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International Journal of Molecular... Jun 2024The gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result...
The gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: mRNA expression could be shown for primary CD4 T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect transcripts for CD14 monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8 T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.
Topics: Cystic Fibrosis Transmembrane Conductance Regulator; Humans; RNA, Messenger; Leukocytes, Mononuclear; Blotting, Western; Real-Time Polymerase Chain Reaction; Cystic Fibrosis; Killer Cells, Natural; Flow Cytometry; CD4-Positive T-Lymphocytes
PubMed: 38928073
DOI: 10.3390/ijms25126367 -
International Journal of Molecular... Jun 2024Maturity-onset diabetes of the young (MODY) is part of the heterogeneous group of monogenic diabetes (MD) characterized by the non-immune dysfunction of pancreatic...
Maturity-onset diabetes of the young (MODY) is part of the heterogeneous group of monogenic diabetes (MD) characterized by the non-immune dysfunction of pancreatic β-cells. The diagnosis of MODY still remains a challenge for clinicians, with many cases being misdiagnosed as type 1 or type 2 diabetes mellitus (T1DM/T2DM), and over 80% of cases remaining undiagnosed. With the introduction of modern technologies, important progress has been made in deciphering the molecular mechanisms and heterogeneous etiology of MD, including MODY. The aim of our study was to identify genetic variants associated with MODY in a group of patients with early-onset diabetes/prediabetes in whom a form of MD was clinically suspected. Genetic testing, based on next-generation sequencing (NGS) technology, was carried out either in a targeted manner, using gene panels for monogenic diabetes, or by analyzing the entire exome (whole-exome sequencing). -MODY 2 was the most frequently detected variant, but rare forms of -MODY 13, specifically, -MODY 1, were also identified. We have emphasized the importance of genetic testing for early diagnosis, MODY subtype differentiation, and genetic counseling. We presented the genotype-phenotype correlations, especially related to the clinical evolution and personalized therapy, also emphasizing the particularities of each patient in the family context.
Topics: Humans; Diabetes Mellitus, Type 2; Genetic Testing; Male; Female; Genetic Counseling; Adult; Precision Medicine; High-Throughput Nucleotide Sequencing; Adolescent; Potassium Channels, Inwardly Rectifying; Young Adult; Child; Hepatocyte Nuclear Factor 4; Exome Sequencing; Genetic Predisposition to Disease; Mutation
PubMed: 38928025
DOI: 10.3390/ijms25126318 -
International Journal of Molecular... Jun 2024The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and...
The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca concentration in hippocampal neurons. Ca responses to the E protein are due to Ca release from intracellular stores at the ER. Moreover, E protein-induced Ca release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca, probably acting like a viroporin, thus producing Ca store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.
Topics: Animals; Rats; Neurons; Hippocampus; Calcium; Endoplasmic Reticulum; SARS-CoV-2; Coronavirus Envelope Proteins; COVID-19; Cells, Cultured; Apoptosis; Primary Cell Culture; Cell Death; Viroporin Proteins
PubMed: 38928009
DOI: 10.3390/ijms25126304 -
Genes Jun 2024Dysfunction in ion channels or processes involved in maintaining ionic homeostasis is thought to lower the threshold for cortical spreading depression (CSD), and plays a...
Dysfunction in ion channels or processes involved in maintaining ionic homeostasis is thought to lower the threshold for cortical spreading depression (CSD), and plays a role in susceptibility to associated neurological disorders, including pathogenesis of a migraine. Rare pathogenic variants in specific ion channels have been implicated in monogenic migraine subtypes. In this study, we further examined the channelopathic nature of a migraine through the analysis of common genetic variants in three selected ion channel or transporter genes: , , and . Using the Agena MassARRAY platform, 28 single-nucleotide polymorphisms (SNPs) across the three candidate genes were genotyped in a case-control cohort comprised of 182 migraine cases and 179 matched controls. Initial results identified significant associations between migraine and rs3776578 ( = 0.04) and rs16903247 ( = 0.05) genotypes within the gene, which encodes the EAAT1 glutamate transporter. These SNPs were subsequently genotyped in an independent cohort of 258 migraine cases and 290 controls using a high-resolution melt assay, and association testing supported the replication of initial findings-rs3776578 ( = 0.0041) and rs16903247 ( = 0.0127). The polymorphisms are in linkage disequilibrium and localise within a putative intronic enhancer region of . The minor alleles of both SNPs show a protective effect on migraine risk, which may be conferred via influencing the expression of .
Topics: Humans; Polymorphism, Single Nucleotide; Migraine Disorders; Female; Male; Excitatory Amino Acid Transporter 1; Adult; Case-Control Studies; Genetic Predisposition to Disease; Middle Aged; Genetic Association Studies
PubMed: 38927733
DOI: 10.3390/genes15060797 -
Genes Jun 2024In humans, the transient receptor potential vanilloid 1 () gene is activated by exogenous (e.g., high temperatures, irritating compounds such as capsaicin) and...
In humans, the transient receptor potential vanilloid 1 () gene is activated by exogenous (e.g., high temperatures, irritating compounds such as capsaicin) and endogenous (e.g., endocannabinoids, inflammatory factors, fatty acid metabolites, low pH) stimuli. It has been shown to be involved in several processes including nociception, thermosensation, and energy homeostasis. In this study, we investigated the association between gene variants, sensory perception (to capsaicin and PROP), and body composition (BMI and bioimpedance variables) in human populations. By comparing sequences deposited in worldwide databases, we identified two haplotype blocks (herein referred to as H1 and H2) that show strong stabilizing selection signals (MAF approaching 0.50, Tajima's D > +4.5) only in individuals with sub-Saharan African ancestry. We therefore studied the genetic variants of these two regions in 46 volunteers of sub-Saharan descent and 45 Italian volunteers (both sexes). Linear regression analyses showed significant associations between diplotypes and body composition, but not with capsaicin perception. Specifically, in African women carrying the H1-b and H2-b haplotypes, a higher percentage of fat mass and lower extracellular fluid retention was observed, whereas no significant association was found in men. Our results suggest the possible action of sex-driven balancing selection at the non-coding sequences of the gene, with adaptive effects on water balance and lipid deposition.
Topics: Adult; Female; Humans; Male; Middle Aged; Africa South of the Sahara; Black People; Body Composition; Haplotypes; Polymorphism, Single Nucleotide; Sub-Saharan African People; TRPV Cation Channels
PubMed: 38927688
DOI: 10.3390/genes15060752 -
Genes May 2024Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the...
Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the feasibility of earlier prenatal diagnosis by celocentesis in four cases of cystic fibrosis and one case of cystic fibrosis and β-thalassemia co-inherited in the same fetus. Celomic fluids were aspired from the celomic cavity between 8 and 9 weeks of gestation and fetal cells were picked up by micromanipulator. Maternal DNA contamination was tested and target regions of fetal DNA containing parental pathogenetic variants of and genes were amplified and sequenced. Four of the five fetuses resulted as being affected by cystic fibrosis and, in all cases, the women decided to interrupt the pregnancy. In the other case, the fetus presented a healthy carrier of cystic fibrosis. The results were confirmed in three cases on placental tissue. In one case, no abortive tissue was obtained. In the last case, the woman refused the prenatal diagnosis to confirm the celocentesis data; the pregnancy is ongoing without complications. This procedure provides prenatal diagnosis of monogenic diseases at least four weeks earlier than traditional procedures, reducing the anxiety of patients and providing the option for medical termination of the affected fetus at 8-10 weeks of gestation, which is less traumatic and safer than surgical termination in the second trimester.
Topics: Humans; Cystic Fibrosis; Female; Pregnancy; Prenatal Diagnosis; Cystic Fibrosis Transmembrane Conductance Regulator; Adult; beta-Thalassemia; Fetus
PubMed: 38927598
DOI: 10.3390/genes15060662 -
Biomedicines May 2024A sodium current (I) reduction occurs in the setting of many acquired and inherited conditions and is associated with cardiac conduction slowing and increased arrhythmia...
A sodium current (I) reduction occurs in the setting of many acquired and inherited conditions and is associated with cardiac conduction slowing and increased arrhythmia risks. The sodium channel blocker mexiletine has been shown to restore the trafficking of mutant sodium channels to the membrane. However, these studies were mostly performed in heterologous expression systems using high mexiletine concentrations. Moreover, the chronic effects on I in a non-diseased cardiomyocyte environment remain unknown. In this paper, we investigated the chronic and acute effects of a therapeutic dose of mexiletine on I and the action potential (AP) characteristics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of a healthy individual. Control hiPSC-CMs were incubated for 48 h with 10 µM mexiletine or vehicle. Following the wash-out of mexiletine, patch clamp analysis and immunocytochemistry experiments were performed. The incubation of hiPSC-CMs for 48 h with mexiletine (followed by wash-out) induced a significant increase in peak I of ~75%, without any significant change in the voltage dependence of (in)activation. This was accompanied by a significant increase in AP upstroke velocity, without changes in other AP parameters. The immunocytochemistry experiments showed a significant increase in membrane Na1.5 fluorescence following a 48 h incubation with mexiletine. The acute re-exposure of hiPSC-CMs to 10 µM mexiletine resulted in a small but significant increase in AP duration, without changes in AP upstroke velocity, peak I density, or the I voltage dependence of (in)activation. Importantly, the increase in the peak I density and resulting AP upstroke velocity induced by chronic mexiletine incubation was not counteracted by the acute re-administration of the drug. In conclusion, the chronic administration of a clinically relevant concentration of mexiletine increases I density in non-diseased hiPSC-CMs, likely by enhancing the membrane trafficking of sodium channels. Our findings identify mexiletine as a potential therapeutic strategy to enhance and/or restore I and cardiac conduction.
PubMed: 38927420
DOI: 10.3390/biomedicines12061212 -
Biomolecules Jun 2024Abdominal aortic aneurysm (AAA) is a chronic aortic disease that lacks effective pharmacological therapies. This study was performed to determine the influence of...
Abdominal aortic aneurysm (AAA) is a chronic aortic disease that lacks effective pharmacological therapies. This study was performed to determine the influence of treatment with the gasdermin D inhibitor necrosulfonamide on experimental AAAs. AAAs were induced in male apolipoprotein E-deficient mice by subcutaneous angiotensin II infusion (1000 ng/kg body weight/min), with daily administration of necrosulfonamide (5 mg/kg body weight) or vehicle starting 3 days prior to angiotensin II infusion for 30 days. Necrosulfonamide treatment remarkably suppressed AAA enlargement, as indicated by reduced suprarenal maximal external diameter and surface area, and lowered the incidence and reduced the severity of experimental AAAs. Histologically, necrosulfonamide treatment attenuated medial elastin breaks, smooth muscle cell depletion, and aortic wall collagen deposition. Macrophages, CD4 T cells, CD8 T cells, and neovessels were reduced in the aneurysmal aortas of necrosulfonamide- as compared to vehicle-treated angiotensin II-infused mice. Atherosclerosis and intimal macrophages were also substantially reduced in suprarenal aortas from angiotensin II-infused mice following necrosulfonamide treatment. Additionally, the levels of serum interleukin-1β and interleukin-18 were significantly lower in necrosulfonamide- than in vehicle-treated mice without affecting body weight gain, lipid levels, or blood pressure. Our findings indicate that necrosulfonamide reduced experimental AAAs by preserving aortic structural integrity as well as reducing mural leukocyte accumulation, neovessel formation, and systemic levels of interleukin-1β and interleukin-18. Thus, pharmacologically inhibiting gasdermin D activity may lead to the establishment of nonsurgical therapies for clinical AAA disease.
Topics: Animals; Angiotensin II; Aortic Aneurysm, Abdominal; Mice; Male; Sulfonamides; Apolipoproteins E; Phosphate-Binding Proteins; Disease Models, Animal; Mice, Inbred C57BL; Macrophages; Indoles; Mice, Knockout, ApoE; Gasdermins
PubMed: 38927129
DOI: 10.3390/biom14060726 -
Biomolecules Jun 2024pH homeostasis is crucial for spermatogenesis, sperm maturation, sperm physiological function, and fertilization in mammals. HCO and H are the most significant factors... (Review)
Review
pH homeostasis is crucial for spermatogenesis, sperm maturation, sperm physiological function, and fertilization in mammals. HCO and H are the most significant factors involved in regulating pH homeostasis in the male reproductive system. Multiple pH-regulating transporters and ion channels localize in the testis, epididymis, and spermatozoa, such as HCO transporters (solute carrier family 4 and solute carrier family 26 transporters), carbonic anhydrases, and H-transport channels and enzymes (e.g., Na-H exchangers, monocarboxylate transporters, H-ATPases, and voltage-gated proton channels). Hormone-mediated signals impose an influence on the production of some HCO or H transporters, such as NBCe1, SLC4A2, MCT4, etc. Additionally, ion channels including sperm-specific cationic channels for Ca (CatSper) and K (SLO3) are directly or indirectly regulated by pH, exerting specific actions on spermatozoa. The slightly alkaline testicular pH is conducive to spermatogenesis, whereas the epididymis's low HCO concentration and acidic lumen are favorable for sperm maturation and storage. Spermatozoa pH increases substantially after being fused with seminal fluid to enhance motility. In the female reproductive tract, sperm are subjected to increasing concentrations of HCO in the uterine and fallopian tube, causing a rise in the intracellular pH (pH) of spermatozoa, leading to hyperpolarization of sperm plasma membranes, capacitation, hyperactivation, acrosome reaction, and ultimately fertilization. The physiological regulation initiated by SLC26A3, SLC26A8, NHA1, sNHE, and CFTR localized in sperm is proven for certain to be involved in male fertility. This review intends to present the key factors and characteristics of pH regulation in the testes, efferent duct, epididymis, seminal fluid, and female reproductive tract, as well as the associated mechanisms during the sperm journey to fertilization, proposing insights into outstanding subjects and future research trends.
Topics: Male; Hydrogen-Ion Concentration; Humans; Spermatozoa; Animals; Fertilization; Fertility; Female; Spermatogenesis; Homeostasis; Sperm Motility
PubMed: 38927088
DOI: 10.3390/biom14060685 -
Biomolecules Jun 2024This retrospective begins with Galvani's experiments on frogs at the end of the 18th century and his discovery of 'animal electricity'. It goes on to illustrate the... (Review)
Review
This retrospective begins with Galvani's experiments on frogs at the end of the 18th century and his discovery of 'animal electricity'. It goes on to illustrate the numerous contributions to the field of physical chemistry in the second half of the 19th century (Nernst's equilibrium potential, based on the work of Wilhelm Ostwald, Max Planck's ion electrodiffusion, Einstein's studies of Brownian motion) which led Bernstein to propose his membrane theory in the early 1900s as an explanation of Galvani's findings and cell excitability. These processes were fully elucidated by Hodgkin and Huxley in 1952 who detailed the ionic basis of resting and action potentials, but without addressing the question of where these ions passed. The emerging question of the existence of ion channels, widely debated over the next two decades, was finally accepted and, a decade later, many of them began to be cloned. This led to the possibility of modelling the activity of individual neurons in the brain and then that of simple circuits. Taking advantage of the remarkable advances in computer science in the new millennium, together with a much deeper understanding of brain architecture, more ambitious scientific goals were dreamed of to understand the brain and how it works. The retrospective concludes by reviewing the main efforts in this direction, namely the construction of a digital brain, an in silico copy of the brain that would run on supercomputers and behave just like a real brain.
Topics: Animals; Humans; Brain; Ion Channels; History, 20th Century; History, 19th Century; Electricity; History, 18th Century; Models, Neurological
PubMed: 38927086
DOI: 10.3390/biom14060684