Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8% identity for nucleotides and 96.8% identity for amino acids) and more similar to the BCoV-ENT strain (98.7% for nucleotides and 98.7% for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Transcranial direct current stimulation and repetitive transcranial magnetic stimulation in consultation-liaison psychiatry

Patients with clinical diseases often present psychiatric conditions whose pharmacological treatment is hampered due to hazardous interactions with the clinical treatment and/or disease. This is particularly relevant for major depressive disorder, the most common psychiatric disorder in the general hospital. In this context, nonpharmacological interventions could be useful therapies; and, among those, noninvasive brain stimulation (NIBS) might be an interesting option. The main methods of NIBS are repetitive transcranial magnetic stimulation (rTMS), which was recently approved as a nonresearch treatment for some psychiatric conditions, and transcranial direct current stimulation (tDCS), a technique that is currently limited to research scenarios but has shown promising results. Therefore, our aim was to review the main medical conditions associated with high depression rates, the main obstacles for depression treatment, and whether these therapies could be a useful intervention for such conditions. We found that depression is an important and prevalent comorbidity in a variety of diseases such as epilepsy, stroke, Parkinson's disease, myocardial infarction, cancer, and in other conditions such as pregnancy and in patients without enteral access. We found that treatment of depression is often suboptimal within the above contexts and that rTMS and tDCS therapies have been insufficiently appraised. We discuss whether rTMS and tDCS could have a significant impact in treating depression that develops within a clinical context, considering its unique characteristics such as the absence of pharmacological interactions, the use of a nonenteral route, and as an augmentation therapy for antidepressants.

Estragole blocks neuronal excitability by direct inhibition of Na+ channels

Estragole is a volatile terpenoid, which occurs naturally as a constituent of the essential oils of many plants. It has several pharmacological and biological activities. The objective of the present study was to investigate the mechanism of action of estragole on neuronal excitability. Intact and dissociated dorsal root ganglion neurons of rats were used to record action potential and Na+ currents with intracellular and patch-clamp techniques, respectively. Estragole blocked the generation of action potentials in cells with or without inflexions on their descendant (repolarization) phase (Ninf and N0 neurons, respectively) in a concentration-dependent manner. The resting potentials and input resistances of Ninf and N0 cells were not altered by estragole (2, 4, and 6 mM). Estragole also inhibited total Na+ current and tetrodotoxin-resistant Na+ current in a concentration-dependent manner (IC50 of 3.2 and 3.6 mM, respectively). Kinetic analysis of Na+ current in the presence of 4 mM estragole showed a statistically significant reduction of fast and slow inactivation time constants, indicating an acceleration of the inactivation process. These data demonstrate that estragole blocks neuronal excitability by direct inhibition of Na+ channel conductance activation. This action of estragole is likely to be relevant to the understanding of the mechanisms of several pharmacological effects of this substance.

Novel retrograde puncture method to establish preperitoneal space for laparoscopic direct inguinal hernia repair with internal ring suturing

The aim of this study was to explore the clinical efficacy of a novel retrograde puncture approach to establish a preperitoneal space for laparoscopic direct inguinal hernia repair with inguinal ring suturing. Forty-two patients who underwent laparoscopic inguinal hernia repair with retrograde puncture for preperitoneal space establishment as well as inguinal ring suturing between August 2013 and March 2014 at our hospital were enrolled. Preperitoneal space was successfully established in all patients, with a mean establishment time of 6 min. Laparoscopic repairs were successful in all patients, with a mean surgical time of 26±15.1 min. Mean postoperative hospitalization duration was 3.0±0.7 days. Two patients suffered from postoperative local hematomas, which were relieved after puncturing and drainage. Four patients had short-term local pain. There were no cases of chronic pain. Patients were followed up for 6 months to 1 year, and no recurrence was observed. Our results demonstrate that preperitoneal space established by the retrograde puncture technique can be successfully used in adult laparoscopic hernioplasty to avoid intraoperative mesh fixation, and thus reduce medical costs.