Genaro Patino | Hospital Infantil de México Federico Gómez (original) (raw)

Papers by Genaro Patino

Frontiers in Immunology, Aug 18, 2022

Mucosal innate immunity functions as the first line of defense against invading pathogens. Member... more Mucosal innate immunity functions as the first line of defense against invading pathogens. Members of the IL-1 family are key cytokines upregulated in the inflamed mucosa. Inflammatory cytokines are regulated by limiting their function and availability through their activation and secretion mechanisms. IL-1 cytokines secretion is affected by the lack of a signal peptide on their sequence, which prevents them from accessing the conventional protein secretion pathway; thus, they use unconventional protein secretion pathways. Here we show in mouse macrophages that LPS/ATP stimulation induces cytokine relocalization to the plasma membrane, and conventional secretion blockade using monensin or Brefeldin A triggers no IL-36g accumulation within the cell. In silico modeling indicates IL-36g can pass through both the P2X7R and Gasdermin D pores, and both IL-36g, P2X7R and Gasdermin D mRNA are upregulated in inflammation; further, experimental blockade of these receptors' limits IL-36g release. Our results demonstrate that IL-36g is secreted mainly by an unconventional pathway through membrane pores formed by P2X7R and Gasdermin D.

Journal of Immunology, Apr 1, 2009

bioRxiv (Cold Spring Harbor Laboratory), Jun 16, 2022

Protein tyrosine phosphatase 1B (PTP1B) plays a key role in developing different types of cancer.... more Protein tyrosine phosphatase 1B (PTP1B) plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify possible molecular targets of PTP1B that mediate its positive role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify the Cyclin-dependent kinase 3 (Cdk3) as a novel PTP1B substrate. Molecular docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at Tyrosine residue 15 and interacts with it in the nuclear envelope of HEK293-T cells and the nucleus and cytoplasm of human glioblastoma (GB) cells. Finally, we found that the pharmacological inhibition of PTP1B leads to a cell cycle progression delay with the diminished activity of Cdk3, the consequent hypophosphorylation of Rb, and the down-regulation of E2F and its target genes Cdk1, Cyclin A, and Cyclin E1. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells and suggest new therapeutic strategies for treating these tumors. .

Methods in molecular biology, 2019

microRNAs are noncoding RNAs of 20-24 nucleotides (nt) in length that act as repressors of genes ... more microRNAs are noncoding RNAs of 20-24 nucleotides (nt) in length that act as repressors of genes and are important in key developmental processes in the entire life cycle of plants. To determine the function of a microRNA, the first step is to resolve its expression pattern; this can be achieved by in situ hybridization, RNA blot assays, or quantitative PCR. However, the study of the expression of a MIR gene is straightforward with the use of reporter proteins such as β-D-glucuronidase (GUS), GFP, or mCherry. To do this, it is necessary to clone the promoter region of the MIR gene and place it upstream of the reporter gene; in this way the activity of the promoter will be a direct reflection of the expression of the MIR gene. Here, we indicate step by step how to make transcriptional fusion constructs from the cloning of a promoter region of a MIR gene fused to the classical reporter proteins GUS and mCherry, the latter with codon optimization for better expression in Arabidopsis thaliana. This method is particularly useful to dissect the promoter region of a MIR gene and to find its expression pattern in a tissue and developmental specific manner.

Clinical Immunology, 2009

Type 1 diabetes (T1D) is an autoimmune disease characterized by the cell-mediated destruction of ... more Type 1 diabetes (T1D) is an autoimmune disease characterized by the cell-mediated destruction of insulin-S145 Abstracts

Scientific Reports, Oct 22, 2021

Cell spreading and phagocytosis are notably regulated by small GTPases and GAP proteins. TBC1D10C... more Cell spreading and phagocytosis are notably regulated by small GTPases and GAP proteins. TBC1D10C is a dual inhibitory protein with GAP activity. In immune cells, TBC1D10C is one of the elements regulating lymphocyte activation. However, its specific role in macrophages remains unknown. Here, we show that TBC1D10C engages in functions dependent on the cytoskeleton and plasma membrane reorganization. Using ex vivo and in vitro assays, we found that elimination and overexpression of TBC1D10C modified the cytoskeletal architecture of macrophages by decreasing and increasing the spreading ability of these cells, respectively. In addition, TBC1D10C overexpression contributed to higher phagocytic activity against Burkholderia cenocepacia and to increased cell membrane tension. Furthermore, by performing in vitro and in silico analyses, we identified 27 TBC1D10C-interacting proteins, some of which were functionally classified as protein complexes involved in cytoskeletal dynamics. Interestingly, we identified one unreported TBC1D10C-intrinsically disordered region (IDR) with biological potential at the cytoskeleton level. Our results demonstrate that TBC1D10C shapes macrophage activity by inducing reorganization of the cytoskeleton-plasma membrane in cell spreading and phagocytosis. We anticipate our results will be the basis for further studies focused on TBC1D10C. For example, the specific molecular mechanism in Burkholderia cenocepacia phagocytosis and functional analysis of TBC1D10C-IDR are needed to further understand its role in health and disease. Abbreviations BMDM Bone marrow-derived macrophage GAP GTPase-activating protein KO Knockout WT Wild type Burkholderia cenocepacia B. cenocepacia IDR Intrinsically disordered region PRR Pattern recognition receptor DN Dominant negative CA Constitutively active average Avg TBC1D10C (also known as Epi64C or Carabin) is an abundant protein in spleen and peripheral blood leukocytes. It is a member of the EPI64 subfamily, a group of related proteins (TBC1D10A/EPI64A, TBC1D10B/EPI64B,

Developmental and Comparative Immunology, Feb 1, 2010

CRTAM was reported as a novel receptor expressed in activated NKT and CD8 T lymphocytes. However,... more CRTAM was reported as a novel receptor expressed in activated NKT and CD8 T lymphocytes. However, we have recently shown that it is also expressed in several non-immune tissues. In opposition to what has been stated for lymphoid cells, CRTAM expression is constitutive in epithelia, suggesting a role in cell-cell interactions. Given the importance of cell interactions during T lymphocyte development, we evaluated CRTAM during T lymphocyte ontogeny. Here we show that CRTAM has an unexpected constitutive expression in adult thymocytes and, remarkably, it is sustained during all stages of thymocyte development. CRTAM expression is restricted to CD8 and all DN subpopulations, with a consistent pattern from E13.5 stage to adult mice. Blocking CRTAM interaction with CADM1 impairs thymus growth, uncovering a novel role in thymus development, with a consequent impact in thymocyte maturation. Thus, CRTAM interaction with CADM1 is involved in structural maintenance of the thymic lobes.

Molecular Immunology, Oct 1, 2009

Class-I MHC-restricted T-cell associated molecule (CRTAM) is a member of the Nectin-like adhesion... more Class-I MHC-restricted T-cell associated molecule (CRTAM) is a member of the Nectin-like adhesion molecule family. It is rapidly induced in NK, NKT and CD8 + T cells. Interaction with its ligand Nectin-like 2 results in increased secretion of IFN-␥ by activated CD8 + T lymphocytes. Through sequential bioinformatic analyses of the upstream region of the human CRTAM gene, we detected cis-elements potentially important for CRTAM gene transcription. Analyzing 2 kb upstream from the ATG translation codon by mutation analysis in conjunction with luciferase reporter assays, electrophoretic mobility shify assay (EMSA) and supershift assays, we identified an AP-1 binding site, located at 1.4 kb from the ATG translation codon of CRTAM gene as an essential element for CRTAM expression in activated but not resting human CD8 + T cells. CRTAM expression was reduced in activated CD8 + T cells treated with the JNK inhibitor SP600125, indicating that CRTAM expression is driven by the JNK-AP-1 signaling pathway. This study represents the first CRTAM gene promoter analysis in human T cells and indicates that AP-1 is a positive transcriptional regulator of this gene, a likely important finding because CRTAM has recently been shown to play a role in IFN-␥ and IL-17 production and T cell proliferation.

Boletín médico del Hospital Infantil de México, Jun 23, 2023

Background: Myosin 1g (Myo1g) has recently been identified as a potential diagnostic biomarker in... more Background: Myosin 1g (Myo1g) has recently been identified as a potential diagnostic biomarker in childhood acute lymphocytic leukemia (ALL). Case report: We describe the case of a 1-year-old Mexican female patient. Although initially studied for hepatomegaly, an infectious or genetic etiology was excluded. Liver biopsy showed infiltration by neoplastic B-cell precursors (BCPs), and bone marrow (BM) aspirate showed 14.5% of BCPs. In a joint session of the oncology, hematology, and pathology departments, low-risk (LR) BCP-ALL of hepatic origin with aberrant myeloid markers was diagnosed. Although treatment was initiated, the patient presented early with BM relapse. Modest overexpression of Myo1g was observed from the onset. However, at the end of the steroid window, expression increased significantly and remained elevated during this first relapse to BM. The parents refused hematopoietic stem cell transplantation, but she continued chemotherapy. After a second BM relapse at 5 years of age, the phenotype switched to myeloid. Her parents then opted for palliative care, and the patient died two months later at home. Conclusions: This case shows the potential use of Myo1g in clinical practice as a high-risk indicator. Myo1g monitoring may reveal a high risk and relapse trend, even when typical parameter values are not altered: Myo1g could be used to classify patients from low to high risk from diagnosis, allowing patients to promptly receive the best treatment and potentially modifying prognosis and survival.

Boletín médico del Hospital Infantil de México, May 1, 2017

Human Immunology, Aug 1, 2022

PLOS ONE, Aug 15, 2022

Severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 infection in children and adolescent... more Severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 infection in children and adolescents primarily causes mild or asymptomatic coronavirus disease 2019 (COVID-19), and severe illness is mainly associated with comorbidities. However, the worldwide prevalence of COVID-19 in this population is only 1%-2%. In Mexico, the prevalence of COVID-19 in children has increased to 10%. As serology-based studies are scarce, we analyzed the clinical features and serological response (SARS-CoV-2 structural proteins) of children and adolescents who visited the Hospital Infantil de Mé xico Federico Gó mez (October 2020-March 2021). The majority were 9-year-old children without comorbidities who were treated as outpatients and had mild-to-moderate illness. Children aged 6-10 years and adolescents aged 11-15 years had the maximum number of symptoms, including those with obesity. Nevertheless, children with comorbidities such as immunosuppression, leukemia, and obesity exhibited the lowest antibody response, whereas those aged 1-5 years with heart disease had the highest levels of antibodies. The SARS-CoV-2 spike receptor-binding

Frontiers in Microbiology, Jan 14, 2022

World Health Organization (WHO) has prioritized the infectious emerging diseases such as Coronavi... more World Health Organization (WHO) has prioritized the infectious emerging diseases such as Coronavirus Disease (COVID-19) in terms of research and development of effective tests, vaccines, antivirals, and other treatments. Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), the etiological causative agent of COVID-19, is a virus belonging to risk group 3 that requires Biosafety Level (BSL)-3 laboratories and the corresponding facilities for handling. An alternative to these BSL-3/-4 laboratories is to use a pseudotyped virus that can be handled in a BSL-2 laboratory for study purposes. Recombinant Vesicular Stomatitis Virus (VSV) can be generated with complementary DNA from complete negative-stranded genomic RNA, with deleted G glycoprotein and, instead, incorporation of other fusion protein, like SARS-CoV-2 Spike (S protein). Accordingly, it is called pseudotyped VSV-SARS-CoV-2 S. In this review, we have described the generation of pseudotyped VSV with a focus on the optimization and application of pseudotyped VSV-SARS-CoV-2 S. The application of this pseudovirus has been addressed by its use in neutralizing antibody assays in order to evaluate a new vaccine, emergent SARS-CoV-2 variants (delta and omicron), and approved vaccine efficacy against variants of concern as well as in viral fusion-focused treatment analysis that can be performed under BSL-2 conditions.

Pathogens, Feb 6, 2020

Urinary tract infection (UTI) is a relevant public health problem, economically and socially affe... more Urinary tract infection (UTI) is a relevant public health problem, economically and socially affecting the lives of patients. The increase of antimicrobial bacterial resistance significantly hinders the treatment of UTIs, raising the need to search for alternative therapies. Bacterial lysates (BL) obtained from Escherichia coli and other pathogens have been used to treat different infectious diseases with promising results. This work aims to evaluate the effect and composition of an autologous BL for the treatment and control of recurrent UTIs in adults. The results show remission in 70% of the patients within the first three months after the administration of BL, while the infection is maintained under control for 6-12 months. The analysis by liquid chromatography-mass spectrometry (LC-MS) of the BL fractions recognized by the sera of patients shows the presence of cytosolic proteins, fimbriae, OMPs, and LPS. Our study demonstrates that the autologous BL contributed to the treatment and control of recurrent UTIs in adults, and its composition shows that different surface components of E. coli are potential immunogens that could be used to create a polyvalent protective vaccine.

Journal of Proteome Research, May 28, 2008

Lymphocyte microvilli mediate initial adhesion to endothelium during lymphocyte transition from b... more Lymphocyte microvilli mediate initial adhesion to endothelium during lymphocyte transition from blood into tissue but their molecular organization is incompletely understood. We modified a shearbased procedure to prepare biochemical fractions enriched for membrane/microvilli (MMV) from both human peripheral blood T-lymphocytes (PBT) and a mouse pre-B lymphocyte line (300.19). Enrichment of proteins in MMV relative to post nuclear lysate was determined by LC/MS/MS analysis and label-free quantitation. Subsequent analysis emphasized the 291 proteins shared by PBT and 300.19 and estimated by MS peak area to be highest abundance. Validity of the label-free quantitation was confirmed by many internal consistencies and by comparison with Western blot analyses. The MMV fraction was enriched primarily for subsets of cytoskeletal proteins, transmembrane proteins and G-proteins, with similar patterns in both lymphoid cell types. The most enriched cytoskeletal proteins were microfilament-related proteins NHERF1, Ezrin/Radixin/Moesin (ERMs), ADF/cofilin and Myosin1G. Other microfilament proteins such as talin, gelsolin, myosin II and profilin were markedly reduced in MMV, as were intermediate filament-and microtubulerelated proteins. Heterotrimeric G-proteins and some small G-proteins (especially Ras and Rap1) were enriched in the MMV preparation. Two notable general observations also emerged. There was less overlap between the two cells in their transmembrane proteins than in other classes of proteins, consistent with a special role of plasma membrane proteins in differentiation. Second, unstimulated primary T-lymphocytes have an unusually high concentration of actin and other microfilament related proteins, consistent with the singular role of actin-mediated motility in the immunological surveillance performed by these primary cells. Lymphocyte microvilli initiate adhesion to endothelium during movement from blood into tissue. Using LC/MS/MS and label-free quantitation, we identify proteins enriched in membrane/microvilli (MMV) fractions from lymphocytes (primary human T-lymphocytes and mouse pre-B lymphocyte line). The cytoskeletal proteins most enriched in both lymphocyte types are microfilament-related proteins NHERF1, Ezrin/Radixin/Moesin (ERMs), ADF/cofilin and Myosin1G. Heterotrimeric Gproteins and some small G-proteins (especially Ras and Rap1) are also enriched. Complementary approaches provide confirmation.

European Journal of Immunology, Jan 16, 2014

Myosin 1g (Myo1g) is a hematopoietic-specific myosin expressed mainly by lymphocytes. Here, we re... more Myosin 1g (Myo1g) is a hematopoietic-specific myosin expressed mainly by lymphocytes. Here, we report the localization of Myo1g in B-cell membrane compartments such as lipid rafts, microvilli, and membrane extensions formed during spreading. By using Myo1gdeficient mouse B cells, we detected abnormalities in the adhesion ability and chemokineinduced directed migration of these lymphocytes. We also assessed a role for Myo1g in phagocytosis and exocytosis processes, as these were also irregular in Myo1g-deficient B cells. Taken together, our results show that Myo1g acts as a main regulator of different membrane/cytoskeleton-dependent processes in B lymphocytes.

Journal of Biological Chemistry, Mar 1, 2010

Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spect... more Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spectrometric analysis of lymphocytes identified two short tail forms: (Myo1G and Myo1C) and one long tail (Myo1F). We investigated Myo1G, the most abundant in T-lymphocytes, and compared key findings with Myo1C and Myo1F. Myo1G localizes to the plasma membrane and associates in an ATP-releasable manner to the actin-containing insoluble pellet. The IQ؉tail region of Myo1G (Myo1C and Myo1F) is sufficient for membrane localization, but membrane localization is augmented by the motor domain. The minimal region lacks IQ motifs but includes: 1) a PH-like domain; 2) a "Pre-PH" region; and 3) a "Post-PH" region. The Pre-PH predicted ␣ helices may contribute electrostatically, because two conserved basic residues on one face are required for optimal membrane localization. Our sequence analysis characterizes the divergent PH domain family, Myo1PH, present also in long tail myosins, in eukaryotic proteins unrelated to myosins, and in a probable ancestral protein in prokaryotes. The Myo1G Myo1PH domain utilizes the classic lipid binding site for membrane association, because mutating either of two basic residues in the "signature motif" destroys membrane localization. Mutation of each basic residue of the Myo1G Myo1PH domain reveals another critical basic residue in the ␤3 strand, which is shared only by Myo1D. Myo1G differs from Myo1C in its phosphatidylinositol 4,5-bisphosphate dependence for membrane association, because membrane localization of phosphoinositide 5-phosphatase releases Myo1C from the membrane but not Myo1G. Thus Myo1PH domains likely play universal roles in myosin I membrane association, but different isoforms have diverged in their binding specificity.

Cell, Jul 1, 2014

To mount an immune response, T lymphocytes must successfully search for foreign material bound to... more To mount an immune response, T lymphocytes must successfully search for foreign material bound to the surface of antigen-presenting cells. How T cells optimize their chances of encountering and responding to these antigens is unknown. T cell motility in tissues resembles a random or Levy walk and is regulated in part by external factors including chemokines and lymph-node topology, but motility parameters such as speed and propensity to turn may also be cell intrinsic. Here we found that the unconventional myosin 1g (Myo1g) motor generates membrane tension, enforces cell-intrinsic meandering search, and enhances T-DC interactions during lymph-node surveillance. Increased turning and meandering motility, as opposed to ballistic motility, is enhanced by Myo1g. Myo1g acts as a ''turning motor'' and generates a form of cellular ''flâ nerie.'' Modeling and antigen challenges show that these intrinsically programmed elements of motility search are critical for the detection of rare cognate antigen-presenting cells.