIO(2) = 0.4 group, postoperative atelectasis was smaller (2.6% +/- 1.1% of total lung surface, P < 0.05) than in the FIO(2) = 1.0 group (8.3% +/- 6.2%) and in the VC maneuver + FIO(2) = 1.0 group (6.8% +/- 3.4%). Oxygen 100% at the end of general anesthesia promotes postoperative atelectasis. A safety margin in terms of oxygenation during tracheal extubation is essential, and further studies should therefore evaluate whether atelectasis formation could be prevented despite the use of 100% oxygen. For safety reasons, it is common to ventilate patients with 100% oxygen before tracheal extubation. This study demonstrates that this practice favors postoperative atelectasis.


A Cone-Beam Computed Tomography Scanning of the Root Canal System of Permanent Teeth among the Moscow Population.
Successful endodontic treatment requires a significant knowledge of root canal anatomy. The aim of this study was to evaluate the root and root canal number of permanent teeth among the Moscow population using cone-beam computed tomography (CBCT) scanning. 300 CBCT images of subjects were analyzed to study the anatomy of roots and root canal system of each tooth. The collected data were analyzed using IBM SPSS statistics software 22.0 version. The maxillary incisors and canines had one root with one canal in 100%. Maxillary premolars had one root with one or two canals and two roots with two canals, while mandibular premolars were single-rooted with one or two canals. Maxillary first and second molar had three separated roots, and the prevalence of four canals was more often in first molars. Mandibular molars had two roots with different number of canals. The root canal system varies greatly among populations and even in different individuals within the same population; thus, using CBCT scanning is an effective technique in investigating the root canal system.


Physiological consequences of membrane-initiated estrogen signaling in the brain.
Many of the actions of 17beta-estradiol (E2) in the central nervous system (CNS) are mediated via the classical nuclear steroid receptors, ER(alpha) and ERbeta, which interact with the estrogen response element to modulate gene expression. In addition to the nuclear-initiated estrogen signaling, E2 signaling in the brain can occur rapidly within minutes prior to any sufficient effects on transcription of relevant genes. These rapid, membrane-initiated E2 signaling mechanisms have now been characterized in many brain regions, most importantly in neurons of the hypothalamus and hippocampus. Furthermore, our understanding of the physiological effects of membrane-initiated pathways is now a major field of interest in the hypothalamic control of reproduction, energy balance, thermoregulation and other homeostatic functions as well as the effects of E2 on physiological and pathophysiological functions of the hippocampus. Membrane signaling pathways impact neuronal excitability, signal transduction, cell death, neurotransmitter release and gene expression. This review will summarize recent findings on membrane-initiated E2 signaling in the hypothalamus and hippocampus and its contribution to the control of physiological and behavioral functions.


Omega-3 fatty acids in ileal effluent after consuming different foods containing microencapsulated fish oil powder - an ileostomy study.
The intestinal absorption of omega-3 long chain polyunsaturated fatty acids (ω3 LCPUFA), [eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)], after consuming fish oil gelatine capsules or different food products fortified with microencapsulated fish oil, was determined using human ileostomates. The total amount of ω3 LCPUFA consumed per dose of fish oil capsule was 266 mg while that for fortified orange juice, yoghurt and cereal bar was 284 mg per serving of food product. In a time course experiment ω3 LCPUFA was measured in ileal effluent over 24 h post ingestion. Only 0.58-0.73% of the total ω3 LCPUFA dose was recovered in the ileal effluent irrespective of whether the fish oil was delivered in a gelatine capsule or in the form of a microencapsulated powder incorporated into fortified foods. Excretion of ω3 LCPUFA was detected in the 2-18 h effluent collections with none detected at 0 h or 24 h. post ingestion. The transit time of the minimal amount of ω3 LCPUFA that remained in the ileal effluent was dependent on the method of delivery of the fish oil. The ω3 LCPUFA content in the ileal effluent peaked at 2-8 h and declined after 10 h after consumption of fish oil capsules and fortified orange juice. In contrast, two peaks in ω3 LCPUFA content were observed in the ileal effluent, first at 2-8 h and again at 14-16 h, after consumption of fortified yoghurt and cereal bar. The highest recovery of the small amount of ω3 LCPUFA in the ileal effluent at 14-16 h was obtained when fortified cereal bar was consumed. The results suggest that the delivery of fish oil through food products fortified with microencapsulated fish oil does not compromise the bioavailability of the ω3 LCPUFA as evidence by no statistical differences detected in the remaining portion of ω3 LCPUFA in the ileal effluent (p = 0.58). However, the food matrix in which the microencapsulated oil was delivered may alter the transit kinetics of the ω3 LCPUFA through the small intestine.


Abscisic acid mediates barley rhizosheath formation under mild soil drying by promoting root hair growth and auxin response.
Soil drying enhances root ABA accumulation and rhizosheath formation, but whether ABA mediates rhizosheath formation is unclear. Here, we used the ABA-deficient mutant Az34 to investigate molecular and morphological changes by which ABA could affect rhizosheath formation. Mild soil drying with intermittent watering increased rhizosheath formation by promoting root and root hair elongation. Attenuated root ABA accumulation in Az34 barley constrained the promotion of root length and root hair length by drying soil, such that Az34 had a smaller rhizosheath. Pharmacological experiments of adding fluridone (an ABA biosynthesis inhibitor) and ABA to drying soil restricted and enhanced rhizosheath formation respectively in Az34 and wild-type Steptoe barley. RNA sequencing suggested that ABA accumulation mediates auxin synthesis and responses and root and root hair elongation in drying soil. In addition, adding indole-3-acetic acid (IAA) to drying soil increased rhizosheath formation by promoting root and root hair elongation in Steptoe and Az34 barley. Together, these results show that ABA accumulation induced by mild soil drying enhance barley rhizosheath formation, which may be achieved through promoting auxin response.


Biogenesis of lysosome-related organelles complex-1 (BORC) regulates late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate.
Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.


Quantity of IgG response to SARS-CoV-2 spike glycoprotein predicts pulmonary recovery from COVID-19.
The CovILD study is a prospective, multicenter, observational cohort study to systematically follow up patients after coronavirus disease-2019 (COVID-19). We extensively evaluated 145 COVID-19 patients at 3 follow-up visits scheduled for 60, 100, and 180 days after initial confirmed diagnosis based on typical symptoms and a positive reverse transcription-polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We employed comprehensive pulmonary function and laboratory tests, including serum concentrations of IgG against the viral spike (S) glycoprotein, and compared the results to clinical data and chest computed tomography (CT). We found that at the 60 day follow-up, 131 of 145 (90.3%) participants displayed S-specific serum IgG levels above the cut-off threshold. Notably, the highly elevated IgG levels against S glycoprotein positively correlated with biomarkers of immune activation and negatively correlated with pulmonary function and the extent of pulmonary CT abnormalities. Based on the association between serum S glycoprotein-specific IgG and clinical outcome, we generated an S-specific IgG-based recovery score that, when applied in the early convalescent phase, accurately predicted delayed pulmonary recovery after COVID-19. Therefore, we propose that S-specific IgG levels serve as a useful immunological surrogate marker for identifying at-risk individuals with persistent pulmonary injury who may require intensive follow-up care after COVID-19.


Role of calcium-sensing receptor, Galectin-3, Cyclin D1, and Ki-67 immunohistochemistry to favor in the diagnosis of parathyroid carcinoma.
As histopathological findings of parathyroid carcinoma are not certain, the diagnosis of tumors with degenerative changes may be difficult. In these cases, immunohistochemical markers are beneficial. We aimed to research the acceptability of calcium-sensing receptor (CaSR), Galactin-3, Cyclin D1, and Ki-67 as helpful markers in parathyroid tumors in cases which are difficult to diagnose. Those cases who had been diagnosed with atypical parathyroid adenoma and parathyroid carcinoma between 2010 and 2015 were reevaluated. İmmunohistochemical markers were applied to this cases. About 21 cases were parathyroid adenoma, 14 were atypical adenoma, and 10 cases were parathyroid carcinoma. According to the immunohistochemical results, global loss of CaSR staining was seen in 50% (5/10) of the patients with carcinoma while there was no loss of staining in those with parathyroid adenoma (P = 0,001). Global loss of CaSR staining was found in only one out of 14 cases with atypical adenoma. The expression of Galactin-3 was found to be positive in 40% (4/10) of carcinoma cases, 71.4% (10/14) of those with atypical adenoma, and 14.3% (3/21) of those with adenoma (P = 0,002). Cyclin D1 expression was determined to be positive in 70% (7/10) of patients with carcinoma, 71.4% (10/14) of atypical adenoma cases, and 23.8% (5/21) of those with adenoma. The Ki-67 proliferation index was seen to be above 5% in 50% (5/10) of carcinoma cases and 35,7% (5/14) of those with atypical adenoma. In these studies, it has been emphasized that the global loss of CaSR staining was used as a negative marker in the diagnosis of carcinoma. In this study, we have also confirmed that the global loss of CaSR staining is a useful marker to determine potential increased malignancy.


A charge-stabilizing, multimodular, ferrocene-bis(triphenylamine)-zinc-porphyrin-fullerene polyad.
A novel multimodular donor-acceptor polyad featuring zinc porphyrin, fullerene, ferrocene, and triphenylamine entities was designed, synthesized, and studied as a charge-stabilizing, photosynthetic-antenna/reaction-center mimic. The ferrocene and fullerene entities, covalently linked to the porphyrin ring, were distantly separated to accomplish the charge-separation/hole-migration events leading to the creation of a long-lived charge-separated state. The geometry and electronic structures of the newly synthesized compound was deduced by B3LYP/3-21G(*) optimization, while the energy levels for different photochemical events was established using data from the optical absorption and emission, and electrochemical studies. Excitation of the triphenylamine entities revealed singlet-singlet energy transfer to the appended zinc porphyrin. As predicted from the energy levels, photoinduced electron transfer from both the singlet and triplet excited states of the zinc porphyrin to fullerene followed by subsequent hole migration involving ferrocene was witnessed from the transient absorption studies. The charge-separated state persisted for about 8.5 μs and was governed by the distance between the final charge-transfer product, that is, a species involving a ferrocenium cation and a fullerene radical anion, with additional influence from the charge-stabilizing triphenylamine entities located on the zinc-porphyrin macrocycle.


Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells.
Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.


Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line.
Gefitinib (Iressa)-a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase-has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells.


Chromosomal organization and expression of Escherichia coli pabA.
The pabA gene in Escherichia coli and Salmonella typhimurium encodes the glutamine amidotransferase subunit of para-aminobenzoate synthase, which catalyzes the first reaction in the conversion of chorismate to para-aminobenzoate (PABA). We have determined the nucleotide sequences of 1,362 base pairs preceding E. coli pabA and of 981 base pairs preceding S. typhimurium pabA. The nucleotide sequences suggest the presence of two protein-coding regions immediately upstream of pabA, designated orf1 and fic. Transcription analysis indicates that E. coli pabA is encoded by two overlapping transcriptional units. The polycistronic transcriptional unit includes orf1-fic-pabA and is initiated by the promoter designated P2. The monocistronic unit includes only pabA and is initiated by the promoter designated P1, which is located in the fic-coding region. Both promoters transcribe pabA to about the same steady-state level. However, expression analysis using chromosomal pabA-lacZ translational fusions indicated that P1 expressed PabA at least 50-fold more efficiently than P2. pabA-dependent growth rate analysis indicates that P1 is essential and P2 is dispensable for PABA metabolism. In the absence of P1, growth was reduced as a result of insufficient PabA expressed from P2. The significance of these results and possible posttranscriptional control mechanisms which affect PabA expression from the P2-initiated polycistronic unit are discussed.


Profound inhibition of platelet aggregation with monoclonal antibody 7E3 Fab after thrombolytic therapy. Results of the Thrombolysis and Angioplasty in Myocardial Infarction (TAMI) 8 Pilot Study.
This study was undertaken to establish evidence for physiologic activity and to study the safety of murine-derived monoclonal antibody 7E3 Fab (m7E3 Fab) in patients receiving recombinant tissue-type plasminogen activator (rt-PA). Platelet aggregation is believed to be a significant factor in the failure of pharmacologic reperfusion. By binding to the glycoprotein IIb/IIIa receptor, m7E3 Fab inhibits platelet aggregation and has been shown experimentally to decrease the time required for lysis and to prevent reocclusion. However, the safety of profound platelet inhibition after thrombolysis for acute myocardial infarction has not been tested in humans. Sixty patients receiving rt-PA, aspirin and heparin for acute myocardial infarction received m7E3 Fab bolus injections in ascending doses at 3, 6 and 15 h after initiation of the thrombolytic infusion. Ten patients treated with rt-PA but not m7E3 Fab were studied as control subjects. Receptor site blockade and inhibition of platelet aggregation to 20 mumol/liter adenosine diphosphate were maximal at a dose of 0.25 mg/kg body weight of m7E3 Fab. Fifteen (25%) m7E3 Fab-treated patients and five (50%) control patients had major bleeding; eight of these events in seven m7E3 Fab-treated patients and one in a control patient occurred at the time of aortocoronary bypass surgery. Recurrent ischemia occurred in eight (13%) m7E3 Fab-treated patients and two (20%) control subjects. Coronary angiography was performed in 43 patients; the infarct-related coronary artery was patent in 5 of 9 (56%) control patients and 34 (92%) of 37 patients receiving m7E3 Fab. Profound inhibition of platelet aggregation after thrombolysis was associated with bleeding rates comparable to those in control patients and a low rate of recurrent ischemia. The combination of m7E3 Fab and rt-PA, heparin and aspirin appears to be a promising and safe combination that should be evaluated in further studies of patients with acute myocardial infarction.


N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry.
Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.


Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain.
The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.


Blue Light and Ultraviolet Radiation Exposure from Infant Phototherapy Equipment.
Phototherapy is the use of light for reducing the concentration of bilirubin in the body of infants. Although it has become a mainstay since its introduction in 1958, a better understanding of the efficacy and safety of phototherapy applications seems to be necessary for improved clinical practices and outcomes. This study was initiated to evaluate workers' exposure to Optical Radiation from different types of phototherapy devices in clinical use in Italy. During infant phototherapy the staff monitors babies periodically for around 10 min every hour, and fixation of the phototherapy beam light frequently occurs: almost all operators work within 30 cm of the phototherapy source during monitoring procedures, with most of them commonly working at ≤25 cm from the direct or reflected radiation beam. The results of this study suggest that there is a great variability in the spectral emission of equipments investigated, depending on the types of lamps used and some phototherapy equipment exposes operators to blue light photochemical retinal hazard. Some of the equipment investigated presents relevant spectral emission also in the UVA region. Taking into account that the exposure to UV in childhood has been established as an important contributing factor for melanoma risk in adults and considering the high susceptibility to UV-induced skin damage of the newborn, related to his pigmentary traits, the UV exposure of the infant during phototherapy should be "as low as reasonably achievable," considering that it is unnecessary to the therapy. It is recommended that special safety training be provided for the affected employees: in particular, protective eyewear can be necessary during newborn assistance activities carried out in proximity of some sources. The engineering design of phototherapy equipment can be optimized. Specific requirements for photobiological safety of lamps used in the phototherapy equipment should be defined in the safety product standard for such equipment.


Efficient methodology for the extraction and analysis of lipids from porcine pulmonary artery by supercritical fluid chromatography coupled to mass spectrometry.
Pulmonary artery grafts are needed as cardiovascular bioprosthetics. For successful tissue recellularization after transplantation, lipids have to be removed from the donor artery. Developing a selective process to remove lipids without damaging the extracellular matrix greatly depends on knowing the amount and type of lipid compounds in the specific tissue. Here we present an efficient methodology for the study of lipids present in porcine pulmonary arteries. The performance of six extraction methods to recover lipids from artery was evaluated. For this purpose, a supercritical fluid chromatography method coupled to quadrupole time-of-flight mass spectrometry detection (UHPSFC/QTOF-MS) was adapted. The method enabled separation of lipids of a wide range of polarity according to lipid class in less than 7 min. One dichloromethane-based extraction method was shown to be the most efficient one for the recovery of lipids from pulmonary artery. However, one MTBE-based extraction method was able to show the highest fatty acid extraction yields (to the expense of longer extraction times). Lipids were relative quantified according to class, and the major species within each class were identified. Triacylglycerols and glycerophospholipids were the most abundant classes, followed by sphingomyelins, monoacylglycerols and fatty acyls. The matrix effect exerted no interference on the analytical method, except for some few combinations of extraction method and lipid class. These results are of relevance for lipidomic studies from solid tissue, in particular for studies on pulmonary and cardiovascular diseases. Finally, our work sets the basis for the further development of a selective processes to remove lipids from pulmonary artery without damaging the tissue prior to transplantation.