Experimental and Computational Analysis of Synthesis Conditions of Hybrid Nanoflowers for Lipase Immobilization.

This work presents a framework for evaluating hybrid nanoflowers using Burkholderia cepacia lipase. It was expanded on previous findings by testing lipase hybrid nanoflowers (hNF-lipase) formation over a wide range of pH values (5-9) and buffer concentrations (10-100 mM). The free enzyme activity was compared with that of hNF-lipase. The analysis, performed by molecular docking, described the effect of lipase interaction with copper ions. The morphological characterization of hNF-lipase was performed using scanning electron microscopy. Fourier Transform Infrared Spectroscopy performed the physical-chemical characterization. The results show that all hNF-lipase activity presented values higher than that of the free enzyme. Activity is higher at pH 7.4 and has the highest buffer concentration of 100 mM. Molecular docking analysis has been used to understand the effect of enzyme protonation on hNF-lipase formation and identify the main the main binding sites of the enzyme with copper ions. The hNF-lipase nanostructures show the shape of flowers in their micrographs from pH 6 to 8. The spectra of the nanoflowers present peaks typical of the amide regions I and II, current in lipase, and areas with P-O vibrations, confirming the presence of the phosphate group. Therefore, hNF-lipase is an efficient biocatalyst with increased catalytic activity, good nanostructure formation, and improved stability.

Chromatographic method development using multivariate approaches for organic solvents optimized analysis in [18F]fluorocholine.

A fast and simple method using Gas Chromatography combined with Flame Ionization Detection (GC-FID) was developed for the determination of ethanol, acetonitrile, dibromomethane, dimethylaminoethanol, and dimethyl sulfoxide in [18F]fluorocholine. The combination of fractional factorial design, Doehlert design, and Desirability function was used to evaluate the operational parameters and to establish the best working condition. The validation results revealed that the proposed method has good recovery (85.1-104.1%) and repeatability (RSD ≤8.1%). Correlation coefficients (R ≥ 0.983) indicated good linearity over a wide range. The limit of detection (≤2.5 ppm) and the limit of quantification (≤7.5 ppm) were satisfactory. The proposed method is based on minimum manual operation, sample preparation free, direct injection technique, and short chromatographic separation time. This method is useful for routine analysis of organic solvents in [18F]fluorocholine, feasible for the modernization of specific monograph, and was therefore successfully implemented to assess samples manufactured by Nuclear Technology Development Center (CDTN).

Transcranial direct current stimulation is safe and effective in autoimmune myopathies: a randomised, double-blind, sham-controlled trial.

OBJECTIVES: We aimed to assess the safety and efficacy of transcranial direct current stimulation (tDCS) in patients with systemic autoimmune myopathies (SAMs). METHODS: This prospective, randomised, sham-controlled, double-blind, study included 20 patients with SAMs allocated to receive sham or active tDCS (2mA, 20 minutes, 3 days). Electrodes were positioned with the anode over the C1 or C2, whereas the cathode was placed over the Fp2 or Fp1, respectively. The groups were evaluated in four periods with specific questionnaires and functional tests: pre-stimulation and after 30 minutes, three weeks, and eight weeks post-tDCS. RESULTS: Two patients from the sham group withdrew after the three sessions. The demographic data, type of myositis, disease duration, and disease status were comparable between the active and sham tDCS groups. After interventions, in the active tDCS group, the physical aspects of SF-36 in week eight, mean and better timed up-and-go test at each evaluation, peak torque of stimulated inferior limb extension improved significantly (p<0.05). The emotional aspect of SF-36 decreased only in the active tDCS group (p<0.001). The patients' adherence to the protocol was 100% and no serious adverse event was reported, including disease relapses. CONCLUSIONS: This study evidences the safety of tDCS, as well as its potential efficacy in improving muscle strength and function in SAMs patients. More studies with a larger sample and longer tDCS sessions are necessary to corroborate the results of the present study.

Formaldehyde effects on kanamycin resistance gene of inactivated recombinant Escherichia coli vaccines.

OBJECTIVES: Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. RESULTS: This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. CONCLUSIONS: We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.

Immunodiagnosis of human and canine visceral leishmaniasis using recombinant Leishmania infantum Prohibitin protein and a synthetic peptide containing its conformational B-cell epitope.

In the present study, Leishmania infantum's Prohibitin was cloned and, alongside a synthetic peptide, evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis (CVL and TL, respectively) in dogs and humans. For TL diagnosis, this study analyzed serum samples from cutaneous (n = 20) or mucosal (n = 39) leishmaniasis patients, and from Chagas disease (CD) patients (n = 8) and non-infected patients (n = 45). For CVL diagnosis, serum samples from asymptomatic (n = 14), symptomatic (n = 71), non-infected (n = 116), and Leish-Tec®-vaccinated (n = 79) dogs were examined, as well as T. cruzi (n = 11) and Ehrlichia canis (n = 10) infected animals. An indirect ELISA method using rProhibitin showed diagnostic sensitivity and specificity values of 91.76% and 89.91%, respectively. L. infantum SLA showed 86.11% and 48.24% of specificity and sensitivity, respectively, for CVL serodiagnosis, and 98.31% and 84.91% sensitivity and specificity, respectively for TL diagnosis. L. braziliensis SLA showed 75.47% and 83.05% of specificity and sensitivity, respectively, for TL diagnosis. The synthetic peptide showed a better result in TL than in CVL diagnosis. In conclusion, preliminary results suggest that the detection of antibodies against the rProhibitin protein and the synthetic peptide improves the serodiagnosis of TL and CVL.

Inactivated recombinant Escherichia coli as a candidate vaccine against Clostridium perfringens alpha toxin in sheep.

Clostridium perfringens type A is the causative agent of gas gangrene and gastroenteric ("yellow lamb disease") disease in ruminants, with C. perfringens alpha toxin (CPA) being the main virulence factor in the pathogenesis of these illnesses. In the present study, we have developed recombinant Escherichia coli bacteria expressing rCPA and used it to vaccinate rabbits and sheep. Doses of up to 200 µg of rCPA used for inoculation, induced 13.82 IU.mL-1 of neutralizing antitoxin in rabbits, which is three times higher than that recommended by the USDA (4 IU.mL-1). In sheep, recombinant bacteria induced antitoxin titers of 4 IU.mL-1, 56 days after the first dose. rCPA which was expressed, mainly, in inclusion bodies, was not found to influence the immunogenicity of the vaccine. The recombinant Escherichia coli bacterin, produced simply and safely, is capable of affording protection against diseases caused by C. perfringens CPA. The current findings represent a novel production method for CPA vaccines potentially applicable to veterinary medicine.

Individual Muscle Hypertrophy and Strength Responses to High vs. Low Resistance Training Frequencies.

Damas, F, Barcelos, C, Nóbrega, SR, Ugrinowitsch, C, Lixandrão, ME, Santos, LMEd, Conceição, MS, Vechin, FC, and Libardi, CA. Individual muscle hypertrophy and strength responses to high vs. low resistance training frequencies. J Strength Cond Res 33(4): 897-901, 2019-The aim of this short communication was to compare the individual muscle mass and strength gains with high (HF) vs. low (LF) resistance training (RT) frequencies using data from our previous study. We used a within-subject design in which 20 subjects had one leg randomly assigned to HF (5× per week) and the other to LF (2 or 3× per week). Muscle cross-sectional area and 1 repetition maximum were assessed at baseline and after 8 weeks of RT. HF showed a higher 8-week accumulated total training volume (TTV) (p < 0.0001) compared with LF. Muscle cross-sectional area and 1 repetition maximum values increased significantly and similarly for HF and LF protocols (p > 0.05). This short communication highlights that some individuals showed greater muscle mass and strength gains after HF (31.6 and 26.3% of individuals, respectively), other had greater gains with LF (36.8 and 15.8% of individuals, respectively), and even others showed similar responses between HF and LF, regardless of the consequent higher or lower TTV resulted from HF and LF, respectively. Importantly, individual manipulation of RT frequency can improve the intrasubject responsiveness to training, but the effect is limited to each individual's capacity to respond to RT. Finally, individual response to different frequencies and resulted TTV does not necessarily agree between muscle hypertrophy and strength gains.

Osteologia do membro torácico de Lycalopex gymnocercus Fischer, 1814 (Carnivora, Mammalia): abordagens comparada, radiográfica e osteométrica / Osteology of the thoracic limb in the Lycalopex gymnocercus Fischer, 1814 (Carnivora, Mammalia): comparative, radiograpahic and osteometric approaches

O membro torácico é a parte do esqueleto pós-craniano que mais informações oferece sobre a locomoção e hábitos de vida de uma espécie. Objetivou-se realizar uma descrição macroscópica, comparativa, radiográfica e osteométrica do esqueleto do membro torácico de Lycalopex gymnocercus. Para tal foram analisados ossos de 24 espécimes adultos coletados mortos em rodovias. Foram realizadas descrições macroscópicas e comparadas com as de outros canídeos, imagens radiográficas para reconhecimento dos padrões de trabeculação e topografia óssea, mensurações lineares e tridimensionais e avaliações histológicas das clavículas. O esqueleto do membro torácico de L. gymnocercus apresentou adaptações morfofuncionais em todos os segmentos para a locomoção cursorial especializada e alguma capacidade de pronação e supinação para captura de presas de porte menor. Houve uma tendência de os ossos serem maiores nos machos, especialmente na região distal do úmero, presumivelmente para a fixação de músculos antebraquiais mais fortes. A clavícula vestigial foi encontrada em todos os espécimes, teve natureza predominantemente cartilagínea e foi significativamente maior nos machos. A conformação básica do esqueleto do membro torácico foi semelhante à dos canídeos domésticos e silvestres descritos na literatura, ainda que particularidades de L. gymnocercus sejam encontradas.(AU)

The forelimb is the part of the post-cranial skeleton that provides more information about locomotion and life habits of a species. The objective of this study was to perform a detailed macroscopic, comparative, radiographic and osteometric investigation of the thoracic limb of Lycalopex gymnocercus. Twenty-four adult specimens collected dead from roadkill were studied. Analysis included macroscopic and comparative descriptions to other canids, radiographic images for recognition of trabeculation patterns and topographical arrangement of bones, linear and three-dimensional measurements, and histological evaluation of the clavicles. The forelimb skeleton of L. gymnocercus presented morphological and functional adaptations in every segment for a specialized cursorial locomotion and some ability to pronation and supination for capture of small vertebrates. There was a tendency for the bones to be longer in males, especially in the distal end of humerus, presumably to fixation of stronger antebrachial muscles. The vestigial clavicle bone was found in all the specimens, had a predominantly cartilaginous nature and was significantly larger in males. The basic conformation of the forelimb skeleton was similar to that of domestic and wild canids described in the literature, although peculiar characteristics of L. gymnocercus have been found.(AU)

Prevalence and vertical transmission of human pegivirus among pregnant women infected with HIV.

OBJECTIVE: To determine the prevalence of human pegivirus (HPgV) and factors associated with vertical transmission among pregnant women infected with HIV. METHOD: A retrospective cross-sectional study was conducted among pregnant women treated at an HIV reference service in Rio Grande, Brazil, between January 1, 2010, and January 1, 2015. The polymerase chain reaction was used to diagnose HPgV infection among the women and their neonates. Clinical, obstetric, and neonatal data were obtained from medical records. RESULTS: Infection with HPgV was detected among 16 (25%) of 63 women and 5 (8%) of 63 newborns, corresponding to a vertical transmission rate of 31%. Multivariate analysis demonstrated that the absence of prenatal care was the only risk factor for vertical transmission of HPgV (prevalence ratio 19.61, 95% confidence interval 1.29-297.48; P=0.032). CONCLUSION: Prenatal care could protect against vertical transmission of HPgV among women infected with HIV; however, studies among HIV-negative individuals are still required to verify this correlation.

Distributed encoding of spatial and object categories in primate hippocampal microcircuits.

The primate hippocampus plays critical roles in the encoding, representation, categorization and retrieval of cognitive information. Such cognitive abilities may use the transformational input-output properties of hippocampal laminar microcircuitry to generate spatial representations and to categorize features of objects, images, and their numeric characteristics. Four nonhuman primates were trained in a delayed-match-to-sample (DMS) task while multi-neuron activity was simultaneously recorded from the CA1 and CA3 hippocampal cell fields. The results show differential encoding of spatial location and categorization of images presented as relevant stimuli in the task. Individual hippocampal cells encoded visual stimuli only on specific types of trials in which retention of either, the Sample image, or the spatial position of the Sample image indicated at the beginning of the trial, was required. Consistent with such encoding, it was shown that patterned microstimulation applied during Sample image presentation facilitated selection of either Sample image spatial locations or types of images, during the Match phase of the task. These findings support the existence of specific codes for spatial and numeric object representations in primate hippocampus which can be applied on differentially signaled trials. Moreover, the transformational properties of hippocampal microcircuitry, together with the patterned microstimulation are supporting the practical importance of this approach for cognitive enhancement and rehabilitation, needed for memory neuroprosthetics.

Facilitation of memory encoding in primate hippocampus by a neuroprosthesis that promotes task-specific neural firing.

OBJECTIVE: Memory accuracy is a major problem in human disease and is the primary factor that defines Alzheimer's, ageing and dementia resulting from impaired hippocampal function in the medial temporal lobe. Development of a hippocampal memory neuroprosthesis that facilitates normal memory encoding in nonhuman primates (NHPs) could provide the basis for improving memory in human disease states. APPROACH: NHPs trained to perform a short-term delayed match-to-sample (DMS) memory task were examined with multi-neuron recordings from synaptically connected hippocampal cell fields, CA1 and CA3. Recordings were analyzed utilizing a previously developed nonlinear multi-input multi-output (MIMO) neuroprosthetic model, capable of extracting CA3-to-CA1 spatiotemporal firing patterns during DMS performance. MAIN RESULTS: The MIMO model verified that specific CA3-to-CA1 firing patterns were critical for the successful encoding of sample phase information on more difficult DMS trials. This was validated by the delivery of successful MIMO-derived encoding patterns via electrical stimulation to the same CA1 recording locations during the sample phase which facilitated task performance in the subsequent, delayed match phase, on difficult trials that required more precise encoding of sample information. SIGNIFICANCE: These findings provide the first successful application of a neuroprosthesis designed to enhance and/or repair memory encoding in primate brain.

Persistent hyperdopaminergia decreases the peak frequency of hippocampal theta oscillations during quiet waking and REM sleep.

Long-term changes in dopaminergic signaling are thought to underlie the pathophysiology of a number of psychiatric disorders. Several conditions are associated with cognitive deficits such as disturbances in attention processes and learning and memory, suggesting that persistent changes in dopaminergic signaling may alter neural mechanisms underlying these processes. Dopamine transporter knockout (DAT-KO) mice exhibit a persistent five-fold increase in extracellular dopamine levels. Here, we demonstrate that DAT-KO mice display lower hippocampal theta oscillation frequencies during baseline periods of waking and rapid-eye movement sleep. These altered theta oscillations are not reversed via treatment with the antidopaminergic agent haloperidol. Thus, we propose that persistent hyperdopaminergia, together with secondary alterations in other neuromodulatory systems, results in lower frequency activity in neural systems responsible for various cognitive processes.

Baseline hippocampal theta oscillation speeds correlate with rate of operant task acquisition.

Many lines of evidence indicate that theta rhythm, a prominent neural oscillatory mode found in the mammalian hippocampus, plays a key role in the acquisition, processing, and retrieval of memories. However, a predictive neurophysiological feature of the baseline theta rhythm that correlates with the learning rate across different animals has yet to be identified. Here we show that the mean theta rhythm speed observed during baseline periods of immobility has a strong positive correlation with the rate at which rats learn an operant task. This relationship is observed across rats, during both quiet waking (r=0.82; p<0.01) and paradoxical sleep (r=0.83; p<0.01), suggesting that the basal theta frequency relates to basic neurological processes that are important in the acquisition of operant behavior.

Dopaminergic control of sleep-wake states.

Dopamine depletion is involved in the pathophysiology of Parkinson's disease, whereas hyperdopaminergia may play a fundamental role in generating endophenotypes associated with schizophrenia. Sleep disturbances are known to occur in both schizophrenia and Parkinson's disease, suggesting that dopamine plays a role in regulating the sleep-wake cycle. Here, we show that novelty-exposed hyperdopaminergic mice enter a novel awake state characterized by spectral patterns of hippocampal local field potentials that resemble electrophysiological activity observed during rapid-eye-movement (REM) sleep. Treatment with haloperidol, a D2 dopamine receptor antagonist, reduces this abnormal intrusion of REM-like activity during wakefulness. Conversely, mice acutely depleted of dopamine enter a different novel awake state characterized by spectral patterns of hippocampal local field potentials that resemble electrophysiological activity observed during slow-wave sleep (SWS). This dopamine-depleted state is marked by an apparent suppression of SWS and a complete suppression of REM sleep. Treatment with D2 (but not D1) dopamine receptor agonists recovers REM sleep in these mice. Altogether, these results indicate that dopamine regulates the generation of sleep-wake states. We propose that psychosis and the sleep disturbances experienced by Parkinsonian patients result from dopamine-mediated disturbances of REM sleep.