Isolation and primary structure of the major toxin from sea snake, Acalyptophis peronii, venom.
The major neurotoxin from the venom of Acalyptophis peronii captured in the Gulf of Thailand was isolated. Although there are two toxic fractions in the venom, the most toxic and abundant fraction was selected for purification and chemical characterization. The LD50 of the major toxin is 0.125 micrograms/g mice, indicating an extremely toxic nature. The toxin consists of 60 amino acid residues with methionine as the amino-terminal and asparagine as the carboxy-terminal end. It contains nine half-cystine residues. There is 1 mol each of tryptophan, tyrosine, methionine, valine, aspartic acid, leucine, and alanine, and there is no phenylalanine. The molecular weight calculated from the amino acid sequence determination was 6600. The toxin replaces alpha-bungarotoxin in binding with the acetylcholine receptor, indicating that the A. peronii major neurotoxin competes with alpha-bungarotoxin for the same binding site of the acetylcholine receptor.


[The correlation between the derivatives of the arachidonic acid cascade and oral pathology. A review of the literature].
The effects of several metabolites deriving from the arachidonic acid cascade on oral tissues are reviewed. These include the effects on the pulp, tissues and gingiva, and on the development of radicular cysts and epithelial tumours of the oral cavity.


Methodological issues of the electronic health records' use in the context of epidemiological investigations, in light of missing data: a review of the recent literature.
Electronic health records (EHRs) are widely accepted to enhance the health care quality, patient monitoring, and early prevention of various diseases, even when there is incomplete or missing information in them. The present review sought to investigate the impact of EHR implementation on healthcare quality and medical decision in the context of epidemiological investigations, considering missing or incomplete data. Google scholar, Medline (via PubMed) and Scopus databases were searched for studies investigating the impact of EHR implementation on healthcare quality and medical decision, as well as for studies investigating the way of dealing with missing data, and their impact on medical decision and the development process of prediction models. Electronic searches were carried out up to 2022. EHRs were shown that they constitute an increasingly important tool for both physicians, decision makers and patients, which can improve national healthcare systems both for the convenience of patients and doctors, while they improve the quality of health care as well as they can also be used in order to save money. As far as the missing data handling techniques is concerned, several investigators have already tried to propose the best possible methodology, yet there is no wide consensus and acceptance in the scientific community, while there are also crucial gaps which should be addressed. Through the present thorough investigation, the importance of the EHRs' implementation in clinical practice was established, while at the same time the gap of knowledge regarding the missing data handling techniques was also pointed out.


Modelling breast cancer: one size does not fit all.
Breast cancer is not a single disease, but is instead a collection of diseases that have distinct histopathological features, genetic and genomic variability, and diverse prognostic outcomes. Thus, no individual model would be expected to completely recapitulate this complex disease. Here, the models commonly used to investigate breast cancer including cell lines, xenografts and genetically engineered mice, are discussed to help address the question: what is the most powerful way to investigate this heterogeneous disease?


Heparin-binding EGF-like growth factor: characterization of rat and mouse cDNA clones, protein domain conservation across species, and transcript expression in tissues.
Clones were obtained that encode the rat and mouse forms of heparin-binding EGF-like growth factor (HB-EGF), a potent mitogen for smooth muscle cells, fibroblasts and keratinocytes that is proposed to be derived from a transmembrane precursor. Within the HB-EGF precursor sequences predicted from these cDNAs, the region corresponding to the secreted ("mature") factor was found to represent one of the least well conserved areas when compared to human or monkey HB-EGF (73-76% sequence identity). Regions of high sequence conservation included the proposed juxtamembrane and transmembrane domains, as well as a proposed heparin-binding region within the "mature" factor. Northern blotting experiments using the HB-EGF clones as probes revealed HB-EGF transcript expression in multiple tissues, particularly lung, skeletal muscle, brain, and heart.


Influence of counter-irritation by carrageenin on inflammatory reactions induced by prostaglandins, serotonin, histamine, compound 48/80, carrageenin and Freund's complete adjuvant.
The carrageenin edema of rat paw and the primary phase of rat adjuvant arthritis were significantly inhibited by carrageenin whereas the increase of skin capillary permeability induced by prostaglandins (PGs) and further irritants as well as the secondary phase of adjuvant arthritis were mostly not significantly affected. Inhibitory effects after single carrageenin injection do not seem to be mediated by the complement system because total complement activity remained unaffected.


A Community-Based Sensory Training Program Leads to Improved Experience at a Local Zoo for Children with Sensory Challenges.
Sensory processing difficulties are common among many special needs children, especially those with autism spectrum disorder (ASD). The sensory sensitivities often result in interference of daily functioning and can lead to social isolation for both the individual and family unit. A quality improvement (QI) project was undertaken within a local zoo to systematically implement a sensory training program targeted at helping special needs individuals with sensory challenges, including those with ASD, Down's syndrome, attention-deficit/hyperactivity disorder, and speech delay. We piloted the program over a 2-year period. The program consisted of staff training, provision of sensory bags and specific social stories, as well as creation of quiet zones. Two hundred family units were surveyed before and after implementation of the sensory training program. In this pilot QI study, families reported increased visitation to the zoo, improved interactions with staff members, and the overall quality of their experience. In conclusion, we are able to demonstrate that a sensory training program within the community zoo is feasible, impactful, and has the potential to decrease social isolation for special needs individuals and their families.


Prevalence and correlates of suicide attempts in young patients with first-episode and drug-naïve major depressive disorder: A large cross-sectional study.
Few studies in China have reported factors influencing suicide attempt in young first-episode drug-free (FEDN) MDD patients. This study aimed to investigate the incidence and potential relevant factors of suicide attempt among young Chinese patients with FEDN MDD to prevent suicidal behavior in this population. We recruited 1076 FEDN MDD outpatients aged 18-45 years. Patients' mental states were measured by the Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD), Positive and Negative Syndrome Scale (PANSS) positive subscale, and Clinical Global Impression Severity Scale (CGIS). Fasting blood glucose, lipid levels, and thyroid function parameters were also measured. The prevalence of suicide attempt for FEDN MDD patients was 18.31 %. Compared to patients without suicide attempt, patients with suicide attempt had an older age of onset, higher HAMA, HAMD, PANSS-positive subscale and CGI-S scores, higher blood pressure, fasting blood glucose, thyroid peroxidases antibody (A-TPO), anti-thyroglobulin (A-TG), thyroid stimulating hormone (TSH), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC), but lower high-density lipoprotein cholesterol (HDLC) (all p < 0.05). Logistic regression analysis showed that duration of illness, hypertension, PANSS-positive subscale, HAMA and CGI-S scores, and A-TPO, LDL-C, TC, and HDL-C were associated with suicide attempt in patients with MDD. The main limitations are cross-sectional design and inability to control selection bias. This study suggests that young patients with FEDN MDD have a high rate of suicide attempts. Several clinical and metabolic indicators related to lipids and thyroid function may be involved in suicide attempts in FEDN MDD patients.


Inhibitory effects of clonidine on allergic reactions in guinea-pig skin.
An experimental guinea-pig model was used to show that clonidine inhibited dose dependently the wheal and flare reaction to i.d. ovalbumin in sensitized animals. The effect of clonidine was counteracted by an alpha 2-adrenoceptor antagonist, yohimbine, but not by an alpha 1-adrenoceptor antagonist, prazosin. An H1-receptor antagonist, clemastine, initially reduced the wheal and flare reaction but did not influence the effect of clonidine. Cimetidine, an H2-receptor antagonist slightly reduced the effect of clonidine on the wheal and flare reaction. It is suggested that clonidine reduces the wheal and flare reaction by stimulating alpha 2-adrenoceptors, which inhibits the axon reflex of the response.


[Medication reconciliation in patients over 75 years].
To continue with the implementation of the medication reconciliation process on admission, and to analyse potentially inappropriate prescriptions according to the STOPP-START -Screening Tool of Older Person's potentially inappropriate Prescriptions/Screening Tool to Alert doctors to the Right (indicated) Treatment- criteria, and drug interactions. To extend the process of reconciliation at hospital discharge from the Cardiology and Chest Diseases departments. Retrospective observational study that included patients over 75 years old. The "Consensus Document on Terminology and Classification in Medication Reconciliation" was used for the classification of the discrepancies found. Potentially inappropriate prescriptions and drug interactions were reviewed. For reconciliation at discharge, a "Drug information program at discharge" leaflet and a "Patient information sheet" was developed. The number of reconciled patients was 818, with a level of coverage of 42.3% (target patients: 1,932). Reconciliation errors by patient were 23.4% (191) on admission and 22.5% (9) at discharge. All patients who were reconciled at discharge were given the "Drug information program at discharge" leaflet. Potentially inappropriate prescriptions, according to STOPP/START criteria, affected 6.0% (49) of reconciled patients. The number of clinically relevant interactions affected 11.7% (96) of patients. The implementation of a reconciliation process has detected the existence of errors between chronic and prescribed treatment in the hospital. The STOPP/START criteria applied to this group of patients have helped to find out the most common potentially inappropriate prescriptions. The analysis of drug interactions shows drug combinations that should be avoided in these patients.


Phase II trial of vaginal cuff brachytherapy followed by dose-dense chemotherapy in early stage endometrial cancer patients with enriched, high-intermediate risk factors for recurrence.
To determine the feasibility of vaginal cuff brachytherapy (VCB) followed by 3 cycles of dose dense paclitaxel and carboplatin chemotherapy (ddTC) in enriched, high-intermediate risk (H-IR) patients with early stage endometrial cancer following hysterectomy. A phase II trial of early stage endometrial cancer patients treated with VCB (2100 cGy) followed by three cycles of carboplatin (AUC 6) and dose dense paclitaxel (80 mg/m2) weekly within 12-weeks of surgery was conducted. The primary endpoint was the proportion of patients completing both VCB and ddTC. Secondary outcomes include short and long-term toxicities, recurrence rate and sites, and progression free survival. Toxicity assessments were patient reported as well as those resulting in delays or dose modifications. A total of 32 evaluable patients with median age of 64.5 years were included. Most patients were endometrioid histology (18/32, 56.3%) and fully staged (21/32, 65.6%) to stage Ib (18/32, 56.3%). In total, 27/32 (84.4%) patients completed treatment per protocol. Protocol non-completion included renal insufficiency, paclitaxel reaction, and treatment refusal. Median time to VCB completion was 11 days with all patients completing three fractions of VCB. Acute toxicities with VCB included grade 1 and 2 gastrointestinal, genitourinary and fatigue symptoms. Acute toxicities associated with ddTC included infusion reaction and neutropenia. Most reported long-term toxicities were grade 1 or 2 and resolved with time. Treatment with VCB followed by three cycles of ddTC is well-tolerated with promising utility for treatment in enriched high-intermediate risk endometrial cancer patients. Recurrence-free and overall survival outcomes are not yet mature.


Terminations of LHRH-immunoreactive fibers in the subfornical organ of the opossum: an ultrastructural study.
Electron microscopic immunocytochemical approaches were used to analyze LHRH-containing elements in the subfornical organ of the opossum, a species in which this input to the subfornical organ is prominent. Not only were LHRH synaptic specializations easily demonstrated in the subfornical organ, forming axo-dendritic and axo-axonal contacts, but also LHRH-immunoreactive fibers contacted astrocytic end-feet on fenestrated capillaries and were found in the subependymal layer. LHRH-carrying elements in the subfornical organ may be important for relating reproductive functions to body fluid balance.


Effect of light intensity on the degree of ammonia toxicity on PSII activity of Arthrospira platensis and Chlorella vulgaris.
Herein the effect of increasing light intensity on the degree of ammonia toxicity and its impact on the photosynthetic performance of Arthrospira and Chlorella was investigated using Chl fluorescence as a technique to characterize their photosystem II (PSII) activity. The results revealed that the increase of light intensity amplifies the ammonia toxicity on PSII. Chl fluorescence transients shown that at a given free ammonia (FA) concentration (100mg-N/L), the photochemistry potential decreased by increasing light intensity. The inhibition of the PSII was not reversible either by re-incubating the cells under dark or under decreased FA concentration. Moreover, the decrease of photochemical and non-photochemical quenching (NPQ) of fluorescence suggest that ammonia toxicity decreases the open available PSII centers, as well the inability of PSII to transfer the generated electrons beyond QA. The collapse of NPQ suggests that ammonia toxicity inhibits the photoprotection mechanism(s) and hence renders PSII more sensitive to photoinhibition.


Sigma Receptors and Substance Use Disorders.
Thanks to advances in neuroscience, addiction is now recognized as a chronic brain disease with genetic, developmental, and cultural components. Drugs of abuse, including alcohol, are able to produce significant neuroplastic changes responsible for the profound disturbances shown by drug addicted individuals. The current lack of efficacious pharmacological treatments for substance use disorders has encouraged the search for novel and more effective pharmacotherapies. Growing evidence strongly suggests that Sigma Receptors are involved in the addictive and neurotoxic properties of abused drugs, including cocaine , methamphetamine , and alcohol. The present chapter will review the current scientific knowledge on the role of the Sigma Receptor system in the effects of drugs and alcohol, and proposes that this receptor system may represent a novel therapeutic target for the treatment of substance use disorders and associated neurotoxicity.


Antibacterial properties of lansoprazole alone and in combination with antimicrobial agents against Helicobacter pylori.
The activities of various types of anti-ulcer agents against Helicobacter pylori (Hp) strains were determined using an agar dilution method. Among the compounds tested, proton pump inhibitors were found to exhibit significant activity against this organism. The activity of lansoprazole was four times more potent than that of omeprazole and bismuth subsalicylate, with MICs ranging from 1.56 to 25 micrograms/ml. Exposure of Hp to lansoprazole led to extensive loss of viability and suppression of virulence factors such as motility, adhesiveness to epithelial cells, and urease activity. Lansoprazole produced aberrant bacterial morphology characterized by elongation and constriction of the cells and collapse of cell surface structures. The combination of lansoprazole with antimicrobial agents such as penicillins, cephalosporins, macrolides, tetracyclines, aminoglycosides, quinolones, and metronidazole produced an additive or synergistic growth inhibition of Hp.


Novel carbon-based composites enriched with Fe and Mn as effective and eco-friendly adsorbents of heavy metals in multicomponent solutions.
With increasing demand for adsorbents highly effective in pollutant removal, carbon-based porous materials are becoming more and more popular. In this work, a new approach to the synthesis of such solids using an environmentally friendly, two-step preparation method is presented. A series of hybrid porous silica-containing carbon composites was synthesized, namely: metal free (C/SiO2), enriched with manganese (C/Mn/SiO2), as well as iron (C/Fe/SiO2). The effect of additives on the structure and morphology of the composites was evaluated using X-ray photoelectron spectroscopy (XPS), nitrogen adsorption/desorption and scanning electron microscope (SEM). The as-synthesized carbons were used as effective adsorbents for the simultaneous removal of heavy metals, including lead (Pb(II)) and zinc (Zn(II)) ions. In particular, it was determined that C/Mn/SiO2 sample demonstrated the highest adsorption capacity towards Pb(II) and Zn(II) ions. It was equal to 211.60 mg/g for Pb(II) and 74.95 mg/g for Zn(II). Zeta potential and surface charge density of the solids, with and without metals, were investigated to determine electrical double layer structure, whereas stability studies and aggregate size measurements were performed to estimate solid aggregation under selected conditions. It was established that solids with adsorbed metals formed suspensions with lower stability than those without ions. This, in turn, facilitates their separation from aqueous solutions.


γ-protocadherins are enriched and transported in specialized vesicles associated with the secretory pathway in neurons.
Gamma protocadherins (Pcdh-γs) resemble classical cadherins and have the potential to engage in cell-cell interactions with homophilic properties. Emerging evidence suggests non-conventional roles for some protocadherins in neural development. We sought to determine whether Pcdh-γ trafficking in neurons is consistent with an intracellular role for these molecules. Here we show that, in contrast to the largely surface localization of classical cadherins, endogenous Pcdh-γs are primarily intracellular in rat neurons in vivo and are equally distributed within organelles of subsynaptic dendritic and axonal compartments. A strikingly higher proportion of Pcdh-γ-containing organelles in synaptic compartments was observed at postnatal day 16. To determine the origin of Pcdh-γ-trafficking organelles, we isolated organelles with Pcdh-γ antibody-coupled magnetic beads from brain organelle suspensions. Vesicles with high levels of COPII and endoplasmic reticulum-Golgi intermediate compartment (ERGIC) components were isolated with the Pcdh-γ antibody but not with the classical cadherin antibody. In cultured hippocampal neurons, Pcdh-γ immunolabeling partially overlapped with calnexin- and COPII-positive puncta in dendrites. Mobile Pcdh-γ-GFP profiles dynamically codistributed with a DsRed construct coupled to ER retention signals by live imaging. Pcdh-γ expression correlated with accumulations of tubulovesicular and ER-like organelles in dendrites. Our results are consistent with the possibility that Pcdh-γs could have a unique function within the secretory pathway in addition to their documented surface roles.


Professionally designed information materials and telephone reminders improved consent response rates: evidence from an RCT nested within a cohort study.
To investigate whether different study-to-participant communication methods increase response, increase response from hard-to-engage individuals, and influence participants' consent decisions. A randomized controlled trial within the Avon Longitudinal Study of Parents and Children. Cohort members were invited to re-enroll at age 18 and consent to linkage to their health and administrative records. Participants were randomized to receive one of eight combinations of three interventions: a prior-notification postcard or no contact, a standard or professionally designed consent pack, and a phone or postal reminder. The primary outcome was return of the consent form ("response"), with consent decision being the secondary outcome. Of 1,950 participants, 806 (41%) responded. Response rates were 2.7% higher (95% confidence interval: -0.06, 5.5%; P = 0.06) among those receiving designed packs than among those receiving standard packs and 6.4% higher (2.3, 10.6%; P = 0.002) among those receiving phone reminders (compared with postal reminders). The prior-notification postcard did not influence response rates [difference = 0% (-2.8, 2.8%; P = 1.0)], and we found no evidence that the communication method influenced consent decision. This trial provides evidence that communication material design can influence response rates and that phone reminders have superior cost/benefit returns over designed materials. Experimental evaluation of communications strategies and dissemination of findings may benefit cohort studies.


Reduction of traumatic secondary shoulder dislocations with lidocaine.
The aim of the present study was to evaluate the value of local anaesthesia versus the commonly used intravenous pethidine/diazepam in the reduction of acute secondary shoulder dislocations. Patients with a traumatic secondary dislocation of the shoulder were randomized to either locally injected lidocaine or intravenously injected pethidine/diazepam. The local method was performed with 20 ml of 1% lidocaine. The patients were observed for any complication during and after the procedure, and the methods used were evaluated with a visual analogue scale (VAS). From November 1991 to September 1993, 62 patients were admitted to our departments of whom 52 were included in the study. Average age was 47 years (range 18-89 years) with 24 men and 28 women. Twenty-six patients were randomized to pethidine/diazepam; 22 had a successful reduction, and 4 were failures. Twenty-six patients received lidocaine, of whom 18 were successful and 8 not. Three patients treated with the intravenous method suffered respiratory depression, and one required an antidote. No systemic or local side-effects, no neurovascular damage and no early or late superficial or deep infection were recorded in the lidocaine group. There was no statistical difference between the average VAS value in the two groups. Lidocaine used to reduce acute secondary dislocations of the shoulder is a simple and safe method. It is as effective as the standard intravenous method and is well accepted by patients.


Pruritus in chronic cholestatic liver disease.
Pruritus is a troublesome complication in patients with cholestatic liver disease. Several links to its pathogenesis have been proposed, including the role of bile acids, endogenous opioid and serotonins, and lysophosphatidic acid. The management of pruritus in cholestasis is challenging. Medical treatment of the underlying cholestatic condition may provide benefit. Extracorporeal albumin dialysis can be pursued for those who have a poor quality of life and failed the various therapeutic interventions, while awaiting liver transplantation. Experimental interventions, and the management of pruritus in certain conditions such as intrahepatic cholestasis of pregnancy and benign recurrent intrahepatic cholestasis, are also briefly reviewed.


Viral infection and cancer.
Infection with specific viruses has a role in the pathogenesis of some cancers in human beings. However, the incidence of such cancers is much lower than the frequency of virus infection, suggesting either that infection alone does not result in cancer and that cellular events in addition to the presence of the virus must occur, or that cancer occurs only if viral proteins are expressed in an appropriate cell type or in an immunocompromised host. Molecular analysis of viruses found in association with cancer has revealed that they function, at least in part, by encoding proteins which can associate with and subvert the function of host cell-encoded tumour suppressor proteins which regulate pathways of growth arrest and apoptosis. Better understanding of the mechanisms underlying this association will have diagnostic, prognostic, and therapeutic implications in the near future.


PTPN11 variant may be a prognostic indicator of IDH-wildtype glioblastoma in a comprehensive genomic profiling cohort.
Glioblastoma (GBM) is the most common type of primary malignant brain tumor and has a poor prognosis. Identifying novel targets and stratification strategies is urgently needed to improve patient survival. The present study aimed to identify clinically relevant genomic alterations in IDH-wildtype GBM using data from comprehensive genomic profiling (CGP) assays performed nationwide in Japan. The CGP assay results of 392 IDH-wildtype GBM cases performed between October 2019 and February 2023 obtained from the Center for Cancer Genomics and Advanced Therapeutics were retrospectively analyzed. The median patient age was 52.5 years, and 207 patients (53%) were male. In the 286 patients for whom survival information was available, a protein-tyrosine phosphatase non-receptor type 11 (PTPN11) variant detected in 20 patients (6.8%) was extracted as the gene associated with significantly shorter overall survival (p = 0.002). Multivariate analysis demonstrated that the PTPN11 variant and poor performance status were independent prognostic indicators. In contrast, no prognostic impact was observed in the cohort in The Cancer Genome Atlas data. The discrepancy in the prognostic impact of the PTPN11 variant from these two pools might have resulted from differences in the biases affecting the survival of patients who underwent a CGP assay, including left-truncation and right-censored bias. However, survival simulation done to adjust for these biases showed that the prognostic impact of the PTPN11 variant was also significant. The PTPN11 variant was a negative prognostic indicator of IDH-wildtype GBM in the patient cohort with the CGP assay.


Tetramer-Guided Epitope Mapping: A Rapid Approach to Identify HLA-Restricted T-Cell Epitopes from Composite Allergens.
Tetramer-guided epitope mapping (TGEM) is a technique in immunology that permits the rapid identification of allergenic epitopes through peptide screening procedures utilizing human lymphocyte antigen (HLA) class II tetramers as staining reagents for detection. The identification of allergenic epitopes is a prerequisite for the accurate characterization of allergen-specific CD4+ T cells without in vitro stimulation. Additionally, these MHC-II/peptide complexes that interact with T-cell receptors (TCR) of pathogenic CD4+ T cells are compatible with a different number of assays like Intracelullar Cytokine Staining (ICS), and Carboxyfluorescein succinimidyl ester (CFSE) making it a robust technology to study the functionality of allergen-specific CD4+ T cells.


A simple technique for assessing the propensity for crystallization of calcium oxalate and brushite in urine from the increment in oxalate or calcium necessary to elicit precipitation.
In an effort to develop a simple and reliable method with which to assess the propensity for spontaneous nucleation of calcium oxalate and brushite in urine, the permissible increment of oxalate and calcium was calculated. This represented the additional amount of oxalate or calcium that could be added to urine in three hours before spontaneous precipitation of calcium oxalate or brushite was initiated. The permissible increment of oxalate inversely correlated (P less than 0.001) with the formation-product ratio-activity-product ratio discriminant score of calcium oxalate, which was previously shown to reflect a quantitative measure of the likelihood for spontaneous nucleation. Similarly, the permissible increment of calcium inversely correlated (P less than 0.001) with the formation-product ratio-activity-product ratio discriminant score of brushite. The permissible increments in oxalate and calcium were significantly lower (P less than 0.001) in patients with renal stones than in control subjects. Moreover, treatment with thiazides, allopurinol, sodium cellulose phosphate, orthophosphate, and diphosphonate significantly raised the permissible increment of oxalate in patients with stones. Thus, the permissible increment was reliable in discriminating "stone-forming" from control urine and in assessing response to treatment.


Long-term bone outcomes in Italian patients with Gaucher disease type 1 or type 3 treated with imiglucerase: A sub-study from the International Collaborative Gaucher Group (ICGG) Gaucher Registry.
Gaucher disease (GD) is a lysosomal storage disorder. We evaluated the "real-world" effectiveness of first-line imiglucerase on long-term bone outcomes in Italian patients in the International Collaborative Gaucher Group (ICGG) Gaucher Registry. Patients treated with imiglucerase for ≥2 years and with bone assessments at baseline and during follow-up were selected. Data on bone pain, bone crises, marrow infiltration, avascular necrosis, infarction, lytic lesions, Erlenmeyer flask deformity, bone fractures, mineral density, and imiglucerase dosage were evaluated. Data on bone manifestations were available for 73 of 229 patients (31.9 %). Bone crises frequency decreased significantly from baseline to the most recent follow-up (p < 0.001), with some improvement observed in bone pain prevalence. Bone pain and bone crises prevalence decreased significantly from baseline at 2 to <4 and 4 to <6 years (all p < 0.05). A low median (25th, 75th percentile) baseline imiglucerase dosage was identified in patients reporting bone pain or bone crises (15.0 [13.7, 30.0] and 22.8 [17.5, 36.0] U/kg once every 2 weeks, respectively). Our study suggests that the management of GD in Italy, with regards to imiglucerase dosage, is suboptimal and confirms the need for clinicians to monitor and correctly treat bone disease according to best practice guidelines.


Reorganization of striatal inhibitory microcircuits leads to pathological synchrony in the Basal Ganglia.
Neural synchronization plays an important role in information flow in the nervous system under healthy and pathological conditions. In this issue of Neuron, Gittis et al. show that reorganization of striatal microcircuits promotes synchronous activity and may underlie the pathological network oscillations at the root of motor symptoms described in Parkinson's disease.


Local density changes and carbonate rotation enable Ba incorporation in amorphous calcium carbonate.
Incorporation of a Ba impurity in amorphous calcium carbonate (ACC) is shown with ab initio molecular dynamics simulations to have a long-range effect on its atomic-level structure and to be energetically favoured relative to incorporation in crystalline calcium carbonate polymorphs. The ability of carbonate ions to rotate and of ACC to undergo local density changes explain ACC's propensity for incorporating divalent metal impurities with a wide range of ionic radii. These findings provide an atomic-level basis for understanding the significant effects of low concentrations of impurities on the structure of ACC.


Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.
Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles. The measured P,/Pd> 1, low Ea and inhibition of Pd by pCMBS provide strong evidence that water transport in the proximal tubule basolateral membrane is facilitated by a specialized aqueous pore or narrow channel.


Aripiprazole-Associated Hypoprolactinemia in the Clinical Setting.
The increase in prolactin (PRL) levels is a common adverse effect that occurs when using conventional and atypical antipsychotic drugs. Aripiprazole (ARI) is beneficial for antipsychotic-associated hyperprolactinemia but has been reported to decrease PRL secretion. Therefore, we investigated blood levels of PRL in patients who had taken ARI alone or in combination with other antipsychotics. Retrospective information was obtained from 25 psychiatric patients who were prescribed ARI, and the blood levels of PRL were measured. The incidence of hypoprolactinemia in the current study was 44.0% (11/25). Eighteen patients were treated with ARI alone and 7 received ARI in combination with other antipsychotics. The PRL value of patients who took ARI alone was significantly lower than those who were also taking other antipsychotics (5.45 ± 3.93 vs 10.85 ± 5.53, P = 0.02; mean ± SD). There was no significant correlation of the PRL levels and dose of ARI used in the 18 patients who had taken ARI alone. This was a retrospective study, and the data were obtained from a small number of psychiatric patients treated with ARI. Monitoring of PRL levels in patients treated with ARI may be useful in minimizing hypoprolactinemia, which has the potential to negatively impact patients. In particular, hypoprolactinemia as a consequence of taking ARI should be discussed with patients of childbearing age and those with immune deficiencies.


Predicting the initiation of substance abuse in Iranian adolescents.
Limited information is available on adolescent's substance abuse in Iran. The aims of this study are to estimate incidence rate of substance abuse among the sample of 10-grade male students in Tabriz City (northwestern region of Iran), and to evaluate the associated factors. Of all grade-10 male students in Tabriz, 1785 (13.7%) were randomly sampled, and were assessed twice. A self-administered questionnaire with 48 questions was distributed to students in February and March 2005. After one year (February and March 2006), another questionnaire with 10 items was distributed to those students to determine the incidence rate of substance abuse. The influence of different factors on substance abuse was evaluated with a logistic regression model. The mean age of the subjects was 16.3+/-0.87 (min. 15, max. 19) years. At the end of follow-up, 10.1% and 2.2% of students reported incidence of alcohol use and drug abuse respectively. This study has shown low prevalence of substance abuse, but higher incidence rate of substance abuse.


Variation in children's cognitive and behavioural adjustment between different types of place in the British National Child Development Study.
Several recent studies suggest that there is a link between children's development and the types of neighbourhood in which they live. This paper examines the relationship of a classification of different types of neighbourhood to children's cognitive and behavioural outcomes using British data on children who have a parent who is a member of the 1958 Birth Cohort Study. Substantial variations in children's outcomes were observed between the neighbourhood types. Part of the variation between neighbourhood types was accounted for by various family risk factors but children living in neighbourhoods classified as Deprived City Areas continued to have higher levels of behaviour problems and lower cognitive test scores than average after adjustment for these factors. Significant adjusted associations were also found between living in neighbourhoods described as Middling Britain and lower cognitive test scores and neighbourhoods described as Prosperous Areas and better cognitive test scores. For children's cognitive outcomes, both the size and statistical significance of coefficients on the neighbourhood categories were smaller than those of family-level measures. In contrast, residence in a Deprived City Area was as significant as the family factors in predicting higher levels of behaviour problems. The association between behaviour problems and family risk factors was mediated by the home environment and cohort member parent's psychological health. Mediated effects were stronger for the family risk factors behavioural problems association than for the neighbourhood classification.


[Study on preparation of phenols gastric floating tablet].
To study the preparation of phenols gastric floating tablet. The tablets which were prepared using Eudragit IV, HPMC(K4M), MCC101 and Octadecanol as excipients were evaluated by vitro floatation and releasing performance. The pressure of preparationg was also study to select the optimal preparation. The tablets were successfully prepared in which the drug, Eudragit IV, Octadecanol were 31% respectively,and MCC101 was 7%. And 3-4 kg was found to be the eligible pressure. The study was found to be effective in the process of phenols gastric floating tablet.


Relation between duration of smoking cessation and bronchial inflammation in COPD.
Chronic obstructive pulmonary disease (COPD) is associated with airway inflammation. Although smoking cessation improves symptoms and the decline in lung function in COPD, it is unknown whether bronchial inflammation in patients with established COPD varies with the duration of smoking cessation. 114 patients (99 men) with COPD of mean (SD) age 62 (8) years, a median (IQR) smoking history of 42 (31-55) pack years, no inhaled or oral corticosteroids, all current or ex-smokers (n = 42, quit >1 month, median cessation duration 3.5 years), post-bronchodilator FEV(1) 63 (9)% predicted, and FEV(1)/IVC 48 (9)% were studied cross sectionally. The numbers of subepithelial T lymphocytes (CD3, CD4, CD8), neutrophils, macrophages, eosinophils, mast cells, and plasma cells were measured in bronchial biopsy specimens (median (IQR)/0.1 mm(2)) using fully automated image analysis. Ex-smokers with COPD had higher CD3+, CD4+, and plasma cell numbers than current smokers with COPD (149 (88-225) v 108 (61-164), p = 0.036; 58 (32-90) v 40 (25-66), p = 0.023; and 9.0 (5.5-20) v 7.5 (3.1-14), p = 0.044, respectively), but no difference in other inflammatory cells. Short term ex-smokers (<3.5 years) had higher CD4+ and CD8+ cell numbers than current smokers (p = 0.017, p = 0.023; respectively). Conversely, long term ex-smokers (quit > or =3.5 years) had lower CD8+ cell numbers than short term ex-smokers (p = 0.009), lower CD8/CD3 ratios than both current smokers and short-term ex-smokers (p = 0.012, p = 0.003; respectively), and higher plasma cell numbers than current smokers (p = 0.003). With longer duration of smoking cessation, CD8 cell numbers decrease and plasma cell numbers increase. This indicates that bronchial T lymphocyte and plasma cell counts, but not other inflammatory cells, are related to duration of smoking cessation in patients with COPD.


A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations.
Overlapping cDNA clones from the translocation breakpoint region (TBR) gene, recently discovered at the neurofibromatosis type 1 locus and found to be interrupted by deletions and a t(17;22) translocation, have been sequenced. A 4 kb sequence of the transcript of the TBR gene has been compared with sequences of genomic DNA, identifying a number of small exons. Identification of splice junctions and a large open reading frame indicates that the gene is oriented with its 5' end toward the centromere, in opposition to the three known active genes in the region. PCR amplification of a subset of the exons, followed by electrophoresis of denatured product on native gels, identified six variant conformers specific to NF1 patients, indicating base pair changes in the gene. Sequencing revealed that one mutant allele contains a T----C transition changing a leucine to a proline; another NF1 allele harbors a C----T transition changing an arginine to a stop codon. These results establish the TBR gene as the NF1 gene and provide a description of a major segment of the gene.


Is apoptosis involved in mechanisms to eliminate Onchocerca ochengi during Simulium damnosum s.l. immune response?
Co-injection of the parasite Onchocerca ochengi and the caspase inhibitors z-VAD.fmk and boc-D.fmk into the natural vector Simulium damnosum s.l. led to significantly increased survival of the parasites. Subsequent in situ apoptosis detection assays demonstrated that in the case of boc-D.fmk the enhanced survival was due to a diminished apoptosis level of the microfilariae in vivo. Additional assays using O. ochengi microfilariae which were coinjected with serine protease inhibitors into S. damnosum s.l. revealed that certain serine protease inhibitors can reduce the level of apoptosis.


Downregulation of the major histocompatibility complex class I molecules by human herpesvirus type 8 and impaired natural killer cell activity in primary effusion lymphoma development.
Primary effusion lymphomas (PELs) are invariably infected by human herpesvirus type 8 (HHV8) and often co-infected by Epstein-Barr virus (EBV). We found that expression of major histocompatibility complex class I (MHC-I) surface molecules was significantly decreased in PEL cells when compared with HHV8 negative lymphomas, irrespective of EBV infection. MHC-I downregulation rendered PEL cells sensitive to recognition and killing by natural killer (NK) cells. Intriguingly, analysis of MHC-I non-restricted cytotoxicity in two PEL patients indicated a reduced NK cell activity when compared with healthy individuals. These data suggest that PEL outgrowth may require an impaired NK cell function.


Effects of high-frequency atrial pacing in atypical atrial flutter and atrial fibrillation.
Atypical atrial flutter has, hitherto, been relatively refractory to termination by rapid atrial pacing. High-frequency pacing (HFP) in the atrium, for termination of atrial flutter or atrial fibrillation (AF), and the electrophysiologic effects related to it have not been examined. We examined the clinical efficacy, safety, and electrophysiologic mechanisms of HFP using 50-Hz bursts at 10 mA applied at the high right atrium in patients with atypical atrial flutter (group 1) or AF (group 2), using a prospective randomized study protocol. Four burst durations (500, 1000, 2000, and 4000 ms) were applied at the high right atrium repetitively in random sequence in 22 patients with spontaneous atrial flutter or AF. Local and distant right and left atrial electrogram recordings were analyzed during and after HFP. HFP resulted in local and distant right and left atrial electrogram acceleration in 8 of 10 patients (80%) in group 1 but caused less frequent local atrial electrogram acceleration (6 of 12 patients) and no distant atrial electrogram effects in group 2 (p < .05 versus group 1). The HFP protocol was effective in arrhythmia termination in 6 of 10 patients in group 1 but in no patient in group 2 (p < .05 versus group 1). Standard HFP protocol applied at the high right atrium can frequently alter atrial activation in both atria and can terminate atypical atrial flutter. Efficacy in AF is limited, probably due to limited electrophysiologic actions beyond the local pacing site.


HIV-1 drug-resistance and drug-dependence.
In this review, we will describe several recent HIV-1 studies in which a drug-dependent virus variant was selected. A common evolutionary route to the drug-dependence phenotype is proposed. First, the selection of a drug-resistance mutation that also affects the function of the targeted viral protein. Second, a compensatory mutation that repairs the protein function, but in the presence of the drug, which becomes an intrinsic part of the mechanism. The clinical relevance of drug-dependent HIV-1 variants is also discussed.


Natural selection and functional diversification of the epidermal growth factor receptor EGFR family in vertebrates.
Genes that have been subject to adaptive evolution can produce varying degrees of pathology or differing symptomatology. ErbB family receptor activation will initiate a number of downstream signaling pathways, such as mitogen-activated protein kinase (MAPK), activator of transcription (STAT), the modulation of calcium channels, and so on, all of which lead to aggressive tumor behavior. However, the evolutionary mechanisms operating in the retention of ErbB family genes and the changes in selection pressures are not clear. Sixty-two full-length cDNA sequences from 27 vertebrate species were extracted from the UniProt protein database, NCBI's GenBank and the Ensembl database. The result of phylogenetic analysis showed that the four ErbB family members in vertebrates might be formed by gene duplication. In order to determine the mode of evolution in vertebrates, selection analysis and functional divergence analysis were combined to explain the relationship of the site-specific evolution and functional divergence in the vertebrate ErbB family. Our results indicate that the acceleration of asymmetric evolutionary rates and purifying selection together were the main force for the production of ErbBs, and positive selections were detected in the ErbB family. An evolutional phylogeny of 27 vertebrates was presented in our study; the tree showed that the genes have evolved through duplications followed by purifying selection, except for seven sites, which evolved by positive selection. There was one common site with positive selection and functional divergence. In the process of functional differentiation evolving through gene duplication, relaxed selection may play an important part.


Missed lesions at CT colonography: lessons learned.
Misinterpretation at CT colonography (CTC) can result in either a colorectal lesion being missed (false-negative) or a false-positive diagnosis. This review will largely focus on potential missed lesions-and ways to avoid such misses. The general causes of false-negative interpretation at CTC can be broadly characterized and grouped into discrete categories related to suboptimal study technique, specific lesion characteristics, anatomic location, and imaging artifacts. Overlapping causes further increase the likelihood of missing a clinically relevant lesion. In the end, if the technical factors of bowel preparation, colonic distention, and robust CTC software are adequately addressed on a consistent basis, and the reader is aware of all the potential pitfalls at CTC, important lesions will seldom be missed.


Evaluation of the effect of targeted Mass Drug Administration and Reactive Case Detection on malaria transmission and elimination in Eastern Hararghe zone, Oromia, Ethiopia: a cluster randomized control trial.
Reactive and proactive case detection measures are widely implemented by national malaria elimination programs globally. Ethiopia decided to include Reactive Case Detection (RCD) and targeted Mass Drug Administration (tMDA) approaches as part of their elimination strategy along with rigorous evaluation. The purpose of this study is to compare the impact of RCD and tMDA on malaria elimination over the 2-year study period, by looking at the annual parasite incidence before and after the intervention. The study will be conducted in the East Hararghe zone of Ethiopia. Malaria transmission in the area is low to moderate. This study will deploy a community-based, three-arm, cluster-randomized control trial implemented over 2 years. Forty-eight clusters (16 clusters per arm) will be selected based on the annual number of confirmed malaria cases seen in the cluster. All clusters will receive the current standard of care in terms of malaria elimination interventions provided by the national malaria control program. In addition, following the identification of malaria parasite infection, individuals who reside within a 100-m radius of the index case will receive a diagnosis for malaria and treatment if positive in the RCD arm or presumptive treatment in the tMDA arm. The primary effectiveness endpoint will be measured at baseline and endline for each intervention arm and compared to the control arm using a difference in difference approach. This randomized controlled trial will provide evidence of the impact of the proposed intervention approaches for malaria elimination. ClinicalTrials.gov NCT04241705 . Registration date: January 27, 2020.


Using a stress reservoir to improve urine leakage after artificial urinary sphincter implantation.
Although the longest efficacy record, some patients report about urinary leakage during higher intra-abdominal pressure after Artificial Urinary Sphincter (AUS) implantation. To improve the continent results, we placed in addition to the occluding cuff and the pressure regulating balloon, in a second procedure a stress-relief reservoir in lower abdomen, for additional passive pressure transmission to the occluding cuff. In this retrospective monocentric data analysis between 2011 and 2018, 80 patients with persistence incontinence after AUS implantation were included. Stress-relief reservoir was indicated in 12 patients with involuntary leakage of urine, that occurred when intra-abdominal pressure raised. In all 12 cases, the stress reservoir was easily implanted and there were no intraoperative complications. In a mean follow-up time of 53 months, the pad per day usage (p/d) improved from 3 (± 1.2) to 1.7 (± 1.5) (p = 0.001). Two patients with multiple previous abdominal surgeries used an equal number of pads after SRR; however, an improvement during physical exertion was reported. Continence situation was evaluated with a questionnaire and was rated as "excellent", "good", or "satisfactory" by 11 (92%) patients. The persistence of urinary incontinence after AUS is a challenging topic. Implantation of a stress reservoir in carefully selected patients with urinary leakage during higher intra-abdominal pressure is minimally invasive and offers new options to improve the proven long-term record of AUS. Certainly, more investigations are needed to determine the clinical relevance of this approach.


Effect of splinting scan bodies on the trueness of complete-arch digital implant scans with 5 different intraoral scanners.
The absence of fixed reference points can affect the trueness of complete-arch intraoral digital implant scans. The effect of splinting intraoral scan bodies (ISBs) or the inclusion of artificial landmarks (AL) on the trueness of complete-arch digital implant scans is still unclear. The purpose of this study was to analyze the effect of splinting ISBs or the inclusion of AL on the trueness of complete-arch digital implant scans with 5 intraoral scanners (IOSs). Six tissue-level dental implants (Straumann Tissue Level) were placed in an edentulous patient, and the correspondent definitive cast was digitized with a desktop scanner (IScan4D LS3i) to obtain the reference digital cast. Digital scans (n=10) were performed with 5 IOSs: TRIOS 4, Virtuo Vivo, Medit i700, iTero Element 5D, and Cerec Primescan. Three different scanning techniques were evaluated: conventional (cIOSs), splinted (sIOSs), and AL (AL-IOSs). The scan data obtained were imported into a metrology software program and superimposed to the reference digital cast by using a best-fit algorithm. The overall deviations of the positions of the ISBs were evaluated by using the root-mean-square (RMS) error (α=.05). The mean ±standard deviation trueness values for the cIOSs, sIOSs, and AL-IOSs groups were 48 ±8 µm, 53 ±7 µm, and 49 ±11 µm, respectively, with no statistically significant differences (P=.06). Significant differences were found for the IOSs used with each technique (P<.001). Primescan (27 ±4 µm cIOSs; 28 ±3 µm sIOSs; 31 ±3 µm AL-IOSs) showed significantly higher trueness than iTero 5D (47 ±5 µm cIOSs; 47 ±4 µm sIOSs; 50 ±6 µm AL-IOSs) (P=.002) and TRIOS 4 (93 ±18 µm cIOSs; 76 ±18 µm sIOSs; 107 ±13 µm AL-IOSs) (P=.001) for all techniques. In addition, no significant differences were found between the techniques by using iTero 5D or Primescan (P=.348 and P=.059, respectively). The cIOSs, sIOSs, and AL-IOSs techniques showed similar trueness. The IOS used influenced the trueness of complete-arch digital implant scans.


High density lipoprotein cholesterol in relation to food consumption and running distance.
The effects of diet and exercise on serum triglycerides (Trig), total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), and the HDL to TC ratio (HDL/TC) were evaluated in 45 men training continuously for a duration of 6 months. Body mass index (BMI), percentage body fat, and serum values for Trig, TC, HDL, and HDL/TC were constant throughout the observation period. Bivariate correlations indicated that Trig, TC, and LDL were linearly related and HDL and HDL/TC were related to HDL and HDL/TC. Multiple regression analysis suggested that BMI was the best predictor of Trig, TC, and LDL, while distance run per month was the best predictor of HDL and HDL/TC and second best predictor of Trig. The weekly intakes of fish, bread, cooked cereal, wine, beer, soft drinks, and vegetables were less significant predictors of lipoprotein levels.


Isoelectric focusing of apo- and holo-transcobalamin present in human blood. Identification of a protein complexing with transcobalamin.
By means of isoelectric focusing we studied the factors influencing the isoelectric point of transcobalamin. Upon binding of cobalamin, the transcobalamin isopeptides M, X and S increase their isoelectric points by 0.4 pH units. Serum protein different from gammaglobulin changes the isoelectric point of transcobalamin, but the attachment of the transcobalamin-cobalamin complex to its acceptor is unchanged. Transcobalamin bound to auto-antibodies shows a characteristic pattern upon isoelectric focusing.


Preferential inhibition of the mRNA expression of p38 mitogen-activated protein kinase regulated cytokines in psoriatic skin by anti-TNFα therapy.
Anti-TNFα therapies are well established for severe psoriasis; however, their mechanism of action in disease resolution is not fully understood. p38 mitogen-activated protein kinase (MAPK) is a kinase known to play a key role in the pathogenesis of psoriasis. To elucidate the early effects of adalimumab, a human monoclonal anti-TNFα antibody, on the expression of interleukins in psoriatic skin. Biopsies from patients with psoriasis were examined before and after the start of adalimumab therapy. mRNA expression of cytokines were measured with quantitative polymerase chain reaction. p38 MAPK and signal transducer and activator of transcription 3 (STAT3) were analysed by Western blotting and immunofluorescence analyses, and IL-17A and IL-17C were examined with immunohistochemistry. The increased mRNA level of IL-1β, IL-8, IL-17C and IL-20 in lesional psoriatic skin was already significantly reduced 4 days after the start of adalimumab treatment, i.e. before clinical and histological improvement was detectable. The mRNA expression of the Th17-derived cytokines IL-17A, IL-17F and IL-22 as well as the dendritic cell product IL-23/IL-12 (p40) were not significantly reduced until 2 weeks after the start of treatment, whereas the mRNA expression of IL-23 (p19) and the Th1 cytokines IFN-γ and IL-2 were reduced late in disease resolution. IL-1β, IL-8 and IL-20 are all known to be regulated by p38 MAPK. IL-17C was produced by cultured human keratinocytes and this production was also mediated by a p38 MAPK dependent mechanism. Moreover, the early effects of adalimumab included the phosphorylation of p38 MAPK, but not STAT3 phosphorylation. This study indicates that an important mechanism of action of anti-TNFα therapy in psoriasis is a reduction in p38 MAPK phosphorylation and a subsequent decrease in the expression of p38 MAPK regulated genes.


Gemcitabine-releasing polymeric films for covered self-expandable metallic stent in treatment of gastrointestinal cancer.
Non-vascular drug-eluting stents have been studied for the treatment of gastrointestinal cancer and cancer-related stenosis. In this study, we designed and evaluated a gemcitabine (GEM)-eluting covered nonvascular stent. Polyurethane (PU)/polytetrafluoroethylene (PTFE) film was selected for the drug loading and eluting membrane. The membrane was fabricated by dip-coating on a Teflon bar (∅; 10mm), air-dried, peeled off and applied to a self-expanding Nitinol stent. Various amounts of poloxamer 407 (PL, Lutrol F127, BASF) (8%, 10%, or 12% of PU by weight) were added to control the release of GEM from membranes. The membrane containing 12% PL (GEM-PU-PL12%) showed the most favourable release properties; 70% of the loaded GEM released within 35 days, including the 35% released during the initial burst. The biological activities of GEM-PU-PL12% were evaluated using human cholangiocarcinoma cells (SK-ChA-1). GEM-PU-PL12% most efficiently inhibited the proliferation of cholangiocarcinoma cells and most highly induced pro-inflammatory cytokines (TNF-α, IL-1β and IL-12) and p38 MAPKs in the cells. Subtumoural insertion of the GEM-PU-PL12% membrane more efficiently inhibited the growth of CT-26 colon cancer than other membranes. In this study, the GEM-eluting metal stents covered with PU-PL12% showed considerable feasibility for the treatment of malignant gastrointestinal cancer as well as cancer-related stenosis.


Metals profile in deep-sea sediment from an active tectonic region around Simeulue Island, Aceh, Indonesia.
Simeulue waters are adjacent to the northern part of Sumatra Island, which is undergoing massive land-use transformations; moreover, the waters are located in an active tectonic region. Land changes and tectonic activity might affect the metal pollution profile in this deep sea area. Thus, this study aimed to investigate the vertical profile and assess the sediment quality from the deep-sea marine sediment around Simeulue Island based on metal concentration. Seventy-six bottom sediment samples were collected from eight cores at a water depth of up to 2800 m in the Simeulue waters, Indonesia, in November 2017. Metals Cd, Cu, Fe, Ni, Pb, and Zn were quantified from the cores and multivariate analyses were carried out to understand the process. Metals distributions are analogous to the grain size parameters and LOI550 distribution pattern, while Sumatra and Simeulue islands influenced grain size and LOI550 spatial distribution. The vertical grain size profile exhibited no extreme oscillation in the investigated cores. Thus, sediment transport from the Island was the main suspect for these metals' profiles in the deep water, and the tectonic activity had a minor impact. Cu, Ni, Pb, and Zn tend to rise in the collected cores, suggesting that the accumulation of the metals is growing. While Fe tended to be stable and Cd oscillated in the cores. Indices were computed to assess the metal contamination profile. The cores were dominated by EF class 1 (none to slight enrichment) status and Igeo class 1 (unpolluted). Cd was the metal of concern in the study since a high Cd was observed in some layers (maximum EF = 26.45 and maximum Igeo = 3.81). Thus, this study can be used as a database to improve the regulation formulation for improved environmental managerial efforts in the region.


Structural Neuroimaging of Anorexia Nervosa: Future Directions in the Quest for Mechanisms Underlying Dynamic Alterations.
Anorexia nervosa (AN) is a serious eating disorder characterized by self-starvation and extreme weight loss. Pseudoatrophic brain changes are often readily visible in individual brain scans, and AN may be a valuable model disorder to study structural neuroplasticity. Structural magnetic resonance imaging studies have found reduced gray matter volume and cortical thinning in acutely underweight patients to normalize following successful treatment. However, some well-controlled studies have found regionally greater gray matter and persistence of structural alterations following long-term recovery. Findings from diffusion tensor imaging studies of white matter integrity and connectivity are also inconsistent. Furthermore, despite the severity of AN, the number of existing structural neuroimaging studies is still relatively low, and our knowledge of the underlying cellular and molecular mechanisms for macrostructural brain changes is rudimentary. We critically review the current state of structural neuroimaging in AN and discuss the potential neurobiological basis of structural brain alterations in the disorder, highlighting impediments to progress, recent developments, and promising future directions. In particular, we argue for the utility of more standardized data collection, adopting a connectomics approach to understanding brain network architecture, employing advanced magnetic resonance imaging methods that quantify biomarkers of brain tissue microstructure, integrating data from multiple imaging modalities, strategic longitudinal observation during weight restoration, and large-scale data pooling. Our overarching objective is to motivate carefully controlled research of brain structure in eating disorders, which will ultimately help predict therapeutic response and improve treatment.


Hot weather hazard analysis over India.
Heat waves are often termed as the silent killer and have become even more important as recent studies suggest that the heat wave have become second most devastating extreme weather events in terms of human deaths and losses. It is also been largely realised by scientific community that it is not just the high temperatures which are responsible for the gruesome effect of heat waves but several other meteorological parameters play a vital role in aggravating the impact and causing much more damages. In view of the above the attention of scientific community, weather forecasters as well as disaster managers has shifted to also take into account the different meteorological parameters like maximum and minimum temperatures, relative humidity, wind speed, duration/spell of heat waves and its intensity which are aggravating the impact of heat stress. In this background, this study is undertaken as an attempt to quantify the effect of different meteorological parameters on heat wave on different regions of India for different summer months (March, April, May and June). In this study the impact of individual meteorological parameter as well their cumulative effect is studied based on data of 30 years (1981-2010) for 300 stations. The effect of different meteorological parameters is identified for different months for different regions of the country. Also the cumulative scores are calculated for different regions considering different meteorological parameters, as a first initiative to perform heat hazard analysis and zonation over the entire country. This could serve as initial step for planning mitigation and adaptation strategies throughout the country. These scores as thresholds for different regions may be also useful for operational forecaster's for early impact based warning services as well as for the disaster managers, for taking effective and timely actions.


Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.
The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.


Exploration of the α-syn/T199678/miR-519-3p/KLF9 pathway in a PD-related α-syn pathology.
Kruppel-like factor 9 (KLF9) plays a key role as an inducer of cellular oxidative stress in the modulation of cell death and in oxidant-dependent tissue injury. Our previous study indicated that lncRNA-T199678 (T199678) affected the expression of KLF9 in an α-synuclein (α-syn) induced cellular model. However, the roles of interactions among α-syn, T199678, KLF9 and related microRNAs (miRNAs) in the Parkinson's disease (PD)-related α-syn pathology are unclear and were therefore investigated in this study. An α-syn-injected mouse model and an α-syn exposed SY-SH5Y cellular model were used in this study. We confirmed the utility of these established models with morphological and behavioral methods. We checked how expression of T199678 and KLF9 were affected by α-syn and demonstrated their interaction by fluorescence in situ hybridization (FISH) staining and western blots. We analyzed expression in ROS+ cells by immunohistochemistry. Finally, we obtained seven miRNAs through bioinformatic analysis simultaneously affected by T199678 and α-syn and verified these with RT-PCR. We found that expression of KLF9 was regulated by T199678, whereas expression of T199678 was not affected by KLF9 in the α-syn exposed SY-SH5Y cells. These findings suggest that KLF9 is the downstream gene regulated by T199678, whereas miR-519-3p may play a contributing role. We also confirmed that α-syn injection upregulated the expression of ROS, which could be downregulated by upregulation of T199678, indicating an anti-oxidative role of T199678 in the α-syn-related mechanisms. Our results indicate the existence of a potential α-syn/T199678/miR-519-3p /KLF9 pathway in PD-related α-syn pathology. This pathway might explain oxidative stress processes in α-syn-related mechanisms, which requires further verification.


Systematic Review of Human Dental Pulp Stem Cells for Cartilage Regeneration.
Background: Symptomatic cartilage lesions and early osteoarthritis produce significant clinical and economic burdens. Cartilage repair can improve the symptoms and delay arthroplasty. The complete healing of damaged cartilage with the consistent reproduction of normal hyaline cartilage has not yet been achieved. The choice of harvesting site might influence the cells' abilities to modulate immunologic and inflammatory responses. Recently, dental pulp has been shown to contain a stem cell niche consisting of dental pulp stem cells (DPSCs) that maintain their self-renewal capacity due to the active environment in the dental pulp of deciduous teeth. Objective: The aim of this study was to critically review the current literature on the potential and limitations of the use of dental pulp-derived mesenchymal stem cells in cell-based therapies for cartilage regeneration. Methods: An electronic, customized search of scientific articles was conducted using the PubMed/MEDLINE and EMBASE databases from their inception to December 2018. The inclusion criteria were applied, and the articles that described the use of DPSC in cartilage treatment were selected for complete evaluation. The articles were classified according to the scaffold used, experimental model, chondrogenic differentiation features, defect location, cartilage evaluation, and results. After the application of the eligibility criteria, a total of nine studies were selected and fully analyzed. Results: A variety of animal models were used, including mice, rats, rabbits, and miniature pigs, to evaluate the quality and safety of human DPSCs in the repair of cartilage defects. Among the articles, two studies focused on preclinical models of cartilage tissue engineering. Five studies implanted DPSCs in other animal sites. Conclusion: The use of DPSCs is a potential new stem cell therapy for articular cartilage repair. The preclinical evidence discussed in this article provides a solid foundation for future clinical trials. Impact statement Osteoarthritis presents an ever-increasing clinical and socioeconomic burden. While cartilage repair has the potential to improve symptoms and delay joint replacement, complete regeneration of hyaline cartilage has been an elusive goal. Dental pulp has been shown to contain a niche that protects dental pulp stem cells (DPSCs) from the cumulative effects of genetic and environmental factors and maintains their self-renewal capacity due to the active environment. Transplantation and preclinical trials have demonstrated the strong potential of regenerative tissue-engineering protocols using DPSCs.


Natural attenuation of sulfometuron-methyl in seawater: Kinetics, intermediates, toxicity change and ecological risk assessment.
This research aims to evaluate the environmental feasibility of sulfometuron-methyl (SM) as a growth inhibitor for restricting the growth of Spartina alterniflora. To achieve this purpose, the natural attenuation characteristics, ecological risk, degradation pathway, and comprehensive toxicity changes of SM in seawater were investigated under the simulated marine environmental conditions of Jiaozhou Bay, China. The natural attenuation of SM in seawater followed first-order reaction kinetics with a rate constant (K) of 0.0694 d-1 and a half-life of 9.99 days. When photolysis, hydrolysis, and biodegradation pathways act alone, the rate constants K of SM were 0.0167, 0.0143, and 0.0099 d-1 respectively, indicating that their contributions to the total removal of SM decreased in turn. The calculation results of risk quotient (RQ) showed that the seawater containing 10 mg/L of SM demonstrated a very high risk to marine diatom Skeletonema costatum before and after 21 days of attenuation with RQ values of 24.46 and 6.32, respectively, however, the risk to other marine organisms (fish, crustaceans, and bivalves) decreased from moderate (RQ < 1) to low (RQ < 0.01). Four attenuation products of SM were identified and two degradation pathways of SM in seawater were proposed. Based on the rate of inhibition of bioluminescence, SM in seawater was not harmful to Photobacterium phosphoreum T3, whereas the toxicity of seawater containing SM increased with the extension of attenuation time, suggesting the formation of intermediate products with high aquatic toxicity. According to the toxicity values predicted by ECOSAR, the toxicity of one identified attenuation product was higher than that of SM. To the best of our knowledge, this is the first report on the attenuation characteristics and toxicity changes of SM in seawater. The results indicated that the toxicity of both SM and its degradation products to non-target marine organisms should be considered in evaluating the feasibility of SM in controlling coastal Spartina alterniflora.


Athletes and novices are differently capable to recognize feint and non-feint actions.
Fast motor reactions in sports often require the ability to predict the intended action of an opponent as early as possible. Therefore, the present paper investigates whether beach volleyball athletes are able to recognize different attack hits (i.e. smash vs. poke shot) at an earlier stage of the movement than novices. Beach volleyball athletes and novices took part in a response priming experiment (Experiment 1). Participants had to decide whether a presented target picture depicts a smash or a poke shot. Importantly, the preceding prime pictures were taken from different stages of the movements varying between the jump (beginning of the movements) and the hand-ball contact (end of the movements). Diverging response congruency effects was found for athletes and novices. Athletes were able to recognize at an earlier movement stage than novices which kind of attack hit was shown at the prime picture. It is suggested that athletes might implicitly read movement-related patterns in the depicted athlete's body posture (e.g. the angle of the elbow). In contrast, novices might use information which is easier to access (e.g. hand-ball relation). In a second experiment, novice participants received a visual training to test for a potential perceptual source of the priming effects. Notably, participants did not improve their ability to differentiate the volleyball techniques, indicating that a better recognition performance in athletes is based on motor and not on perceptual expertise.


Uncoupling of the synthesis of edited and unedited COIII RNA in Trypanosoma brucei.
RNA editing, a novel and unexpected type of information processing, was first demonstrated in the kinetoplasts of certain protozoans. It is a remarkable phenomenon: certain species of messenger RNA have nucleotide sequences that differ greatly from the sequences of the genes from which they are presumably transcribed. The differences are usually due to addition of uridylate residues, but occasionally also to their deletion. The most spectacular case of editing known occurs in the mRNA of the mitochondrial gene for subunit III of cytochrome oxidase (COIII) in Trypanosoma brucei. This mRNA is twice the length of its gene owing to the addition of several hundred uridylate residues and a few deletions, spread over the entire length of the RNA molecule. Whereas unedited RNA molecules, the nucleotide sequences of which correspond to the genomic sequence, have been isolated, no DNA template corresponding to the edited RNA sequence has been detected in either the mitochondrial or nuclear genome. It was suggested, therefore, that unedited mRNAs are transcribed from mitochondrial DNA and then edited post-transcriptionally by endonucleolytic cleavage of the primary transcript at specific sites, followed by insertion or deletion of uridylate residues and religation. We have now examined the general nature of the RNA editing process to determine whether it involves the insertion of nucleotide residues into pre-existing molecules or the continuous de novo synthesis of edited mRNA. The results of our experiments rule out an insertion mechanism and strongly indicate that edited mRNA is synthesized as a unit.


Flame curtain kilns produce biochar from dry biomass with minimal methane emissions.
Flame curtain kilns have emerged as the preferred biochar technology for smallholders but reported methane emissions (30 g kg-1 biochar) have impeded carbon certification. Here, for flame curtain kilns we show almost no methane (0-3.6 g kg-1 biochar) emissions for dry (<15 % moisture) feedstock consisting of twigs and leaves. Wet feedstock (>40 % moisture) however generated significant methane (>500 g kg-1 biochar), underscoring that feedstock preparation is decisive for the carbon balance. Even for dry feedstock, both aerosol and CO emissions were significant (21-82 and 40-118 g kg-1 biochar, respectively). The data demonstrate that certification of low-tech biochar made from dry twigs and leaves should not be objected to on the grounds of methane. Careful selection of feedstock and potential after-combustion of the syn-gases are probably needed to avoid CO and aerosol emissions. More data are needed on methane emissions of other dry feedstocks.


Differences in the management of inflammatory bowel disease in children and adolescents compared to adults.
Managing IBD in children and adolescents requires attention to issues unique to these age groups. The spectrum of presenting signs and symptoms is broad and often, subtle. Physician awareness of intestinal and extra-intestinal features prompts earlier diagnosis and intervention. The focus of treatment is not limited to intestinal symptoms, but also involves assessing weight and height gains, sexual maturation, extra-intestinal manifestations and psychosocial well-being. Differences in selecting drugs for pediatric versus adult patients are based on: 1. lack of prospective trials establishing effective doses for different ages; 2. inability to swallow capsules; 3. importance of nutrition in promoting growth; 4. paucity of data regarding the long-term safety of medications; 5. untoward cosmetic effects of corticosteroids, and 6. the need to develop coping mechanisms for a chronic illness. While sulfasalazine and mesalamine are useful in mild disease, corticosteroids are necessary for moderate and severe disease. Metronidazole and ciprofloxacin are effective in perianal CD. Elemental and polymeric formulas induce and maintain remission in active CD and reverse growth failure. Immunomodulatory agents (azathioprine and 6-mercaptopurine) enable physicians to reduce steroids and hospitalization. In practice, combination therapy is recommended to control symptoms and limit drug-induced side effects.


Genome Sequence of the Autotrophic Acetogen Clostridium magnum DSM 2767.
Here we report the draft genome sequence (6.6 Mbp) of the type strain Clostridium magnum, an acetogen with two operons coding for two separate Rnf complexes. C. magnum grows on a broad range of organic substrates and converts CO2 and H2 to acetate using the Wood-Ljungdahl pathway.


Magnetic delivery of antitumor carboplatin by using PEGylated-Niosomes.
To improve the efficiency of niosomal drug delivery, here we employed two tactics. First, niosomes were magnetized using Fe3O4@SiO2 mangnetic nanoparticles, and second, their surface was modified by PEGylation. PEGylation was intended for increasing the bioavailability of niosomes, and magnetization was used for rendering them capable of targeting specific tissues. These PEGylated magnetic niosomes were also loaded with Carboplatin, an antitumor drug. Next, these niosomes were studied in terms of size, morphology, zeta potential, and drug entrapment efficiency. Then, the in vitro drug release from these modified niosomes was compared to that of both naked and nonmagnetized niosomes. Interestingly, although loading of naked-niosomes with magnetic particles lead to an increase in the rate of drug release, PEGylation of these magnetized niosomes caused a more sustained drug release. Thus, PEGylation of magnetic niosomes, besides improving their bioavailability, caused a slower and sustained release of the drug over time. Finally, studying the in vitro effectives of niosomal formulations towards MCF-7, a breast cancer cell line, showed that PEGylated magnetic niosomes had a satisfactory toxicity towards these cells in the presence of an external magnetic field. In conclusion, PEGylated magnetic niosomes showed enhanced qualities regarding the controlled release and delivery of drug. Graphical abstract .


SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage ( Brassica rapa ssp. pekinensis).
Clubroot disease, caused by Plasmodiophora brassicae Wor., is highly damaging for Chinese cabbage. The CR (clubroot resistant) Shinki DH (doubled haploid) line of Chinese cabbage carries a single dominant gene, CRb, which confers resistance to the P. brassicae races 2, 4, and 8. An F(2) population derived from a cross between the CR Shinki DH line and a susceptible line, 94SK, was used to map the CRb gene. Inoculation of F(3) families with SSI (single-spore isolate) resulted in a 1:2:1 segregation ratio. Use of the AFLP technique combined with bulked segregant analysis allowed five co-dominant AFLP markers, and four and seven dominant AFLP markers linked in coupling and repulsion, respectively, to be identified. Six of the 16 AFLP markers showing low frequencies of recombination with the CRb locus among 138 F(2) lines were cloned. A reliable conversion procedure allowed five AFLP markers to be successfully converted into CAPS and SCAR markers. An F(2) population (143 plants) was analyzed with these markers and a previously identified SCAR marker, and a genetic map around CRb covering a total distance of 6.75 cM was constructed. One dominant marker, TCR09, was located 0.78 cM from CRb. The remaining markers (TCR05, TCR01, TCR10, TCR08, and TCR03) were located on the other side of CRb, and the nearest of these was TCR05, at a distance of 1.92 cM.


Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer.
Our previous study demonstrated that fascin homolog 1 (FSCN1) might have an oncogenic function in bladder cancer (BC) and that its expression was regulated by specific microRNAs (miRNAs). Recently, LIM and SH3 protein 1 (LASP1) as well as FSCN1 have been reported as actin filament bundling proteins in the same complexes attached to the inner surfaces of cell membranes. We hypothesize that LASP1 as well as FSCN1 have an oncogenic function and that is regulated by miRNAs targeting LASP1 mRNA. The expression levels of LASP1 mRNA in 86 clinical samples were evaluated by real-time RT-PCR. LASP1-knockdown BC cell lines were transfected by siRNA in order to examine cellular viability by XTT assay, wound healing assay, and matrigel invasion assay. We employed web-based software in order to search for candidate miRNAs targeting LASP1 mRNA, and we focused on miR-1, miR-133a, miR-145, and miR-218. The luciferase reporter assay was used to confirm the actual binding sites between the miRNAs and LASP1 mRNA. Real-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P < 0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P < 0.0005), cell migration inhibition (P < 0.0001), and a decrease in the number of invading cells (P < 0.005) in the transfectants compared with the controls. Transient transfection of three miRNAs (miR-1, miR-133a, and miR-218), which were predicted as the miRNAs targeting LASP1 mRNA, repressed the expression levels of mRNA and protein levels of LASP1. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1, miR-133a, and miR-218 transfectants (P < 0.05), suggesting that these miRNAs have actual target sites in the 3' untranslated region of LASP1 mRNA. Furthermore, significant cell viability inhibitions occurred in miR-218, miR-1, and miR-133a transfectants (P < 0.001). Our data indicate that LASP1 may have an oncogenic function and that it might be regulated by miR-1, miR-133a, and miR-218, which may function as tumor suppressive miRNAs in BC.


Association of blood eicosapentaenoic acid levels with intracerebral hemorrhage during the COVID-19 pandemic: preliminary experience from a single-center in Japan.
The COVID-19 pandemic has forced lockdowns and declarations of states of emergency, resulting in marked changes to daily life such as dietary habits in many countries. Though serum omega-3 polyunsaturated fatty acids levels have been shown to be useful markers for recurrent vascular events or worse prognosis in cardiovascular diseases and ischemic stroke, the relationship between serum omega-3 PUFA levels and the occurrence of intracerebral hemorrhage has essentially been unknown. We explored the association of serum omega-3 polyunsaturated fatty acids with intracerebral hemorrhage during the COVID-19 pandemic. Participants comprised patients admitted to Juntendo University Hospital (Tokyo, Japan) with intracerebral hemorrhage between January 1, 2016 and April 30, 2020. Clinical characteristics including serum omega-3 polyunsaturated fatty acid levels were compared between patients developing intracerebral hemorrhage during the period from January 1, 2016 to February 29, 2020, and the subsequent COVID-19 pandemic period (March 1 to April 30, 2020). Clinical characteristics independently related to intracerebral hemorrhage during the COVID-19 pandemic were analyzed by comparing these two cohorts of intracerebral hemorrhage patients in different periods. A total of 103 patients (age, 67.0 ± 13.9 years; 67 males) with intracerebral hemorrhage were enrolled. Intracerebral hemorrhage developed in 91 patients before and 12 patients during the COVID-19 pandemic. Monthly averages of intracerebral hemorrhage patients admitted to our hospital during and before the COVID-19 pandemic were 6 and 1.82, respectively. Serum eicosapentaenoic acid levels were significantly lower in intracerebral hemorrhage patients during the COVID-19 pandemic than before (31.87 ± 12.93 μg/ml vs. 63.74 ± 43.29 μg/ml, p = 0.007). Multiple logistic regression analysis showed that, compared to before the COVID-19 pandemic, dyslipidemia (odds ratio 0.163, 95% confidence interval 0.031-0.852; p = 0.032) and eicosapentaenoic acid levels (odds ratio 0.947, 95% confidence interval 0.901-0.994; p = 0.029) were associated with intracerebral hemorrhage during the COVID-19 pandemic. From our preliminary results, low eicosapentaenoic acid levels were linked with intracerebral hemorrhage during the COVID-19 pandemic. Low levels of eicosapentaenoic acid might be an endogenous surrogate marker for intracerebral hemorrhage during the COVID-19 pandemic.


Acetylcholine-induced Na+ influx in the mouse lacrimal gland acinar cells: demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements.
In the isolated, superfused mouse lacrimal gland, intracellular Na+ activities (aNai) of the acinar cells were directly measured with double-barreled Na+-selective microelectrodes. In the nonstimulated condition aNai was 6.5 +/- 0.5 mM and membrane potential (Vm) was -38.9 +/- 0.4 mV. Addition of 1 mM ouabain or superfusion with a K+-free solution slightly depolarized the membrane and caused a gradual increase in aNai. Stimulation with acetylcholine (ACh, 1 microM) caused a membrane hyperpolarization by about 20 mV and an increase in aNai by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase in aNai by approximately 50%, without affecting Vm and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH4Cl or by replacement of Tris+-buffer saline solution with HCO3-/CO2-buffered solution increased aNai by a few mM. Superfusion with a Cl(-)-free NO3- solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the resting aNai levels, however, it reduced the ACh-induced increase in aNai by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the resting aNai and the ACh-induced increase in aNai. The present results suggest the presence of multiple Na+ entry mechanisms activated by ACh, namely, Na+/H+ exchange, Na-K-Cl cotransport and organic substrate-coupled Na+ transport mechanisms.


Fibrinogen deficiency, but not plasminogen deficiency, increases mortality synergistically in combination with sickle hemoglobin SAD in transgenic mice.
Patients with sickle cell disease exhibit both acute and chronic activation of the coagulation and fibrinolytic systems. To test the relationship between sickle cell pathology and activation of the hemostatic system, mice with targeted deletions of plasminogen (Plg) or fibrinogen (Fib) were crossed with transgenic mice expressing Hb SAD [beta(6Glu-Val) (HbS), beta(23Val-Ile) (HbAntilles), and beta(121Glu-Gln) (HbD-Punjab)]. Fibrinogen deficiency dramatically reduced the survival of mice with Hb SAD to a much greater degree than mice with normal hemoglobin. The combination of Hb SAD and fibrinogen deficiency had a greater effect on mortality than that obtained by adding the mortality risks of each defect alone. The deleterious effect of the combination of Hb SAD and fibrinogen deficiency on mortality was accelerated by hypoxia. The excess mortality associated with plasminogen deficiency was identical in SAD and control mice. The adverse effect of fibrinogen deficiency on mortality in SAD mice is not consistent with the simple hypothesis that fibrin deposition is uniformly deleterious in the context of vaso-occlusive sickle cell disease. Rather, our findings suggest that the contribution of fibrinogen to tissue repair may in some contexts limit sickle cell disease pathophysiology.


Effect of temperature on the velocity of erythrocyte aggregation.
The velocity of the aggregation of human erythrocytes was examined in the range of 5-43 degrees C with a rheoscope combined with a video camera, an image analyzer and a computer. (1) With increasing temperature, the velocity of erythrocyte aggregation induced by fibrinogen, immunoglobulin G and artificial macromolecules (dextran of 70 kDa and poly(glutamic acid) of 50 kDa) increased. However, the relationship between the velocity of erythrocyte aggregation and the temperature was different among these macromolecules. (2) In 70% autologous plasma, the velocity of erythrocyte aggregation was minimum at 15-18 degrees C, and increased at both higher and lower temperatures. (3) The shape of erythrocyte aggregates in 12 mumol/l fibrinogen (containing 770 mumol/l albumin) and in 70% autologous plasma was dependent on temperature: three-dimensional below 15-18 degrees C and one-dimensional (mainly rouleaux) above 15-18 degrees C. However, the shape of aggregates in 27 mumol/l immunoglobulin G (containing 770 mumol/l albumin) was three-dimensional in all temperature ranges. (4) The temperature dependency of erythrocyte aggregation was discussed in terms of the changes of medium viscosity, of erythrocyte properties and of bridging macromolecules.


Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.
Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.


In vivo measurement of the redox state.
As part of an aerobic life, we oxidize a large pool of biomolecules to obtain chemical energy. During this process, several intermediates are formed; some are chemically unstable and are referred to as free radicals (FR). FR tend to react quickly with their surrounding biological environment; depending on the nature of the molecule attacked, different reactions can occur, i.e., lipid peroxidation, protein oxidation, or DNA oxidation products. As aerobic life has evolved, antioxidant defense systems against FR have developed. When an imbalance between production of FR (oxidants) and defense systems against them (antioxidants) happens, a situation of oxidative stress occurs. This can lead to irreversible biochemical changes, with subsequent tissue damage and disease. Establishing the involvement of FR in the pathogenesis of a disease has been difficult because of the lack of sensitive and specific methodology to detect them. No ideal biomarkers for in vivo FR-induced damage are available as yet. However, some reliable indices of FR formation are now available, and in some pathologic conditions, evidence is accumulating to show that FR damage might play a functional role. The task for the near future will be to try to simplify the analytical methodology and elucidate the molecular mechanisms underlying the formation, disposition, and kinetics of FR marker molecules.


A percutaneous needle biopsy technique for sampling the supraclavicular brown adipose tissue depot of humans.
Brown adipose tissue (BAT) has been proposed as a potential target tissue against obesity and its related metabolic complications. Although the molecular and functional characteristics of BAT have been intensively studied in rodents, only a few studies have used human BAT specimens due to the difficulty of sampling human BAT deposits. We established a novel positron emission tomography and computed tomography-guided Bergström needle biopsy technique to acquire human BAT specimens from the supraclavicular area in human subjects. Forty-three biopsies were performed on 23 participants. The procedure was tolerated well by the majority of participants. No major complications were noted. Numbness (9.6%) and hematoma (2.3%) were the two minor complications noted, which fully resolved. Thus, the proposed biopsy technique can be considered safe with only minimal risk of adverse events. Adoption of the proposed method is expected to increase the sampling of the supraclavicular BAT depot for research purposes so as to augment the scientific knowledge of the biology of human BAT.


Trial of oral physostigmine in amyotrophic lateral sclerosis.
We evaluated a double-blind, placebo-controlled, and double-crossover trial of oral physostigmine salicylate for a 9-month period in 13 of 25 patients with sporadic amyotrophic lateral sclerosis (ALS). A large dropout rate of 48% was secondary to eight deaths and four exclusions attributed to the incapability to swallow the tablets (physostigmine) and capsules (lecithin) or to attend the clinic. Parameters used for assessment of the drug efficacy included body weight, ALS score, Jamar grip strength, forced vital capacity, and maximum voluntary ventilation. It revealed slight benefit in reduced loss of grip strength compared with the pretrial and placebo periods. However, the rates of decline for body weight, ALS score, forced vital capacity, maximum voluntary ventilation, and megascore did not differ significantly between the pretrial, placebo, and physostigmine periods. We therefore concluded that overall no significant alteration in the clinical course was gained by oral physostigmine therapy in the 13 patients with ALS who were included in this study.