NCS-1 protein regulates TRPA1 channel through the PI3K pathway in breast cancer and neuronal cells.

The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.

PTEN loss in glioma cell lines leads to increased extracellular vesicles biogenesis and PD-L1 cargo in a PI3K-dependent manner.

Phosphatase and Tensin Homologue (PTEN) is one of the most frequently lost tumor suppressors in cancer and the predominant negative regulator of the PI3K/AKT signaling axis. A growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1), an altered tumor derived secretome that drives an immunosuppressive tumor immune microenvironment (TIME), and resistance to certain immunotherapies. Given their roles in immunosuppression and tumor growth, we examined whether the loss of PTEN would impact the biogenesis, cargo, and function of extracellular vesicles (EVs) in the context of the anti-tumor associated cytokine interferon-γ (IFN-γ). Through genetic and pharmacological approaches, we show that PD-L1 expression is regulated by JAK/STAT signaling, not PI3K signaling. Instead, we observe that PTEN loss positively upregulates cell surface levels of PD-L1 and enhances the biogenesis of EVs enriched with PD-L1 in a PI3K-dependent manner. We demonstrate that because of these changes, EVs derived from glioma cells lacking PTEN have a greater ability to suppress T cell receptor (TCR) signaling. Taken together, these findings provide important new insights into how the loss of PTEN can contribute to an immunosuppressive TIME, facilitate immune evasion, and highlight a novel role for PI3K signaling in the regulation of EV biogenesis and the cargo they contain.

Expression of placental glycans and its role in regulating peripheral blood NK cells during preeclampsia: a perspective.

Preeclampsia is a pregnancy-related multisystem disorder characterized by altered trophoblast invasion, oxidative stress, exacerbation of systemic inflammatory response, and endothelial damage. The pathogenesis includes hypertension and mild-to-severe microangiopathy in the kidney, liver, placenta, and brain. The main mechanisms involved in its pathogenesis have been proposed to limit trophoblast invasion and increase the release of extracellular vesicles from the syncytiotrophoblast into the maternal circulation, exacerbating the systemic inflammatory response. The placenta expresses glycans as part of its development and maternal immune tolerance during gestation. The expression profile of glycans at the maternal-fetal interface may play a fundamental role in physiological pregnancy changes and disorders such as preeclampsia. It is unclear whether glycans and their lectin-like receptors are involved in the mechanisms of maternal-fetal recognition by immune cells during pregnancy homeostasis. The expression profile of glycans appears to be altered in hypertensive disorders of pregnancy, which could lead to alterations in the placental microenvironment and vascular endothelium in pregnancy conditions such as preeclampsia. Glycans with immunomodulatory properties at the maternal-fetal interface are altered in early-onset severe preeclampsia, implying that innate immune system components, such as NK cells, exacerbate the systemic inflammatory response observed in preeclampsia. In this article, we discuss the evidence for the role of glycans in gestational physiology and the perspective of glycobiology on the pathophysiology of hypertensive disorders in gestation.

Paclitaxel Regulates TRPA1 Function and Expression Through PKA and PKC.

Paclitaxel (PTX) is a frequently used anticancer drug that causes peripheral neuropathy. Transient receptor potential ankyrin 1 (TRPA1), a plasma membrane calcium channel, has been associated with PTX toxicity and with other chemotherapy agents such as oxaliplatin and vincristine. However, the effect of PTX on the functional expression and calcium currents of TRPA1 has not been determined. The present study shows the effect of PTX on TRPA1 activity in a neuronal cell line (SH-SY5Y). The effect of PTX on the expression of TRPA1 was assessed through quantitative PCR and Western blot analyses to determine the relative mRNA and protein expression levels. To assess the effect on calcium flux and currents, cells were exposed to PTX; simultaneously, a specific agonist and antagonist of TRPA1 were added to evaluate the differential response in exposed versus control cells. To assess the effect of PKA, PKC and PI3K on PTX-induced TRPA1 increased activity, selective inhibitors were added to these previous experiments. PTX increased the mRNA and protein expression of TRPA1 as well as the TRPA1-mediated Ca2+ currents and intracellular Ca2+ concentrations. This effect was dependent on AITC (a selective specific agonist) and was abolished with HC-030031 (a selective specific antagonist). The inhibition of PKA and PKC reduced the effect of PTX on the functional expression of TRPA1, whereas the inhibition of PI3K had no effects. PTX-induced neuropathy involves TRPA1 activity through an increase in functional expression and is regulated by PKA and PKC signaling. These findings support the role of the TRPA1 channel in the mechanisms altered by PTX, which can be involved in the process that lead to chemotherapy-induced neuropathy.

The COVID-19 Pandemic in a Hispanic Population: A Primary Care Perspective.

INTRODUCTION: The COVID-19 global pandemic has affected all ethnic and minority groups although not equally. The goals of the present study are twofold: describe the diverse COVID-19-related care needs Hispanic patients presenting to a primary care facility and the symptom clusters and socioeconomic factors that may impact their wellbeing. METHODS: This is a retrospective cohort of Hispanic patients in an outpatient clinic serving an urban lower socioeconomic demographic, between May 9 and July 31, 2020. COVID-19 infection was confirmed by polymerase chain reaction or rapid antibody test. Student's t-test was used for means and the chi2 was used for comparisons of proportions. RESULTS: A total of 6616 patients visited Alivio, 409 were triaged to a containment area, and 378 were tested for COVID-19; 230 with, 148 without symptoms. Of those tested, 161(42.6%) were positive, representing 2.4% of total patients seen. Age, temperature, and pulse rate were all significantly higher in patients with symptoms compared with those without. Symptoms were grouped into 5 clusters: constitutional, n = 143(62%), respiratory, n = 136 (59%), and somatic, n = 97(42%) were most common. No single cluster was particularly diagnostic of COVID-19, although those with symptoms in multiple clusters were more likely to test positive, P < .001. The majority worked in essential jobs, were uninsured, and had more than half had prolonged symptoms. CONCLUSIONS: Hispanic patients have diverse reasons for seeking health care and for testing in a pandemic. COVID-19 is a syndromic disease as evidenced from the clustering of symptoms. Essential workers and uninsured health status may lead to more prolonged disease course.

Self-Perceived Emotional Intelligence Levels in Nursing Students in Times of a Pandemic: Multivariate Representation.

Self-perceived emotional intelligence in healthcare personnel is not just an individual skill but a work tool, which is even more necessary in times of crisis. This article aimed to determine emotional intelligence as perceived by students studying nursing at the University of Colima, Mexico, a year after the start of the COVID-19 pandemic. A cross-sectional survey of an academic year stratified population of 349 students was conducted, using the Trait Meta-Mood Scale-24 instrument. A global descriptive analysis was performed for each school year. Additionally, an ANOVA was performed, and a Multiple Correspondence Analysis was executed. It is essential to highlight the high percentages for emotional attention within the results. However, a large percentage of students required improvement in emotional attention, clarity, and repair. According to their school year, significant differences were observed among student groups within the three emotional intelligence subscales (p < 0.05). Second-year students had low levels in the three subscales of emotional intelligence, while fourth-year students had adequate levels. We established that the scores were different depending on the school year, with a significant decrease in second-year students. The implementation of educational programs could aid in the development of emotional skills in students from the health field, especially in times of crisis.

Media Agenda and Press Conferences on COVID-19 in Mexico: An Analysis of Journalists' Questions.

The impact of the COVID-19 pandemic has highlighted the need to strengthen health communication in times of crisis. This study aims to analyze the media agenda of press conferences on COVID-19 in Mexico during the first two phases of the pandemic, based on journalists' questions. The study is based on framing theory. The method used was content analysis from a quantitative perspective. This method was explicitly applied to the final section of the conferences, which dealt with "questions from the press." The results show that at the beginning of the pandemic, the press was more interested in the government's management of the health crisis than in issues such as the prevention of the disease itself or the economic impact of the crisis on the country. Moreover, the main characteristic of the questions was that they were generally socially relevant. In conclusion, we found that in the media agenda of the Mexican conference, the frame of attribution of responsibility was prominent but in combination with the frames of conflict, human interest, morality, and economic consequences.

Role of TRPV4 Channel in Human White Adipocytes Metabolic Activity.

BACKGROUND: Intracellular calcium (Ca2+) homeostasis plays an essential role in adipocyte metabolism and its alteration is associated with obesity and related disorders. Transient receptor potential vanilloid 4 (TRPV4) channels are an important Ca2+ pathway in adipocytes and their activity is regulated by metabolic mediators such as insulin. In this study, we evaluated the role of TRPV4 channels in metabolic activity and adipokine secretion in human white adipocytes. METHODS: Human white adipocytes were freshly cultured and the effects of the activation and inhibition of TRPV4 channels on lipolysis, glucose uptake, lactate production, and leptin and adiponectin secretion were evaluated. RESULTS: Under basal and isoproterenol-stimulated conditions, TRPV4 activation by GSK1016709A decreased lipolysis whereas HC067047, an antagonist, increased lipolysis. The activation of TRPV4 resulted in increased glucose uptake and lactate production under both basal conditions and insulin-stimulated conditions; in contrast HC067047 decreased both parameters. Leptin production was increased, and adiponectin production was diminished by TRPV4 activation and its inhibition had the opposite effect. CONCLUSION: Our results suggested that TRPV4 channels are metabolic mediators involved in proadipogenic processes and glucose metabolism in adipocyte biology. TRPV4 channels could be a potential pharmacological target to treat metabolic disorders.

Functional Interaction between Transient Receptor Potential V4 Channel and Neuronal Calcium Sensor 1 and the Effects of Paclitaxel.

Neuronal calcium sensor 1 (NCS1), a calcium-binding protein, and transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, are fundamental in the regulation of calcium homeostasis. The interactions of these proteins and their regulation by paclitaxel (PTX) were investigated using biochemical, pharmacological, and electrophysiological approaches in both a breast cancer epithelial cell model and a neuronal model. TRPV4 and NCS1 reciprocally immunoprecipitated each other, suggesting that they make up a signaling complex. The functional consequence of this physical association was that TRPV4 currents increased with increased NCS1 expression. Calcium fluxes through TRPV4 correlated with the magnitude of TRPV4 currents, and these calcium fluxes depended on NCS1 expression levels. Exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes but decreased the expression of NCS1. These findings augment the understanding of the properties of TRPV4, the role of NCS1 in the regulation of TRPV4, and the cellular mechanisms of PTX-induced neuropathy. SIGNIFICANCE STATEMENT: TRPV4 and NCS1 physically and functionally interact. Increased expression of NCS1 enhances TRPV4-dependent currents, which are further amplified by treatment with the chemotherapeutic drug paclitaxel, an effect associated with adverse effects of chemotherapy, including neuropathy.

Decellularization and In Vivo Recellularization of Abdominal Porcine Fascial Tissue.

BACKGROUND: Tissue decellularization has evolved as a promising approach for tissue engineering applications. METHODS: In this study, we harvested fascial tissue from porcine anterior abdominal wall and the samples were decellularized with a combination of agents such as Triton X-100, trypsin and DNAase. Afterwards, we evaluated cell removal by histological analysis and DNA quantification. Mechanical functionality was evaluated by applying a range of hydrostatic pressures. A sample of decellularized fascia was transplanted into a rabbit and after 15 days a biopsy of this tissue was examined; the animal was observed during 6 months after surgery. RESULTS: The extracellular matrix was retained with a complete decellularization as evidenced by histologic examination. The DNA content was significantly reduced. The scaffold preserved its tensile mechanical properties. The graft was incorporated into a full thickness defect made in the rabbit abdominal wall. This tissue was infiltrated by granulation and inflammatory cells and the histologic structure was preserved 15 days after surgery. The animal did not develop hernias, infections or other complications, after a 6-months of follow up. CONCLUSIONS: The protocol of decellularization of fascial tissue employed in this study proved to be efficient. The mechanical test demonstrated that the samples were not damaged and maintained its physical characteristics; clinical evolution of the rabbit, recipient of the decellularized fascia, demonstrated that the graft was effective as a replacement of native tissue.In conclusion, a biological scaffold derived from porcine fascial tissue may be a suitable candidate for tissue engineering applications.

Effect of flipped classroom methodology on the student performance of gastrointestinal and renal physiology entrants and repeaters.

BACKGROUND: Physiology is a subject that is considered difficult; it is associated with academic failure and causes high levels of stress and anxiety in students. METHODS: This study compared the effectiveness of a traditional lecture-based methodology with that of a flipped classroom scheme focusing on cooperative ludic learning among gastrointestinal and renal physiology students. Two groups were subjected to these two different methods to teach gastrointestinal and renal physiology content divided into 14 topics. Additionally, two subgroups were identified in each group: entrants and repeaters. There were no differences in age or gender between the subgroups. RESULTS: Levels of self-perceived stress (measured by the SISCO scale), biological stress (measured by awakening salivary cortisol levels), and anxiety (measured by the Zung scale) were high in all of the students; the cortisol levels increased in the entrants and some of the scores in SISCO scale increased in the repeaters, throughout the study. The self-reported study time was longer in the students subjected to the flipped classroom-based method. The final exam results were better only in the new students facing the flipped methodology, but not in the repeaters, who scored lower on the final evaluation. The quantitative and qualitative assessments completed by the participants regarding the different aspects of the flipped-classroom-based methodology were favorable; however, the participants believed that traditional lectures should be maintained for specific topics. CONCLUSIONS: A methodology based on flipped teaching was an effective strategy to improve academic performance ingastrointestinal and renal physiology, but only in new students.

Fungal infection, decline and persistence in the only obligate troglodytic Neotropical salamander.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is implicated in global mass die-offs and declines in amphibians. In Mesoamerica, the Bd epidemic wave hypothesis is supported by detection of Bd in historic museum specimens collected over the last century, yet the timing and impact of the early stages of the wave remain poorly understood. Chiropterotriton magnipes, the only obligate troglodytic Neotropical salamander, was abundant in its small range in the decade following its description in 1965, but subsequently disappeared from known localities and was not seen for 34 years. Its decline is roughly coincident with that of other populations of Neotropical salamanders associated with the invasion and spread of Bd. To determine the presence and infection intensity of Bd on C. magnipes and sympatric amphibian species (which are also Bd hosts), we used a noninvasive sampling technique and qPCR assay to detect Bd on museum specimens of C. magnipes collected from 1952 to 2012, and from extant populations of C. magnipes and sympatric species of amphibians. We also tested for the presence of the recently discovered Batrachochytrium salamandivorans (Bsal), another fungal chytridiomycete pathogen of salamanders, using a similar technique specific for Bsal. We did not detect Bd in populations of C. magnipes before 1969, while Bd was detected at low to moderate prevalence just prior to and during declines. This pattern is consistent with Bd-caused epizootics followed by host declines and extirpations described in other hosts. We did not detect Bsal in any extant population of C. magnipes. We obtained one of the earliest positive records of the fungus to date in Latin America, providing additional historical evidence consistent with the Bd epidemic wave hypothesis. Genotyping results show that at least one population is currently infected with the Global Panzootic Lineage of Bd, but our genotyping of the historical positive samples was unsuccessful. The lack of large samples from some years and the difficulty in genotyping historical Bd samples illustrate some of the difficulties inherent in assigning causality to historical amphibian declines. These data also provide an important historical baseline for actions to preserve the few known remaining populations of C. magnipes.

Modulating TRPV4 channels with paclitaxel and lithium.

Transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, is implicated as a contributor to the initiation of chemotherapy-induced peripheral neuropathy (CIPN). Paclitaxel (PTX) is a commonly used anticancer drug that causes CIPN and lithium has been shown to prevent CIPN. However, the direct effect of PTX and lithium on TRPV4 is not clear. This study investigated these actions using biochemical, pharmacological, and electrophysiological approaches using a neuronal cell line (SH-SY5Y). The addition of pharmacologically appropriate levels of PTX increased the expression of TRPV4, TRPV4 currents, and TRPV4-dependent calcium fluxes. Prolonged exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes. Pretreatment with lithium (1 mM) decreased TRPV4 currents and calcium fluxes in the absence and presence of PTX. These findings enhance our understanding of the properties and regulation of TRPV4, the cellular mechanisms of PTX-induced neuropathy, and the mechanism of lithium for prevention of CIPN.

The molecular basis of selective DNA binding by the BRG1 AT-hook and bromodomain.

The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in >20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts. We previously identified a composite domain in the BRG1 ATPase subunit that is capable of associating with both histones and DNA in a multivalent manner. Mapping the DNA binding pocket revealed that it contains several cancer mutations. Here, we utilize SELEX-seq to investigate the DNA specificity of this composite domain and NMR spectroscopy and molecular modelling to determine the structural basis of DNA binding. Finally, we demonstrate that cancer mutations in this domain alter the mode of DNA association.

New insights into extracellular vesicle biogenesis and function.

It is becoming increasingly evident that most cell types are capable of forming and releasing multiple distinct classes of membrane-enclosed packages, referred to as extracellular vesicles (EVs), as a form of intercellular communication. Microvesicles (MVs) represent one of the major classes of EVs and are formed by the outward budding of the plasma membrane. The second major class of EVs, exosomes, are produced as components of multivesicular bodies (MVBs) and are released from cells when MVBs fuse with the cell surface. Both MVs and exosomes have been shown to contain proteins, RNA transcripts, microRNAs and even DNA that can be transferred to other cells and thereby trigger a broad range of cellular activities and biological responses. However, EV biogenesis is also frequently de-regulated in different pathologies, especially cancer, where MVs and exosomes have been suggested to promote tumor cell growth, therapy resistance, invasion and even metastasis. In this Review, we highlight some of the recent advances in this rapidly emerging and exciting field of cell biology, focusing on the underlying mechanisms that drive MV and exosome formation and release, with a particular emphasis on how EVs potentially impact different aspects of cancer progression and stem cell biology.

Síndrome antifosfolipídico: una aproximación desde la glicobiología / Antiphospholipid syndrome: an approach from the glycobiology

Introducción: La glicosilación es una modificación postraduccional que tiene un papel protagónico en los procesos de comunicación célula-célula, célula-matrix extracelular, por lo tanto modula la interacción con antígenos y el sistema inmune. La inmunoglobulina G (IgG) tiene un sitio de N-glicosilación en la posición 297. Los sacáridos terminales de este glicano impactan la función efectora de la proteína, dirigiendo la respuesta hacia un efecto proinflamatorio o antiinflamatorio. Maverakis. et al (2015), propusieron que cada enfermedad autoinmune tiene una única marca de glicanos, especialmente en las diferentes clases de inmunoglobulinas (teoría de la alteración de los glicanos en la autoinmunidad).

DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes.

BRG1 and BRM, central components of the BAF (mSWI/SNF) chromatin remodelling complex, are critical in chromatin structure regulation. Here, we show that the human BRM (hBRM) bromodomain (BRD) has moderate specificity for H3K14ac. Surprisingly, we also find that both BRG1 and hBRM BRDs have DNA-binding activity. We demonstrate that the BRDs associate with DNA through a surface basic patch and that the BRD and an adjacent AT-hook make multivalent contacts with DNA, leading to robust affinity and moderate specificity for AT-rich elements. Although we show that the BRDs can bind to both DNA and H3K14ac simultaneously, the histone-binding activity does not contribute substantially to nucleosome targeting in vitro. In addition, we find that neither BRD histone nor DNA binding contribute to the global chromatin affinity of BRG1 in mouse embryonic stem cells. Together, our results suggest that association of the BRG1/hBRM BRD with nucleosomes plays a regulatory rather than targeting role in BAF activity.

Effects of a Humor Therapy Program on Stress Levels in Pediatric Inpatients.

OBJECTIVE: Disease and hospitalization generate stress, which can affect the response to treatment. Humor has been used in many hospitals to decrease stress. The aim of this study was to evaluate the impact of a humor therapy program on stress levels in pediatric inpatients. METHODS: In the first phase, an intervention and a control group were studied over 2 consecutive 3-month periods; the interventions were performed by a team of artists trained in humor therapy. Salivary cortisol levels were measured by enzyme-linked immunosorbent assay, and the Weisz test, a pictorial chart that determines subjective stress perception, and the Parker test, which assesses objective stress, were applied. In the second phase, salivary cortisol levels were measured and the Weisz test was administered before and after the interventions. RESULTS: A total of 306 patients were recruited into this study: 198 in the first phase (94 in the intervention group and 104 in the nonintervention group) and 108 in the second phase. There were no differences between groups regarding age, sex, or medical diagnosis. The children in the intervention group presented lower cortisol levels, lower scores on the Parker test, and higher scores on the Weisz test than children in the nonintervention group. In the second phase, the children showed lower salivary cortisol levels and higher scores on the Weisz test after the intervention. CONCLUSIONS: Humor therapy has beneficial effects on stress and cortisol levels in pediatric inpatients. This supports the implementation and reinforcement of these therapies in pediatric hospitals.

Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.