A peptide dataset for target analysis of human complement system proteins.

The targeted LC-MS/MS method has been widely applied for peptide quantification, offering sensibility, specificity, and reproducibility to the analysis. However, it requires the prior selection of targets, including the construction of a spectral library. Here, we present a dataset comprising peptide mass spectra for targeted LC-MS/MS method setup, applied to a set of human complement system proteins. Additionally, we selected a group of peptides and demonstrated their stability and reproducibility in quantification. This dataset is invaluable for studies aiming at the quantification of the complement system proteins by targeted LC-MS/MS, as it provides data for spectral library construction and a list of selected peptides.

Generation of an induced pluripotent stem cell line from a patient with epileptic encephalopathy caused by the CYFIP2 R87C variant.

Induced pluripotent stem cells (iPSCs) opened the possibility to use patient cells as a model for several diseases. iPSCs can be reprogrammed from somatic cells collected in a non-invasive way, and then differentiated into any other cell type, while maintaining the donor´s genetic background. CYFIP2 variants were associated with the onset of an early form of epileptic encephalopathy. Studies with patients showed that the R87C variant seems to be one of the variants that causes more severe disease, however, to date there are no studies with a human cell model that allows investigation of the neuronal phenotype of the R87C variant. Here, we generated an iPSC line from a patient with epileptic encephalopathy caused by the CYFIP2 R87C variant. We obtained iPSC clones by reprogramming urinary progenitor cells from a female patient. The generated iPSC line presented a pluripotent stem cell morphology, normal karyotype, expressed pluripotency markers and could be differentiated into the three germ layers. In further studies, this cell line could be used as model for epileptic encephalopathy disease and drug screening studies.

Molecular Dynamics of CYFIP2 Protein and Its R87C Variant Related to Early Infantile Epileptic Encephalopathy.

The CYFIP2 protein (cytoplasmic FMR1-interacting protein 2) is part of the WAVE regulatory complex (WRC). CYFIP2 was recently correlated to neurological disorders by the association of the R87C variant with early infantile epileptic encephalopathy (EIEE) patients. In this set of syndromes, the epileptic spasms and seizures since early childhood lead to impaired neurological development in children. Inside the WRC, the variant residue is at the CYFIP2 and WAVE1 protein interface. Thus, the hypothesis is that the R87C modification weakens this interaction, allowing the WRC complex's constant activation. This work aimed to investigate the impacts of the mutation on the structure of the WRC complex through molecular dynamics simulation. For that, we constructed WRC models containing WAVE1-NCKAP1 proteins complexed with WT or R87C CYFIP2. Our simulations showed a flexibilization of the loop comprising residues 80-110 due to the loss of contacts between internal residues in the R87C CYFIP2 as well as the key role of residues R/C87, E624, and E689 in structural modification. These data could explain the mechanism by which the mutation impairs the stability and proper regulation of the WRC.

Selecting Genetic Variants and Interactions Associated with Amyotrophic Lateral Sclerosis: A Group LASSO Approach.

Amyotrophic lateral sclerosis (ALS) is a multi-system neurodegenerative disease that affects both upper and lower motor neurons, resulting from a combination of genetic, environmental, and lifestyle factors. Usually, the association between single-nucleotide polymorphisms (SNPs) and this disease is tested individually, which leads to the testing of multiple hypotheses. In addition, this classical approach does not support the detection of interaction-dependent SNPs. We applied a two-step procedure to select SNPs and pairwise interactions associated with ALS. SNP data from 276 ALS patients and 268 controls were analyzed by a two-step group LASSO in 2000 iterations. In the first step, we fitted a group LASSO model to a bootstrap sample and a random subset of predictors (25%) from the original data set aiming to screen for important SNPs and, in the second step, we fitted a hierarchical group LASSO model to evaluate pairwise interactions. An in silico analysis was performed on a set of variables, which were prioritized according to their bootstrap selection frequency. We identified seven SNPs (rs16984239, rs10459680, rs1436918, rs1037666, rs4552942, rs10773543, and rs2241493) and two pairwise interactions (rs16984239:rs2118657 and rs16984239:rs3172469) potentially involved in nervous system conservation and function. These results may contribute to the understanding of ALS pathogenesis, its diagnosis, and therapeutic strategy improvement.

Modulation and function of Pumilio proteins in cancer.

Post-transcriptional regulation is involved in tumorigenesis, and in this control, RNA-binding proteins are the main protagonists. Pumilio proteins are highly conserved RNA-binding proteins that regulate many aspects of RNA processing. The dysregulation of Pumilio expression is associated with different types of cancer. This review summarizes the roles of Pumilio 1 and Pumilio 2 in cancer and discusses the factors that account for their distinct biological functions. Pumilio levels seem to be related to tumor progression and poor prognoses in some kinds of tumors, such as lung, pancreatic, prostate, and cervical cancers. Pumilio 1 is associated with cancer proliferation, migration, and invasion, and so is Pumilio 2, although there are contradictory reports regarding the latter. Furthermore, the circular RNA, circPUM1, has been described as a miRNAs sponge, regulating miRNA involved in the cell cycle. The expression and function of Pumilio proteins depend on the fine adjustment of a set of modulators, including miRNAs, lncRNAs, and circRNAs; this demonstrates that Pumilio plays an important role in tumorigenesis through a variety of regulatory axes.

Safety and long-term improvement of mesenchymal stromal cell infusion in critically COVID-19 patients: a randomized clinical trial.

BACKGROUND: COVID-19 is a multisystem disease that presents acute and persistent symptoms, the postacute sequelae (PASC). Long-term symptoms may be due to consequences from organ or tissue injury caused by SARS-CoV-2, associated clotting or inflammatory processes during acute COVID-19. Various strategies are being chosen by clinicians to prevent severe cases of COVID-19; however, a single treatment would not be efficient in treating such a complex disease. Mesenchymal stromal cells (MSCs) are known for their immunomodulatory properties and regeneration ability; therefore, they are a promising tool for treating disorders involving immune dysregulation and extensive tissue damage, as is the case with COVID-19. This study aimed to assess the safety and explore the long-term efficacy of three intravenous doses of UC-MSCs (umbilical cord MSCs) as an adjunctive therapy in the recovery and postacute sequelae reduction caused by COVID-19. To our knowledge, this is one of the few reports that presents the longest follow-up after MSC treatment in COVID-19 patients. METHODS: This was a phase I/II, prospective, single-center, randomized, double-blind, placebo-controlled clinical trial. Seventeen patients diagnosed with COVID-19 who require intensive care surveillance and invasive mechanical ventilation-critically ill patients-were included. The patient infusion was three doses of 5 × 105 cells/kg UC-MSCs, with a dosing interval of 48 h (n = 11) or placebo (n = 6). The evaluations consisted of a clinical assessment, viral load, laboratory testing, including blood count, serologic, biochemical, cell subpopulation, cytokines and CT scan. RESULTS: The results revealed that in the UC-MSC group, there was a reduction in the levels of ferritin, IL-6 and MCP1-CCL2 on the fourteen day. In the second month, a decrease in the levels of reactive C-protein, D-dimer and neutrophils and an increase in the numbers of TCD3, TCD4 and NK lymphocytes were observed. A decrease in extension of lung damage was observed at the fourth month. The improvement in all these parameters was maintained until the end of patient follow-up. CONCLUSIONS: UC-MSCs infusion is safe and can play an important role as an adjunctive therapy, both in the early stages, preventing severe complications and in the chronic phase with postacute sequelae reduction in critically ill COVID-19 patients. Trial registration Brazilian Registry of Clinical Trials (ReBEC), UTN code-U1111-1254-9819. Registered 31 October 2020-Retrospectively registered, https://ensaiosclinicos.gov.br/rg/RBR-3fz9yr.

Interleukin 21 Receptor Affects Adipogenesis of Human Adipose-Derived Stem/Stromal Cells.

Dysfunctions in adipose tissue cells are responsible for several obesity-related metabolic diseases. Understanding the process of adipocyte formation is thus fundamental for understanding these diseases. The adipocyte differentiation of adipose-derived stem/stromal cells (ADSCs) showed a reduction in the mRNA level of the interleukin 21 receptor (IL21R) during this process. Although the receptor has been associated with metabolic diseases, few studies have examined its function in stem cells. In this study, we used confocal immunofluorescence assays to determine that IL21R colocalizes with mitochondrial protein ATP5B, ALDH4A1, and the nucleus of human ADSCs. We demonstrated that silencing and overexpression of IL21R did not affect the cell proliferation and mitochondrial activity of ADSCs. However, IL21R silencing did reduce ADSC adipogenic capacity. Further studies are needed to understand the mechanism involved between IL21R and the adipogenic differentiation process.

Accessing the Human Pluripotent Stem Cell Translatome by Polysome Profiling.

Polysome profiling is a technique that uses sucrose density gradient ultracentrifugation to separate complexes of mRNAs associated with one or more ribosomes. Here we describe polysome profiling analysis in human pluripotent stem cells (hPSCs) using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR or large-scale sequencing analysis, used to establish the translational status of specific mRNAs and identify the role of noncoding RNA in translation.

Alternative Methods as Tools for Obesity Research: In Vitro and In Silico Approaches.

The study of adipogenesis is essential for understanding and treating obesity, a multifactorial problem related to body fat accumulation that leads to several life-threatening diseases, becoming one of the most critical public health problems worldwide. In this review, we propose to provide the highlights of the adipogenesis study based on in vitro differentiation of human mesenchymal stem cells (hMSCs). We list in silico methods, such as molecular docking for identification of molecular targets, and in vitro approaches, from 2D, more straightforward and applied for screening large libraries of substances, to more representative physiological models, such as 3D and bioprinting models. We also describe the development of physiological models based on microfluidic systems applied to investigate adipogenesis in vitro. We intend to identify the main alternative models for adipogenesis evaluation, contributing to the direction of preclinical research in obesity. Future directions indicate the association of in silico and in vitro techniques to bring a clear picture of alternative methods based on adipogenesis as a tool for obesity research.

Methodological Appraisal of Literature Concerning the Analysis of Genetic Variants or Protein Levels of Complement Components on Susceptibility to Infection by Trypanosomatids: A Systematic Review.

Background: Trypanosomatids are protozoa responsible for a wide range of diseases, with emphasis on Chagas Disease (CD) and Leishmaniasis, which are in the list of most relevant Neglected Tropical Diseases (NTD) according to World Health Organization (WHO). During the infectious process, immune system is immediately activated, and parasites can invade nucleated cells through a broad diversity of receptors. The complement system - through classical, alternative and lectin pathways - plays a role in the first line of defense against these pathogens, acting in opsonization, phagocytosis and lysis of parasites. Genetic modifications in complement genes, such as Single Nucleotide Polymorphisms (SNPs), can influence host susceptibility to these parasites and modulate protein expression. Methods: In March and April 2021, a literature search was conducted at the PubMed and Google Scholar databases and the reference lists obtained were verified. After applying the inclusion and exclusion criteria, the selected studies were evaluated and scored according to eleven established criteria regarding their thematic approach and design, aiming at the good quality of publications. Results: Twelve papers were included in this systematic review: seven investigating CD and five focusing on Leishmaniasis. Most articles presented gene and protein approaches, careful determination of experimental groups, and adequate choice of experimental techniques, although several of them were not up-to-date. Ten studies explored the association of polymorphisms and haplotypes with disease progression, with emphasis on lectin complement pathway genes. Decreased and increased patient serum protein levels were associated with susceptibility to CD and Visceral Leishmaniasis, respectively. Conclusion: This systematic review shows the influence of genetic alterations in complement genes on the progression of several infectious diseases, with a focus on conditions caused by trypanosomatids, and contributes suggestions and evidence to improve experimental design in future research proposals.

Cytoplasmic FMR1 interacting protein (CYFIP) family members and their function in neural development and disorders.

In humans, the cytoplasmic FMR1 interacting protein (CYFIP) family is composed of CYFIP1 and CYFIP2. Despite their high similarity and shared interaction with many partners, CYFIP1 and CYFIP2 act at different points in cellular processes. CYFIP1 and CYFIP2 have different expression levels in human tissues, and knockout animals die at different time points of development. CYFIP1, similar to CYFIP2, acts in the WAVE regulatory complex (WRC) and plays a role in actin dynamics through the activation of the Arp2/3 complex and in a posttranscriptional regulatory complex with the fragile X mental retardation protein (FMRP). Previous reports have shown that CYFIP1 and CYFIP2 may play roles in posttranscriptional regulation in different ways. While CYFIP1 is involved in translation initiation via the 5'UTR, CYFIP2 may regulate mRNA expression via the 3'UTR. In addition, this CYFIP protein family is involved in neural development and maturation as well as in different neural disorders, such as intellectual disabilities, autistic spectrum disorders, and Alzheimer's disease. In this review, we map diverse studies regarding the functions, regulation, and implications of CYFIP proteins in a series of molecular pathways. We also highlight mutations and their structural effects both in functional studies and in neural diseases.

Combined Use of Tocilizumab and Mesenchymal Stromal Cells in the Treatment of Severe Covid-19: Case Report.

The coronavirus pandemic is one of the most significant public health events in recent history. Currently, no specific treatment is available. Some drugs and cell-based therapy have been tested as alternatives to decrease the disease's symptoms, length of hospital stay, and mortality. We reported the case of a patient with a severe manifestation of COVID-19 in critical condition who did not respond to the standard procedures used, including six liters of O2 supplementation under a nasal catheter and treatment with dexamethasone and enoxaparin in prophylactic dose. The patient was treated with tocilizumab and an advanced therapy product based on umbilical cord-derived mesenchymal stromal cells (UC-MSC). The combination of tocilizumab and UC-MSC proved to be safe, with no adverse effects, and the results of this case report prove to be a promising alternative in the treatment of patients with severe acute respiratory syndrome due to SARS-CoV-2.

Adipogenesis, Osteogenesis, and Chondrogenesis of Human Mesenchymal Stem/Stromal Cells: A Comparative Transcriptome Approach.

Adipogenesis, osteogenesis and chondrogenesis of human mesenchymal stem/stromal cells (MSC) are complex and highly regulated processes. Over the years, several studies have focused on understanding the mechanisms involved in the MSC commitment to the osteogenic, adipogenic and/or chondrogenic phenotypes. High-throughput methodologies have been used to investigate the gene expression profile during differentiation. Association of data analysis of mRNAs, microRNAs, circular RNAs and long non-coding RNAs, obtained at different time points over these processes, are important to depict the complexity of differentiation. This review will discuss the results that were highlighted in transcriptome analyses of MSC undergoing adipogenic, osteogenic and chondrogenic differentiation. The focus is to shed light on key molecules, main signaling pathways and biological processes related to different time points of adipogenesis, osteogenesis and chondrogenesis.

Effects of PUMILIO1 and PUMILIO2 knockdown on cardiomyogenic differentiation of human embryonic stem cells culture.

Posttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA-binding proteins (RBPs) that orchestrate mRNA expression. A family of RBPs, which is known as the Pumilio-FBF (PUF) family, is highly conserved among different species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first assessed the influence of the silencing of PUM1 and PUM2 on pluripotency genes and found that the knockdown of Pumilio genes significantly decreased the OCT4 and NANOG mRNA levels and reduced the amount of nuclear OCT4, which suggests that Pumilio proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that PUM1-and-PUM2-silenced hESCs exhibited improved efficiency of in vitro cardiomyogenic differentiation. Through an in silico analysis, we identified mRNA targets of PUM1 and PUM2 that are expressed at the early stages of cardiomyogenesis, and further investigation will determine whether these target mRNAs are active and involved in the progression of cardiomyogenesis. Our findings contribute to the understanding of the role of Pumilio proteins in hESC maintenance and differentiation.

Data describing the experimental design and quality control of RNA-Seq of human adipose-derived stem cells undergoing early adipogenesis and osteogenesis.

An important tool to study the regulation of gene expression is the sequencing and the analysis of different RNA fractions: total, ribosome-free, monosomal and polysomal. By comparing these different populations, it is possible to identity which genes are differentially expressed and to get information on how transcriptional and translational regulation modulates cellular function. Therefore, we used this strategy to analyze the regulation of gene expression of human adipose-derived stem cells during the triggering of the adipogenic and osteogenic differentiation. Here, we have focused on analyzing the differential expression of mRNAs during early adipogenic and osteogenic differentiation, and presented the detailed data concerning the experimental design, the RNA-Seq quality data, the raw data obtained and the RT-qPCR validation data. This information is important to confirm the accuracy of the data considering a future reuse of the data provided. Moreover, this study may be used as groundwork for future characterization of the transcriptome and the translatome regulation of different cell types.

Cell cycle genes are downregulated after adipogenic triggering in human adipose tissue-derived stem cells by regulation of mRNA abundance.

The adipogenic process is characterized by the expression of adipocyte differentiation markers that lead to changes in cell metabolism and to the accumulation of lipid droplets. Moreover, during early adipogenesis, cells undergo a strong downregulation of translational activity with a decrease in cell size, proliferation and migration. In the present study, we identified that after 24 hours of adipogenic induction, human adipose tissue-derived stem cells (hASCs) undergo a G1-cell cycle arrest consistent with reduced proliferation, and this effect was correlated with a shift in polysome profile with an enrichment of the monosomal fraction and a reduction of the polysomal fraction. Polysome profiling analysis also revealed that this change in the monosomal/polysomal ratio was related to a strong downregulation of cell cycle and proliferation genes, such as cyclins and cyclin-dependent kinases (CDKs). Comparing total and polysome-associated mRNA sequencing, we also observed that this downregulation was mostly due to a reduction of cell cycle and proliferation transcripts via control of total mRNA abundance, rather than by translational control.

Crosstalk between Hedgehog pathway and energy pathways in human adipose-derived stem cells: A deep sequencing analysis of polysome-associated RNA.

Adult stem cells are considered promising candidates for cellular therapies due to their capacity to differentiate and self-renew. Differentiation leads to changes in the metabolism, structure, and gene expression patterns of cells. Hedgehog is one of the pathways that is involved in the enhancement of osteogenesis and chondrogenesis in adult stem cells, but its mechanisms are poorly understood. In this study, we treated adipose tissue-derived stem cells (ADSC) with two well-characterized drugs, purmorphamine (Hedgehog pathway activator) and cyclopamine (Hedgehog pathway inhibitor), and identified mRNAs associated with polysomes in each treatment group to determine the post transcriptional genetic networks governed by the Hedgehog pathway. Activation of the Hedgehog pathway by purmorphamine results in significant upregulation of mRNAs associated with cellular communication and signal transduction. Furthermore, our experiments show that cyclopamine acts late downregulating GLI1 expression in ADSCs but promotes the upregulation of mRNAs associated with energy pathways and metabolism at early times. Through in silico analysis, we identified some miRNAs, such as miR-355, that could regulate these mRNAs association with polysomes and thereby modulate the Hedgehog pathway. Our results suggest that activation of the Hedgehog pathway by purmorphamine also results in a negative regulation of mRNAs in the protein translation machinery.

Gene expression analysis of human adipose tissue-derived stem cells during the initial steps of in vitro osteogenesis.

Mesenchymal stem cells (MSCs) have been widely studied with regard to their potential use in cell therapy protocols and regenerative medicine. However, a better comprehension about the factors and molecular mechanisms driving cell differentiation is now mandatory to improve our chance to manipulate MSC behavior and to benefit future applications. In this work, we aimed to study gene regulatory networks at an early step of osteogenic differentiation. Therefore, we analyzed both the total mRNA and the mRNA fraction associated with polysomes on human adipose tissue-derived stem cells (hASCs) at 24 h of osteogenesis induction. The RNA-seq results evidenced that hASC fate is not compromised with osteogenesis at this time and that 21 days of continuous cell culture stimuli are necessary for full osteogenic differentiation of hASCs. Furthermore, early stages of osteogenesis induction involved gene regulation that was linked to the management of cell behavior in culture, such as the control of cell adhesion and proliferation. In conclusion, although discrete initial gene regulation related to osteogenesis occur, the first 24 h of induction is not sufficient to trigger and drive in vitro osteogenic differentiation of hASCs.

lncRNAs are associated with polysomes during adipose-derived stem cell differentiation.

Over the past few years, an increasing number of long noncoding RNAs (lncRNAs) have been identified in mammalian genomes. Most of these lncRNAs are expressed at low levels in different human cell types. lncRNAs are found not only in the nucleus but are also enriched in the cytosolic fraction and are associated with translating polysomes. Expression of lncRNAs that have putative roles in cell differentiation has been identified in embryonic and adult stem cells. Nevertheless, the mechanisms by which lncRNAs operate in the cell are still poorly understood.Here, we studied the expression of the subpopulation of lncRNAs that are associated with polysomes in adipose-derived stem cells (hASCs) during their commitment to adipocytes. We established that lncRNAs and protein coding genes have similar expression levels. The relatively comparable expression of these transcripts could be a particular feature of hASCs. We then show that lncRNAs are associated with polysomes in undifferentiated and early differentiating cells, which was confirmed by quantitative RT-PCR. The association of lncRNAs with polysomes was also comparable to that of mRNAs. Our results suggest that the presence of lncRNAs in the polysomal RNA fraction is not the result of random association. We observed that a high percentage of lncRNAs are actively mobilized to or from polysomes during early stages of adipogenesis. Moreover, we found several lncRNAs that can potentially target miRNAs relevant to adipogenesis.