The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM), a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM) displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB) cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets.

OBJECTIVE: Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables. METHODS: Pancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre-coated standard Seahorse or Resipher microplates. Oxygen consumption was evaluated using a Seahorse Extracellular Flux Analyzer or a Resipher Real-time Cell Analyzer. RESULTS: We provide a detailed protocol with all steps to optimize islet isolation with high yield and functionality. Our method requires a few islets per replicate; both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators and glucose. The technique was validated by other functional assays, which show these cells present conserved calcium influx and insulin secretion in response to glucose. We also show that our dispersed islets maintain robust basal respiration levels, in addition to maintaining up to 89% viability after five days in dispersed cultures. Furthermore, OCRs can be measured in Seahorse analyzers and in other plate respirometry systems, using standard materials. CONCLUSIONS: Overall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic ß-cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.

Effects of cold stress on physiologic metabolism in the initial phase and performance of broiler rearing.

This study aimed to investigate the effects of 8 h of cold stress (18 °C) every day in broiler chicks during the first 7 days of rearing on crop filling analysis, yolk sac consumption, digestive and immune organs weights, and physiological metabolism at seven days and performance between 1 and 35 days. Cobb500 male broiler chickens (n = 274) were randomly assigned to two treatments. The treatments consisted of varying environmental temperatures during the first week post-housing. Chicks were reared at a thermoneutral temperature (32 °C) or under cold stress (18 °C) for 8 h/day during the first week, and both groups were subsequently reared at a thermoneutral temperature for 8-35 days. The thermoneutral group reached 90% full crop after 48 h of housing (P < 0.05), while the cold-stressed group had more empty crops at 2 h and 48 h after housing (P < 0.05). The chick cloacal temperature was not affected by the treatments (P > 0.05). Additionally, the treatment did not affect serum amylase and corticosterone levels, feed intake, body weight gain, or feed conversion ratio (P > 0.05, while the cold-stressed group had elevated heterophil/lymphocyte count at day 7 (P < 0.05). The thermoneutral group showed higher viability (%) at 7 and 35 days and a higher production factor at 35 days (P < 0.05). Broiler chickens under cyclic cold stress experienced decreased yolk sac absorption during the first week and increased feed intake and feed conversion ratio after 35 days of rearing. Viability was also lower in the cold-stressed group. An appropriate strategy to minimize these adverse effects is to rear the chicks in a thermoneutral environment during the first week.

A molecular landscape of quiescence and proliferation highlights the role of Pten in mammary gland acinogenesis.

Cell context is key for cell state. Using physiologically relevant models of laminin-rich extracellular matrix (lrECM) induction of mammary epithelial cell quiescence and differentiation, we provide a landscape of the key molecules for the proliferation-quiescence decision, identifying multiple layers of regulation at the mRNA and protein levels. Quiescence occurred despite activity of Fak (also known as PTK2), Src and phosphoinositide 3-kinases (PI3Ks), suggesting the existence of a disconnecting node between upstream and downstream proliferative signalling. Pten, a lipid and protein phosphatase, fulfils this role, because its inhibition increased proliferation and restored signalling via the Akt, mTORC1, mTORC2 and mitogen-activated protein kinase (MAPK) pathways. Pten and laminin levels were positively correlated in developing murine mammary epithelia, and Pten localized apicolaterally in luminal cells in ducts and near the nascent lumen in terminal end buds. Consistently, in three-dimensional acinogenesis models, Pten was required for triggering and sustaining quiescence, polarity and architecture. The multilayered regulatory circuitry that we uncovered provides an explanation for the robustness of quiescence within a growth-suppressive microenvironment, which could nonetheless be disrupted by perturbations in master regulators such as Pten.

Co-Design Dedicated System for Efficient Object Tracking Using Swarm Intelligence-Oriented Search Strategies.

The template matching technique is one of the most applied methods to find patterns in images, in which a reduced-size image, called a target, is searched within another image that represents the overall environment. In this work, template matching is used via a co-design system. A hardware coprocessor is designed for the computationally demanding step of template matching, which is the calculation of the normalized cross-correlation coefficient. This computation allows invariance in the global brightness changes in the images, but it is computationally more expensive when using images of larger dimensions, or even sets of images. Furthermore, we investigate the performance of six different swarm intelligence techniques aiming to accelerate the target search process. To evaluate the proposed design, the processing time, the number of iterations, and the success rate were compared. The results show that it is possible to obtain approaches capable of processing video images at 30 frames per second with an acceptable average success rate for detecting the tracked target. The search strategies based on PSO, ABC, FFA, and CS are able to meet the processing time of 30 frame/s, yielding average accuracy rates above 80% for the pipelined co-design implementation. However, FWA, EHO, and BFOA could not achieve the required timing restriction, and they achieved an acceptance rate around 60%. Among all the investigated search strategies, the PSO provides the best performance, yielding an average processing time of 16.22 ms coupled with a 95% success rate.

Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis.

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.

Predictive factors for obstructive sleep apnea in adults with severe asthma receiving biologics: a single-center cross-sectional study.

PURPOSE: To evaluate the prevalence and potential predictors of obstructive sleep apnea (OSA) in a cohort of adults with severe asthma. METHODS: From March 2021 to December 2021, this cross-sectional study enrolled patients with severe asthma receiving biologics, who were consecutively referred for sleep evaluation irrespective of sleep-related symptoms. Clinical and functional data, including three OSA screening instruments (GOAL, STOP-Bang, and NoSAS) were recorded. All participants underwent a portable sleep test (ApneaLink Air™). OSA diagnosis was based on the respiratory disturbance index ≥ 5.0/h and subclassified according to severity thresholds. Data were subjected to logistic regression tests to identify possible predictors for OSA. Discrimination was estimated from the area under the curve (AUC). RESULTS: Overall, 56 outpatients were included (80% females): 54% with any OSA, 13% with moderate-to-severe OSA, and 4% with severe OSA. In the multivariate analysis, no parameter emerged as an independent predictor for OSA: age (p = 0.080), body mass index (p = 0.060), loud snoring (p = 0.130), and hypertension (p = 0.848). No screening instrument was useful to predict any OSA: GOAL (AUC: 0.714; 95% confidence interval (CI): 0.579-0.849), NoSAS (AUC: 0.645; 95% CI: 0.497-0.793), and STOP-Bang (AUC: 0.640; 95% CI: 0.493-0.788). Similarly, no screening tool was also useful for predicting moderate-to-severe OSA or severe OSA. CONCLUSION: Patients with evere asthma receiving biologics exhibit a high prevalence of OSA. However, no clinical, functional, or OSA screening instrument showed acceptable discriminatory ability to predict the presence of OSA in these patients with severe asthma.

Long-term respiratory outcomes after COVID-19: a Brazilian cohort study.

Objective: To investigate the prevalence and risk factors for persistent symptoms up to 12 months after hospital discharge in COVID-19 survivors. Methods: This prospective cohort study included patients with COVID-19 discharged from a university hospital in Brazil. Follow-up was performed 2, 6, and 12 months after discharge. Lung function tests and chest computed tomography (CT) were performed 2 months after discharge and were repeated if abnormal. The primary outcomes were the symptoms present, work status, and limitations in daily activities. Results: Eighty-eight patients were included. Dyspnea (54.5%), fatigue (50.0%), myalgia, and muscle weakness (46.6%) were the most common symptoms, which decreased over time. Anxiety was frequent (46.6%) and remained unchanged. One year after discharge, 43.2% of the patients reported limitations in daily activities, and 17.6% had not returned to work. Corticosteroid use was significantly associated with dyspnea and limitations in daily activities. Females had an increased risk of fatigue at the 12-month assessment, with marginal significance after multivariable adjustment. Young age and bronchial wall thickening on admission CT were also risk factors for dyspnea at follow-up. The most common lung function abnormalities were reduced diffusion capacity and small airway disease, which partially improved over time. Conclusions: One year after hospital discharge, more than one-third of patients still had persistent COVID-19-related symptoms, remarkable dyspnea, fatigue, and limitations in daily activities, regardless of acute disease severity. Age, female sex, corticosteroid use during hospitalization, and bronchial thickening on admission CT were associated with an increased risk of sequelae.

Long-term respiratory outcomes after COVID-19: a Brazilian cohort study

[ABSTRACT]. Objective. To investigate the prevalence and risk factors for persistent symptoms up to 12 months after hospi- tal discharge in COVID-19 survivors. Methods. This prospective cohort study included patients with COVID-19 discharged from a university hos- pital in Brazil. Follow-up was performed 2, 6, and 12 months after discharge. Lung function tests and chest computed tomography (CT) were performed 2 months after discharge and were repeated if abnormal. The primary outcomes were the symptoms present, work status, and limitations in daily activities. Results. Eighty-eight patients were included. Dyspnea (54.5%), fatigue (50.0%), myalgia, and muscle weak- ness (46.6%) were the most common symptoms, which decreased over time. Anxiety was frequent (46.6%) and remained unchanged. One year after discharge, 43.2% of the patients reported limitations in daily activ- ities, and 17.6% had not returned to work. Corticosteroid use was significantly associated with dyspnea and limitations in daily activities. Females had an increased risk of fatigue at the 12-month assessment, with mar- ginal significance after multivariable adjustment. Young age and bronchial wall thickening on admission CT were also risk factors for dyspnea at follow-up. The most common lung function abnormalities were reduced diffusion capacity and small airway disease, which partially improved over time. Conclusions. One year after hospital discharge, more than one-third of patients still had persistent COVID-19- related symptoms, remarkable dyspnea, fatigue, and limitations in daily activities, regardless of acute disease severity. Age, female sex, corticosteroid use during hospitalization, and bronchial thickening on admission CT were associated with an increased risk of sequelae.

[RESUMEN]. Objetivo. Investigar la prevalencia y los factores de riesgo de los síntomas persistentes de la COVID-19 hasta 12 meses después del alta hospitalaria en pacientes sobrevivientes de esta enfermedad. Métodos. Este estudio prospectivo de cohorte incluyó pacientes con COVID-19 que recibieron el alta de un hospital universitario en Brasil. El seguimiento se hizo a los 2, 6 y 12 meses del alta. Se realizaron pruebas de función pulmonar y tomografía computarizada de tórax dos meses después del alta y se repitieron si los resul- tados eran anormales. Los resultados primarios investigados fueron síntomas presentes, situación laboral y limitaciones en las actividades diarias. Resultados. Se incluyeron 88 pacientes. Los síntomas más comunes fueron disnea (54,5%), fatiga (50,0%), mialgia y debilidad muscular (46,6%), que disminuyeron con el tiempo. La ansiedad fue frecuente (46,6%) y se mantuvo sin cambios. Un año después del alta, 43,2% de los pacientes notificaron limitaciones en las actividades diarias, y 17,6% no se había reincorporado al trabajo. El consumo de corticosteroides se asoció significativamente con disnea y limitaciones en las actividades diarias. Las mujeres tuvieron un mayor riesgo de fatiga en la evaluación a los 12 meses, con una importancia marginal después del ajuste multivariable. También fueron factores de riesgo de disnea en el seguimiento: edad temprana y engrosamiento de las pare- des bronquiales en la tomografía computarizada al momento del ingreso hospitalario. Las anomalías más comunes de la función pulmonar fueron la reducción de la capacidad de difusión y la enfermedad de las vías respiratorias pequeñas, que mejoraron parcialmente con el tiempo. Conclusiones. Un año después del alta hospitalaria, más de un tercio de los pacientes todavía tenían sín- tomas persistentes relacionados con la COVID-19, disnea notable, fatiga y limitaciones en las actividades diarias, independientemente de la gravedad aguda de la enfermedad. La edad, el sexo femenino, el uso de corticosteroides durante la hospitalización y el engrosamiento bronquial en la tomografía computarizada al momento del ingreso hospitalario se asociaron con un mayor riesgo de secuelas.

[RESUMO]. Objetivo. Investigar a prevalência e os fatores de risco para sintomas persistentes por até 12 meses após a alta hospitalar entre sobreviventes da COVID-19. Métodos. Este estudo de coorte prospectivo incluiu pacientes com COVID-19 que receberam alta de um hos- pital universitário do Brasil. O acompanhamento foi realizado 2, 6 e 12 meses após a alta. Testes de função pulmonar e tomografia computadorizada (TC) do tórax foram realizados 2 meses após a alta hospitalar e repetidos em caso de resultados alterados. Os desfechos primários foram os sintomas presentes, a situação de trabalho e as limitações nas atividades diárias. Resultados. Foram incluídos 88 pacientes. Dispneia (54,5%), fadiga (50,0%), mialgia e fraqueza muscular (46,6%) foram os sintomas mais comuns, que diminuíram com o tempo. A ansiedade era frequente (46,6%) e permaneceu inalterada. Um ano após a alta, 43,2% dos pacientes relatavam limitações nas atividades diárias e 17,6% não haviam retornado ao trabalho. O uso de corticosteroides estava significativamente asso- ciado à dispneia e a limitações nas atividades diárias. Pacientes do sexo feminino tinham um risco maior de fadiga na avaliação de 12 meses, com significância marginal após ajuste multivariado. A idade jovem e o espessamento da parede brônquica na TC de admissão também eram fatores de risco para dispneia no acompanhamento. As alterações mais comuns da função pulmonar foram capacidade de difusão reduzida e doença das pequenas vias aéreas, que melhoraram parcialmente com o tempo. Conclusões. Um ano após a alta hospitalar, mais de um terço dos pacientes ainda apresentava sintomas persistentes relacionados à COVID-19, dispneia marcante, fadiga e limitações nas atividades diárias, inde- pendentemente da gravidade da doença aguda. A idade, o sexo feminino, o uso de corticosteroides durante a internação e o espessamento brônquico na TC de admissão estavam associados a um maior risco de sequelas.

Extracellular matrix stiffness regulates degradation of MST2 via SCF ßTrCP.

The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFßTrCP E3 ubiquitin ligase and silencing ßTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that ßTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.

The role of cellular quiescence in cancer - beyond a quiet passenger.

Quiescence, the ability to temporarily halt proliferation, is a conserved process that initially allowed survival of unicellular organisms during inhospitable times and later contributed to the rise of multicellular organisms, becoming key for cell differentiation, size control and tissue homeostasis. In this Review, we explore the concept of cancer as a disease that involves abnormal regulation of cellular quiescence at every step, from malignant transformation to metastatic outgrowth. Indeed, disrupted quiescence regulation can be linked to each of the so-called 'hallmarks of cancer'. As we argue here, quiescence induction contributes to immune evasion and resistance against cell death. In contrast, loss of quiescence underlies sustained proliferative signalling, evasion of growth suppressors, pro-tumorigenic inflammation, angiogenesis and genomic instability. Finally, both acquisition and loss of quiescence are involved in replicative immortality, metastasis and deregulated cellular energetics. We believe that a viewpoint that considers quiescence abnormalities that occur during oncogenesis might change the way we ask fundamental questions and the experimental approaches we take, potentially contributing to novel discoveries that might help to alter the course of cancer therapy.

Sleep-Disordered Breathing in Adults with Precapillary Pulmonary Hypertension: Prevalence and Predictors of Nocturnal Hypoxemia.

PURPOSE: To evaluate the frequency of sleep-disordered breathing (SDB) and predictors of the presence of nocturnal desaturation in adults with pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. METHODS: Outpatients with a hemodynamic diagnosis of precapillary pulmonary hypertension who underwent portable polysomnography were evaluated. Diagnosis and severity of SDB were assessed using three well-established respiratory disturbance index (RDI) thresholds: 5.0/h, 15.0/h, and 30.0/h, while nocturnal hypoxemia was defined by the average oxygen saturation (SpO2) < 90%. Multiple linear regression analysis evaluated the potential relationships among explanatory variables with the dependent variable (average SpO2 values), with comparisons based on the standardized regression coefficient (ß). The R-squared (R2; coefficient of determination) was used to evaluate the goodness-of-fit measure for the linear regression model. RESULTS: Thirty-six adults were evaluated (69.4% females). The majority of the participants (75.0%) had SDB (26 with obstructive sleep apnea [OSA] and one with central sleep apnea [CSA]); while 50% of them had nocturnal hypoxemia. In the linear regression model (R2 = 0.391), the mean pulmonary artery pressure [mPAP] (ß - 0.668; p = 0.030) emerged as the only independent parameter of the average SpO2. CONCLUSION: Our study found that the majority of the participants had some type of SDB with a marked predominance of OSA over CSA, while half of them had nocturnal desaturation. Neither clinical and hemodynamic parameters nor the RDI was a predictor of nocturnal desaturation, except for mPAP measured during a right heart catheterization, which emerged as the only independent and significant predictor of average SpO2.

Geographic distribution of human leishmaniasis and phlebotomine sand flies in the State of Mato Grosso do Sul, Brazil.

BACKGROUND: In the State of Mato Grosso do Sul, Brazil, sand flies and cases of visceral (VL) and cutaneous (CL) leishmaniases have been reported in almost all municipalities. The aim of this study was to analyze the geographic distribution of VL and CL in relation the sand fly species found in the municipalities of Mato Grosso do Sul. METHODS: We analyzed VL and CL cases from 2001 to 2018 using data from the Notifiable Diseases Information System (SINAN). Data collected since 2003 on the presence of sand fly vectors (proven or suspected) were provided by the State Health Secretariat. RESULTS: A total of 3566 and 3030 cases of VL and CL, respectively, were reported from 2001 to 2018. The municipalities with the most reported cases of VL were Campo Grande (2495), Três Lagoas (442), Corumbá (140) and Aquidauana (136); and those for CL were Campo Grande (635) and Bodoquena (197). The following sand fly species with vector potential were found in 59 municipalities (74.7%): Lutzomyia longipalpis, Lutzomyia cruzi, Nyssomyia whitmani, Migonemyia migonei, Nyssomyia neivai, Pintomyia pessoai, Bichromomyia flaviscutellata and Pintomyia fischeri. Sand flies were present in six municipalities where no cases of VL were reported and in two municipalities where no cases of CL were reported. CONCLUSIONS: Our results indicate that the geographical distribution of VL and CL in Mato Grosso do Sul expanded during the study period, and highlight the presence of sand fly vectors in municipalities where these diseases are currently considered to be non-endemic.

Prognostic value of integrin αV expression and localization pattern in invasive breast carcinomas.

Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.

Structural analysis of TrkA mutations in patients with congenital insensitivity to pain reveals PLCγ as an analgesic drug target.

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

Real-life Effectiveness of Omalizumab in Patients with Severe Allergic Asthma: RELIEF Study.

Introduction: Patients with severe allergic asthma (SAA) are at risk of severe exacerbations. Omalizumab is recommended as an add-on treatment for patients with uncontrolled SAA, despite high-dose inhaled corticosteroids and long acting ß2-agonist combination therapy (standard therapy).RELIEF was a prospective, open label, multicenter study conducted to assess the real-life effectiveness of omalizumab co-administered with standard therapy in patients with SAA for 24 months. Methods: A total of 347 patients aged ≥ 6 years with SAA were enrolled, 285 of whom (8 pediatrics and 277 adolescents and adults) completed this 24-month study. Compared with the 12 months prior to baseline, the mean number of exacerbations was reduced in the overall population at any time interval during the study. Proportion of patients with no exacerbations increased to 77.7% at 24 months from 32.6% at 12 months prior to baseline. A reduction in healthcare resource utilization was also observed. The mean number of specialist visits reduced from baseline (5.8 visits) to 2.4 visits at Month 24. Results: The mean asthma control test score was >19 at every time-point during the study. The rate of Global Evaluation of Treatment Effectiveness (GETE) for asthma response significantly increased at Months 18 and 24 (P <0.05) compared to baseline. Pulmonary function remained relatively stable for the overall study population. There were no new or unexpected safety findings in the study. Conclusion: RELIEF study showed that add-on therapy with omalizumab is effective in reducing exacerbations, healthcare utilization, and improving GETE score in patients with SAA uncontrolled by standard therapy.

Long-term respiratory outcomes after COVID-19: a Brazilian cohort study / Resultados respiratorios a largo plazo tras la COVID-19: estudio de cohorte en Brasil / Desfechos respiratórios de longo prazo pós-COVID-19: estudo de coorte brasileiro

ABSTRACT Objective. To investigate the prevalence and risk factors for persistent symptoms up to 12 months after hospital discharge in COVID-19 survivors. Methods. This prospective cohort study included patients with COVID-19 discharged from a university hospital in Brazil. Follow-up was performed 2, 6, and 12 months after discharge. Lung function tests and chest computed tomography (CT) were performed 2 months after discharge and were repeated if abnormal. The primary outcomes were the symptoms present, work status, and limitations in daily activities. Results. Eighty-eight patients were included. Dyspnea (54.5%), fatigue (50.0%), myalgia, and muscle weakness (46.6%) were the most common symptoms, which decreased over time. Anxiety was frequent (46.6%) and remained unchanged. One year after discharge, 43.2% of the patients reported limitations in daily activities, and 17.6% had not returned to work. Corticosteroid use was significantly associated with dyspnea and limitations in daily activities. Females had an increased risk of fatigue at the 12-month assessment, with marginal significance after multivariable adjustment. Young age and bronchial wall thickening on admission CT were also risk factors for dyspnea at follow-up. The most common lung function abnormalities were reduced diffusion capacity and small airway disease, which partially improved over time. Conclusions. One year after hospital discharge, more than one-third of patients still had persistent COVID-19-related symptoms, remarkable dyspnea, fatigue, and limitations in daily activities, regardless of acute disease severity. Age, female sex, corticosteroid use during hospitalization, and bronchial thickening on admission CT were associated with an increased risk of sequelae.

RESUMEN Objetivo. Investigar la prevalencia y los factores de riesgo de los síntomas persistentes de la COVID-19 hasta 12 meses después del alta hospitalaria en pacientes sobrevivientes de esta enfermedad. Métodos. Este estudio prospectivo de cohorte incluyó pacientes con COVID-19 que recibieron el alta de un hospital universitario en Brasil. El seguimiento se hizo a los 2, 6 y 12 meses del alta. Se realizaron pruebas de función pulmonar y tomografía computarizada de tórax dos meses después del alta y se repitieron si los resultados eran anormales. Los resultados primarios investigados fueron síntomas presentes, situación laboral y limitaciones en las actividades diarias. Resultados. Se incluyeron 88 pacientes. Los síntomas más comunes fueron disnea (54,5%), fatiga (50,0%), mialgia y debilidad muscular (46,6%), que disminuyeron con el tiempo. La ansiedad fue frecuente (46,6%) y se mantuvo sin cambios. Un año después del alta, 43,2% de los pacientes notificaron limitaciones en las actividades diarias, y 17,6% no se había reincorporado al trabajo. El consumo de corticosteroides se asoció significativamente con disnea y limitaciones en las actividades diarias. Las mujeres tuvieron un mayor riesgo de fatiga en la evaluación a los 12 meses, con una importancia marginal después del ajuste multivariable. También fueron factores de riesgo de disnea en el seguimiento: edad temprana y engrosamiento de las paredes bronquiales en la tomografía computarizada al momento del ingreso hospitalario. Las anomalías más comunes de la función pulmonar fueron la reducción de la capacidad de difusión y la enfermedad de las vías respiratorias pequeñas, que mejoraron parcialmente con el tiempo. Conclusiones. Un año después del alta hospitalaria, más de un tercio de los pacientes todavía tenían síntomas persistentes relacionados con la COVID-19, disnea notable, fatiga y limitaciones en las actividades diarias, independientemente de la gravedad aguda de la enfermedad. La edad, el sexo femenino, el uso de corticosteroides durante la hospitalización y el engrosamiento bronquial en la tomografía computarizada al momento del ingreso hospitalario se asociaron con un mayor riesgo de secuelas.

RESUMO Objetivo. Investigar a prevalência e os fatores de risco para sintomas persistentes por até 12 meses após a alta hospitalar entre sobreviventes da COVID-19. Métodos. Este estudo de coorte prospectivo incluiu pacientes com COVID-19 que receberam alta de um hospital universitário do Brasil. O acompanhamento foi realizado 2, 6 e 12 meses após a alta. Testes de função pulmonar e tomografia computadorizada (TC) do tórax foram realizados 2 meses após a alta hospitalar e repetidos em caso de resultados alterados. Os desfechos primários foram os sintomas presentes, a situação de trabalho e as limitações nas atividades diárias. Resultados. Foram incluídos 88 pacientes. Dispneia (54,5%), fadiga (50,0%), mialgia e fraqueza muscular (46,6%) foram os sintomas mais comuns, que diminuíram com o tempo. A ansiedade era frequente (46,6%) e permaneceu inalterada. Um ano após a alta, 43,2% dos pacientes relatavam limitações nas atividades diárias e 17,6% não haviam retornado ao trabalho. O uso de corticosteroides estava significativamente associado à dispneia e a limitações nas atividades diárias. Pacientes do sexo feminino tinham um risco maior de fadiga na avaliação de 12 meses, com significância marginal após ajuste multivariado. A idade jovem e o espessamento da parede brônquica na TC de admissão também eram fatores de risco para dispneia no acompanhamento. As alterações mais comuns da função pulmonar foram capacidade de difusão reduzida e doença das pequenas vias aéreas, que melhoraram parcialmente com o tempo. Conclusões. Um ano após a alta hospitalar, mais de um terço dos pacientes ainda apresentava sintomas persistentes relacionados à COVID-19, dispneia marcante, fadiga e limitações nas atividades diárias, independentemente da gravidade da doença aguda. A idade, o sexo feminino, o uso de corticosteroides durante a internação e o espessamento brônquico na TC de admissão estavam associados a um maior risco de sequelas.

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling.

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.

Increased circulating levels of angiotensin-(1-7) in severely ill COVID-19 patients.

This letter reports an unexpected increase of the ACE2 product angiotensin-(1-7) and a parallel decrease of its substrate angiotensin II, suggesting a dysregulation of the renin-angiotensin system towards angiotensin-(1-7) formation in #COVID19 patients https://bit.ly/3xFXuTU.