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The application of Peptide Nucleic Acids (PNAs), mimics of DNA lacking the sugar-phosphate backbone, for antisense/anti-gene therapy and gene editing is limited by their low uptake by cells. Currently, no simple and efficient delivery systems and methods are available to solve this open issue. One of the most promising approach is the modification of the PNA structure through the covalent linkage of poliarginine tails, but this means that every PNA intended to be internalized must be modified. Herein we report the results relative to the delivery ability of a macrocyclic multivalent tetraargininocalix[4]arene ( 1 ) used as non-covalent vector for anti-miR-221-3p PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity and ease preparation of the transfection formulation, simply attained by mixing PNA and calixarene, candidate this vector as universal delivery system for this class of nucleic acid analogues.

Garlic (Allium sativum L.) is a species of the onion family (Alliaceae) widely used as a food and a folk medicine. The objective of this study was to determine the effects of AGE (aged garlic extract) on pro-inflammatory genes relevant to COVID-19. To this aim, we treated bronchial epithelial IB3-1 cells with SARS-CoV-2 spike protein (S-protein) or with the COVID-19 BNT162b2 vaccine in the absence or in the presence of AGE. The results obtained demonstrated that AGE is a potent inhibitor of the S-protein-induced expression of the IL-1β, IL-6 and IL-8 genes. Bio-Plex analysis demonstrated that AGE reduced release of IL-6 and IL-8, which were highly induced by S-protein. No inhibition of cells’ growth, toxicity and pro-apoptotic effects were found in AGE-treated cells. The effects of one of the major AGE constituents (S-allyl cysteine, SAC) were studied on the same experimental model systems. SAC was able to inhibit the S-protein-induced expression of IL-1β, IL-6 and IL-8 genes and extracellular release of IL-6 and IL-8, confirming that S-allyl-cysteine is one of the constituents of AGE that is responsible for inhibiting S-protein-induced pro-inflammatory genes. Docking experiments suggest that a possible mechanism of action of SAC is an interference with the activity of Toll-like receptors (TLRs), particularly TLR4, thereby inhibiting NF-κB- and NF-κB-regulated genes, such as IL-1β, IL-6 and IL-8 genes. These results suggest that both AGE and SAC deserve further experimental efforts to verify their effects on pro-inflammatory genes in SARS-CoV-2-infected cells.

Since the discovery and classification of non-coding RNAs, their roles have gained great attention. In this respect, microRNAs and long non-coding RNAs have been firmly demonstrated to be linked to regulation of gene expression and onset of human diseases, including rare genetic diseases; therefore they are suitable targets for therapeutic intervention. This issue, in the context of rare genetic diseases, is being considered by an increasing number of research groups and is of key interest to the health community. In the case of rare genetic diseases, the possibility of developing personalized therapy in precision medicine has attracted the attention of researchers and clinicians involved in developing “orphan medicinal products” and proposing these to the European Medicines Agency (EMA) and to the Food and Drug Administration (FDA) Office of Orphan Products Development (OOPD) in the United States. The major focuses of these activities are the evaluation and development of products (drugs, biologics, devices, or medical foods) considered to be promising for diagnosis and/or treatment of rare diseases or conditions, including rare genetic diseases. In an increasing number of rare genetic diseases, analysis of microRNAs and long non-coding RNAs has been proven a promising strategy. These diseases include, but are not limited to, Duchenne muscular dystrophy, cystic fibrosis, Rett syndrome, and β-thalassemia. In conclusion, a large number of approaches based on targeting microRNAs and long non-coding RNAs are expected in the field of molecular diagnosis and therapy, with a facilitated technological transfer in the case of rare genetic diseases, in virtue of the existing regulation concerning these diseases.

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a “combo-therapy” associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). We suggest that this laboratory practical exercises should be considered for face-to-face teaching especially if the emergency related to the COVID-19 pandemic is maintained. The teaching protocol here described might be considered in order to perform fast but meaningful in-presence teaching, making feasible the division of crowded classes in low-number cohorts of students, allowing the maintenance of the required social distance.

Gastric cancer (GC) represents one of the most common and lethal cancers worldwide. The lack of early detection methods and wide heterogeneity from both histopathological and molecular points of view contribute to the high mortality associated with GC. Nowadays, the growing interest in the epigenetic landscape appears to represent a promising approach for improving early diagnosis, disease monitoring, therapy selection, and drug response. The knowledge of the epigenetic landscape in cancerous and, especially in precancerous lesions, remain scarce and controversial. The limitations must be identified due to the lack of standardization in experimental settings which introduce bias and lead to discrepancies in final conclusions. This review aims to provide an overview of epigenetics in the diagnostic setting of gastric cancer and in preneoplastic conditions, illustrating the available experimental approaches and therapeutic management in the era of epigenetics.

BackgroundHuman epidermal growth factor receptor 2 (HER2; ERBB2) is overexpressed or amplified in 15–20% of gastric cancers (HER2+ GC). Within individual HER2+ GCs, HER2/ERBB2 expression is often variable. Although HER2 therapeutic targeting improves outcomes for HER2+ GC patients, acquired resistance is frequent.ObjectiveTo spatially interrogate HER2+ GC interpatient and intrapatient heterogeneity and resistance mechanisms associated with HER2-targeting agents (trastuzumab, trastuzumab deruxtecan (T-DXd)).DesignSpatial transcriptomic analysis (GeoMx Digital Spatial Profiler) was applied to >1500 regions of interest in 30 GCs—these contained 15 HER2+ GCs treated with trastuzumab and T-DXd subsequently. Analysis of patient-matched samples with acquired trastuzumab or T-DXd resistance revealed escape mechanisms. Results were validated by immunohistochemistry, independent cohorts and patient-derived xenografts and organoids.ResultsHER2+ tumours exhibited PD-L1 expression within the spatial tumour microenvironment. We observed increased expression of CLDN18.2, a promising therapeutic target, in trastuzumab-resistant tumours. One-third of HER2+ GC patients developed epithelial-mesenchymal transition (EMT) on trastuzumab resistance, associated with PD-L1 and CCL2 upregulation. Another third of trastuzumab-resistant HER2+ GC patients activated the endoplasmic reticulum-associated degradation (ERAD) pathway including genes such as GOLM1. HLA loss and increases in oxidative phosphorylation pathways were observed in T-DXd-resistant GCs.ConclusionOur results delineate multiple acquired resistance mechanisms to trastuzumab and T-DXd in HER2+ GC in vivo. This information may guide trials combining trastuzumab or T-DXd with new agents to enhance the efficacy and durability of HER2 blockade.

Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis.

Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level.

The biological activity of a combined treatment of U251, U373 and T98G glioma cell lines with two anti-miR PNAs, directed against miR-221 and miR-222 and conjugated with an ocataarginine tail (R8-PNA-a221 and R8-PNA-a222) for efficient cellular delivery, was determined. Apoptosis was analyzed, and the effect of the combined treatment of glioma cells with either or both PNAs on the reversion of drug-resistance phenotype was assessed in the temozolomide-resistant T98G glioma cell line. Selectivity of PNA/miRNA interactions was studied by surface plasmon resonance (SPR)-based Biacore analysis. Specificity of the PNA effects at the cellular level was analyzed by RT-qPCR. These experiments support the concept that the effects of R8-PNA-a221 and R8-PNA-a222 are specific. The studies on apoptosis confirmed that the R8-PNA-a221 induces apoptosis and demonstrated the pro-apoptotic effects of R8-PNA-a222. Remarkably, increased pro-apoptotic effects were obtained with the co-administration of both anti-miR-221 and anti-miR-222 PNAs. In addition, co-administration of R8-PNA-a221 and R8-PNA-a222 induced apoptosis of TMZ-treated T98G cells at a level higher than that obtained following singular administration of R8-PNA-a221 or R8-PNA-a222.