Interleukin 3 Inhibits Glutamate-Cytotoxicity in Neuroblastoma Cell Line.

Interleukin 3 (IL-3) is a well-known pleiotropic cytokine that regulates the proliferation and differentiation of hematopoietic progenitor cells, triggering classical signaling pathways such as JAK/STAT, Ras/MAPK, and PI3K/Akt to carry out its functions. Interestingly, the IL-3 receptor is also expressed in non-hematopoietic cells, playing a crucial role in cell survival. Our previous research demonstrated the expression of the IL-3 receptor in neuron cells and its protective role in neurodegeneration. Glutamate, a principal neurotransmitter in the central nervous system, can induce cellular stress and lead to neurotoxicity when its extracellular concentrations surpass normal levels. This excessive glutamate presence is frequently observed in various neurological diseases. In this study, we uncover the protective role of IL-3 as an inhibitor of glutamate-induced cell death, analyzing the cytokine's signaling pathways during its protective effect. Specifically, we examined the relevance of JAK/STAT, Ras/MAPK, and PI3 K signaling pathways in the molecular mechanism triggered by IL-3. Our results show that the inhibition of JAK, ERK, and PI3 K signaling pathways, using pharmacological inhibitors, effectively blocked IL-3's protective role against glutamate-induced cell death. Additionally, our findings suggest that Bcl-2 and Bax proteins may be involved in the molecular mechanism triggered by IL-3. Our investigation into IL-3's ability to protect neuronal cells from glutamate-induced damage offers a promising therapeutic avenue with potential clinical implications for several neurological diseases characterized by glutamate neurotoxicity.

Revealing the Salmo salar NLRP3 Inflammasome: Insights from Structural Modeling and Transcriptome Analysis.

The NLRP3, one of the most heavily studied inflammasome-related proteins in mammals, remains inadequately characterized in Atlantic salmon (Salmo salar), despite the significant commercial importance of this salmonid. The NLRP3 inflammasome is composed of the NLRP3 protein, which is associated with procaspase-1 via an adapter molecule known as ASC. This work aims to characterize the Salmo salar NLRP3 inflammasome through in silico structural modeling, functional transcript expression determination in the SHK-1 cell line in vitro, and a transcriptome analysis on Atlantic salmon. The molecular docking results suggested a similar arrangement of the ternary complex between NLRP3, ASC, and caspase-1 in both the Atlantic salmon and the mammalian NLRP3 inflammasomes. Moreover, the expression results confirmed the functionality of the SsNLRP3 inflammasome in the SHK-1 cells, as evidenced by the lipopolysaccharide-induced increase in the transcription of genes involved in inflammasome activation, including ASC and NLRP3. Additionally, the transcriptome results revealed that most of the inflammasome-related genes, including ASC, NLRP3, and caspase-1, were down-regulated in the Atlantic salmon following its adaptation to seawater (also known as parr-smolt transformation). This is correlated with a temporary detrimental effected on the immune system. Collectively, these findings offer novel insights into the evolutionarily conserved role of NLRP3.

Discovery of BbX transcription factor in the patagonian blennie: Exploring expression changes following combined bacterial and thermal stress exposure.

High-Mobility Group (HMG) proteins are involved in different processes such as transcription, replication, DNA repair, and immune response. The role of HMG proteins in the immune response of fish has been studied mainly for HMGB1, where its expression can be induced by the stimulation of viral/bacterial PAMPs and can act as a proinflammatory mediator and as a global regulator of transcription in response to temperature. However, for BbX this role remains to be discovered. In this work, we identified the BbX of E. maclovinus and evaluated the temporal expression levels after simultaneous challenge with P. salmonis and thermal stress. Phylogenetic analysis does not significantly deviate from the expected organismal relationships suggesting orthologous relationships and that BbX was present in the common ancestor of the group. BbX mRNA expression levels were very high in the intestinal tissue of E. maclovinus (foregut, midgut, and hindgut). Nevertheless, the protein levels analyzed by WB showed the highest levels of BbX protein in the liver (constitutive expression). On the other hand, the mRNA expression levels of BbX in the liver of E. maclovinus injected with P. salmonis and subjected to thermal stress showed an increase at days 16 and 20 in all treatments applied at 12 °C and 18 °C. Meanwhile, the protein levels quantified by WB showed a statistically significant increase in the HMG-Bbx at all experimental times (4, 8, 12, 16, and 20 dpi). However, at 4 dpi the HMG-Bbx protein levels were much higher than the other days evaluated. The results suggest that BbX protein may be implicated in the response mechanism to temperature and bacterial stimulation in the foregut, midgut, hindgut, and liver, according to our findings at the level of mRNA and protein. Furthermore, our WB analysis suggests an effect of P. salmonis on the expression of this protein that can be observed in condition C+ 12 °C compared to C- 12 °C. Then, there is an effect of temperature that can be evidenced in the condition AM 18 °C and SM 18 °C, compared to AB 18 °C and SB 18 °C at 4, 8, and 12 dpi. We found not differences in the levels of this protein if the thermal stress is achieved through acclimatization or shock. More research is necessary to clarify the importance of this type of HMG in the immune response and thermal tolerance in fish.

Immune Transcriptional Response in Head Kidney Primary Cell Cultures Isolated from the Three Most Important Species in Chilean Salmonids Aquaculture.

Fish cell culture is a common in vitro tool for studies in different fields such as virology, toxicology, pathology and immunology of fish. Fish cell cultures are a promising help to study how to diagnose and control relevant viral and intracellular bacterial infections in aquaculture. They can also be used for developing vaccines and immunostimulants, especially with the ethical demand aiming to reduce and replace the number of fish used in research. This study aimed to isolate head kidney primary cell cultures from three Chilean salmonids: Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss, and characterize the response to bacterial and viral stimuli by evaluating various markers of the innate and adaptive immune response. Specifically, the primary cell cultures of the head kidney from the three salmonids studied were cultured and exposed to two substances that mimic molecular patterns of different pathogens, i.e., Lipopolysaccharide (LPS) (bacterial) and Polyinosinic: polycytidylic acid (POLY I:C). Subsequently, we determined the mRNA expression profiles of the TLR-1, TLR-8, IgM, TLR-5, and MHC II genes. Head kidney primary cell cultures from the three species grown in vitro responded differently to POLY I:C and LPS. This is the first study to demonstrate and characterize the expression of immune genes in head kidney primary cell culture isolated from three salmonid species. It also indicates their potential role in developing immune responses as defense response agents and targets of immunoregulatory factors.

How Many Sirtuin Genes Are Out There? Evolution of Sirtuin Genes in Vertebrates With a Description of a New Family Member.

Studying the evolutionary history of gene families is a challenging and exciting task with a wide range of implications. In addition to exploring fundamental questions about the origin and evolution of genes, disentangling their evolution is also critical to those who do functional/structural studies to allow a deeper and more precise interpretation of their results in an evolutionary context. The sirtuin gene family is a group of genes that are involved in a variety of biological functions mostly related to aging. Their duplicative history is an open question, as well as the definition of the repertoire of sirtuin genes among vertebrates. Our results show a well-resolved phylogeny that represents an improvement in our understanding of the duplicative history of the sirtuin gene family. We identified a new sirtuin gene family member (SIRT3.2) that was apparently lost in the last common ancestor of amniotes but retained in all other groups of jawed vertebrates. According to our experimental analyses, elephant shark SIRT3.2 protein is located in mitochondria, the overexpression of which leads to an increase in cellular levels of ATP. Moreover, in vitro analysis demonstrated that it has deacetylase activity being modulated in a similar way to mammalian SIRT3. Our results indicate that there are at least eight sirtuin paralogs among vertebrates and that all of them can be traced back to the last common ancestor of the group that existed between 676 and 615 millions of years ago.

Metabolic changes induced by DNA damage in Ramos cells: exploring the role of mTORC1 complex.

DNA damage induces the activation of many different signals associated with repair or cell death, but it is also connected with physiological events, such as adult neurogenesis and B-cell differentiation. DNA damage induces different signaling pathways, some of them linked to important metabolic changes. The mTORC1 pathway has a central role in the regulation of growth processes and cell division in response to environmental changes and also controls protein synthesis, lipid biogenesis, nucleotide synthesis, and expression of glycolytic genes. Here, we report that double-strand breaks induced with etoposide affect the expression of genes encoding different enzymes associated with specific metabolic pathways in Ramos cells. We also analyzed the role of mTOR signaling, demonstrating that double-strand breaks induce downregulation of mTOR signaling. Specific inhibition of mTORC1 using rapamycin also induced changes in the expression of metabolic genes. Finally, we demonstrated that DNA damage and rapamycin can regulate glucose uptake. In summary, our findings show that etoposide and rapamycin affect the expression of metabolic genes as well as apoptotic and proliferation markers in Ramos cells, increasing our understanding of cancer metabolism.

Identification of multiple TAR DNA binding protein retropseudogene lineages during the evolution of primates.

The TAR DNA Binding Protein (TARDBP) gene has become relevant after the discovery of its several pathogenic mutations. The lack of evolutionary history is in contrast to the amount of studies found in the literature. This study investigated the evolutionary dynamics associated with the retrotransposition of the TARDBP gene in primates. We identified novel retropseudogenes that likely originated in the ancestors of anthropoids, catarrhines, and lemuriformes, i.e. the strepsirrhine clade that inhabit Madagascar. We also found species-specific retropseudogenes in the Philippine tarsier, Bolivian squirrel monkey, capuchin monkey and vervet. The identification of a retropseudocopy of the TARDBP gene overlapping a lncRNA that is potentially expressed opens a new avenue to investigate TARDBP gene regulation, especially in the context of TARDBP associated pathologies.

Independent duplications of the Golgi phosphoprotein 3 oncogene in birds.

Golgi phosphoprotein 3 (GOLPH3) was the first reported oncoprotein of the Golgi apparatus. It was identified as an evolutionarily conserved protein upon its discovery about 20 years ago, but its function remains puzzling in normal and cancer cells. The GOLPH3 gene is part of a group of genes that also includes the GOLPH3L gene. Because cancer has deep roots in multicellular evolution, studying the evolution of the GOLPH3 gene family in non-model species represents an opportunity to identify new model systems that could help better understand the biology behind this group of genes. The main goal of this study is to explore the evolution of the GOLPH3 gene family in birds as a starting point to understand the evolutionary history of this oncoprotein. We identified a repertoire of three GOLPH3 genes in birds. We found duplicated copies of the GOLPH3 gene in all main groups of birds other than paleognaths, and a single copy of the GOLPH3L gene. We suggest there were at least three independent origins for GOLPH3 duplicates. Amino acid divergence estimates show that most of the variation is located in the N-terminal region of the protein. Our transcript abundance estimations show that one paralog is highly and ubiquitously expressed, and the others were variable. Our results are an example of the significance of understanding the evolution of the GOLPH3 gene family, especially for unraveling its structural and functional attributes.

Piscirickettsia salmonis-Triggered Extracellular Traps Formation as an Innate Immune Response of Atlantic Salmon-Derived Polymorphonuclear Neutrophils.

Extracellular traps (ETs) are webs of DNA, citrullinated histones, anti-microbial peptides, and proteins that were not previously reported in Atlantic salmon (Salmo salar). ETs are mainly released from polymorphonuclear neutrophils (PMN) and are considered a novel PMN-derived effector mechanism against different invasive pathogens. Here, we showed that Atlantic salmon-derived PMN released ETs-like structures in vitro in response to highly pathogenic facultative intracellular rickettsial bacteria Piscirickettsia salmonis. PMN were isolated from pre-smolt Atlantic salmon and stimulated in vitro with oleic acid and P. salmonis. Extracellular DNA was measured using the PicoGreen™ dye, while immunofluorescence image analysis was used to confirm the classical components of salmonid-extruded ETs. Future studies are required to better understand the role of Atlantic salmon-derived ETs orchestrating innate/adaptive immunity and the knowledge on regulation pathways involved in this cell death process. Thus, comprehension of salmonid-derived ETs against P. salmonis might represent novel alternative strategies to improve host innate defense mechanisms of farmed salmon against closely related rickettsial bacteria, as a complement to disease prevention and control strategies.

Vincristine, carboplatin and cisplatin increase oxidative burst induced by PAF in canine neutrophils.

Myelosupression resulting from chemotherapy has been widely described in veterinary medicine; however, there is limited information relating to alterations in neutrophil function after chemotherapy in dogs with cancer. The aim of this study was to determine the non-proliferative effects of vincristine, carboplatin, and cisplatin on canine neutrophils by evaluating activation of oxidative and non-oxidative responses. Neutrophils were isolated from venous blood. Levels of reactive oxygen species (ROS) and metalloproteinase 9 (MMP-9) were measured in vitro during neutrophil exposure to these chemotherapeutic agents for 15 min followed by stimulation with platelet activating factor (PAF). ROS production was detected via luminescence, and MMP- 9 liberation was determined by zymography. The chemotherapeutic agents caused an increase in PAF-induced ROS production, but no change in the non-oxidative response was observed. These results suggest that these chemotherapeutic agents may act as priming agents by increasing the oxidative response. These effects could be beneficial for dogs with cancer by supporting their immune systems; however, excessive ROS liberation has been associated with inflammation, neutrophil-mediated cell injury, carcinogenesis, and metastasis. Clinical studies are necessary to evaluate the significance of these findings.

Intermediary metabolic response and gene transcription modulation on the Sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1930) injected with two strains of Piscirickettsia salmonis.

Pathogen interactions with cultured fish populations are well studied, but their effects on native fishes have not been characterized. In Chile, the disease caused by bacterial species Piscirickettsia salmonis represents one of the main issues and is considered to be one of the important pathogens in the field of aquaculture. They have been found to infect native fish. Therefore, it is necessary to understand the impact of P. salmonis on native species of local commercial value, as well as the potential impact associated with the emergence of antibiotic-resistant strains of P. salmonis. Due to this purpose, the native fish Eleginops maclovinus was used in our study. Fish were randomly distributed in tanks and intraperitoneally inoculated with two strains of P. salmonis. No mortality was recorded during the experiment. Cortisol, glucose and total α-amino acid levels increased in fish injected with AUSTRAL-005 strain compared to sham-injected and LF-89-inoculated fish. Moreover, results showed an increase in the activity of carbohydrates and lipids metabolism in liver; and an increase in the carbohydrates, lipids and total α-amino acid metabolism in muscle after injection with AUSTRAL-005. Our results suggest that P. salmonis modulates the physiology of E. maclovinus and the physiological impact increase in the presence of the antibiotic-resistant strain AUSTRAL-005.

Early differential gene expression in beef Longissimus thoracis muscles from carcasses with normal (<5.8) and high (>5.9) ultimate pH.

The objective of this study was to investigate early postmortem (0.5 h) gene expression in beef Longissimus thoracis (LT) muscles from carcasses with NORMAL (<5.8) and HIGH (>5.9) ultimate pH (pHu). A total of 53 transcripts were differentially expressed (P-value <.05): 40 showed up-regulation and 13 showed down-regulation in HIGH pHu carcasses. Four up-regulated (PDK4, GADD45B, MAOA, METTL21C) genes were confirmed (P < .05) by q-PCR. HIGH pHu samples resulted with lower values in glycolytic potential and AMP-activated protein kinase activity compared to NORMAL at 0.5 and 24 h postmortem (P < .05). Functional pathway analysis showed that calcium transport and GADD45 signaling pathways are associated with the development of HIGH pH meat. Genes involved in stress-related signaling, such as GADD45B, METTL21C and MAOA were overexpressed. These genes are involved in stress signaling that might be affecting Ca2+ transport and oxidative metabolism pathways in HIGH pH muscles.

Delphinidin Reduces Glucose Uptake in Mice Jejunal Tissue and Human Intestinal Cells Lines through FFA1/GPR40.

Anthocyanins are pigments with antihyperglycemic properties, and they are potential candidates for developing functional foods for the therapy or prevention of Diabetes mellitus type 2 (DM2). The mechanism of these beneficial effects of anthocyanins are, however, hard to explain, given their very low bioavailability due to poor intestinal absorption. We propose that free fatty acid receptor 1 (FFA1, also named GPR40), is involved in an inhibitory effect of the anthocyanidin delphinidin over intestinal glucose absorption. We show the direct effects of delphinidin on the intestine using jejunum samples from RF/J mice, and the human intestinal cell lines HT-29, Caco-2, and NCM460. By the use of specific pharmacological antagonists, we determined that delphinidin inhibits glucose absorption in both mouse jejunum and a human enterocytic cell line in a FFA1-dependent manner. Delphinidin also affects the function of sodium-glucose cotransporter 1 (SGLT1). Intracellular signaling after FFA1 activation involved cAMP increase and cytosolic Ca2+ oscillations originated from intracellular Ca2+ stores and were followed by store-operated Ca2+ entry. Taken together, our results suggest a new GPR-40 mediated local mechanism of action for delphinidin over intestinal cells that may in part explain its antidiabetic effect. These findings are promising for the search for new prevention and pharmacological treatment strategies for DM2 management.

Natural recombination in alphaherpesviruses: Insights into viral evolution through full genome sequencing and sequence analysis.

Recombination in alphaherpesviruses was first described more than sixty years ago. Since then, different techniques have been used to detect recombination in natural (field) and experimental settings. Over the last ten years, next-generation sequencing (NGS) technologies and bioinformatic analyses have greatly increased the accuracy of recombination detection, particularly in field settings, thus contributing greatly to the study of natural alphaherpesvirus recombination in both human and veterinary medicine. Such studies have highlighted the important role that natural recombination plays in the evolution of many alphaherpesviruses. These studies have also shown that recombination can be a safety concern for attenuated alphaherpesvirus vaccines, particularly in veterinary medicine where such vaccines are used extensively, but also potentially in human medicine where attenuated varicella zoster virus vaccines are in use. This review focuses on the contributions that NGS and sequence analysis have made over the last ten years to our understanding of recombination in mammalian and avian alphaherpesviruses, with particular focus on attenuated live vaccine use.

Identification and expressional analysis of NLRC5 inflammasome gene in smolting Atlantic salmon (Salmo salar).

The NOD-like receptors (NLRs) were recently identified as an intracellular pathogen recognition receptor family in vertebrates. While the immune system participation of NLRs has been characterized and analyzed in various mammalian models, few studies have considered NLRs in teleost species. Therefore, this study analyzed the Atlantic salmon (Salmo salar) NLRC5. Structurally, Atlantic salmon NLRC5 presented leucine-rich repeat subfamily genes. Phylogenetically, NLRC5 was moderately conserved between S. salar and other species. Real-time quantitative PCR revealed NLRC5 expression in almost all analyzed organs, with greatest expressions in the head kidney, spleen, and hindgut. Furthermore, NLRC5 gene expression decreased during smolt stage. These data suggest that NLRC5 participates in the Atlantic salmon immune response and is regulated, at least partly, by the smoltification process, suggesting that there is a depression of immune system from parr at smolt stage. This is the first report on the NLRC5 gene in salmonid smolts.

Voltage-gated proton (H(v)1) channels, a singular voltage sensing domain.

The main role of voltage-gated proton channels (Hv1) is to extrude protons from the intracellular milieu when, mediated by different cellular processes, the H(+) concentration increases. Hv1 are exquisitely selective for protons and their structure is homologous to the voltage sensing domain (VSD) of other voltage-gated ion channels like sodium, potassium, and calcium channels. In clear contrast to the classical voltage-dependent channels, Hv1 lacks a pore domain and thus permeation necessarily occurs through the voltage sensing domain. Hv1 channels are activated by depolarizing voltages, and increases in internal proton concentration. It has been proposed that local conformational changes of the transmembrane segment S4, driven by depolarization, trigger the molecular rearrangements that open Hv1. However, it is still unclear how the electromechanical coupling is achieved between the VSD and the potential pore, allowing the proton flux from the intracellular to the extracellular side. Here we provide a revised view of voltage activation in Hv1 channels, offering a comparative scenario with other voltage sensing channels domains.

Voltage-dependent BK and Hv1 channels expressed in non-excitable tissues: New therapeutics opportunities as targets in human diseases.

Voltage-gated ion channels are the molecular determinants of cellular excitability. This group of ion channels is one of the most important pharmacological targets in excitable tissues such as nervous system, cardiac and skeletal muscle. Moreover, voltage-gated ion channels are expressed in non-excitable cells, where they mediate key cellular functions through intracellular biochemical mechanisms rather than rapid electrical signaling. This review aims at illustrating the pharmacological impact of these ion channels, highlighting in particular the structural details and physiological functions of two of them - the high conductance voltage- and Ca(2+)-gated K(+) (BK) channels and voltage-gated proton (Hv1) channels- in non-excitable cells. BK channels have been implicated in a variety of physiological processes ranging from regulation of smooth muscle tone to modulation of hormone and neurotransmitter release. Interestingly, BK channels are also involved in modulating K(+) transport in the mammalian kidney and colon epithelium with a potential role in the hyperkalemic phenotype observed in patients with familial hyperkalemic hypertension type 2, and in the pathophysiology of hypertension. In addition, BK channels are responsible for resting and stimulated Ca(2+)-activated K(+) secretion in the distal colon. Hv1 channels have been detected in many cell types, including macrophages, blood cells, lung epithelia, skeletal muscle and microglia. These channels have a central role in the phagocytic system. In macrophages, Hv1 channels participate in the generation of reactive oxygen species in the respiratory burst during the process of phagocytosis.

The first transmembrane domain (TM1) of ß2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels.

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary ß-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in ß-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the ß2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the ß2-subunit physically binds to the transmembrane S1 domain of the α-subunit.

Allosteric interactions and the modular nature of the voltage- and Ca2+-activated (BK) channel.

The high conductance voltage- and Ca(2+)-activated K(+) channel is one of the most broadly expressed channels in mammals. This channel is named BK for 'big K' because of its single-channel conductance that can be as large as 250 pS in 100 mm symmetrical K(+). BK channels increase their activity by membrane depolarization or an increase in cytosolic Ca(2+). One of the key features that defines the behaviour of BK channels is that neither Ca(2+) nor voltage is strictly necessary for channel activation. This and several other observations led to the idea that both Ca(2+) and voltage increase the open probability by an allosteric mechanism. In this type of mechanism, the processes of voltage sensor displacement, Ca(2+) binding and pore opening are independent equilibria that interact allosterically with each other. These allosteric interactions in BK channels reside in the structural characteristics of the BK channel in the sense that voltage and Ca(2+) sensors and the pore need to be contained in different structures or 'modules'. Through electrophysiological, mutagenesis, biochemical and fluorescence studies these modules have been identified and, more important, some of the interactions between them have been unveiled. In this review, we have covered the main advances achieved during the last few years in the elucidation of the structure of the BK channel and how this is related with its function as an allosteric protein.

Cell volume and membrane stretch independently control K+ channel activity.

A number of potassium channels including members of the KCNQ family and the Ca(2+) activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of -5.0 +/- 0.1 mmHg the increase amounted to 381 +/- 146% (mean +/- S.E.M., n = 6, P < 0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms.