ICAN (TRPM4) Contributes to the Intrinsic Excitability of Prefrontal Cortex Layer 2/3 Pyramidal Neurons.

Pyramidal neurons in the medial prefrontal cortical layer 2/3 are an essential contributor to the cellular basis of working memory; thus, changes in their intrinsic excitability critically affect medial prefrontal cortex (mPFC) functional properties. Transient Receptor Potential Melastatin 4 (TRPM4), a calcium-activated nonselective cation channel (CAN), regulates the membrane potential in a calcium-dependent manner. In this study, we uncovered the role of TRPM4 in regulating the intrinsic excitability plasticity of pyramidal neurons in the mouse mPFC layer of 2/3 using a combination of conventional and nystatin perforated whole-cell recordings. Interestingly, we found that TRPM4 is open at resting membrane potential, and its inhibition increases input resistance and hyperpolarizes membrane potential. After high-frequency stimulation, pyramidal neurons increase a calcium-activated non-selective cation current, increase the action potential firing, and the amplitude of the afterdepolarization, these effects depend on intracellular calcium. Furthermore, pharmacological inhibition or genetic silencing of TRPM4 reduces the firing rate and the afterdepolarization after high frequency stimulation. Together, these results show that TRPM4 plays a significant role in the excitability of mPFC layer 2/3 pyramidal neurons by modulating neuronal excitability in a calcium-dependent manner.

EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cationic channel involved in a wide variety of physiologic and pathophysiological processes. Bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative motif for interaction with end-binding (EB) proteins, which are microtubule plus-end tracking proteins. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. We show that mutations of the putative EB binding motif abolish the TRPM4-EB interaction, leading to a reduced expression of the mature population of the plasma membrane channel and instead display an endoplasmic reticulum-associated distribution. Furthermore, we demonstrate that EB1 and EB2 proteins are required for TRPM4 trafficking and functional activity. Finally, we demonstrated that the expression of a soluble fragment containing the EB binding motif of TRPM4 reduces the plasma membrane expression of the channel and affects TRPM4-dependent focal adhesion disassembly and cell invasion processes.-Blanco, C., Morales, D., Mogollones, I., Vergara-Jaque, A., Vargas, C., Álvarez, A., Riquelme, D., Leiva-Salcedo, E., González, W., Morales, D., Maureira, D., Aldunate, I., Cáceres, M., Varela, D., Cerda, O. EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel.

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca²âº-activated non-selective cation channel TRPM4.

Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.

A retrospective study suggests 55 days of persistence of SARS-CoV-2 during the first wave of the pandemic in Santiago de Chile.

Background: As the COVID-19 pandemic persists, infections continue to surge globally. Presently, the most effective strategies to curb the disease and prevent outbreaks involve fostering immunity, promptly identifying positive cases, and ensuring their timely isolation. Notably, there are instances where the SARS-CoV-2 virus remains infectious even after patients have completed their quarantine. Objective: Understanding viral persistence post-quarantine is crucial as it could account for localized infection outbreaks. Therefore, studying and documenting such instances is vital for shaping future public health policies. Design: This study delves into a unique case of SARS-CoV-2 persistence in a 60-year-old female healthcare worker with a medical history of hypertension and hypothyroidism. The research spans 55 days, marking the duration between her initial and subsequent diagnosis during Chile's first COVID-19 wave, with the analysis conducted using RT-qPCR. Results: Genomic sequencing-based phylogenetic analysis revealed that the SARS-CoV-2 detected in both Nasopharyngeal swab samples (NPSs) was consistent with the 20B clade of the Nextstrain classification, even after a 55-day interval. Conclusion: This research underscores the need for heightened vigilance concerning cases of viral persistence. Such instances, albeit rare, might be pivotal in understanding sporadic infection outbreaks that occur post-quarantine.

Neuregulin directly decreases voltage-gated sodium current in hippocampal ErbB4-expressing interneurons.

The Neuregulin 1 (NRG1)/ErbB4 signaling pathway has been genetically and functionally implicated in the etiology underlying schizophrenia, and in the regulation of glutamatergic pyramidal neuron function and plasticity. However, ErbB4 receptors are expressed in subpopulations of GABAergic interneurons, but not in hippocampal or cortical pyramidal neurons, indicating that NRG1 effects on principal neurons are indirect. Consistent with these findings, NRG1 effects on hippocampal long-term potentiation at CA1 pyramidal neuron synapses in slices are mediated indirectly by dopamine. Here we studied whether NRG/ErbB signaling directly regulates interneuron intrinsic excitability by pharmacologically isolating ErbB4-expressing neurons in rat dissociated hippocampal cultures, which lack dopaminergic innervation. We found that NRG1 acutely attenuates ErbB4-expressing interneuron excitability by depolarizing the firing threshold; neurons treated with the pan-ErbB inhibitor PD158780 or negative for ErbB4 were unaffected. These effects of NRG1 are primarily attributable to decreased voltage-gated sodium channel activity, as current density was attenuated by ∼60%. In stark contrast, NRG1 had minor effects on whole-cell potassium currents. Our data reveal the direct actions of NRG1 signaling in ErbB4-expressing interneurons, and offer novel insight into how NRG1/ErbB4 signaling can impact hippocampal activity.

The importance of the NRG-1/ErbB4 pathway for synaptic plasticity and behaviors associated with psychiatric disorders.

Neuregulin 1 (NRG-1) and its receptor ErbB4 have emerged as biologically plausible schizophrenia risk factors, modulators of GABAergic and dopaminergic neurotransmission, and as potent regulators of glutamatergic synaptic plasticity. NRG-1 acutely depotentiates LTP in hippocampal slices, and blocking ErbB kinase activity inhibits LTP reversal by theta-pulse stimuli (TPS), an activity-dependent reversal paradigm. NRG-1/ErbB4 signaling in parvalbumin (PV) interneurons has been implicated in inhibitory transmission onto pyramidal neurons. However, the role of ErbB4, in particular in PV interneurons, for LTP reversal has not been investigated. Here we show that ErbB4-null (ErbB4(-/-)) and PV interneuron-restricted mutant (PV-Cre;ErbB4) mice, as well as NRG-1 hypomorphic mice, exhibit increased hippocampal LTP. Moreover, both ErbB4(-/-) and PV-Cre;ErbB4 mice lack TPS-mediated LTP reversal. A comparative behavioral analysis of full and conditional ErbB4 mutant mice revealed that both exhibit hyperactivity in a novel environment and deficits in prepulse inhibition of the startle response. Strikingly, however, only ErbB4(-/-) mice exhibit reduced anxiety-like behaviors in the elevated plus maze task and deficits in cued and contextual fear conditioning. These results suggest that aberrant NRG-1/ErbB4 signaling in PV interneurons accounts for some but not all behavioral abnormalities observed in ErbB4(-/-) mice. Consistent with the observation that PV-Cre;ErbB4 mice exhibit normal fear conditioning, we find that ErbB4 is broadly expressed in the amygdala, largely by cells negative for PV. These findings are important to better understand ErbB4's role in complex behaviors and warrant further analysis of ErbB4 mutant mice lacking the receptor in distinct neuron types.

Modulation of GABAA receptors and of GABAergic synapses by the natural alkaloid gelsemine.

The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABAA receptors (GABAARs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAARs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAARs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAARs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAARs but support a role of GABAARs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system.

First Identification of Reinfection by a Genetically Different Variant of SARS-CoV-2 in a Homeless Person from the Metropolitan Area of Santiago, Chile.

The identification and tracking of SARS-CoV-2 infected patients in the general population are essential components of the global strategy to limit the COVID-19 viral spread, specifically for maintaining traceability and suppressing the resurgence of local outbreaks. Public health programs that include continuous RT-qPCR testing for COVID-19 in the general population, viral sequencing, and genomic surveillance for highly contagious forms of the virus have allowed for the identification of SARS-CoV-2 infections and reinfections. This work identified SARS-CoV-2 reinfection in a homeless person, which occurred 58 days after the first COVID-19 diagnosis. Genomic sequencing identified a different Nextstrain classification clade (20A and 20B) and PANGO lineage, with a divergence of 4 single nucleotide variants (SNVs) in S and ORF1ab genes, suggesting reinfection by different viral variants. This study is the first from the great metropolitan area of Santiago, Chile, one of the top ten countries in the world to live during the COVID-19 pandemic. We support the importance of performing intensive genomic surveillance programs in the whole population and high-risk groups, such as homeless people, nearly 20 thousand people in Chile, and have limited access to health care services and poor viral traceability.

Comparison of the First and Second Wave of Infections by SARS-CoV-2: A Retrospective and Longitudinal Study From a Primary Health Care Center in Santiago of Chile.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many countries have reported the experience of at least two contagion waves, describing associated mortality rates and population behavior. The analysis of the effect of this pandemic in different localities can provide valuable information on the key factors to consider in the face of future massive infectious diseases. This work describes the first retrospective and comparative study about behavior during the first and second waves of the COVID-19 pandemic in Chile from a primary Healthcare Center. From 19,313 real-time quantitative PCR (RT-qPCR) tests assessed, the selected 1,694 positive diagnostics showed a decrease in mortality rate in the second wave (0.6%) compared with the first (4.6%). In addition, we observed that infections in the second wave were mainly in young patients with reduced comorbidities. The population with a complete vaccination schedule shows a decrease in the duration of symptoms related to the disease, and patients with more comorbidities tend to develop severe illness. This report provides evidence to partially understand the behavior and critical factors in the severity of the COVID-19 pandemic in the population of Santiago of Chile.

Hydrogen peroxide removes TRPM4 current desensitization conferring increased vulnerability to necrotic cell death.

Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H(2)O(2) induces a sustained activity of TRPM4, a Ca(2+)-activated, Ca(2+)-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H(2)O(2) was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys(1093) residue is crucial for the H(2)O(2)-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H(2)O(2) elicited necrosis as well as apoptosis. H(2)O(2)-mediated necrosis but not apoptosis was abolished by replacement of external Na(+) ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H(2)O(2)-induced necrotic cell death. In addition, HeLa cells exposed to H(2)O(2) displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown.

P2X4 activation modulates volume-sensitive outwardly rectifying chloride channels in rat hepatoma cells.

Volume-sensitive outwardly rectifying (VSOR) Cl(-) channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H(2)O(2) plays an essential role in the activation of these channels and that H(2)O(2) per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H(2)O(2)-induced and hypotonicity-mediated VSOR Cl(-) activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H(2)O(2)-induced and hypotonicity-mediated VSOR Cl(-) current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl(-) current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 microm H(2)O(2) VSOR Cl(-) current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 microm H(2)O(2), exogenous addition of ATP in the presence of extracellular Ca(2+) resulted in a decrease in the half-time for VSOR Cl(-) current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl(-) current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl(-) current onset in a extracellular Ca(2+)-dependent manner.

Lipopolysaccharide inhibits the channel activity of the P2X7 receptor.

The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.

TRPM4 Expression During Postnatal Developmental of Mouse CA1 Pyramidal Neurons.

TRPM4 is a non-selective cation channel activated by intracellular calcium and permeable to monovalent cations. This channel participates in the control of neuronal firing, neuronal plasticity, and neuronal death. TRPM4 depolarizes dendritic spines and is critical for the induction of NMDA receptor-dependent long-term potentiation in CA1 pyramidal neurons. Despite its functional importance, no subcellular localization or expression during postnatal development has been described in this area. To examine the localization and expression of TRPM4, we performed duplex immunofluorescence and patch-clamp in brain slices at different postnatal ages in C57BL/6J mice. At P0 we found TRPM4 is expressed with a somatic pattern. At P7, P14, and P35, TRPM4 expression extended from the soma to the apical dendrites but was excluded from the axon initial segment. Patch-clamp recordings showed a TRPM4-like current active at the resting membrane potential from P0, which increased throughout the postnatal development. This current was dependent on intracellular Ca2+ (I CAN ) and sensitive to 9-phenanthrol (9-Ph). Inhibiting TRPM4 with 9-Ph hyperpolarized the membrane potential at P14 and P35, with no effect in earlier stages. Together, these results show that TRPM4 is expressed in CA1 pyramidal neurons in the soma and apical dendrites and associated with a TRPM4-like current, which depolarizes the neurons. The expression, localization, and function of TRPM4 throughout postnatal development in the CA1 hippocampal may underlie an important mechanism of control of membrane potential and action potential firing during critical periods of neuronal development, particularly during the establishment of circuits.

Adenosine triphosphate, polymyxin B and B16 cell-derived immunization induce anticancer response.

Aim: Whole dead tumor cells can be used as antigen source and the induction of protective immune response could be enhanced by damage-associated molecular patterns. Materials & methods: We generated whole dead tumor cells called B16-immunogenic cell bodies (ICBs) from B16 melanoma cells by nutrient starvation and evaluated the in vivo antitumor effect of B16-ICBs plus ATP and polymyxin B (PMB). Results: The subcutaneous immunization with B16-ICBs + PMB + ATP a 50% of tumor-free animals and induced a significant delay in tumor growth in a prophylactic approach. These results correlated with maturation of bone marrow-derived dendritic cells and activation of T CD8+ lymphocytes in vitro. Conclusion: Altogether, ICB + ATP + PMB is efficient in inducing the antitumor efficacy of the whole dead tumor cells vaccine.

P2X7 receptor is essential for cross-dressing of bone marrow-derived dendritic cells.

T cell activation requires the processing and presentation of antigenic peptides in the context of a major histocompatibility complex (MHC complex). Cross-dressing is a non-conventional antigen presentation mechanism, involving the transfer of preformed peptide/MHC complexes from whole cells, such as apoptotic cells (ACs) to the cell membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This is an essential mechanism for the induction of immune response against viral antigens, tumors, and graft rejection, which until now has not been clarified. Here we show for first time that the P2X7 receptor (P2X7R) is crucial to induce cross-dressing between ACs and Bone-Marrow DCs (BMDCs). In controlled ex vivo assays, we found that the P2X7R in both ACs and BMDCs is required to induce membrane and fully functional peptide/MHC complex transfer to BMDCs. These findings show that acquisition of ACs-derived preformed antigen/MHC-I complexes by BMDCs requires P2X7R expression.

In Vivo Antitumor Effect against Murine Cells of CT26 Colon Cancer and EL4 Lymphoma by Autologous Whole Tumor Dead Cells.

Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long-lasting antigen-specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost-efficient starvation-protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice's immunization with ICBs-E.G7 and ICBs-CT26 generate 18% and 25% of tumor-free animals, respectively. On the other hand, all carrying tumor-animals and immunized with ICBs, including ICBs-EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T-bet+ and CD4+ROR-γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell-based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors.

Evaluation of the Immune Response Induced by CoronaVac 28-Day Schedule Vaccination in a Healthy Population Group.

CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chile, the effectiveness of preventing hospitalization is higher than 80% with a vaccination schedule. However, to date, there are no data about immune response induction or specific memory. For this reason, we recruited 15 volunteers without previous suspected/diagnosed COVID-19 and with negative PCR over time to evaluate the immune response to CoronaVac 28 and 90 days after the second immunization (dpi). The CoronaVac administration induces total and neutralizing anti-spike antibodies in all vaccinated volunteers at 28 and 90 dpi. Furthermore, using ELISpot analysis to assay cellular immune responses against SARS-CoV-2 spike protein, we found an increase in IFN-gamma- and Granzyme B-producing cells in vaccinated volunteers at 28 and 90 dpi. Together, our results indicate that CoronaVac induces a robust humoral immune response and cellular immune memory of at least 90 dpi.

K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity.

Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca2+ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca2+ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca2+ signaling, respectively.

Lithraea caustic (Litre) Extract Promotes an Antitumor Response Against B16 Melanoma.

Melanoma immunotherapy, specifically the autotransplant of dendritic cells charged with tumors antigens, has shown promising results in clinical trials. The positive clinical effects of this therapy have been associated to increased Th17 response and delayed-type hypersensitivity (DTH) against to tumor antigens. Some synthetic compounds, such as diphenylcyclopropenone (DPCP), are capable of triggering a DTH response in cutaneous malignancies and also to induce clinically relevant effects against melanoma. In this work, we evaluated Litre extract (LExT), a standardized extract of a Chilean stinging plant, Lithraea caustic (Litre). As Litre plant is known to induce DTH, we used a murine B16 melanoma model to compare the topical and intratumor efficacy of LExT with synthetic DTH inducers (DPCP and 2,4-dinitrochlorobenzene [DNCB]). LExt contained mainly long chain catechols and sesquiterpenes. The intratumor injection of LExT induced a significant delay in tumor growth, similarly topical treatment of an established tumor with 0.1% LExT ointment induced a growth delay and even tumor regression in 15% of treated animals. No significant changes were observed on the T-cell populations associated to LExT treatment, and neither DNCB nor DPCP were capable to induce none of the LExT-induced antitumoral effects. Interestingly, our results indicate that LExT induces an antitumor response against melanoma in a mouse model and could bring a new -and affordable- treatment for melanoma in humans.