Expression analysis of miRNA hsa-let7b-5p in naso-oropharyngeal swabs of COVID-19 patients supports its role in regulating ACE2 and DPP4 receptors.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the novel coronavirus responsible for worldwide coronavirus disease (COVID-19). We previously observed that Angiotensin-converting enzyme 2 (ACE2) and Dipeptidyl peptidase-4 (DPP4) are significantly overexpressed in naso-oropharyngeal swabs (NPS) of COVID-19 patients, suggesting their putative functional role in the disease progression. ACE2 and DPP4 overexpression in COVID-19 patients may be associated to epigenetic mechanism, such as miRNA differential expression. We investigated if hsa-let7b-5p, reported to target both ACE2 and DPP4 transcripts, could be involved in the regulation of these genes. We verified that the inhibition and overexpression of hsa-let7b-5p matched to a modulation of both ACE2 and DPP4 levels. Then, we observed a statistically significant downregulation (FC = -1.5; p < 0.05) of hsa-let7b-5p in the same COVID-19 and control samples of our previous study. This is the first study that shows hsa-let7b-5p low expression in naso-oropharyngeal swabs of COVID-19 patients and demonstrates a functional role of this miR in regulating ACE2 and DPP4 levels. These data suggest the involvement of hsa-let7b-5p in the regulation of genes necessary for SARS-CoV-2 infections and its putative role as a therapeutic target for COVID-19.

Application of CRISPR/Cas9 to human-induced pluripotent stem cells: from gene editing to drug discovery.

Human-induced pluripotent stem cells (hiPSCs) and CRISPR/Cas9 gene editing system represent two instruments of basic and translational research, which both allow to acquire deep insight about the molecular bases of many diseases but also to develop pharmacological research.This review is focused to draw up the latest technique of gene editing applied on hiPSCs, exploiting some of the genetic manipulation directed to the discovery of innovative therapeutic strategies. There are many expediencies provided by the use of hiPSCs, which can represent a disease model clinically relevant and predictive, with a great potential if associated to CRISPR/Cas9 technology, a gene editing tool powered by ease and precision never seen before.Here, we describe the possible applications of CRISPR/Cas9 to hiPSCs: from drug development to drug screening and from gene therapy to the induction of the immunological response to specific virus infection, such as HIV and SARS-Cov-2.

Modelling the pathogenesis of Myotonic Dystrophy type 1 cardiac phenotype through human iPSC-derived cardiomyocytes.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease, autosomal dominant, caused by a CTG repeat expansion in DMPK gene. We assessed the appropriateness of patient-specific induced pluripotent stem cell-derived cardiomyocytes (CMs) as a model to recapitulate some aspects of the pathogenetic mechanism involving cardiac manifestations in DM1 patients. Once obtained in vitro, CMs have been characterized for their morphology and their functionality. CMs DM1 show intranuclear foci and transcript markers abnormally spliced respect to WT ones, as well as several irregularities in nuclear morphology, probably caused by an unbalanced lamin A/C ratio. Electrophysiological characterization evidences an abnormal profile only in CMs DM1 such that the administration of antiarrythmic drugs to these cells highlights even more the functional defect linked to the disease. Finally, Atomic Force Measurements reveal differences in the biomechanical behaviour of CMs DM1, in terms of frequencies and synchronicity of the beats. Altogether the complex phenotype described in this work, strongly reproduces some aspects of the human DM1 cardiac phenotype. Therefore, the present study provides an in vitro model suggesting novel insights into the mechanisms leading to the development of arrhythmogenesis and dilatative cardiomyopathy to consider when approaching to DM1 patients, especially for the risk assessment of sudden cardiac death (SCD). These data could be also useful in identifying novel biomarkers effective in clinical settings and patient-tailored therapies.

A surge of late-occurring meiotic double-strand breaks rescues synapsis abnormalities in spermatocytes of mice with hypomorphic expression of SPO11.

Meiosis is the biological process that, after a cycle of DNA replication, halves the cellular chromosome complement, leading to the formation of haploid gametes. Haploidization is achieved via two successive rounds of chromosome segregation, meiosis I and II. In mammals, during prophase of meiosis I, homologous chromosomes align and synapse through a recombination-mediated mechanism initiated by the introduction of DNA double-strand breaks (DSBs) by the SPO11 protein. In male mice, if SPO11 expression and DSB number are reduced below heterozygosity levels, chromosome synapsis is delayed, chromosome tangles form at pachynema, and defective cells are eliminated by apoptosis at epithelial stage IV at a spermatogenesis-specific endpoint. Whether DSB levels produced in Spo11 (+/-) spermatocytes represent, or approximate, the threshold level required to guarantee successful homologous chromosome pairing is unknown. Using a mouse model that expresses Spo11 from a bacterial artificial chromosome, within a Spo11 (-/-) background, we demonstrate that when SPO11 expression is reduced and DSBs at zygonema are decreased (approximately 40 % below wild-type level), meiotic chromosome pairing is normal. Conversely, DMC1 foci number is increased at pachynema, suggesting that under these experimental conditions, DSBs are likely made with delayed kinetics at zygonema. In addition, we provide evidences that when zygotene-like cells receive enough DSBs before chromosome tangles develop, chromosome synapsis can be completed in most cells, preventing their apoptotic elimination.

LOX-1 and Its Splice Variants: A New Challenge for Atherosclerosis and Cancer-Targeted Therapies.

Alternative splicing (AS) is a process in which precursor messenger RNA (pre-mRNA) splicing sites are differentially selected to diversify the protein isoform population. Changes in AS patterns have an essential role in normal development, differentiation and response to physiological stimuli. It is documented that AS can generate both "risk" and "protective" splice variants that can contribute to the pathogenesis of several diseases including atherosclerosis. The main endothelial receptor for oxidized low-density lipoprotein (ox-LDLs) is LOX-1 receptor protein encoded by the OLR1 gene. When OLR1 undergoes AS events, it generates three variants: OLR1, OLR1D4 and LOXIN. The latter lacks exon 5 and two-thirds of the functional domain. Literature data demonstrate a protective role of LOXIN in pathologies correlated with LOX-1 overexpression such as atherosclerosis and tumors. In this review, we summarize recent developments in understanding of OLR1 AS while also highlighting data warranting further investigation of this process as a novel therapeutic target.

Early Hippocampal i-LTP and LOX-1 Overexpression Induced by Anoxia: A Potential Role in Neurodegeneration in NPC Mouse Model.

Niemann-Pick type C disease (NPCD) is an autosomal recessive storage disorder, characterized by abnormal sequestration of unesterified cholesterol within the late endo-lysosomal compartment of cells. In the central nervous system, hypoxic insults could result in low-density lipoprotein (LDL) oxidation and Lectin-like oxidized LDL receptor-1 (LOX-1) induction, leading to a pathological hippocampal response, namely, ischemic long-term potentiation (i-LTP). These events may correlate with the progressive neural loss observed in NPCD. To test these hypotheses, hippocampal slices from Wild Type (WT) and NPC1-/- mice were prepared, and field potential in the CA1 region was analyzed during transient oxygen/glucose deprivation (OGD). Moreover, LOX-1 expression was evaluated by RT-qPCR, immunocytochemical, and Western blot analyses before and after an anoxic episode. Our results demonstrate the development of a precocious i-LTP in NPC1-/- mice during OGD application. We also observed a higher expression of LOX-1 transcript and protein in NPC1-/- mice with respect to WT mice; after anoxic damage to LOX-1 expression, a further increase in both NPC1-/- and WT mice was observed, although the protein expression seems to be delayed, suggesting a different kinetic of induction. These data clearly suggest an elevated susceptibility to neurodegeneration in NPC1-/- mice due to oxidative stress. The observed up-regulation of LOX-1 in the hippocampus of NPC1-/- mice may also open a new scenario in which new biomarkers can be identified.

Adenosine Triphosphate stimulates differentiation and mineralization in human osteoblast-like Saos-2 cells.

In the last years adenosine triphosphate (ATP) and subsequent purinergic system activation through P2 receptors were investigated highlighting their pivotal role in bone tissue biology. In osteoblasts ATP can regulate several activities like cell proliferation, cell death, cell differentiation and matrix mineralization. Since controversial results exist, in this study we analyzed the ATP effects on differentiation and mineralization in human osteoblast-like Saos-2 cells. We showed for the first time the altered functional activity of ATP receptors. Despite that, we found that ATP can reduce cell proliferation and stimulate osteogenic differentiation mainly in the early stages of in vitro maturation as evidenced by the enhanced expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and Osteocalcin (OC) genes and by the increased ALP activity. Moreover, we found that ATP can affect mineralization in a biphasic manner, at low concentrations ATP always increases mineral deposition while at high concentrations it always reduces mineral deposition. In conclusion, we show the osteogenic effect of ATP on both early and late stage activities like differentiation and mineralization, for the first time in human osteoblastic cells.

SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression.

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.

A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

Cell Electrokinetic Fingerprint: A Novel Approach Based on Optically Induced Dielectrophoresis (ODEP) for In-Flow Identification of Single Cells.

A novel optically induced dielectrophoresis (ODEP) system that can operate under flow conditions is designed for automatic trapping of cells and subsequent induction of 2D multi-frequency cell trajectories. Like in a "ping-pong" match, two virtual electrode barriers operate in an alternate mode with varying frequencies of the input voltage. The so-derived cell motions are characterized via time-lapse microscopy, cell tracking, and state-of-the-art machine learning algorithms, like the wavelet scattering transform (WST). As a cell-electrokinetic fingerprint, the dynamic of variation of the cell displacements happening, over time, is quantified in response to different frequency values of the induced electric field. When tested on two biological scenarios in the cancer domain, the proposed approach discriminates cellular dielectric phenotypes obtained, respectively, at different early phases of drug-induced apoptosis in prostate cancer (PC3) cells and for differential expression of the lectine-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcript levels in human colorectal adenocarcinoma (DLD-1) cells. The results demonstrate increased discrimination of the proposed system and pose an additional basis for making ODEP-based assays addressing cancer heterogeneity for precision medicine and pharmacological research.

From cue to meaning: The involvement of POLD1 gene in DNA replication, repair and aging.

Aging is an extremely complex biological process. Aging, cancer and inflammation represent a trinity, object of many interesting researches. The accumulation of DNA damage and its consequences progressively interfere with cellular function and increase susceptibility to developing aging condition. DNA Polymerase delta (Pol δ), encoded by POLD1 gene (MIM#174761) on 19q13.3, is well implicated in many steps of the replication program and repair. Thanks to its exonuclease and polymerase activities, the enzyme is involved in the regulation of the cell cycle, DNA synthesis, and DNA damage repair processes. Damaging variants within the exonuclease domain predispose to cancers, while those occurring in the polymerase active site cause the autosomal dominant Progeroid Syndrome called MDPL, Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy Since DNA damage represents the main cause of ageing and age-related pathologies, an overview of critical Pol δ activities will allow to better understand the associations between DNA damage and nearly every aspect of the ageing process, helping the researchers to counteract all the ageing-pathologies at the same time.

COVID-19: S-Peptide RBD 484-508 Induces IFN-γ T-Cell Response in Naïve-to-Infection and Unvaccinated Subjects with Close Contact with SARS-CoV-2-Positive Patients.

Despite the availability on the market of different anti-SARS-CoV-2 vaccines, there are still unanswered questions on whether they can stimulate long-lasting protection. A deep understanding of adaptive immune response to SARS-CoV-2 is important for optimizing both vaccine development and pandemic control measures. Among cytokines secreted by lymphocytes in response to viral infection, IFN-γ plays a pivotal role both in innate and adaptive immunity. In this study, we report on 28 naïve-to-SARS-Cov-2-infection and unvaccinated subjects, having reported a close and prolonged contact with COVID-19-positive patients. Samples were tested for defective genetic variants in interferon pathway genes by whole exome sequencing and anti-IFN autoantibodies production was investigated. Subject T-cells were cultured and infected with pseudotype particles bearing the S proteins and in parallel stimulated with two S-peptides designed on the RBD region of the spike protein. Our results showed that one of these peptides, RBD 484-508, induces a significant increase in IFN-γ gene expression and protein production in T-cells, comparable to those obtained in cells infected by SARS-CoV-2 pseudovirus. This work deepens our understanding of immune response and highlights the selected peptide as a reasonable approach to induce broad, potent, and variant concern-independent T-cell responses.

IPLEX administration improves motor neuron survival and ameliorates motor functions in a severe mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disorder and the first genetic cause of death in childhood. SMA is caused by low levels of survival motor neuron (SMN) protein that induce selective loss of α-motor neurons (MNs) in the spinal cord, resulting in progressive muscle atrophy and consequent respiratory failure. To date, no effective treatment is available to counteract the course of the disease. Among the different therapeutic strategies with potential clinical applications, the evaluation of trophic and/or protective agents able to antagonize MNs degeneration represents an attractive opportunity to develop valid therapies. Here we investigated the effects of IPLEX (recombinant human insulinlike growth factor 1 [rhIGF-1] complexed with recombinant human IGF-1 binding protein 3 [rhIGFBP-3]) on a severe mouse model of SMA. Interestingly, molecular and biochemical analyses of IGF-1 carried out in SMA mice before drug administration revealed marked reductions of IGF-1 circulating levels and hepatic mRNA expression. In this study, we found that perinatal administration of IPLEX, even if does not influence survival and body weight of mice, results in reduced degeneration of MNs, increased muscle fiber size and in amelioration of motor functions in SMA mice. Additionally, we show that phenotypic changes observed are not SMN-dependent, since no significant SMN modification was addressed in treated mice. Collectively, our data indicate IPLEX as a good therapeutic candidate to hinder the progression of the neurodegenerative process in SMA.

Organoid factory: The recent role of the human induced pluripotent stem cells (hiPSCs) in precision medicine.

During the last decades, hiPSC-derived organoids have been extensively studied and used as in vitro models for several applications among which research studies. They can be considered as organ and tissue prototypes, especially for those difficult to obtain. Moreover, several diseases can be accurately modeled and studied. Hence, patient-derived organoids (PDOs) can be used to predict individual drug responses, thus paving the way toward personalized medicine. Lastly, by applying tissue engineering and 3D printing techniques, organoids could be used in the future to replace or regenerate damaged tissue. In this review, we will focus on hiPSC-derived 3D cultures and their ability to model human diseases with an in-depth analysis of gene editing applications, as well as tumor models. Furthermore, we will highlight the state-of-the-art of organoid facilities that around the world offer know-how and services. This is an increasing trend that shed the light on the need of bridging the publicand the private sector. Hence, in the context of drug discovery, Organoid Factories can offer biobanks of validated 3D organoid models that can be used in collaboration with pharmaceutical companies to speed up the drug screening process. Finally, we will discuss the limitations and the future development that will lead hiPSC-derived technology from bench to bedside, toward personalized medicine, such as maturity, organoid interconnections, costs, reproducibility and standardization, and ethics. hiPSC-derived organoid technology is now passing from a proof-of-principle to real applications in the clinic, also thanks to the applicability of techniques, such as CRISPR/Cas9 genome editing system, material engineering for the scaffolds, or microfluidic systems. The benefits will have a crucial role in the advance of both basic biological and translational research, particularly in the pharmacological field and drug development. In fact, in the near future, 3D organoids will guide the clinical decision-making process, having validated patient-specific drug screening platforms. This is particularly important in the context of rare genetic diseases or when testing cancer treatments that could in principle have severe side effects. Therefore, this technology has enabled the advancement of personalized medicine in a way never seen before.

Mitochondrial dysfunction in mandibular hypoplasia, deafness and progeroid features with concomitant lipodystrophy (MDPL) patients.

Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy is a rare, genetic, premature aging disease named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. In previous in vitro studies, we have already described several hallmarks of aging, including genetic damage, telomere shortening, cell senescence and proliferation defects. Since a clear connection has been reported between telomere shortening and mitochondria malfunction to initiate the aging process, we explored the role that mitochondrial metabolism and activity play in pathogenesis of MDPL Syndrome, an aspect that has not been addressed yet. We thus evaluated mtDNA copy number, assessing a significant decrease in mutated cells. The expression level of genes related to mitochondrial biogenesis and activity also revealed a significant reduction, highlighting a mitochondrial dysfunction in MDPL cells. Even the expression levels of mitochondrial marker SOD2, as assessed by immunofluorescence, were reduced. The decrease in this antioxidant enzyme correlated with increased production of mitochondrial ROS in MDPL cells, compared to WT. Consistent with these data, Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) analysis revealed in MDPL cells fewer mitochondria, which also displayed morphological abnormalities. Accordingly, we detected autophagic vacuoles containing partially digested mitochondria. Overall, our results demonstrate a dramatic impairment of mitochondrial biogenesis and activity in MDPL Syndrome. Administration of Metformin, though unable to restore mitochondrial impairment, proved efficient in rescuing nuclear abnormalities, suggesting its use to specifically ameliorate the premature aging phenotype.

Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids.

The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide homologous to dipeptidyl peptidase-4 (DPP4) receptor on host cells. Both could represent powerful immunotherapeutic candidates for COVID-19 treatment. The infection begins in the proximal airways, namely the alveolar type 2 (AT2) cells of the distal lung, which express both ACE2 and DPP4 receptors. Thus, to evaluate the efficacy of both approaches, we developed three-dimensional (3D) complex lung organoid structures (hLORGs) derived from human-induced pluripotent stem cells (iPSCs) and resembling the in vivo organ. Afterward, hLORGs were infected by different SARS-CoV-2 S pseudovirus variants and treated by the Ab15033-7 or DPP4 peptide. Using both approaches, we observed a significant reduction of viral entry and a modulation of the expression of genes implicated in innate immunity and inflammatory response. These data demonstrate the efficacy of such approaches in strongly reducing the infection efficiency in vitro and, importantly, provide proof-of-principle evidence that hiPSC-derived hLORGs represent an ideal in vitro system for testing both therapeutic and preventive modalities against COVID-19.

Indole-3-carbinol in vitro antiviral activity against SARS-Cov-2 virus and in vivo toxicity.

The effects of indole-3-carbinol (I3C) compound have been described deeply as antitumor drug in multiple cancers. Herein, I3C compound was tested for toxicity and antiviral activity against SARS-CoV-2 infection. Antiviral activity was assessed in vitro in both in VeroE6 cell line and human Lung Organoids (hLORGs) where I3C exhibited a direct anti-SARS-CoV-2 replication activity with an antiviral effect and a modulation of the expression of genes implicated in innate immunity and inflammatory response was observed at 16.67 µM. Importantly, we further show the I3C is also effective against the SARS-CoV-2 Omicron variant. In mouse model, instead, we assessed possible toxicity effects of I3C through two different routes of administration: intragastrically (i.g.) and intraperitoneally (i.p.). The LD50 (lethal dose 50%) values in mice were estimated to be: 1410 and 1759 mg/kg i.g.; while estimated values for i.p. administration were: 444.5 mg/kg and 375 mg/kg in male and female mice, respectively. Below these values, I3C (in particular at 550 mg/kg for i.g. and 250 mg/kg for i.p.) induces neither death, nor abnormal toxic symptoms as well as no histopathological lesions of the tissues analysed. These tolerated doses are much higher than those already proven effective in pre-clinical cancer models and in vitro experiments. In conclusion, I3C exhibits a significant antiviral activity, and no toxicity effects were recorded for this compound at the indicated doses, characterizing it as a safe and potential antiviral compound. The results presented in this study could provide experimental pre-clinical data necessary for the start of human clinical trials with I3C for the treatment of SARS-CoV-2 and beyond.

Effects of Simulated Microgravity on Wild Type and Marfan hiPSCs-Derived Embryoid Bodies.

BACKGROUND: Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including the inhibition of regenerative stem cell differentiation. In this work, we investigate the effects of microgravity simulation on early lineage commitment of hiPSCs from healthy and Marfan Syndrome (MFS; OMIM #154700) donors, using the embryoid bodies model of tissue differentiation and evaluating their ultra-structural conformation. MFS model involves an anomalous organization of the extracellular matrix for a deficit of fibrillin-1, an essential protein of connective tissue. METHODS: In vitro models require the use of embryoid bodies derived from hiPSCs. A DRPM was used to simulate microgravity conditions. RESULTS: Our data suggest an increase of the stemness of those EBs maintained in SMG condition. EBs are still capable of external migration, but are less likely to distinguish, providing a measure of the remaining progenitor or stem cell populations in the earlier stage. The microgravity response appears to vary between WT and Marfan EBs, presumably as a result of a cell structural component deficiency due to fibrillin-1 protein lack. In fact, MFS EBs show a reduced adaptive capacity to the environment of microgravity that prevented them from reacting and making rapid adjustments, while healthy EBs show stem retention, without any structural changes due to microgravity conditions. CONCLUSION: EBs formation specifically mimics stem cell differentiation into embryonic tissues, this process has also significant similarities with adult stem cell-based tissue regeneration. The use of SMG devices for the maintenance of stem cells on regenerative medicine applications is becoming increasingly more feasible. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00680-1.

Clinical Features of LMNA-Related Cardiomyopathy in 18 Patients and Characterization of Two Novel Variants.

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed myocardial performance in the absence of hypertension, valvular, congenital, or ischemic heart disease. Mutations in LMNA gene, encoding for lamin A/C, account for 10% of familial DCM. LMNA-related cardiomyopathies are characterized by heterogeneous clinical manifestations that vary from a predominantly structural heart disease, mainly mild-to-moderate left ventricular (LV) dilatation associated or not with conduction system abnormalities, to highly pro-arrhythmic profiles where sudden cardiac death (SCD) occurs as the first manifestation of disease in an apparently normal heart. In the present study, we select, among 77 DCM families referred to our center for genetic counselling and molecular screening, 15 patient heterozygotes for LMNA variants. Segregation analysis in the relatives evidences other eight heterozygous patients. A genotype-phenotype correlation has been performed for symptomatic subjects. Lastly, we perform in vitro functional characterization of two novel LMNA variants using dermal fibroblasts obtained from three heterozygous patients, evidencing significant differences in terms of lamin expression and nuclear morphology. Due to the high risk of SCD that characterizes patients with lamin A/C cardiomyopathy, genetic testing for LMNA gene variants is highly recommended when there is suspicion of laminopathy.

Peptide Platform as a Powerful Tool in the Fight against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic causing over 195 million infections and more than 4 million fatalities as of July 2021.To date, it has been demonstrated that a number of mutations in the spike glycoprotein (S protein) of SARS-CoV-2 variants of concern abrogate or reduce the neutralization potency of several therapeutic antibodies and vaccine-elicited antibodies. Therefore, the development of additional vaccine platforms with improved supply and logistic profile remains a pressing need. In this work, we have validated the applicability of a peptide-based strategy focused on a preventive as well as a therapeutic purpose. On the basis of the involvement of the dipeptidyl peptidase 4 (DPP4), in addition to the angiotensin converting enzyme 2 (ACE2) receptor in the mechanism of virus entry, we analyzed peptides bearing DPP4 sequences by protein-protein docking and assessed their ability to block pseudovirus infection in vitro. In parallel, we have selected and synthetized peptide sequences located within the highly conserved receptor-binding domain (RBD) of the S protein, and we found that RBD-based vaccines could better promote elicitation of high titers of neutralizing antibodies specific against the regions of interest, as confirmed by immunoinformatic methodologies and in vivo studies. These findings unveil a key antigenic site targeted by broadly neutralizing antibodies and pave the way to the design of pan-coronavirus vaccines.