Duchenne muscular dystrophy (DMD) is the most common fatal muscle disease, with an estimated incidence of 1/3500–1/5000 male births, and it is associated with mutations in the X-linked DMD gene encoding dystrophin, the largest known human gene. There is currently no cure for DMD. The large size of the DMD gene hampers exogenous gene addition and delivery. The genetic correction of DMD patient-derived induced pluripotent stem cells (DMD-iPSCs) and differentiation into suitable cells for transplantation is a promising autologous therapeutic strategy for DMD. In this study, using CRISPR/Cas9, the full-length dystrophin coding sequence was reconstructed in an exon-50-deleted DMD-iPSCs by the targeted addition of exon 50 at the junction of exon 49 and intron 49 via homologous-directed recombination (HDR), with a high targeting efficiency of 5/15, and the genetically corrected iPSCs were differentiated into cardiomyocytes (iCMs). Importantly, the full-length dystrophin expression and membrane localization were restored in genetically corrected iPSCs and iCMs. Thus, this is the first study demonstrating that full-length dystrophin can be restored in iPSCs and iCMs via targeted exon addition, indicating potential clinical prospects for DMD gene therapy. Full article

Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3⁺  Escherichia coli strain. The fosA3⁺  E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3⁺  E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3⁺ bacteria. Full article

Cerebral ischemia in humans and animals is a life-threatening neuropathological event and leads to the development of dementia with the Alzheimer’s disease phenotype [...] Full article

Kidney transplantation from a donor with latent BKPyV might be the cause of serious complications, such as BK virus-associated nephropathy. The aim of the study was to determine the prevalence of BKPyV infection in donors after brain death (DBDs), to analyse the molecular variation of BKPyV and to compare clinical and inflammation parameters of DBDs infected with various genotypes of BKPyV. BKPyV was investigated in blood and urine samples of 103 DBDs using PCR followed by sequencing and bioinformatic analysis, and the viral load was assessed by qPCR. Clinical parameters, including cellular markers of inflammation were assessed. The results confirm high prevalence of BKPyV (48%),and genotype IV (49%) over genotype I (43%) and the co-infection with genotypes I and IV in 8.2%. Viral load ranged from 10² to 10⁷ copies/mL, with an average of 1.92 × 10⁶ copies/mL. No specific markers for BKPyV infection were detected among the parameters tested. Infection with genotype I may be associated with the adverse impact on thekidney function, while infection with genotype IV was associated with the anemia Not only the viral load but also the genotype of BKPyV may have an impact on the course of infection. Full article

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd₁-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins. Full article

During the first wave of COVID-19 infection in Italy, the number of cases and the mortality rates were among the highest compared to the rest of Europe and the world. Several studies demonstrated a severe clinical course of COVID-19 associated with old age, comorbidities, and male gender. However, there are cases of virus infection resistance in subjects living in close contact with infected subjects. Thus, to explain the predisposition to virus infection and to COVID-19 disease progression, we must consider, in addition to the genetic variability of the virus and other environmental or comorbidity conditions, the allelic variants of specific human genes, directly or indirectly related to the life cycle of the virus. Here, we analyzed three human genetic polymorphisms belonging to the TMPRSS2 and CCR5 genes in a sample population from Sicily (Italy) to investigate possible correlations with the resistance to viral infection and/or to COVID-19 disease progression as recently described in other human populations. Our results did not show any correlations of the rs35074065, rs12329760, and rs333 polymorphisms with SARS-CoV-2 infection or with COVID-19 disease severity. Further studies on other human genetic polymorphisms should be performed to identify the major human determinants of SARS-CoV-2 viral resistance. Full article

The transient outward potassium current (I_tof) is generated by the activation of K_V4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by K_V4.3/KChIP2, K_V4.3/KChIP2/DPP6 and K_V4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (K_V4.3/KChIP2, K_V4.3/KChIP2/DPP6 or K_V4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of K_V4.3/KChIP2, K_V4.3/KChIP2/DPP6 and K_V4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in K_V4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing K_V4.3/KChIP2 and K_V4.3/KChIP2/KCNE2, but not in K_V4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of K_V4.3/KChIP2 channels to IQM-266. Full article

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal dominantly inherited progressive disorders with degeneration and dysfunction of the cerebellum. Although different subtypes of SCAs are classified according to the disease-associated causative genes, the clinical syndrome of the ataxia is shared, pointing towards a possible convergent pathogenic pathway among SCAs. In this review, we summarize the role of SCA-associated gene function during cerebellar Purkinje cell development and discuss the relationship between SCA pathogenesis and neurodevelopment. We will summarize recent studies on molecules involved in SCA pathogenesis and will focus on the mGluR1-PKCγ signaling pathway evaluating the possibility that this might be a common pathway which contributes to these diseases. Full article

Despite all the important advances in its diagnosis and treatment, acute myocardial infarction (AMI) is still one of the most prominent causes of morbidity and mortality worldwide. Early identification of patients at high risk of poor outcomes through the measurement of various biomarker concentrations might contribute to more accurate risk stratification and help to guide more individualized therapeutic strategies, thus improving prognoses. The aim of this article is to provide an overview of the role and applications of cardiac biomarkers in risk stratification and prognostic assessment for patients with myocardial infarction. Although there is no ideal biomarker that can provide prognostic information for risk assessment in patients with AMI, the results obtained in recent years are promising. Several novel biomarkers related to the pathophysiological processes found in patients with myocardial infarction, such as inflammation, neurohormonal activation, myocardial stress, myocardial necrosis, cardiac remodeling and vasoactive processes, have been identified; they may bring additional value for AMI prognosis when included in multi-biomarker strategies. Furthermore, the use of artificial intelligence algorithms for risk stratification and prognostic assessment in these patients may have an extremely important role in improving outcomes. Full article

Exogenously applied double-stranded RNA (dsRNA) can induce potent host specific gene knockdown and mortality in insects. The deployment of RNA-interference (RNAi) technologies for pest suppression is gaining traction in both agriculture and horticulture, but its implementation in forest systems is lagging. While numerous forest pests have demonstrated susceptibility to RNAi mediated gene silencing, including the southern pine beetle (SPB), Dendroctonus frontalis, multiple barriers stand between laboratory screening and real-world deployment. One such barrier is dsRNA delivery. One possible delivery method is through host plants, but an understanding of exogenous dsRNA movement through plant tissues is essential. Therefore, we sought to understand the translocation and persistence of dsRNAs designed for SPB throughout woody plant tissues after hydroponic exposure. Loblolly pine, Pinus taeda, seedlings were exposed to dsRNAs as a root soak, followed by destructive sampling. Total RNA was extracted from different tissue types including root, stem, crown, needle, and meristem, after which gel electrophoresis confirmed the recovery of the exogenous dsRNAs, which were further verified using Sanger sequencing. Both techniques confirmed the presence of the exogenously applied target dsRNAs in each tissue type after 1, 3, 5, and 7 d of dsRNA exposure. These findings suggest that root drench applications of exogenous dsRNAs could provide a viable delivery route for RNAi technology designed to combat tree feeding pests. Full article

Polymer/layered silicate composites have gained huge attention in terms of research and industrial applications. Traditional nanocomposites contain particles regularly dispersed in a polymer matrix. In this work, a strategy for the formation of a composite thin film on the surface of a polycaprolactone (PCL) matrix was developed. In addition to the polymer, the composite layer was composed of the particles of saponite (Sap) modified with alkylammonium cations and functionalized with methylene blue. The connection between the phases of modified Sap and polymer was achieved by fusing the chains of molten polymer into the Sap film. The thickness of the film of several μm was confirmed using electron microscopy and X-ray tomography. Surfaces of precursors and composite materials were analyzed in terms of structure, composition, and surface properties. The penetration of polymer chains into the silicate, thus joining the phases, was confirmed by chemometric analysis of spectral data and changes in some properties upon PCL melting. Ultimately, this study was devoted to the spectral properties and photoactivity of methylene blue present in the ternary composite films. The results provide directions for future research aimed at the development of composite materials with photosensitizing, photodisinfection, and antimicrobial surfaces. Full article

Lead (Pb), due to its high toxicity and bioaccumulation tendency, is one of the top three pollutants of concern for both humans and wildlife and occupies second place in the Priority List of Hazardous Substances. In freshwater fish, Pb is mainly absorbed through the gills, where the greatest accumulation occurs. Despite the crucial role of gills in several physiological functions such as gas exchange, water balance, and osmoregulation, no studies evaluated the effects of environmentally relevant concentrations of Pb on this organ, and existing literature only refers to high levels of exposure. Herein we investigated for the first time the molecular and morphological effects induced by two low and environmentally relevant concentrations of Pb (2.5 and 5 μg/L) on the gills of Danio rerio, a model species with a high translational value for human toxicity. It was demonstrated that Pb administration at even low doses induces osmoregulatory dysfunctions by affecting Na⁺/K⁺-ATPase and AQP3 expression. It was also shown that Pb upregulates MTs as a protective response to prevent cell damage. Modulation of SOD confirms that the production of reactive oxygen species is an important toxicity mechanism of Pb. Histological and morphometric analysis revealed conspicuous pathological changes, both dose- and time-dependent. Full article

Hypovitaminosis D is involved in various inflammatory, infectious and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Moreover, the active form of vitamin D, calcitriol, has been shown to modulate the immune response, playing an anti-inflammatory effect. However little is known about the mechanisms underlying this anti-inflammatory effect and the potential sex differences of calcitriol immune regulation. Hence, the aim of this study was to investigate whether calcitriol could act differently in modulating T cell immunity of age-matched male and female healthy donors. We analyzed the effects of calcitriol in T lymphocytes from healthy women and men on the expression levels of the vitamin D receptor (VDR) and pro- and anti-inflammatory cytokine production. We showed that a treatment with calcitriol induced a significant increase in the VDR expression levels of activated T lymphocytes from male and female healthy subjects. Moreover, we found that calcitriol significantly reduced the expression level of pro-inflammatory cytokines IL-17, INF-γ and TNF-α in the T lymphocytes of both sexes. Notably, we observed that calcitriol induced a significant increase in the expression level of anti-inflammatory cytokine IL-10 only in the T lymphocytes from female healthy donors. In conclusion, our study provides new insights regarding the sex-specific anti-inflammatory role of calcitriol in T cell immunity. Full article

The emergence of resistant bacteria takes place, endangering the effectiveness of antibiotics. A reason for antibiotic resistance is the presence of lactamases that catalyze the hydrolysis of β-lactam antibiotics. An inhibitor of serine-β-lactamases such as clavulanic acid binds to the active site of the enzymes, thus solving the resistance problem. A pressing issue, however, is that the reaction mechanism of metallo-β-lactamases (MBLs) hydrolyzing β-lactam antibiotics differs from that of serine-β-lactamases due to the existence of zinc ions in the active site of MBLs. Thus, the development of potential inhibitors for MBLs remains urgent. Here, the ability to inhibit MBL from Bacillus anthracis (Bla2) was investigated in silico and in vitro using compounds possessing two hydroxamate functional groups such as 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6). In silico docking and molecular dynamics simulations revealed that both Compounds 4 and 6 were coordinated with zinc ions in the active site, suggesting that the hydroxamate group attached to the aromatic ring of the compound plays a crucial role in the coordination to the zinc ions. In vitro kinetic analysis demonstrated that the mode of inhibitions for Compounds 4 and 6 were a competitive inhibition with Ki values of 6.4 ± 1.7 and 4.7 ± 1.4 kcal/mol, respectively. The agreement between in silico and in vitro investigations indicates that compounds containing dihyroxamate moieties may offer a new avenue to overcome antibiotic resistance to bacteria. Full article

A new ligand 5-((1-methyl-pyrrol-2-yl) methyl)-4-(naphthalen-1-yl)-1,2,4-triazoline-3-thione (C15) and its metal complexes with formulae: Mn(C15)Cl₂MeOH (1), Fe(C15)Cl₂MeOH (2), Ni(C15)Cl₂MeOH (3), Cu(C15)₂Cl₂ (4) and Zn(C15)₄Cl₂ (5) have been synthesized. The C15 ligand and complexes were characterized by NMR, elemental analysis, FT-IR, EPR, magnetic and TGA studies. The anticancer activities of the organic ligand (C15) and complexes (1–5) were evaluated against human colon adenocarcinoma (HT29) and human lung (A549) cancer cell lines. The complex (1) exhibited potential activity at concentration of 794.37 μM (A549) and 654.31 μM (HT29) in both cancer cells. The complex (3) showed significant activity against the HT29 cancer cell line with an IC₅₀ value of 1064.05 μM. This article highlights some of the metals that have become important in the development of new coordination complexes and the treatment of cancer. Additionally, for C15, the toxicity was predicted by ADMET analysis and molecular docking. Full article