NMR studies of DNA (R+)n.(Y-)n.(Y+)n triple helices in solution: imino and amino proton markers of T.A.T and C.G.C+ base-triple formation

High-resolution exchangeable proton two-dimensional NMR spectra have been recorded on 11-mer DNA triple helices containing one oligopurine (R)n and two oligopyrimidine (Y)n strands at acidic pH and elevated temperatures. Our two-dimensional nuclear Overhauser effect studies have focused on an 11-mer triplex where the third oligopyrimidine strand is parallel to the oligopurine strand. The observed distance connectivities establish that the third oligopyrimidine strand resides in the major groove with the triplex stabilized through formation of T.A.T and C.G.C+ base triples. The T.A.T base triple can be monitored by imino protons of the thymidines involved in Watson-Crick (13.65-14.25 ppm) and Hoogsteen (12.9-13.55 ppm) pairing, as well as the amino protons of adenosine (7.4-7.7 ppm). The amino protons of the protonated (8.5-10.0 ppm) and unprotonated (6.5-8.3 ppm) cytidines in the C.G.C+ base triple provide distinct markers as do the imino protons of the guanosine (12.6-13.3 ppm) and the protonated cytidine (14.5-16.0 ppm). The upfield chemical shift of the adenosine H8 protons (7.1-7.3 ppm) establishes that the oligopurine strand adopts an A-helical base stacking conformation in the 11-mer triplex. These results demonstrate that oligonucleotide triple helices can be readily monitored by NMR at the individual base-triple level with distinct markers differentiating between Watson-Crick and Hoogsteen pairing. Excellent exchangeable proton spectra have also been recorded for (R+)n.(Y-)n.(Y+)n 7-mer triple helices with the shorter length permitting spectra to be recorded at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fertility in dairy cows following presynchronization and administering twice the luteolytic dose of prostaglandin F2α as one or two injections in the 5-day timed artificial insemination protocol

The objectives were to evaluate pregnancy per AI (P/AI) of dairy cows subjected to the 5-day timed AI protocol under various synchronization and luteolytic treatments. Cows were either presynchronized or received supplemental progesterone during the synchronization protocol, and received a double luteolytic dose of PGF_2α, either as one or two injections. In Experiment 1, dairy cows (n = 737; Holstein = 250, Jersey = 80, and crossbred = 407) in two seasonal grazing dairy farms were randomly assigned to one of four treatments in a 2 × 2 factorial arrangement. The day of AI was considered study Day 0. Half of the cows were presynchronized (G6G: PGF_2α on Day −16 and GnRH on Day −14) and received the 5-day timed AI protocol using 1 mg of cloprostenol, either as a single injection (G6G-S: GnRH on Day −8, PGF_2α on Day −3, and GnRH + AI on Day 0) or divided into two injections of 0.5 mg each (G6G-T: GnRH on Day −8, PGF_2α on Day −3 and −2, and GnRH + AI on Day 0). The remaining cows were not presynchronized and received a controlled internal drug-release (CIDR) insert containing progesterone from GnRH to the first PGF_2α injection of the 5-day timed AI protocol, and 1 mg of cloprostenol either as a single injection on Day -3 (CIDR-S) or divided into two injections of 0.5 mg each on Days -3 and -2 (CIDR-T). Ovaries were examined by ultrasonography on Days −8 and −3 and plasma progesterone concentrations were determined on Days −3 and 0. In Experiment 2, 655 high-producing Holstein cows had their estrous cycle presynchronized with PGF_2α at 46 ± 3 and 60 ± 3 days postpartum and were randomly assigned to receive 50 mg of dinoprost during the 5-day timed AI protocol, either as a single injection or divided into two injections of 25 mg each. Pregnancies per AI were determined on Days 35 and 64 after AI in both experiments. In Experiment 1, presynchronization with G6G increased the proportion of cows with a CL on Day −8 (80.6 vs. 58.8%), ovulation to the first GnRH of the protocol (64.2 vs. 50.2%), and the presence (95.6 vs. 88.4%) and number (1.79 vs. 1.30) of CL at PGF_2α compared with CIDR cows. Luteolysis was greater for two injections compared to a single PGF_2α injection (two PGF_2α = 95.9 vs. single PGF_2α = 72.2%), especially in presynchronized cows (G6G-T = 96.2 vs. G6G-S = 61.7%). For cows not presynchronized, two PGF_2α injections had no effect on P/AI (CIDR-S = 30.2 vs. CIDR-T = 34.3%), whereas for presynchronized cows, it improved P/AI (G6G-S = 28.7 vs. G6G-T = 45.4%). In Experiment 2, the two-PGF_2α injection increased P/AI on Days 35 (two PGF_2α = 44.5 vs. single PGF_2α = 36.4%) and 64 (two PGF_2α = 40.3% vs. single PGF_2α = 32.6%) after AI. Presynchronization and dividing the dose of PGF_2α (either cloprostenol or dinoprost) into two injections increased P/AI in lactating dairy cows subjected to the 5-day timed AI protocol.     

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker,Antibody and Method

Background

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.

Methodology

First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.

Principal findings

EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.

Conclusion

RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.     

Effects of 6-benzylaminopurine on photosystem II functionality and leaf anatomy of <Emphasis Type="Italic">in vitro</Emphasis> cultivated <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis>

Cytokinins (CKs) are often used during the in vitro cultivation of plant species. However, it is not clear how CKs, such as 6-benzylaminopurine (BAP), affect photosystem PS) II functionality and leaf anatomy over a long period of in vitro plant propagation. The aim of this study was to analyze the residual effects of BAP on the photosynthetic performance and leaf anatomy of Aechmea blanchetiana after 120 d without exposure to CKs. Aechmea blanchetiana plants previously grown in vitro were transferred to Murashige and Skoog (MS) culture media containing 0, 5, 10, 15, or 20 μM BAP. After 60 d on the MS medium with BAP, explants were subcultivated twice on the MS medium without growth regulators, first in a stationary liquid medium for 60 d and then in a solidified medium with 6 g dm^-3 agar for 60 d. Leaf anatomy, pigment content, and chlorophyll a fluorescence were assessed for plants from each treatment after 120 d on the CK-free medium. Stomatal density presented a negative linear correlation with BAP concentration. Pigment content decreased in plants subjected to previous BAP exposure. An increase in absorbed energy flux per reaction center (ABS/RC) and a sharp decrease in energy transport flux (ETo/RC) followed by an increase in energy dissipation flux (DIo/RC) also occurred. Furthermore, maximum quantum yield (F_V/F_M) decreased as a function of BAP concentration. Thus, the use of BAP during in vitro propagation of A. blanchetiana induced long-term physiological defects.     

Neonatal Morphine Administration Leads to Changes in Hippocampal BDNF Levels and Antioxidant Enzyme Activity in the Adult Life of Rats

It is know that repeated exposure to opiates impairs spatial learning and memory and that the hippocampus has important neuromodulatory effects after drug exposure and withdrawal symptoms. Thus, the aim of this investigation was to assess hippocampal levels of BDNF, oxidative stress markers associated with cell viability, and TNF-α in the short, medium and long term after repeated morphine treatment in early life. Newborn male Wistar rats received subcutaneous injections of morphine (morphine group) or saline (control group), 5 μg in the mid-scapular area, starting on postnatal day 8 (P8), once daily for 7 days, and neurochemical parameters were assessed in the hippocampus on postnatal days 16 (P16), 30 (P30), and 60 (P60). For the first time, we observed that morphine treatment in early life modulates BDNF levels in the medium and long term and also modulates superoxide dismutase activity in the long term. In addition, it was observed effect of treatment and age in TNF-α levels, and no effects in lactate dehydrogenase levels, or cell viability. These findings show that repeated morphine treatment in the neonatal period can lead to long-lasting neurochemical changes in the hippocampus of male rats, and indicate the importance of cellular and intracellular adaptations in the hippocampus after early-life opioid exposure to tolerance, withdrawal and addiction.     

In silico design of a <Emphasis Type="Italic">Zika virus</Emphasis> non-structural protein 5 aiming vaccine protection against zika and dengue in different human populations

Background

The arboviruses Zika virus (ZIKV) and Dengue virus (DENV) have important epidemiological impact in Brazil and other tropical regions of the world. Recently, it was shown that previous humoral immunity to DENV enhances ZIKV replication in vitro, which may lead to more severe forms of the disease. Thus, traditional approaches of vaccine development aiming to control viral infection through neutralizing antibodies may induce cross-reactive enhancing antibodies. In contrast, cellular immune response was shown to be capable of controlling DENV infection independently of antibodies. The aim of the present study was to design a flavivirus NS5 protein capable of inducing a cellular immune response against DENV and ZIKV.

Methods

A consensus sequence of ZIKV NS5 protein was designed among isolates from various continents. Epitopes were predicted for the most prevalent alleles of class I and II HLA in the Brazilian population. Then, this epitopes were analyzed with regard to their conservation, population coverage and distribution along the whole antigen.

Results

Nineteen epitopes predicted to be more reactive (percentile rank <1) and 100% conserved among ZIKV and DENV serotypes were selected. The distribution of such epitopes along the protein was shown on a three-dimensional model and population coverage was calculated for different regions of the world. The designed protein was predicted to be stable and the distribution of selected epitopes was shown to be homogeneous along domains. The population coverage of selected epitopes was higher than 50% for most of tropical areas of the world.

Conclusion

Such results indicate that the proposed antigen has the potential to induce protective cellular immune response to ZIKV and DENV in different human populations of the world.

     

Morphometric study of the genus Panstrongylus Berg, 1879 (Hemiptera, Reduviidae, Triatominae)

The subfamily Triatominae is actually represented by 137 species distributed among 6 tribes and 19 genera. Within this subfamily, the genus Panstrongylus, Berg 1879, is composed by 13 species widespread in sylvatic, peridomestic, and domestic habitats of Neotropical regions. These species are vectors of Chagas disease and consequently are found associated with its main hosts, such as birds and mammals. Interest in species of this genus has been increasing in the last few years. Reports of several authors indicate these Triatominae to invade and colonize houses, increasing their epidemiological significance. Morphometry was used in this study to investigate correlations among possible closely related species. We measured 224 specimens among 13 species through a set of metric variables of the head. The results indicated that the genus Panstrongylus seems to be homogeneous since 10 of the 14 species were shown to be closely related.     

Mechanics of the maxillary central incisor. Influence of the periodontal ligament represented by beam elements

This study aimed to evaluate the influence of loading on a maxillary central incisor with the periodontal ligament (PDL) represented by 2D elastic beam elements using a 2D finite element analysis. Two models (M) were built varying the PDL representation: Mh (homogeneous PDL) and Mht (heterogeneous PDL with beam3 elements). Stress and displacements were determined for three loading conditions (L): Ll, lingual face loading at 45° with the tooth long axis; Li, perpendicular to the incisal edge; and Lip, on the incisal edge, parallel to the tooth long axis. Evaluation was performed on ANSYS software. Lip provided lower stress variation on the tooth and support structures when compared to Ll and Li. PDL's influence on stress values was lower for Lip. Oblique loading showed stress and displacement not observed in parallel loading condition through PDL's heterogeneous representation and it is probably incompatible with the in vivo condition.     

On‐target ultrasonic digestion of proteins

In the present work, we report a novel on‐target protein cleavage method. The method utilizes ultrasonic energy and allows up to 20 samples to be cleaved in 5 min for protein identification and one sample in 30 s for on‐tissue digestion. The standard proteins were spotted on a conductive glass slide in a volume of 0.5 μL followed by 5 min of ultrasonication after trypsin addition. Controls (5 min, 37°C no ultrasonication) were also assayed. After trypsin addition, digestion of the tissues was enhanced by 30 s of ultrasonication. The samples were analyzed and compared to those obtained by using conventional 3 h heating proteolysis. The low sample volume needed for the digestion and reduction in sample‐handling steps and time are the features that make this method appealing to the many laboratories working with high‐throughput sample treatment.