Zebrafish automatic monitoring system for conditioning and behavioral analysis.

Studies using zebrafish (Danio rerio) in neuro-behavioural research are growing. Measuring fish behavior by computational methods is one of the most efficient ways to avoid human bias in experimental analyses, extending them to various approaches. Sometimes, thorough analyses are difficult to do, as fish can behave unpredictably during an experimental strategy. However, the analyses can be implemented in an automated way, using an online strategy and video processing for a complete assessment of the zebrafish behavior, based on the detection and tracking of fish during an activity. Here, a fully automatic conditioning and detailed analysis of zebrafish behavior is presented. Microcontrolled components were used to control the delivery of visual and sound stimuli, in addition to the concise amounts of food after conditioned stimuli for adult zebrafish groups in a conventional tank. The images were captured and processed for automatic detection of the fish, and the training of the fish was done in two evaluation strategies: simple and complex. In simple conditioning, the zebrafish showed significant responses from the second attempt, learning that the conditioned stimulus was a predictor of food presentation in a specific space of the tank, where the food was dumped. When the fish were subjected to two stimuli for decision-making in the food reward, the zebrafish obtained better responses to red light stimuli in relation to vibration. The behavior change was clear in stimulated fish in relation to the control group, thus, the distances traveled and the speed were greater, while the polarization was lower in stimulated fish. This automated system allows for the conditioning and assessment of zebrafish behavior online, with greater stability in experiments, and in the analysis of the behavior of individual fish or fish schools, including learning and memory studies.

HIV-1 subtypes and drug resistance in children during antiretroviral therapy in Brazil.

We evaluate the genetic characterization of 132 HIV-1 pol sequences from children and adolescents undergoing antiretroviral therapy in Northeast Brazil. Phylogenetic and recombination analyses were performed using the maximum likelihood method using SeaView version 4 and SIMPLOT software. Most individuals harbored HIV-1 B (84.8%) and BF recombinants (9.8%), although other non-B subtypes were detected: HIV-1 C (1.5%), HIV-1 F (2.4%), and BC recombinants (1.5%). Antiretroviral resistance was 47% (95% confidence interval [CI]: 38.7%-55.4%). Non-nucleoside reverse transcriptase inhibitors (NNRTIs) showed higher frequencies of primary mutations, with 40.9% (95% CI: 32.9%-49.4%), followed by nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PIs) with 34.8% (95% CI: 27.3-43.3) and 6.1% (95% CI: 3.1%-11.5%), respectively. Among NRTIs, higher resistance levels were observed for abacavir, emtricitabine, and lamivudine; for NNRTI, nevirapine and efavirenz. The most common primary mutations found were M184V (29.5%), K103N (25%), M41L (9.8%), T215Y (8.3%), and G190A (8.3%). Our findings highlight the importance of surveillance of resistance mutations, which contributes to the continuous updating and implementation of preventive measures to decrease mother-to-child-transmission and transmitted drug resistance.

Zebrafish tracking using YOLOv2 and Kalman filter.

Fish show rapid movements in various behavioral activities or associated with the presence of food. However, in periods of rapid movement, the rate at which occlusion occurs among the fish is quite high, causing inconsistency in the detection and tracking of fish, hindering the fish's identity and behavioral trajectory over a long period of time. Although some algorithms have been proposed to solve these problems, most of their applications were made in groups of fish that swim in shallow water and calm behavior, with few sudden movements. To solve these problems, a convolutional network of object recognition, YOLOv2, was used to delimit the region of the fish heads to optimize individual fish detection. In the tracking phase, the Kalman filter was used to estimate the best state of the fish's head position in each frame and, subsequently, the trajectories of each fish were connected among the frames. The results of the algorithm show adequate performances in the trajectories of groups of zebrafish that exhibited rapid movements.

Is the tryptophan codon of gene vif the Achilles' heel of HIV-1?

To evaluate the impact of hypermutation on the HIV-1 dissemination at the population level we studied 7072 sequences HIV-1 gene vif retrieved from the public databank. From this dataset 854 sequences were selected because they had associated values of CD4+ T lymphocytes counts and viral loads and they were used to assess the correlation between clinical parameters and hypermutation. We found that the frequency of stop codons at sites 5, 11 and 79 ranged from 2.8x10-4 to 4.2x10-4. On the other hand, at codons 21, 38, 70, 89 and 174 the frequency of stop codons ranged from 1.4x10-3 to 2.5x10-3. We also found a correlation between clinical parameters and hypermutation where patients harboring proviruses with one or more stop codons at the tryptophan sites of the gene vif had higher CD4+ T lymphocytes counts and lower viral loads compared to the population. Our findings indicate that A3 activity potentially restrains HIV-1 replication because individuals with hypermutated proviruses tend to have lower numbers of RNA copies. However, owing to the low frequency of hypermutated sequences observed in the databank (44 out of 7072), it is unlikely that A3 has a significant impact to curb HIV-1 dissemination at the population level.

Characterization of HIV-1 genetic diversity and antiretroviral resistance in the state of Maranhão, Northeast Brazil.

The HIV-1 epidemic in Brazil has been growing in northeast and north regions, particularly an increase in AIDS cases among the younger male population has been observed. This study aims to characterize the HIV-1 genetic diversity and to evaluate its antiretroviral resistance profile among individuals presenting virological failure in the state of Maranhão-Brazil. HIV-1 pol gene sequences from 633 patients on antiretroviral therapy were obtained from the Department of Surveillance, Prevention and Control of Sexually Transmitted Infections, HIV/AIDS and Viral Hepatitis of the Brazilian Ministry of Health. Phylogenetic and recombination analyses were performed to characterize viral genetic diversity. The presence of antiretroviral resistance mutations was assessed using the HIV Drug Resistance Database online platform of Stanford University. A predominance of subtype B (84.5%) was observed, followed by recombinant BF (9.5%), where more than half of the sequences were dispersed in 3 clusters. Antiretroviral resistance was detected in 74.1% of the sequences, and it was significantly higher for nucleoside analogue reverse-transcriptase inhibitors (NRTIs) than for non-nucleoside analogue reverse-transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). Inference of putative transmissions clusters identified 11 clusters with 22 query sequences (22/633, 3.5%). Thus, we conclude that continuous monitoring of the molecular epidemiology of HIV-1 is essential for prevention strategies, epidemic control, and treatment adequacy.

Genetic Diversity of HIV-1 Gene vif Among Treatment-Naive Brazilians.

HIV-1 has the Vif protein, which binds to human antiviral proteins APOBEC3 to form complexes to be degraded by cellular proteolysis. To further explore HIV-1 diversity at the population level, we analyzed blood samples from 317 treatment-naive patients in Brazil. In this study, we explored the correlations of Vif polymorphisms with clinical parameters of the patients and found that mutation K22H is associated with low CD4+ cell counts and higher viral loads. Phylogenetic analysis of the vif gene indicated that subtype B was predominant in ∼77% (243/317) of the patients, followed by HIV-1 F ∼18% (56/317), and subtype C ∼4% (12/317); five samples were BF recombinants (∼1% of patients), and one was an AG recombinant. On the basis of the vif gene, we detected the presence of one AG and several previously unknown BF intersubtypes in this population. The global mean diversity, measured by pairwise distances, was 0.0931 ± 0.0006 among sequences of subtype B (n = 243), whereas the mean diversity of subtype C sequences (n = 12) was 0.0493 ± 0.001 and that of subtype F (n = 56) was 0.050 ± 0.001.