Maternal Vitamin D Deficiency Impairs the Development of ß Cells in Offspring Rats in a Sex-Dependent Manner.

Recent studies have shown that maternal vitamin D deficiency (VDD) causes long-term metabolic changes in offspring. However, little is known about the impact of maternal VDD on offspring endocrine pancreas development and insulin secretion in the adult life of male and female animals. Female rats (Wistar Hannover) were fed either control (1000 IU Vitamin D3/kg), VDD (0 IU Vitamin D3/kg), or a Ca2+-enriched VDD diet (0 IU Vitamin D3/kg + Ca2+ and P/kg) for 6 weeks and during gestation and lactation. At weaning, VDD status was confirmed based on low serum calcidiol levels in dams and pups. Next, male and female offspring were randomly separated and fed a standard diet for up to 90 days. At this age, serum calcidiol levels were restored to normal levels in all groups, but serum insulin levels were decreased in VDD males without affecting glucagon levels, glycemia, or glucose tolerance. Islets isolated from VDD males showed lower insulin secretion in response to different glucose concentrations, but this effect was not observed in VDD females. Furthermore, VDD males, but not females, showed a smaller total pancreatic islet area and lower ß cell mass, an effect that was accompanied by reduced gene expression of Ins1, Ins2, Pdx1, and SLC2A2. The decrease in Pdx1 expression was not related to the methylation profile of the promoter region of this gene. Most of these effects were observed in the male VDD+Ca2+ group, indicating that the effects were not due to alterations in Ca2+ metabolism. These data show that maternal VDD selectively impairs the morphology and function of ß cells in adult male offspring rats and that female offspring are fully protected from these deleterious effects.

Impact of Pesticides on Human Health in the Last Six Years in Brazil.

Every year, Brazil intensifies its activity in agriculture and, as a result, it has become one of the biggest consumers of pesticides in the world. The high rate of these substances raises environmental and human health concerns. Therefore, we collected papers from PubMed, Scopus, Scielo, and Web of Science databases, from 2015 to 2021. After a blind selection using the software Rayyan QCRI by two authors, 51 studies were included. Researchers from the South and the Southeast Brazilian regions contributed to most publications, from areas that concentrate agricultural commodity complexes. Among the pesticides described in the studies, insecticides, herbicides, and fungicides were the most frequent. The articles reported multiple toxic effects, particularly in rural workers. The results obtained can be used to direct policies to reduce the use of pesticides, and to protect the health of the population.

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development.

BACKGROUND: The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. RESULTS: Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. CONCLUSIONS: The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

Impact of pesticides on human health in the last six years in Brazil

Every year, Brazil intensifies its activity in agriculture and, as a result, it has become one of the biggest consumers of pesticides in the world. The high rate of these substances raises environmental and human health concerns. Therefore, we collected papers from PubMed, Scopus, Scielo, and Web of Science databases, from 2015 to 2021. After a blind selection using the software Rayyan QCRI by two authors, 51 studies were included. Researchers from the South and the Southeast Brazilian regions contributed to most publications, from areas that concentrate agricultural commodity complexes. Among the pesticides described in the studies, insecticides, herbicides, and fungicides were the most frequent. The articles reported multiple toxic effects, particularly in rural workers. The results obtained can be used to direct policies to reduce the use of pesticides, and to protect the health of the population.

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

Background The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. Results Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. Conclusions The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

The Natterin Proteins Diversity: A Review on Phylogeny, Structure, and Immune Function.

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Natterin an aerolysin-like fish toxin drives IL-1ß-dependent neutrophilic inflammation mediated by caspase-1 and caspase-11 activated by the inflammasome sensor NLRP6.

Natterin is an aerolysin-like pore-forming toxin responsible for the toxic effects of the venom of the medically significant fish Thalassophryne nattereri. Using a combination of pharmacologic and genetic loss-of-function approaches we conduct a systematic investigation of the regulatory mechanisms that control Natterin-induced neutrophilic inflammation in the peritonitis model. Our data confirmed the capacity of Natterin to induce a strong and sustained neutrophilic inflammation leading to systemic inflammatory lung infiltration and revealed overlapping regulatory paths in its control. We found that Natterin induced the extracellular release of mature IL-1ß and the sustained production of IL-33 by bronchial epithelial cells. We confirmed the dependence of both ST2/IL-33 and IL-17A/IL-17RA signaling on the local and systemic neutrophils migration, as well as the crucial role of IL-1α, caspase-1 and caspase-11 for neutrophilic inflammation. The inflammation triggered by Natterin was a gasdermin-D-dependent inflammasome process, despite the cells did not die by pyroptosis. Finally, neutrophilic inflammation was mediated by non-canonical NLRP6 and NLRC4 adaptors through ASC interaction, independent of NLRP3. Our data highlight that the inflammatory process dependent on non-canonical inflammasome activation can be a target for pharmacological intervention in accidents by T. nattereri, which does not have adequate specific therapy.

The natterin proteins diversity: a review on phylogeny, structure, and immune function

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Validação da importância funcional das Natterinas na resposta imune inata em zebrafish por CRISPR/Cas9, um método de loss-of-function

Zebrafish presents Natterin-like isoforms that share identity with the Natterina family, an important aerolysin for membrane penetration and inflammasome activation. The aim of this project is to silence the natterin-like gene in zebrafish using the CRISPR/Cas9 depletion system to assess the molecule's action in the body's immune defense against pathogens and its homeostatic function. Initially, we verified the susceptibility of zebrafish to infection with pathogens that activate the canonical and non-canonical pathways of the inflammasome. Thus, zebrafish larvaes with 1 day post fertilization (dpf) were exposed for 2 h to the bacteria Staphylococcus aureus and to Salmonella typhimurium for analysis of the effect on survival, embryonic development, locomotor activity and activation of the inflammasome immediately after the infection. The larvaes showed defects in embryonic development and behavioral change, and responded to infection by S. aureus and S. typhimurium with the production of mature caspase-a. Then, after synthesizing the sgRNA complex (trcarRNA + natterin-like crRNA, XM_017356964 / 2) for the complementary target in the second exon of the natterin-like gene on chromosome 7 (ID: 795232) the complex was microinjected into 0 embryos hpf with Cas9 enzyme in different concentrations (50 μg / nL: 250 μg / nL, 50: 125, 25: 125 and 12.5: 125) for gene depletion and obtaining KO embryos. The complete absence of natterin-like in the entire body of embryos depleted by CRISPR/Cas9 was confirmed by in situ hybridization. All concentrations delayed embryonic development with changes in the body's morphology with anomalies development and induced persistent mortality up to 144 hpf, dose-dependent. Lastly, WT larvae and depleted larvae of the natterin-like gene by CRISPR/Cas9 (sgRNA 50 ng/μL: Cas9 250 ng/μL) were exposed to infection in 1 dpf by S. aureus and S. typhimurium for 15 min, 30 min, 1 h and 2 h. Immediately after infection, the effect on survival, embryonic development, locomotor activity and activation of the inflammasome was evaluated. The absence of natterin-like in larvae (1 dpf) altered the response to infection by S. aureus and S. typhimurium inducing changes in locomotor activity in zebrafish associated with the decreased production of caspase-a and caspase-b at all times of infection performed. Our results show that natterin-like is crucial for the embryonic development of zebrafish and acts as an effector molecule in the innate immune response against bacterial pathogens activating the inflammasome complex in zebrafish.

Zebrafish apresenta isoformas de Natterin-like que compartilham identidade com a família Natterina, uma aerolisina importante para penetração em membranas e ativação do inflamassoma. O objetivo deste projeto é silenciar o gene da natterin-like em zebrafish utilizando o sistema de depleção CRISPR/Cas9 para avaliar a ação da molécula na defesa imune do organismo contra patógenos e sua função homeostática. Inicialmente verificamos a suscetibilidade do zebrafish para infecção com patógenos que ativam as vias canônica e não-canonica do inflamassoma. Assim, larvas de zebrafish com 1 dia após fertilização (dpf) foram expostas por 2 h às bactérias Staphylococcus aureus e Salmonella typhimurium para análise da sobrevivência, desenvolvimento embrionário, atividade locomotora e ativação do inflamassoma. As larvas apresentaram defeitos no desenvolvimento embrionário e no comportamento locomotor e responderam à infecção por S. aureus (canônico) e S. typhimurium (não- canônico) com a produção de caspase-a madura. Em seguida, após sintetizar o complexo sgRNA (trcarRNA + crRNA natterin-like, XM_017356964/2) para o alvo complementar no segundo exon do gene da natterin-like gene no cromossomo 7 (ID: 795232) o complexo foi microinjetado em embrioes de 0 hpf com a enzima Cas9 em diferentes concentracoes (50 μg/nL:250 μg/nL, 50:125, 25:125 e 12.5:125) para a depleção do gene e obtenção de embrioes depletados KO. A ausência completa de natterin-like no corpo inteiro dos embriões depletados por CRISPR/Cas9 foi confirmada por hibridização in situ. Todas as concentracoes atrasaram o desenvolvimento embrionario com alterações na morfologia do corpo com desenvolvimento de anomalias e induziram mortalidade persistente ate 144 hpf, dose- dependente. Por fim, larvas WT e larvas depletadas do gene natterin-like por CRISPR/Cas9 (sgRNA 50 ng/μL: Cas9 250 ng/μL) foram expostas à infecção em 1 dpf por S. aureus e S. typhimurium durante 15 min, 30 min, 1 h e 2 h. Imediatamente após a infecção foi avaliado o efeito na sobrevivência, no desenvolvimento embrionário, atividade locomotora e na ativação do inflamassoma. A ausência de natterin-like em larvas (1 dpf) alterou a resposta à infecção por S. aureus e S. typhimurium induzindo aumento na mortalidade e hiperatividade locomotora, indicativo de altos níveis de estree ou ansiedade. Confirmamos que a suscetibilidade à infecção nas larvas depletadas de natterin-like está relacionada à expressão deficiente de caspase-a e caspase-b. Podemos concluir que a natterin-like é crucial para o controle da ativação do inflamassoma responsável por garantir uma resposta inflamatória eficiente contra patógenos, visando à eliminação bacteriana e a sobrevivência do próprio hospedeiro.

Natterin an aerolysin-like fish toxin drives IL-1β-dependent neutrophilic inflammation mediated by caspase-1 and caspase-11 activated by the inflammasome sensor NLRP6

Natterin is an aerolysin-like pore-forming toxin responsible for the toxic effects of the venom of the medically significant fish Thalassophryne nattereri. Using a combination of pharmacologic and genetic loss-of-function approaches we conduct a systematic investigation of the regulatory mechanisms that control Natterin-induced neutrophilic inflammation in the peritonitis model. Our data confirmed the capacity of Natterin to induce a strong and sustained neutrophilic inflammation leading to systemic inflammatory lung infiltration and revealed overlapping regulatory paths in its control. We found that Natterin induced the extracellular release of mature IL-1β and the sustained production of IL-33 by bronchial epithelial cells. We confirmed the dependence of both ST2/IL-33 and IL-17A/IL-17RA signaling on the local and systemic neutrophils migration, as well as the crucial role of IL-1α, caspase-1 and caspase-11 for neutrophilic inflammation. The inflammation triggered by Natterin was a gasdermin-D-dependent inflammasome process, despite the cells did not die by pyroptosis. Finally, neutrophilic inflammation was mediated by non-canonical NLRP6 and NLRC4 adaptors through ASC interaction, independent of NLRP3. Our data highlight that the inflammatory process dependent on non-canonical inflammasome activation can be a target for pharmacological intervention in accidents by T. nattereri, which does not have adequate specific therapy.

Action of copper(II) complex with ß-diketone and 1,10-phenanthroline (CBP-01) on sarcoma cells and biological effects under cell death.

This work reports the biological evaluation of a copper complex of the type [Cu(O-O)(N-N)ClO4], in which O-O = 4,4,4-trifluoro-1-phenyl-1,3-butanedione (Hbta) and N-N = 1,10-phenanthroline (phen), whose generic name is CBP-01. The cytotoxic effect of CBP-01 was evaluated by resazurin assay and cell proliferation was determined by MTT assay. DNA fragmentation was analyzed by gel electrophoresis. Cell cycle progression was detected through propidium iodide (PI) staining. Apoptosis and autophagy were determined by, respectively, Annexin V and 7-AAD staining and monodansylcadaverine (MDC) staining. The changes in intracellular reactive oxygen species levels were detected by DCFDA analysis. The copper complex CBP-01 showed in vitro antitumor activity with IC50s values of 7.4 µM against Sarcoma 180 and 26.4 against murine myoblast cells, displaying selectivity toward the tumor cell tested in vitro (SI > 3). An increase in reactive oxygen species (ROS) generation was observed, which may be related to the action mechanism of the complex. The complex CBP-01 may induce DNA damage leading cells to accumulate at G0/G1 checkpoint where, apparently, cells that are not able to recover from the damage are driven to cell death. Evidence has shown that cell death is initiated by autophagy dysfunction, culminating in apoptosis induction. The search for new metal-based drugs is focused on overcoming the drawbacks of already used agents such as acquired resistance and non-specificity; thus, the results obtained with CBP-01 show promising effects on cancer cells.