Upregulation of miR145 and miR126 in EVs from Renal Cells Undergoing EMT and Urine of Diabetic Nephropathy Patients

Simple SummaryDiabetic nephropathy is one of the most frequent complications of diabetes, resulting from diffuse damage to different kidney cells. The identification of subjects at risk is mandatory to prevent its development and provide appropriate therapies reducing the unmanageable evolution towards end-stage kidney disease. The aim of this work was to identify urinary-derived extracellular vesicles (EVs) miRNA cargo to be used as biomarker of kidney damage in diabetic patients. The miRNA profile was then correlated with the molecular mechanism associated with the glomerular and tubular damage using a diabetic-like model. In patients, miR145 and miR126 in urinary EVs increased together with albuminuria. MiR145 and miR126 increased in parallel in EVs from renal epithelial cells undergoing transition to a fibrotic mesenchymal phenotype. These data unveiled a role for miR126 and miR145 as the biomarkers of damage progression and proteinuria development in diabetic nephropathy. AbstractDiabetic nephropathy (DN) is a severe kidney-related complication of type 1 and type 2 diabetes and the most frequent cause of end-stage kidney disease. Extracellular vesicles (EVs) present in the urine mainly derive from the cells of the nephron, thus representing an interesting tool mirroring the kidney’s physiological state. In search of the biomarkers of disease progression, we here assessed a panel of urinary EV miRNAs previously related to DN in type 2 diabetic patients stratified based on proteinuria levels. We found that during DN progression, miR145 and miR126 specifically increased in urinary EVs from diabetic patients together with albuminuria. In vitro, miRNA modulation was assessed in a model of TGF-β1-induced glomerular damage within a three-dimensional perfusion system, as well as in a model of tubular damage induced by albumin and glucose overload. Both renal tubular cells and podocytes undergoing epithelial to mesenchymal transition released EVs containing increased miR145 and miR126 levels. At the same time, miR126 levels were reduced in EVs released by glomerular endothelial cells. This work highlights a modulation of miR126 and miR145 during the progression of kidney damage in diabetes as biomarkers of epithelial to mesenchymal transition.     

Molecular Genetics of GLUT1DS Italian Pediatric Cohort: 10 Novel Disease-Related Variants and Structural Analysis

GLUT1 deficiency syndrome (GLUT1DS1; OMIM #606777) is a rare genetic metabolic disease, characterized by infantile-onset epileptic encephalopathy, global developmental delay, progressive microcephaly, and movement disorders (e.g., spasticity and dystonia). It is caused by heterozygous mutations in the SLC2A1 gene, which encodes the GLUT1 protein, a glucose transporter across the blood-brain barrier (BBB). Most commonly, these variants arise de novo resulting in sporadic cases, although several familial cases with AD inheritance pattern have been described. Twenty-seven Italian pediatric patients, clinically suspect of GLUT1DS from both sporadic and familial cases, have been enrolled. We detected by trios sequencing analysis 25 different variants causing GLUT1DS. Of these, 40% of the identified variants (10 out of 25) had never been reported before, including missense, frameshift, and splice site variants. Their structural mapping on the X-ray structure of GLUT1 strongly suggested the potential pathogenic effects of these novel disease-related mutations, broadening the genotypic spectrum heterogeneity found in the SLC2A1 gene. Moreover, 24% is located in a vulnerable region of the GLUT1 protein that involves transmembrane 4 and 5 helices encoded by exon 4, confirming a mutational hotspot in the SLC2A1 gene. Lastly, we investigated possible correlations between mutation type and clinical and biochemical data observed in our GLUT1DS cohort, revealing that splice site and frameshift variants are related to a more severe phenotype and low CSF parameters.     

Methylome Profiling in Fabry Disease in Clinical Practice: A Proof of Concept

Anderson–Fabry disease (FD) is an X-linked disease caused by a functional deficit of the α-galactosidase A enzyme. FD diagnosis relies on the clinical manifestations and research of GLA gene mutations. However, because of the lack of a clear genotype/phenotype correlation, FD diagnosis can be challenging. Recently, several studies have highlighted the importance of investigating DNA methylation patterns for confirming the correct diagnosis of different rare Mendelian diseases, but to date, no such studies have been reported for FD. Thus, in the present investigation, we analyzed for the first time the genome-wide methylation profile of a well-characterized cohort of patients with Fabry disease. We profiled the methylation status of about 850,000 CpG sites in 5 FD patients, all carrying the same mutation in the GLA gene (exon 6 c.901C>G) and presenting comparable low levels of α-Gal A activity. We found that, although the whole methylome profile did not discriminate the FD group from the unaffected one, several genes were significantly differentially methylated in Fabry patients. Thus, we provide here a proof of concept, to be tested in patients with different mutations and in a larger cohort, that the methylation state of specific genes can potentially identify Fabry patients and possibly predict organ involvement and disease evolution.     

Gut Microbiota,Metabolic Disorders and Breast Cancer: Could Berberine Turn Out to Be a Transversal Nutraceutical Tool? A Narrative Analysis

Metabolic disorders, mainly characterized as the marked alteration of the lipid and carbohydrate profile, in addition to the clinical presence of the direct consequences of these alterations, are pathological conditions that have considerably increased in prevalence in recent years. They are directly linked to the onset of various pathologies, including cancer, particularly breast cancer, and are hormone-responsive. Alongside the known conditions responsible for this scenario, such as nutrition and lifestyle in general, the importance of both the colonic microbiota and the various organs and systems is becoming increasingly evident. In fact, it is now evident that microbial dysbiosis plays a fundamental role in the onset of these metabolic disorders, and therefore how these conditions are indirectly responsible for the onset and progression of neoplasms. Indirect mechanisms such as an altered Firmicutes/Bacteroidetes ratio; the formation of metabolites such as short-chain fatty acids (SCFAs), in particular, butyrate, which is capable of acting as a tumor suppressor; and the glucuronidase activity of estroboloma (bacteria responsible for estrogen metabolism) are just some of the most important mechanisms that contribute to the history of breast cancer. It is therefore understandable that in clinical terms, it is essential to associate the modulation of metabolic disorders and the microbial conditions that contribute to generating them with common therapies, preferably using compounds and solutions that are effective and acceptable for the patient without side effects. Nutraceuticals such as berberine (active both in metabolic scenarios and in the microbiota) and interventions modulating the microbial structure such as the use of probiotics and prebiotics seem to be ideal solutions for these preventive and no-longer-ignorable strategies in the light of numerous data now present in the literature.     

Anticoagulants as Potential SARS-CoV-2 Mpro Inhibitors for COVID-19 Patients: In Vitro,Molecular Docking,Molecular Dynamics,DFT, and SAR Studies

In this article, 34 anticoagulant drugs were screened in silico against the main protease (M^pro) of SARS-CoV-2 using molecular docking tools. Idraparinux, fondaparinux, eptifibatide, heparin, and ticagrelor demonstrated the highest binding affinities towards SARS-CoV-2 M^pro. A molecular dynamics study at 200 ns was also carried out for the most promising anticoagulants to provide insights into the dynamic and thermodynamic properties of promising compounds. Moreover, a quantum mechanical study was also conducted which helped us to attest to some of the molecular docking and dynamics findings. A biological evaluation (in vitro) of the most promising compounds was also performed by carrying out the MTT cytotoxicity assay and the crystal violet assay in order to assess inhibitory concentration 50 (IC₅₀). It is worth noting that ticagrelor displayed the highest intrinsic potential for the inhibition of SARS-CoV-2 with an IC₅₀ value of 5.60 µM and a safety index of 25.33. In addition, fondaparinux sodium and dabigatran showed promising inhibitory activities with IC₅₀ values of 8.60 and 9.40 µM, respectively, and demonstrated safety indexes of 17.60 and 15.10, respectively. Moreover, the inhibitory potential of the SARS-CoV-2 M^pro enzyme was investigated by utilizing the SARS-CoV-2 M^pro assay and using tipranavir as a reference standard. Interestingly, promising SARS-CoV-2 M^pro inhibitory potential was attained for fondaparinux sodium with an IC₅₀ value of 2.36 µM, surpassing the reference tipranavir (IC₅₀ = 7.38 µM) by more than three-fold. Furthermore, highly eligible SARS-CoV-2 M^pro inhibitory potential was attained for dabigatran with an IC₅₀ value of 10.59 µM. Finally, an SAR was discussed, counting on the findings of both in vitro and in silico approaches.     

Highly Charged Ru(II) Polypyridyl Complexes as Photosensitizer Agents in Photodynamic Therapy of Epithelial Ovarian Cancer Cells

Ovarian cancer recurrence is frequent and associated with chemoresistance, leading to extremely poor prognosis. Herein, we explored the potential anti-cancer effect of a series of highly charged Ru(II)-polypyridyl complexes as photosensitizers in photodynamic therapy (PDT), which were able to efficiently sensitize the formation of singlet oxygen upon irradiation (Ru1²⁺ and Ru2²⁺) and to produce reactive oxygen species (ROS) in their corresponding dinuclear metal complexes with the Fenton active Cu(II) ion/s ([CuRu1]⁴⁺ and [Cu₂Ru2]⁶⁺). Their cytotoxic and anti-tumor effects were evaluated on human ovarian cancer A2780 cells both in the absence or presence of photoirradiation, respectively. All the compounds tested were well tolerated under dark conditions, whereas they switched to exert anti-tumor activity following photoirradiation. The specific effect was mediated by the onset of programed cell death, but only in the case of Ru1²⁺ and Ru2²⁺ was preceded by the loss of mitochondrial membrane potential soon after photoactivation and ROS production, thus supporting the occurrence of apoptosis via type II photochemical reactions. Thus, Ru(II)-polypyridyl-based photosensitizers represent challenging tools to be further investigated in the identification of new therapeutic approaches to overcome the innate chemoresistance to platinum derivatives of some ovarian epithelial cancers and to find innovative drugs for recurrent ovarian cancer.     

The Cysteine Protease Giardipain-1 from Giardia duodenalis Contributes to a Disruption of Intestinal Homeostasis

In giardiasis, diarrhoea, dehydration, malabsorption, weight loss and/or chronic inflammation are indicative of epithelial barrier dysfunction. However, the pathogenesis of giardiasis is still enigmatic in many aspects. Here, we show evidence that a cysteine protease of Giardia duodenalis called giardipain-1, contributes to the pathogenesis of giardiasis induced by trophozoites of the WB strain. In an experimental system, we demonstrate that purified giardipain-1 induces apoptosis and extrusion of epithelial cells at the tips of the villi in infected jirds (Meriones unguiculatus). Moreover, jird infection with trophozoites expressing giardipain-1 resulted in intestinal epithelial damage, cellular infiltration, crypt hyperplasia, goblet cell hypertrophy and oedema. Pathological alterations were more pronounced when jirds were infected intragastrically with Giardia trophozoites that stably overexpress giardipain-1. Furthermore, Giardia colonization in jirds results in a chronic inflammation that could relate to the dysbiosis triggered by the protist. Taken together, these results reveal that giardipain-1 plays a key role in the pathogenesis of giardiasis.     

Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants

Rett syndrome caused by MECP2 variants is characterized by a heterogenous clinical spectrum accounted for in 60% of cases by hot-spot variants. Focusing on the most frequent variants, we generated in vitro iPSC-neurons from the blood of RTT girls with p.Arg133Cys and p.Arg255*, associated to mild and severe phenotype, respectively, and of an RTT male harboring the close to p.Arg255*, p.Gly252Argfs*7 variant. Truncated MeCP2 proteins were revealed by Western blot and immunofluorescence analysis. We compared the mutant versus control neurons at 42 days for morphological parameters and at 120 days for electrophysiology recordings, including girls’ isogenic clones. A precocious reduced morphological complexity was evident in neurons with truncating variants, while in p.Arg133Cys neurons any significant differences were observed in comparison with the isogenic wild-type clones. Reduced nuclear size and branch number show up as the most robust biomarkers. Patch clamp recordings on mature neurons allowed the assessment of cell biophysical properties, V-gated currents, and spiking pattern in the mutant and control cells. Immature spiking, altered cell capacitance, and membrane resistance of RTT neurons, were particularly pronounced in the Arg255* and Gly252Argfs*7 mutants. The overall results indicate that the specific markers of in vitro cellular phenotype mirror the clinical severity and may be amenable to drug testing for translational purposes.     

Mechanical regulation of the early stages of angiogenesis

Favouring or thwarting the development of a vascular network is essential in fields as diverse as oncology, cardiovascular disease or tissue engineering. As a result, understanding and controlling angiogenesis has become a major scientific challenge. Mechanical factors play a fundamental role in angiogenesis and can potentially be exploited for optimizing the architecture of the resulting vascular network. Largely focusing on in vitro systems but also supported by some in vivo evidence, the aim of this Highlight Review is dual. First, we describe the current knowledge with particular focus on the effects of fluid and solid mechanical stimuli on the early stages of the angiogenic process, most notably the destabilization of existing vessels and the initiation and elongation of new vessels. Second, we explore inherent difficulties in the field and propose future perspectives on the use of in vitro and physics-based modelling to overcome these difficulties.