Chronic exposure to 2,2'-azobis-2-amidinopropane that induces intestinal damage and oxidative stress in larvae of Drosophila melanogaster.

Embryonic development is exceptionally susceptible to pathogenic, chemistry and mechanical stressors as they can disrupt homeostasis, causing damage and impacted viability. Oxidative stress has the capacity to induce alterations and reshape the environment. However, the specific impacts of these oxidative stress-induced damages in the gastrointestinal tract of Drosophila melanogaster larvae have been minimally explored. This study used 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), a free radical generator, to investigate oxidative stress effects on Drosophila embryo development. The results showed that exposing Drosophila eggs to 30 mM AAPH during 1st instar larva, 2nd instar larva and 3rd instar larva stages significantly reduced hatching rates and pupal generation. It increased the activity of antioxidant enzymes and increased oxidative damage to proteins and MDA content, indicating severe oxidative stress. Morphological changes in 3rd individuals included decreased brush borders in enterocytes and reduced lipid vacuoles in trophocytes, essential fat bodies for insect metabolism. Immunostaining revealed elevated cleaved caspase 3, an apoptosis marker. This evidence validates the impact of oxidative stress toxicity and cell apoptosis following exposure, offering insights into comprehending the chemically induced effects of oxidative stress by AAPH on animal development.

Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oils.

This study addresses the current trend of essential oils in alternative medicine using the non-chordate model Drosophila melanogaster. Following the three R's principles, it proposes non-chordate models to fill knowledge gaps on essential oil toxicity. Copaiba, lavender, and ginger essential oils are evaluated for effects on D. melanogaster lifespan, climbing ability, and brain structure, while their anti-inflammatory properties are also analyzed. Results show dose-related differences: higher concentrations (0.25% v/v) cause brain deterioration and impaired climbing, while lower concentrations (0.0625% v/v for copaiba and ginger; 0.125% for lavender) have no effect on climbing or brain structure. Lavender oil significantly extends lifespan and maintains anti-inflammatory activity when ingested, underscoring its therapeutic potential. These findings highlight the importance of D. melanogaster as a model for studying essential oil properties, potentially replacing chordate models. In addition, this research advances alternative remedies for currently incurable diseases, with lavender oil emerging as a promising candidate for drug discovery.

Interleukin-6, tumor necrosis factor-α, C-reactive protein, and hematological parameters in experimental periodontal disease after ß-adrenergic blockade.

BACKGROUND: Changes in the levels of C-reactive protein (CRP), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) observed during periodontal disease were linked with vascular manifestations. Recent studies showed that the beta-blocker propranolol reduces the pathological parameters associated with certain molecules at sites of bone injury. Hence, in this study, we evaluated the activity of propranolol on hematological parameters and systemic concentrations of inflammatory proteins in a model of experimental periodontitis. MATERIALS AND METHODS: Periodontal disease was induced in rats. After euthanasia, the number of inflammatory cells in each rat was quantified using histopathological assays. In addition, hematological parameters were quantitated using automated analysers, cytokine levels were determined using an enzyme-linked immunosorbent assay, and CRP levels were determined using a high-sensitivity immunoturbidimetric assay. RESULTS: Low doses of propranolol suppressed the systemic production of CRP, TNF-α, and IL-6; however, the hematological parameters were not affected. CONCLUSIONS: ß-adrenergic activation indirectly contributes to the pattern of systemic inflammatory molecules observed in periodontal disease. These molecules may initiate cardiovascular diseases as a consequence of periodontitis.

Micro-RNAs differentially expressed in comparative analysis of sperm samples with high and low efficiency in the in vitro production of bovine (Bos Taurus) embryos / Micro-rnas diferencialmente expressos em análise comparativa de amostras de espermatozoides com alta e baixa efi ciência na produção in vitro de embriões bovinos (bos taurus )

Infertility or subfertility in bovine males may be related to spermatic microRNAs (miRNAs), whose function seems to be associated with the regulation of gene expression, degradation orstorage of messenger RNAs (mRNAs) for later translation into early embryonic development. Thus, the purpose of this study was to identify differentially expressed miRNAs in semen samples from bulls (Bos taurus) with low and high efficiency in the in vitro embryo production (IVEP) and to evaluate if they can be used as markers of semen efficiency for IVEPs. In order to identify miRNA markers of semen efficiency in thein vitro embryo production, eight semen samples from each animal, one bull with high and two bulls with low efficiency in IVEPs were used to perform the RNAseq technique for miRNAs. Initially the samples were washed with PBS to remove the extender semen and subsequently were submitted to RNA extraction protocols performed according to procedures described by mirVana™ miRNA Isolation Kit. Then, the amplification of the miRNAs was carried out, not to mention the preparation of the library (Ion Total RNA-Seq Kit v2), the PCR emulsion reaction, enrichment, as well as the injection of the sample on the chip by the Ion Chef equipment. The sequencing was done on Ion Proton equipment. The comparison between the samples was established using two methodologies for searching for targets to increase the robustness of the analytical procedure: the miRanda program using as cutoff minimum free energy of the hybridization -20 kcal/Mol, 100% of identity between nucleotides 2 and 8 of the miRNA, and the RNAhybrid program, using as cutoff minimum free energy of hybridization -20 kcal/mol. In sum, 1306 miRNAs were identified in the samples. The bta-miR-380-5p, bta-miR-155, bta-miR-30c and bta-miR-34a genes were identified by the Bioinformatics as being strongly differentially expressed between the groups, indicating that these genes may present themselves as possible efficiency markers. However, it has become clear that there is no single miRNA that marks different types and causes of fertility problems.

A infertilidade ou subfertilidade em machos bovinos pode estar relacionada a microRNAs espermáticos (miRNAs), cuja função parece estar associada à regulação da expressão gênica, degradação ou armazenamento de RNAs mensageiros (mRNAs), para posterior tradução no desenvolvimento embrionário inicial. Assim, o objetivo deste estudo foi identificar miRNAs diferencialmente expressos em amostras de sêmen de touros (Bos taurus) com baixa e alta eficiência na produção in vitro de embriões (PIVE) e avaliar se eles podem ser utilizados como marcadores de eficiência do sêmen em PIVEs. Para identificar miRNA marcadores da eficiência de sêmen em PIVE, oito amostras de sêmen de cada animal, sendo um touro com alto e dois touros com baixa eficiência, foram utilizados para realizar a técnica de RNAseq para miRNAs. Inicialmente as amostras foram lavadas com PBS para remover o diluente do sêmen e, posteriormente, foram submetidas a protocolos de extração de RNA realizados de acordo com os procedimentos descritos pelo Kit de isolamento de miRNA mirVana ™. Em seguida, foi realizada a amplificação dos miRNAs, a preparação da biblioteca (Ion RNA-Seq Kit v2), a reação de emulsão de PCR, enriquecimento e a injeção das amostras no chip apropriado utilizando o equipamento Ion. Chef. O sequenciamento foi realizado no equipamento Ion Proton. A comparação entre as amostras foi estabelecida utilizando duas metodologias de busca de alvos para aumentar a robustez do procedimento analítico: o programa miRanda utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / Mol e 100% de identidade entre os nucleotídeos 2 e 8 do miRNA, e o programa RNAhybrid, utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / mol. Em suma, 1306 miRNAs foram identificados nas amostras. Os genes bta-miR-380-5p, bta-miR-155, bta-miR-30c e bta-miR-34a foram identificados pela bioinformática como sendo fortemente diferencialmente expressos entre os grupos, indicando que esses genes podem se apresentar como possíveis marcadores de eficiência. No entanto, ficou claro que não existe um único miRNA que marque diferentes tipos e causas de problemas de fertilidade.

Serological Electrodetection of Rheumatoid Arthritis Using Mimetic Peptide.

BACKGROUND: Rheumatoid arthritis is the most common inflammatory autoimmune disease in the world. Recently new targets for its detection were developed as alternatives to classic biomarkers, including the M-12 peptide, that mimics carbonic anhydrase III. Thus, the application of this peptide for the development of new detection devices is attractive. OBJECTIVE: Our goal was to construct a modified electrode for immobilization of M-12 peptide and detection of a rheumatoid arthritis biomarker in serum of patients. METHODS: 3-Hydroxybenzoic acid was electropolymerized onto graphite electrodes, and M-12 peptide was immobilized by adsorption. Negative and positive serum samples for rheumatoid arthritis were diluted and applied onto the electrode. Detection was carried in potassium ferrocyanide/ ferricyanide solution by differential pulse voltammetry. Atomic force microscopy and scanning electron microscopy were used to evaluate electrode surfaces. RESULTS: Cyclic voltammograms indicated the poly(3-hydroxybenzoic acid) formation and increase of electroactive area. Immobilization of M-12 probe increased current by 1.2 times, and negative serum addition caused no suitable difference. However, positive serum showed expressive decrease in the current signal of about 2.2 times, possibly due to steric hindrance when the anti-CA3 antibody interacts with the M-12 peptide, decreasing the electron transfer. Microscopies images corroborated with the electrochemical detection, showing evident changes in the morphology of the electrode surfaces. CONCLUSION: The bioelectrode was able to discriminate positive and negative serum samples of rheumatoid arthritis by a considerable decrease in the current signal value. Morphological analyses supported the electrochemical results. Thus, the constructed bioelectrode offers a new platform for detection of rheumatoid arthritis.

Ecotoxicological effects of the insecticide fipronil in Brazilian native stingless bees Melipona scutellaris (Apidae: Meliponini).

Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae) is a pollinator of various native and cultivated plants. Because of the expansion of agriculture and the need to ensure pest control, the use of insecticides such as fipronil (FP) has increased. This study aimed to evaluate the effects of sublethal doses of FP insecticide on M. scutellaris at different time intervals (6, 12, and 24 h) after exposure, via individually analyzed behavioral biomarkers (locomotor activity, behavioral change) as well as the effect of FP on different brain structures of bees (mushroom bodies, antennal cells, and optic cells), using sub-individual cell biomarkers (heterochromatin dispersion, total nuclear and heterochromatic volume). Forager bees were collected when they were returning to the nest and were exposed to three different concentrations of FP (0.40, 0.040, and 0.0040 ng a.i/bee) by topical application. The results revealed a reduction in the mean velocity, lethargy, motor difficulty, paralysis, and hyperexcitation in all groups of bees treated with FP. A modification of the heterochromatic dispersion pattern and changes in the total volume of the nucleus and heterochromatin were also observed in the mushroom bodies (6, 12, and 24 h of exposure) and antennal lobes (6 and 12 h) of bees exposed to 0.0040 ng a.i/bee (LD50/100). FP is toxic to M. scutellaris and impairs the essential functions required for the foraging activity.

An antibody-like peptide that recognizes malignancy among thyroid nodules.

There is an urgent need for biomarkers to identify malignant thyroid nodules from indeterminate follicular lesions. We have used a subtractive proteomic strategy to identify novel biomarkers by selecting ligands to goiter tissue from a 12-mer random peptide phage-displayed library using the BRASIL method (Biopanning and Rapid Analysis of Selective Interactive Ligands). After three rounds of selection, two highly reactive clones to the papillary thyroid tumor cell line NPA were further evaluated, and their specific binding to tumor proteins was confirmed using phage-ELISA. The antibody-like peptide CaT12 was tumor-specific, which was further tested by immunohistochemistry against TMAs (tissue microarrays) comprised of 775 human benign and malignant tissues, including 232 thyroid nodular lesions: 15 normal thyroid tissues, 53 nodular goiters (NG), 54 follicular adenomas (FA); 69 papillary thyroid carcinomas (PTC); and 41 follicular carcinomas (FC). CaT12 was able to identify PTC among thyroid nodular lesions with 91.2% sensitivity and 85.1% specificity, despite its non-specificity for thyroid tissues. Additionally, the CaT12 peptide helped characterize follicular lesions distinguishing the follicular variant of PTC (FVPTC) from FA with 91.9% accuracy; FVPTC from NG with 83.1% accuracy; FVPTC from the classic PTC with 57.7% accuracy; and FVPTC from FC with 88.7% accuracy. In conclusion, our strategy to select differentially expressed ligands to thyroid tissue was highly effective and resulted in a useful antibody-like biomarker that recognizes malignancy among thyroid nodules and may help distinguish follicular patterned lesions.

Selection of high affinity peptide ligands for detection of circulating antibodies in neurocysticercosis.

Neurocysticercosis (NC), caused by Taenia solium, is the most common infection caused by helminthes of the human central nervous system. In this study, a random peptide phage display library was used to isolate peptide ligands as potential markers for neurocysticercosis diagnosis, because occurrence of cross-reactions with other helminthes species in the current used markers. We selected different peptides using IgG purified from pooled sera of neurocysticercosis patients. To investigate the diagnostic potential of recombinant peptides, we have tested different panels of serum samples by Phage-ELISA, and 10 phage clones strongly bound to the anti-T. solium IgGs in NC sera, with an accuracy range from 84.2% to 95%. The phage clones, NC(4)1 and NC(2)8, presented the highest sensitivity and specificity (100%), respectively, and most important, some phage clones did not react with patients' sera from Echinococcus granulosus infected patients. The validation with a competitive ELISA assay demonstrated that the selected phages could mimic T. solium epitopes and bind specifically to the pool of NC sera. Finally, the two recombinant antigens may become potential biomarkers for serodiagnosis of NC, and the Phage-ELISA demonstrated to be a very good assay, being reproducible, simple, fast, and low-cost due to its production through Escherichia coli culture, allowing a high throughput screening of NC.

Toxoplasma gondii: effects of neuwiedase, a metalloproteinase from Bothrops neuwiedi snake venom, on the invasion and replication of human fibroblasts in vitro.

The major aim to the present study was to determine the effects of neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom, on invasion and replication of Toxoplasma gondii in human fibroblasts in vitro. Neuwiedase treatment was done on host cells previously infected with T. gondii or on parasite before fibroblast infection. When treatments were done after or before infection, infection rates were inhibited in 71% and 61%, respectively. Considering that therapy protocols currently used in T. gondii infection cause considerable side effects, particularly in immunocompromised individuals and pregnant women, the results of neuwiedase treatment described herein could be taken into account for the development of new synthetic therapeutic agents, mainly due to the capacity of this enzyme to degrade extracellular matrix components, such as laminin, fibronectin and type I collagen, which is important to interfere in T. gondii host cell invasion.

Identification of point mutations in a putative carboxylesterase and their association with acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Chemical control based on the use of pyrethroid and organophosphate compounds has selected resistant genotypes in populations of Rhipicephalus (Boophilus) microplus. Point mutations in esterase-encoding genes represent one of the main resistance mechanisms in this species. In this study, the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique was used to investigate the presence of mutations in a fragment of a putative carboxylesterase in a population of ticks with a history of resistance. The digestion of a fragment of 372 pb with EcoRI revealed three genotypes: W, H and M, observed in different frequencies. The homozygous wild-type genotype (W) was detected only in sensitive strains, with high frequency. The heterozygous genotype (H) was observed in all the strains, albeit with higher frequency in the strains with a moderate resistance, while the homozygous mutant genotype (M) was found only in the moderate resistant strain and resistant strains, with higher frequency in the resistant strains. A comparison of the sequences indicated the presence of other mutations, besides EcoRI polymorphism in the moderate resistant and resistant strains. Also found was the presence of stop codons generating truncated proteins in the sensitive and moderate resistant strains. A domain analysis revealed the presence of additional domains in the resistant strain. These findings suggest that different point mutations, as well as the influence of post-translational modification mechanisms, are altering the activity of the translated proteins and may be associated with resistance.

Analysis in vivo of antitumor activity, cytotoxicity and Interaction between plasmid DNA and the cis-dichloro-tetra-amine-ruthenium(III) chloride.

Several metallic compounds recognized as potent antitumor agents, have been developed and tested in vivo and in vitro. In this work, we evaluated the toxic, therapeutic, and cytotoxic properties of the cis-dichloro-tetra-amine-ruthenium(III) chloride. Transplanted animals with Sarcoma 180 cells were treated with ruthenium(III) complex and injected i.p., at different time intervals. After the 15th day, tumoral postimplant, the animals were sacrificed and their lungs, kidneys, liver, and tumors were removed and processed for histopathological analysis. Blood samples were also taken for haematological and biochemical analyses. Interaction between the ruthenium complex and the DNA was also investigated. Besides being cytotoxic for the S180 cells, the metallic compound induced tumoral volume reduction and increased survival time of the animals treated. Serum levels of LDH, creatinine, and bilirubin increased, but no serious irreversible histopathological alterations were observed in the analyzed tissues. The compound did not cause anemia, but reduced the number of leukocytes in the treated animals. The absence of viable S180 cells, necrotic cells, and the presence of granulation tissue were observed in tumor tissue of treated animals. The Ru(III) complex, in the presence of the reduction agent, caused plasmid DNA to fragment. These results suggest that cis-RuCl(2)(NH(3))(4)Cl compound is a potent antitumoral drug in vitro and in vivo, which seems to involve binding to DNA molecule.

Effect of juvenile hormone III on the ultrastructure of the corpora allata in Melipona quadrifasciata (Hymenoptera,. Apidae, Meliponýný)

Changes in hormonal levels can produce alternative phenotypes. Juvenile hormone III plays an importantrole in the regulation of metamorphosis, caste determination and age in bees. In this work, we examined theultrastructure of corpora allata cells from stingless bees (Melipona quadrifasciata) treated with juvenilehormone during development. The corpora allata cells of M. quadrifasciata queens showed greater activitythan those of workers. The topical application of juvenile hormone III altered the cellular ultrastructureand either delayed development (as shown by fewer mitochondria and greater chromatin condensation) orenhanced development (looser chromatin and numerous mitochondria) when compared to untreated (control)bees. Our results show that corpora allata cells differ in their ultrastructural characteristics and that thecessation of juvenile hormone production by these cells in M. quadrifasciata is not synchronous.

Differential gene expression in Melipona scutellaris (Hymenoptera, Meliponini): effect of juvenile hormone III

The objective of the present study was to determine by differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) the effects of juvenile hormone (JH) III applied during the late larval 3 (L3) phase on gene expression in Melipona scutellaris. A temporal window of expression of feminizing genes exists during the late L3 and pre-defecating larval phases when these genes can be turned on or off by the action of JH, which is able to mediate the differentiation of female larvae into queens. Combination of the HT11A-AP4 primers revealed differential expression in L3 individuals treated with JH III for 1 h, with weak expression of the transcript, while intense expression was observed for controls and individuals treated for 4 h. Combination of the HT11G-AP4 and HT11G-AP5 primers showed suppression of the gene products for each primer combination in 1-h treated larvae compared to untreated control individuals of the same age and individuals treated for 4 h. Differential gene expression was also observed during development. These results demonstrate that the JH III may suppress or alter gene expression profiles during phase L3 of M. scutellaris.