Influence of the COVID-19 pandemic on labor and childbirth care practices in Brazil: a cross-sectional study.

BACKGROUND: It has been hypothesized that the coronavirus disease 2019 (COVID-19) pandemic may have changed the conduct of obstetric practices at the time of labor, delivery, and birth. In Brazil, many practices lacking scientific evidence are implemented in this care, which is charcaterized by excessive use of unnecessary interventions. This scenario may have been worsened by the pandemic. Thus, we analyzed the effects of the pandemic on care during prenatal care and delivery by comparing the results of two surveys (one was administered before the pandemic and the other during the pandemic) in public hospitals in Belo Horizonte - Minas Gerais (MG), Brazil. METHODS: This cross-sectional and comparative study analyzed preliminary data from the study "Childbirth and breastfeeding in children of mothers infected with SARS-CoV-2", which was conducted in three referral maternity hospitals in Belo Horizonte - MG during the pandemic in the first half of 2020 in Brazil. The final sample consisted of 1532 eligible women. These results were compared with data from 390 puerperae who gave birth in the three public hospitals in the study "Birth in Belo Horizonte: labor and birth survey", conducted before the pandemic to investigate the changes in practices of labor and delivery care for the mother and her newborn, with or without COVID-19 infection, before and during the pandemic. In this research, "Birth in Belo Horizonte: labor and birth survey", data collection was performed between November 2011 and March 2013 by previously trained nurses. Between study comparisons were performed using Pearson's chi-square test, with a confidence level of 95%, and using Stata statistical program. RESULTS: We found a significant increase in practices recommended by the World Health Organization during the pandemic including the following: diet offering (48.90 to 98.65%), non-pharmacological pain relief (43.84 to 67.57%), and breastfeeding in the newborn´s first hour of life (60.31 to 77.98%) (p < 0.001). We found a significant reduction of non-recommended interventions, such as routine use of episiotomy (15.73 to 2.09%), the Kristeller maneuver (16.55 to 0.94%), oxytocin infusion misused (45.55 to 28.07%), amniotomy (30.81 to 15.08%), and lithotomy position during labor (71.23 to 6.54%) (p < 0.001). CONCLUSION: Our study revealed a statistically significant increase in the proportion of use of recommended practices and a reduction in non-recommended practices during labor and delivery. However, despite advances in the establishment of World Health Organization recommended practices in labor, delivery, and birth, the predominance of interventionist and medicalized practices persists, which is worsened by events, such as the pandemic.

MCL1 alternative polyadenylation is essential for cell survival and mitochondria morphology.

Alternative polyadenylation in the 3' UTR (3' UTR-APA) is a mode of gene expression regulation, fundamental for mRNA stability, translation and localization. In the immune system, it was shown that upon T cell activation, there is an increase in the relative expression of mRNA isoforms with short 3' UTRs resulting from 3' UTR-APA. However, the functional significance of 3' UTR-APA remains largely unknown. Here, we studied the physiological function of 3' UTR-APA in the regulation of Myeloid Cell Leukemia 1 (MCL1), an anti-apoptotic member of the Bcl-2 family essential for T cell survival. We found that T cells produce two MCL1 mRNA isoforms (pA1 and pA2) by 3' UTR-APA. We show that upon T cell activation, there is an increase in both the shorter pA1 mRNA isoform and MCL1 protein levels. Moreover, the less efficiently translated pA2 isoform is downregulated by miR-17, which is also more expressed upon T cell activation. Therefore, by increasing the expression of the more efficiently translated pA1 mRNA isoform, which escapes regulation by miR-17, 3' UTR-APA fine tunes MCL1 protein levels, critical for activated T cells' survival. Furthermore, using CRISPR/Cas9-edited cells, we show that depletion of either pA1 or pA2 mRNA isoforms causes severe defects in mitochondria morphology, increases apoptosis and impacts cell proliferation. Collectively, our results show that MCL1 alternative polyadenylation has a key role in the regulation of MCL1 protein levels upon T cell activation and reveal an essential function for MCL1 3' UTR-APA in cell viability and mitochondria dynamics.

Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.

Thymic epithelial cells require p53 to support their long-term function in thymopoiesis in mice.

Thymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-κB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissue-restricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral T-cell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life.

Lower limb wounds in diabetic and non-diabetic patients: survival analysis. / Feridas em membros inferiores em diabéticos e não diabéticos: estudo de sobrevida.

OBJECTIVE: To evaluate the survival of wounds in lower limbs of diabetic and non-diabetic patients. METHOD: Retrospective cohort study of patients with lower limb ulcers treated at a specialized center between 2011 and 2013. Outcome: healing of lower limb injuries in days. The survival function of lower limb wounds and the differences between diabetic and non-diabetic were analyzed. The Log-rank test was used to compare the survival curves between the study groups. RESULTS: In up to 600 days, 23% of the diabetic patients presented wound healing, while 63% of the non-diabetic patients had their wounds healed, with a statistically significant difference in survival curves in comparison between the groups. The Hazard Ratios (RH) of healing were lower for diabetic patients (HR = 0.13, 95% CI = 0.02-0.97). CONCLUSION: The results show that there is a delay in wound healing in diabetic patients.

Assessment of liposome disruption to quantify drug delivery in vitro.

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

CD5 expression is regulated during human T-cell activation by alternative polyadenylation, PTBP1, and miR-204.

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses.

Sweetened Soft Drinks Consumption Is Associated with Metabolic Syndrome: Cross-sectional Analysis from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil).

OBJECTIVE: To estimate the association between regular consumption of sweetened soft drinks, natural fruit juice, and coconut water with metabolic syndrome (MetS). METHODS: This was a cross-sectional study including men and women aged 35-74 years from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) Study, excluding patients with type 2 diabetes. The main explanatory variables were beverage consumption and the outcome variable was metabolic syndrome (Adult Treatment Panel III). RESULTS: After adjustments, a daily intake of 250 ml of soft drink increased the chance of metabolic syndrome (odds ratio [OR] = 1.95; 95% confidence interval [CI], 1.60-2.38). There was no association between coconut water and MetS. Moderate consumption of fruit juices has low odds of MetS compared to no consumption. CONCLUSION: Our results add evidence to potential negative effects of sweetened soft drinks on cluster metabolic abnormalities in middle-income countries.

Factors associated with the incidence of pressure ulcer during hospital stay.

OBJECTIVE: Estimating the incidence rate of pressure ulcers and verifying factors associated with this occurrence in a cohort of hospitalized patients. METHOD: This is a cohort study in which the considered outcome was the time until pressure ulcer occurrence. Estimated effect of the variables on the cumulative incidence ratio of the outcome was performed using the Cox proportional hazards model. Variable selection occurred via the Logrank hypothesis test. RESULTS: The sample consisted of 442 adults, with 25 incidents of pressure ulcers. Patients with high scores on the Braden scale presented a higher risk of pressure ulcer incidence when compared to those classified into the low score category. CONCLUSION: These results reinforce the importance of using the Braden Scale to assist in identifying patients more likely to develop pressure ulcers. OBJETIVO: Estimar a taxa de incidência de úlcera por pressão e verificar fatores associados a essa ocorrência em uma coorte de pacientes hospitalizados. MÉTODO: Trata-se de estudo de coorte no qual o desfecho foi a ocorrência da úlcera por pressão. A estimativa do efeito das variáveis para a proporção de incidência acumulada do desfecho foi realizada utilizando o modelo de riscos proporcionais de Cox. A seleção das variáveis ocorreu por meio do teste de hipóteses Logrank. RESULTADOS: A amostra foi composta de 442 adultos, com 25 casos incidentes de úlcera por pressão. Pacientes com altos escores na escala de Braden apresentaram maior risco de incidência de úlcera por pressão quando comparados com aqueles classificados na categoria de baixo escore. CONCLUSÃO: Os resultados reforçam a importância do uso da Escala de Braden para auxiliar na identificação dos pacientes com maior probabilidade de desenvolver úlcera por pressão.

T cell activation regulates CD6 alternative splicing by transcription dynamics and SRSF1.

The T cell-surface glycoprotein CD6 is a modulator of cellular responses and has been implicated in several autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. During Ag presentation, CD6 is targeted to the immunological synapse in a ligand binding-dependent manner, in which CD6 domain 3 directly contacts CD166, expressed on the APC. T cell activation results in the induction of CD6Δd3, an alternatively spliced isoform that lacks the ligand-binding domain and thus no longer localizes at the immunological synapse. In this study, we investigated the molecular mechanisms regulating the expression of CD6Δd3 upon human primary T cell activation. Using chromatin immunoprecipitation, we observed an increase in RNA polymerase II occupancy along the CD6 gene and augmented CD6 transcription. We showed that activation leads to transcription-related chromatin modifications, revealed by higher CD6 acetylation levels. Modulation of chromatin conformation using a histone deacetylase inhibitor that increases transcription rate causes an increase of exon 5 skipping. We further showed that the splicing factor SRSF1 binds to a regulatory element in CD6 intron 4, activating exon 5 splicing and promoting exon 5 inclusion. Concomitant with T cell activation-induced exon 5 skipping, we observed a downregulation of SRSF1. Using RNA immunoprecipitation, we showed that in activated T cells, SRSF1 recruitment to the CD6 transcript is impaired by increased chromatin acetylation levels. We propose that upon T cell activation, SRSF1 becomes limiting, and its function in CD6 exon 5 splicing is countered by an increase in CD6 transcription, dependent on chromatin acetylation.

RNA polymerase II kinetics in polo polyadenylation signal selection.

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.

Peptide Anchor for Folate-Targeted Liposomal Delivery.

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Electrical neuromodulation for the treatment of chronic pain: derivation of the intrinsic barriers, outcomes and considerations of the sustainability of implantable spinal cord stimulation therapies.

INTRODUCTION: For over 60 years, spinal cord stimulation has endured as a therapy through innovation and novel developments. Current practice of neuromodulation requires proper patient selection, risk mitigation and use of innovation. However, there are tangible and intangible challenges in physiology, clinical science and within society. AREAS COVERED: We provide a narrative discussion regarding novel topics in the field especially over the last decade. We highlight the challenges in the patient care setting including selection, as well as economic and socioeconomic challenges. Physician training challenges in neuromodulation is explored as well as other factors related to the use of neuromodulation such as novel indications and economics. We also discuss the concepts of technology and healthcare data. EXPERT OPINION: Patient safety and durable outcomes are the mainstay goal for neuromodulation. Substantial work is needed to assimilate data for larger and more relevant studies reflecting a population. Big data and global interconnectivity efforts provide substantial opportunity to reinvent our scientific approach, data analysis and its management to maximize outcomes and minimize risk. As improvements in data analysis become the standard of innovation and physician training meets demand, we expect to see an expansion of novel indications and its use in broader cohorts.

Application of the iPLUS non-coding sequence in improving biopharmaceuticals production.

The biotechnological landscape has witnessed significant growth in biological therapeutics particularly in the field of recombinant protein production. Here we investigate the function of 3'UTR cis-regulatory elements in increasing mRNA and protein levels in different biological therapeutics and model systems, spanning from monoclonal antibodies to mRNA vaccines. We explore the regulatory function of iPLUS - a universal sequence capable of consistently augmenting recombinant protein levels. By incorporating iPLUS in a vector to express a monoclonal antibody used in immunotherapy, in a mammalian cell line used by the industry (ExpiCHO), trastuzumab production increases by 2-fold. As yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals, we then used iPLUS in tandem (3x) and iPLUSv2 (a variant of iPLUS) to provide proof-of-concept data that it increases the production of a reporter protein more than 100-fold. As iPLUS functions by also increasing mRNA levels, we hypothesize that these sequences could be used as an asset in the mRNA vaccine industry. In fact, by including iPLUSv2 downstream of Spike we were able to double its production. Moreover, the same effect was observed when we introduced iPLUSv2 downstream of MAGEC2, a tumor-specific antigen tested for cancer mRNA vaccines. Taken together, our study provides data (TLR4) showing that iPLUS may be used as a valuable asset in a variety of systems used by the biotech and biopharmaceutical industry. Our results underscore the critical role of non-coding sequences in controlling gene expression, offering a promising avenue to accelerate, enhance, and cost-effectively optimize biopharmaceutical production processes.

CD6 attenuates early and late signaling events, setting thresholds for T-cell activation.

The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6-CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.

Arterial hypertension and associated factors: National Health Survey, 2019.

OBJECTIVE: To analyze the factors associated with self-reported arterial hypertension, as well as its prevalence in the Brazilian adult population. METHODS: Data from 88,531 individuals aged 18 years or older who responded to the 2019 National Health Survey were analyzed. The outcome studied was self-reported arterial hypertension. Sociodemographic variables and clinical and lifestyle conditions were considered as exposures. The prevalence ratio (PR), crude and adjusted for sex, age, and schooling was used as a measure of association to verify the factors related to its prevalence, obtained by Poisson regression with robust variance. RESULTS: The prevalence of self-reported arterial hypertension was of 23.9% (95%CI: 23.4-24.4). When adjusting for age, sex, and schooling, the adjusted Prevalence Ratios (APR) were higher among: regular health self-assessment (APR = 1.6; 95%CI: 1.5-1.6) and bad health self-assessment (APR = 1.7; 95%CI: 1.6-1.8); self-reference to heart disease (APR = 1.7; 95%CI: 1.6-1.7), diabetes (APR = 1.7; 95%CI: 1.6-1.8), high cholesterol (APR = 1.6; 95%CI: 1.6-1.7), overweight (APR = 1.4; 95%CI: 1.4-1.5), and obesity (APR = 2.0; 95%CI: 1.9-2.1); high salt intake (APR = 1.1; 95%CI: 1.0-1.1); higher among former smokers (APR = 1.1; 95%CI: 1.1-1.2) and lower among smokers (APR = 0.9; 95%CI: 0.8-0.9); and consumption of ultra-processed foods (APR = 0.9; 95%CI: 0.8-0.9). CONCLUSION: A quarter of the Brazilian adult population claims to have arterial hypertension, more prevalent among women and associated with older age groups, Black, mixed-race, and others, low schooling, high salt intake, former smoking, presence of comorbidities, and worse health self-assessment.

Awareness of the diagnosis, treatment, and control of diabetes mellitus in Brazil.

OBJECTIVE: To estimate the proportions of awareness, treatment, and control of diabetes mellitus (DM) in the Brazilian adult population. METHOD: This is a cross-sectional study, with data from a representative sample of the Brazilian population, taken from the National Health Survey(PNS 2014/2015). Outcomes were defined based on glycated hemoglobin (HbA1c) measurements, self-reported DM diagnosis, and use of hypoglycemic agents or insulin. The proportion of DM awareness, treatment, and control was estimated according to sociodemographic characteristics, health conditions, and access to health services, and their respective 95% confidence intervals. RESULTS: DM prevalence in the Brazilian population was of 8.6% (95%CI: 7.8-9.3): 68.2% (95%CI: 63.9-72.3) were aware of their diagnosis, 92.2% (95%CI: 88.6-94.7) of those who were aware were undergoing drug treatments, and, of these, 35.8% (95%CI: 30.5-41.6) had controlled HbA1c levels. The proportions of DM awareness, control, and treatment were lower in men aged 18 to 39 years, individuals with low education, without health insurance, and beneficiaries of the Bolsa Família program. CONCLUSION: Approximately one in ten Brazilians has DM. A little more than half of this population is aware of their diagnosis, a condition measured by HbA1c dosage and clinical diagnosis. Among those who know, the vast majority are undergoing drug treatments. However, less than half of these have their HbA1c levels controlled. Worse scenarios were found in subgroups with high social vulnerability.

Investigation of cancer incidence in ELSA-Brasil.

Low- and middle-income countries have high cancer-related mortality rates, and Brazil has one of the highest incidences of all cancers in South America. Since 2008, the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) follows up 15,105 civil adult servants of college education and research institutions in six Brazilian capitals, collecting relevant information on risk factors for the incidence of cancer and other noncommunicable diseases. This study aimed to describe the methodology for surveillance and investigation of the incidence of primary cancers in ELSA-Brasil. The surveillance and investigation involve annual telephone interviews, data collection of medical registries (biopsy records, chemotherapy/radiotherapy reports, hospital records or death certificates/autopsy statements), eligibility and final classification, date and basis of diagnosis, tumor topography and histological type. The cumulative incidence of all types of cancer in ELSA-Brasil during the first five years of follow-up (2009-2013) was 2.27 %. The challenges encountered in the investigation process are related to the lack of data on the occurrence of distant metastasis and essential details regarding diagnoses. However, the quality control and the adequacy of the methods adopted enables the construction of a solid and pioneering database in the country, which will contribute to advance in research on the natural history of the most frequent types of cancer.

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages.

Introduction: Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3'UTR (3'UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, particularly in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells affect 3'UTR-APA and IPA in primary human macrophages was unclear. Methods: In this study, we isolated primary human monocytes from healthy donors, differentiated and polarized them into a pro-inflammatory state and performed indirect co-cultures with CRC cells. ChrRNA-Seq and 3'RNA-Seq was performed to quantify gene expression and characterize new 3'UTR-APA and IPA mRNA isoforms. Results: Our results show that polarization of human macrophages from naïve to a pro-inflammatory state causes a marked increase of proximal polyA site selection in the 3'UTR and IPA events in genes relevant to macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3'UTR-APA and IPA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induces 3'UTR-APA alterations. Notably, some of these gene expression differences were also found in tumor-associated macrophages of CRC patients, indicating that they are physiologically relevant. Upon macrophage pro-inflammatory polarization, SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Discussion: Our results reveal new 3'UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools. Furthermore, our results highlight a function for SRSF12 in pro-inflammatory macrophages, key cells in the tumor response.

A new pathway of CD5 glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase.

Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.