A straightforward molecular strategy to retrospectively investigate the spread of SARS-CoV-2 VOC202012/01 B.1.1.7 variant.

The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of 4 RNA viral genomes demonstrated that those curves were associated with B.1.1.7 variant strains. Our results strongly suggest that B.1.1.7 variant spread has begun in this area at least since January and imply the potential of these analytical methods to track and characterize the spread of B.1.1.7 strains in those areas where Allplex SARS-CoV-2 datasets have been previously recorded

Synthesis, cytotoxicity and antiviral evaluation of new series of imidazo[4,5-g]quinoline and pyrido[2,3-g]quinoxalinone derivatives

Linear aromatic N-tricyclic compounds with promising antiviral activity and minimal cytotoxicity were prepared and analyzed in the last years. Specifically, the pyrido[2,3-g]quinoxalinone nucleus was found endowed with high potency against several pathogenic RNA viruses as etiological agents of important veterinary and human pathologies. Following our research program on new antiviral agents we have designed, synthesized and assayed new series of imidazo[4,5-g]quinoline and pyrido[2,3-g]quinoxalinone derivatives. Lead compounds 1-4 were further modified to enhance their antiviral activity and reduce their cytotoxicity. Thus, different substituents were introduced on N atom at position 1 or the O atom at position 2 of the leads; contextually, several groups were inserted on the nitrogen atom at position 7 of diaminoquinoline intermediates. Title compounds were tested in cell-based assays for cytotoxicity and antiviral activity against RNA virus families containing single-stranded (either positive-sense (ssRNA+) or negative-sense (ssRNA-)), and double-stranded genomes (dsRNA), and against two representatives of DNA virus families. Some derivatives emerged as potential leads for further development as antiviral agents against some viruses of public health significance, such as RSV, Reo, BVDV and HCV. Particularly, compounds 4, 11b, 11c, 13c, 15a, 18 and 21 resulted active against BVDV at concentrations ranging from 1.3 to 5\ua0\u3bcM. Compound 21 was also evaluated for its activity on the BVDV RdRp. Compound 4 was also tested as potential anti-HCV compound in a subgenomic replication assay. Molecular simulation results provided a molecular rationale for the anti-BVDV activity of these compounds

Asymmetric Azidation under Hydrogen Bonding Phase-Transfer Catalysis: A Combined Experimental and Computational Study

[Image: see text] Asymmetric catalytic azidation has increased in importance to access enantioenriched nitrogen containing molecules, but methods that employ inexpensive sodium azide remain scarce. This encouraged us to undertake a detailed study on the application of hydrogen bonding phase-transfer catalysis (HB-PTC) to enantioselective azidation with sodium azide. So far, this phase-transfer manifold has been applied exclusively to insoluble metal alkali fluorides for carbon–fluorine bond formation. Herein, we disclose the asymmetric ring opening of meso aziridinium electrophiles derived from β-chloroamines with sodium azide in the presence of a chiral bisurea catalyst. The structure of novel hydrogen bonded azide complexes was analyzed computationally, in the solid state by X-ray diffraction, and in solution phase by (1)H and (14)N/(15)N NMR spectroscopy. With N-isopropylated BINAM-derived bisurea, end-on binding of azide in a tripodal fashion to all three NH bonds is energetically favorable, an arrangement reminiscent of the corresponding dynamically more rigid trifurcated hydrogen-bonded fluoride complex. Computational analysis informs that the most stable transition state leading to the major enantiomer displays attack from the hydrogen-bonded end of the azide anion. All three H-bonds are retained in the transition state; however, as seen in asymmetric HB-PTC fluorination, the H-bond between the nucleophile and the monodentate urea lengthens most noticeably along the reaction coordinate. Kinetic studies corroborate with the turnover rate limiting event resulting in a chiral ion pair containing an aziridinium cation and a catalyst-bound azide anion, along with catalyst inhibition incurred by accumulation of NaCl. This study demonstrates that HB-PTC can serve as an activation mode for inorganic salts other than metal alkali fluorides for applications in asymmetric synthesis

Expression of HERV Genes as Possible Biomarker and Target in Neurodegenerative Diseases

Human endogenous retroviruses (HERVs) are genetic parasites, in-between genetics and environment. Few HERVs retain some coding capability. Sometimes, the host has the advantage of some HERV genes; conversely, HERVs may contribute to pathogenesis. The expression of HERVs depends on several factors, and is regulated epigenetically by stimuli such as inflammation, viral and microbial infections, etc. Increased expression of HERVs occurs in physiological and pathological conditions, in one or more body sites. Several diseases have been attributed to one or more HERVs, particularly neurological diseases. The key problem is to differentiate the expression of a HERV as cause or effect of a disease. To be used as a biomarker, a correlation between the expression of a certain HERV and the disease onset and/or behavior must be found. The greater challenge is to establish a pathogenic role. The criteria defining causal connections between HERVs and diseases include the development of animal models, and disease modulation in humans, by anti-HERV therapeutic antibody. So far, statistically significant correlations between HERVs and diseases have been achieved for HERV-W and multiple sclerosis; disease reproduction in transgenic animals was achieved for HERV-W and multiple sclerosis, and for HERV-K and amyotrophic lateral sclerosis. Clinical trials for both diseases are in progress

Disruption by SaCas9 Endonuclease of HERV-Kenv, a Retroviral Gene with Oncogenic and Neuropathogenic Potential, Inhibits Molecules Involved in Cancer and Amyotrophic Lateral Sclerosis

The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options

Diagnosi di Laboratorio per Infezione nel Distretto Parodontale, dalla PCR al “Molecular Beacon Array”

I dati forniti dall’ Organizzazione Mondiale della Sanità (OMS) riportano che a fronte del 30% della popolazione italiana che presenta salute parodontale, il restante 70 % è affetta da una forma di patologia che può essere lieve (80%) e grave (5-20%). Si tratta dunque di una patologia che ha un forte impatto sociale, e quindi una notevole incidenza nei costi della spesa sanitaria. Si impone pertanto un corretto approccio in termini di: prevenzione della diagnosi clinica e di laboratorio, delle strategie terapeutiche. L ‘eziologia della malattia parodontale e di tipo infettivo ma rispetto ad altre patologie causate da microorganismi la parodontite presenta notevoli peculiarità, peculiarità che vanno ricercate nelle caratteristiche dei patogeni parodontali, in particolare : (a) sono descritte almeno 300 specie differenti ( la maggior parte non ancora identificate), (b) La maggior parte sono anaerobi, (c) difficoltà nella coltura, (d) crescita molto lenta ( anche 2 settimane), (e) presentano un' organizzazione a ” biofim complesso”. Attualmente, un aiuto importante alla diagnostica di laboratorio in parodontologia, viene dalla biologia molecolare. Le tecniche più efficaci e moderne attualmente in uso sono la Polymerase Chain Reaction (PCR), e la sua versione quantitativa : la PCR real-Time. Queste metodiche hanno come principio lo specifico riconoscimento di una sequenza di DNA/RNA appartenente al microorganismo patogeno. I vantaggi principali vengono rilevati nella sensibilita’ ( un incremento di 10-100 volte) e nel tempo del risultato (1- 6 ore ). Nonostante questi vantaggi notevoli , i metodi citati possono presentare una bassa specificità , dovuta a sequenze di DNA molto simili appartenenti a specie microbiche affini. 2 Recentemente un ulteriore passo nella diagnostica microbiologica molecolare e’ stato raggiunto con i “Molecular Beacon”MB, (Tyagi et al., 1996). I MB, ,rappresentano sonde fluorescenti a DNA con struttura circolare (Fig.1). La sonda presenta una sequenza nucleotidica che si apre (ed emette fluorescenza),solo se trova una regione complementare perfettamente identica nel DNA bersaglio del patogeno parodontale, questo determina il massimo grado di specificità possibile nella diagnostica molecolare, di conseguenza vengono ridotti i possibili falsi positivi della PCR tradizionale. L’utilizzo dei MB in un pannello analitico contenente tutte le specie rappresentative della malattia parodontale “molecular beacon array”, dispone per il clinico elementi più oggettivi e, soprattutto scientificamente/tecnologicamente più significativi, su cui basare la diagnosi di infezione per malattia parodontale

VOLUNTARY ETHANOL DRINKING IN GABAB KNOCK-OUT MICE AND GENE EXPRESSION OF GABAA RECEPTORS

Our goal was to study the role of GABAB receptors in voluntary ethanol drinking behaviour and the possible alterations in GABAA receptor gene expression in GABAB knock-out mice. To achieve this goal we used the original Balb/c strain of GABAB(1) knock-out mice crossed with FVB mice in our laboratory. Male knock-out (KO), heterozygous (ET) and wild type (WT) animals were used and compared in this study. Mice were offered ethanol using the 2 hours, 2 bottle choice drinking para- digm (ethanol 15%). After establishing baseline drinking mice were tested and monitored for 4 weeks. At the end of treatment we measured BAC, by gas chromatography, and GABAA receptor gene expression in the hippocampus, by real time PCR. Our results show that voluntary ethanol con- sumption was significantly increased, starting at the second week of treatment, in both KO and ET mice. Over 4 weeks of treatment the average amount of ethanol consumed during the 2 hour access was 1.45 g/Kg and 1.36 g/kg (P < 0.001) for KO and ET mice respectively, wile WT mice drank only 0.90 g/Kg. Blood samples collected immediately after the last session of ethanol exposure revealed that the BAC average in KO, ET and WT mice were 3.76; 5.29 and 0.48; respectively. Measure- ments of ethanol preference show that both KO and ET mice display a statistically significant greater consumption of alcohol than water (P < 0.001 and 0.05 respectively) compared to WT mice. The lack of GABAB receptors in KO mice dramatically reduced the expression of GABAA receptor delta subunit (-44%; P < 0.001) but not alpha4 (+9%). These patterns of expression were not modified in animals that had free access to ethanol; nevertheless the baseline expression of the alpha 4 subunit was significantly reduced (P < 0.05) in all three genotypes of mice exposed to ethanol. These results show for the first time that the absence or reduced expression of GABAB receptors in KO and ET mice, respectively, significantly increases both the amount of ethanol consumed and the preference to ethanol, and suggests that GABAB receptors may play a pivotal role in controlling ethanol drinking behaviour. Knock-out of the GABAB receptor alters the gene expression of the delta subunit of the GABAA receptor, the putative ethanol sensitive subunit, but this effect is not likely to be involved in the increased ethanol drinking behaviour since was not evident in ET mice that drank similar amounts of ethanol compared to KO mice

Inhibition of Enterovirus A71 by a Novel 2-Phenyl-Benzimidazole Derivative

Enterovirus A71 (EV-A71) infection has emerged as a significant public health concern at the global level. Epidemic events of EV-A71 have been reported worldwide, and this succession of outbreaks has heightened concern that EV-A71 may become a public health threat. In recent years, widespread A71 enterovirus also occurred in European countries. EV-A71 infection causes hand-foot-mouth disease (HFMD), herpangina, and fever. However, it can sometimes induce a variety of neurological complications, including encephalitis, aseptic meningitis, pulmonary edema, and acute flaccid paralysis. We identified new benzimidazole derivatives and described theirin vitrocytotoxicity and broad-spectrum anti-enterovirus activity. Among them, derivative 2b resulted in interesting activity against EV-A71, and therefore it was selected for further investigations. Compound 2b proved to be able to protect cell monolayers from EV-A71-induced cytopathogenicity, with an EC50 of 3 µM. Moreover, Vero-76 cells resulted in being significantly protected from necrosis and apoptosis when treated with 2b at 20 and 80 µM. Compound 2b reduced viral adsorption to Vero-76 cells, and when evaluated in a time-of-addition assay, the derivative had the highest effect when added during the infection period. Moreover, derivative 2b reduced viral penetration into host cells. Besides, 2b did not affect intestinal monolayers permeability, showing no toxic effects. A detailed insight into the efficacy of compound 2b against EV-A71 showed a dose-dependent reduction in the viral titer, also at low concentrations. Mechanism of action investigations suggested that our derivative can inhibit viral endocytosis by reducing viral attachment to and penetration into host cells. Pharmacokinetic and toxicity predictions validated compound 2b as a good candidate for furtherin vivoassays