Gastric Carcinoma in Autoimmune Gastritis: A Histopathologic and Molecular Study.

Patients with autoimmune gastritis (AIG) have a 13-fold risk of developing type-1 neuroendocrine tumors, whereas the risk for gastric adenocarcinoma is still uncertain. Here we describe the clinicopathologic and molecular features of a series of gastric carcinomas (GC) arising in the context of AIG. A total of 26 AIG-associated GC specimens were collected from 4 Italian Institutions. Immunohistochemistry for MUC1, MUC2, MUC5AC, MUC6, CDX2, HER2, PD-L1, CLDN18, mismatch repair (MMR) proteins, and p53 and EBV-encoded RNA (EBER) in situ hybridization were performed. Histologic and immunohistochemical features were jointly reviewed by 5 expert gastrointestinal pathologists. Next-generation sequencing analysis (TrueSight Oncology 500, Illumina) of 523 cancer-related genes was performed on 19 cases. Most tumors were diagnosed as pT1 (52%) and they were located in the corpus/fundus (58%) and associated with operative link for gastritis assessment stage II gastritis (80.8%), absence of parietal cells, complete intestinal metaplasia, and enterochromaffin-like-cell micronodular hyperplasia. Only 4 (15.4%) GCs were diagnosed during follow-up for AIG. The following histotypes were identified: 20 (77%) adenocarcinomas; 3 (11%) mixed neuroendocrine-non-neuroendocrine neoplasms, and 2 (8%) high-grade solid adenocarcinomas with focal neuroendocrine component, 1 (4%) adenocarcinoma with an amphicrine component. Overall, 7 cases (27%) showed MMR deficiency, 3 (12%) were positive (score 3+) for HER2, 6 (23%) were CLDN18 positive, and 11 (42%) had PD-L1 combined positive score ≥ 10. EBER was negative in all cases. Molecular analysis revealed 5/19 (26%) microsatellite instability (MSI) cases and 7 (37%) tumor mutational burden (TMB) high. The most frequently altered genes were TP53 (8/19, 42%), RNF43 (7/19, 37%), ERBB2 (7/19, 37% [2 amplified and 5 mutated cases]), ARID1A (6/19, 32%), and PIK3CA (4/19, 21%). In summary, AIG-associated GCs are often diagnosed at low stage in patients with longstanding misrecognized severe AIG; they often display a neuroendocrine component or differentiation, have relatively higher rates of MMR deficiency, and TMB high.

HER2 expression and genOmic characterization of rESected brain metastases from colorectal cancer: the HEROES study.

BACKGROUND: Little is known about prognostic factors of brain metastases (BM) from colorectal cancer (CRC). HER2 amplification/overexpression (HER2+) was previously described; its impact on prognosis remains uncertain. METHODS: In the translational study HEROES, extensive molecular analysis was performed on primary CRC (prCRC) and their matched resected BM by means of NGS comprehensive genomic profiling and HER2 status as assessed by immunohistochemical/ in situ hybridization. Count of tumour-infiltrating lymphocytes (TILs) was also performed. PRIMARY OBJECTIVE: to describe the molecular landscape of paired BM/prCRC. SECONDARY OBJECTIVES: to search for new prognostic biomarkers of outcome after BM resection: intracranial-only Progression-Free Survival (BM-iPFS), Progression-Free Survival (BM-PFS), and Overall Survival (BM-OS). RESULTS: Out of 22 patients having paired samples of prCRC and BM, HER2+ was found on 4 (18%) BM, 3 (75%) of which also HER2+ in matched prCRC. Lower tumour mutation burden (HR 3.08; 95%CI 1.06-8.93; p = 0.0386) and HER2-negative BM (HER2neg) (HR 7.75;95%CI 1.97-30.40; p = 0.0033) were associated with longer BM-iPFS; HER2neg BM (HR 3.44; 95%CI 1.03-11.53; p = 0.0449) and KRASmut BM (HR 0.31; 95%CI 0.12-0.80; p = 0.0153) conferred longer BM-PFS. Longer BM-OS was found in pts with TILs-enriched (≥1.6/HPF) BM (HR 0.11; 95%CI0.01-0.91; p = 0.0403). CONCLUSIONS: This study shows HER2+ enrichment in both BM and their prCRC. TILs-enriched BM conferred better BM-OS.

Inhibitory effects of SARS-CoV-2 spike protein and BNT162b2 vaccine on erythropoietin-induced globin gene expression in erythroid precursor cells from patients with ß-thalassemia.

During the recent coronavirus disease 2019 (COVID-19) pandemic several patients with ß-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with ß-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a ß-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes were ß+-IVSI-110/ß+-IVSI-110 (one patient),  ß039/ß+-IVSI-110 (3 patients), and ß039/ ß039 (2 patients). After treatment with S-protein or BNT162b2 for 5 days, lysates were analyzed by high performance liquid chromatography (HPLC), for hemoglobin production, and isolated RNA was assayed by RT-qPCR, for detection of globin gene expression. The main conclusions of the results obtained are that SARS-CoV-2 S-protein and BNT162b2 vaccine (a) inhibit fetal hemoglobin (HbF) production by ß-thalassemic ErPCs and (b) inhibit γ-globin mRNA accumulation. In addition, we have performed in silico studies suggesting a high affinity of S-protein to HbF. Remarkably, the binding interaction energy of fetal hemoglobin to S-protein was comparable with that of angiotensin-converting enzyme 2 (ACE2). Our results are consistent with the hypothesis of a relevant impact of SARS-CoV-2 infection and COVID-19 vaccination on the hematopoietic system.

The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from ß-Thalassemia Patients Treated with Sirolimus.

The ß-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the ß-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including ß-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from ß-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for ß-thalassemias.

Effects of Mithramycin on BCL11A Gene Expression and on the Interaction of the BCL11A Transcriptional Complex to γ-Globin Gene Promoter Sequences.

The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from ß-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of ß-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that alterations of BCL11A gene expression and biological functions occur during MTH-mediated erythroid differentiation. Our study demonstrates that one of the mechanisms of action of MTH is a down-regulation of the transcription of the BCL11A gene, while a second mechanism of action is the inhibition of the molecular interactions between the BCL11A complex and specific sequences of the γ-globin gene promoter.

The Long Scientific Journey of Sirolimus (Rapamycin): From the Soil of Easter Island (Rapa Nui) to Applied Research and Clinical Trials on ß-Thalassemia and Other Hemoglobinopathies.

In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to induce γ-globin gene expression and increased production of fetal hemoglobin (HbF) in erythroid cells, including primary erythroid precursor cells (ErPCs) isolated from ß-thalassemia patients. For this reason, rapamycin is considered of great interest for the treatment of ß-thalassemia. In fact, high levels of HbF are known to be highly beneficial for ß-thalassemia patients. The story of rapamycin discovery began in 1964, with METEI, the Medical Expedition to Easter Island (Rapa Nui). During this expedition, samples of the soil from different parts of the island were collected and, from this material, an antibiotic-producing microorganism (Streptomyces hygroscopicus) was identified. Rapamycin was extracted from the mycelium with organic solvents, isolated, and demonstrated to be very active as an anti-bacterial and anti-fungal agent. Later, rapamycin was demonstrated to inhibit the in vitro cell growth of tumor cell lines. More importantly, rapamycin was found to be an immunosuppressive agent applicable to prevent kidney rejection after transplantation. More recently, rapamycin was found to be a potent inducer of HbF both in vitro using ErPCs isolated from ß-thalassemia patients, in vivo using experimental mice, and in patients treated with this compound. These studies were the basis for proposing clinical trials on ß-thalassemia patients.

Assessing the interaction between hemoglobin and the receptor binding domain of SARS-CoV-2 spike protein through MARTINI coarse-grained molecular dynamics.

The emergence of different coronavirus-related diseases in the 2000's (SARS, MERS, and Covid-19) warrants the need of a complete understanding of the pathological, biological, and biochemical behavior of this class of pathogens. Great attention has been paid to the SARS-CoV-2 Spike protein, and its interaction with the human ACE2 has been thoroughly investigated. Recent findings suggested that the SARS-CoV-2 components may interact with different human proteins, and hemoglobin has very recently been demonstrated as a potential target for the Spike protein. Here we have investigated the interaction between either adult or fetal hemoglobin and the receptor binding domain of the Spike protein at molecular level through advanced molecular dynamics techniques and proposed rational binding modes and energy estimations. Our results agree with biochemical data previously reported in literature. We also demonstrated that co-incubation of pulmonary epithelial cells with hemoglobin strongly reduces the pro-inflammatory effects exerted by the concomitant administration of Spike protein.

Cationic Calix[4]arene Vectors to Efficiently Deliver AntimiRNA Peptide Nucleic Acids (PNAs) and miRNA Mimics.

One of the most appealing approaches for regulating gene expression, named the "microRNA therapeutic" method, is based on the regulation of the activity of microRNAs (miRNAs), the intracellular levels of which are dysregulated in many diseases, including cancer. This can be achieved by miRNA inhibition with antimiRNA molecules in the case of overexpressed microRNAs, or by using miRNA-mimics to restore downregulated microRNAs that are associated with the target disease. The development of new efficient, low-toxic, and targeted vectors of such molecules represents a key topic in the field of the pharmacological modulation of microRNAs. We compared the delivery efficiency of a small library of cationic calix[4]arene vectors complexed with fluorescent antimiRNA molecules (Peptide Nucleic Acids, PNAs), pre-miRNA (microRNA precursors), and mature microRNAs, in glioma- and colon-cancer cellular models. The transfection was assayed by cytofluorimetry, cell imaging assays, and RT-qPCR. The calix[4]arene-based vectors were shown to be powerful tools to facilitate the uptake of both neutral (PNAs) and negatively charged (pre-miRNAs and mature microRNAs) molecules showing low toxicity in transfected cells and ability to compete with commercially available vectors in terms of delivery efficiency. These results could be of great interest to validate microRNA therapeutics approaches for future application in personalized treatment and precision medicine.

Effects of Sulforaphane on SARS­CoV­2 infection and NF­κB dependent expression of genes involved in the COVID­19 'cytokine storm'.

Since its spread at the beginning of 2020, the coronavirus disease 2019 (COVID­19) pandemic represents one of the major health problems. Despite the approval, testing, and worldwide distribution of anti­severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) vaccines, the development of specific antiviral agents targeting the SARS­CoV­2 life cycle with high efficiency, and/or interfering with the associated 'cytokine storm', is highly required. A recent study, conducted by the authors' group indicated that sulforaphane (SFN) inhibits the expression of IL­6 and IL­8 genes induced by the treatment of IB3­1 bronchial cells with a recombinant spike protein of SARS­CoV­2. In the present study, the ability of SFN to inhibit SARS­CoV­2 replication and the expression of pro­inflammatory genes encoding proteins of the COVID­19 'cytokine storm' was evaluated. SARS­CoV­2 replication was assessed in bronchial epithelial Calu­3 cells. Moreover, SARS­CoV­2 replication and expression of pro­inflammatory genes was evaluated by reverse transcription quantitative droplet digital PCR. The effects on the expression levels of NF­κB were assessed by western blotting. Molecular dynamics simulations of NF­kB/SFN interactions were conducted with Gromacs 2021.1 software under the Martini 2 CG force field. Computational studies indicated that i) SFN was stably bound with the NF­κB monomer; ii) a ternary NF­kB/SFN/DNA complex was formed; iii) the SFN interacted with both the protein and the nucleic acid molecules modifying the binding mode of the latter, and impairing the full interaction between the NF­κB protein and the DNA molecule. This finally stabilized the inactive complex. Molecular studies demonstrated that SFN i) inhibits the SARS­CoV­2 replication in infected Calu­3 cells, decreasing the production of the N­protein coding RNA sequences, ii) decreased NF­κB content in SARS­CoV­2 infected cells and inhibited the expression of NF­kB­dependent IL­1ß and IL­8 gene expression. The data obtained in the present study demonstrated inhibitory effects of SFN on the SARS­CoV­2 life cycle and on the expression levels of the pro­inflammatory genes, sustaining the possible use of SFN in the management of patients with COVID­19.

Combined Treatment of Cancer Cells Using Allyl Palladium Complexes Bearing Purine-Based NHC Ligands and Molecules Targeting MicroRNAs miR-221-3p and miR-222-3p: Synergistic Effects on Apoptosis.

Combined treatments employing lower concentrations of different drugs are used and studied to develop new and more effective anticancer therapeutic approaches. The combination therapy could be of great interest in the controlling of cancer. Regarding this, our research group has recently shown that peptide nucleic acids (PNAs) that target miR-221 are very effective and functional in inducing apoptosis of many tumor cells, including glioblastoma and colon cancer cells. Moreover, in a recent paper, we described a series of new palladium allyl complexes showing a strong antiproliferative activity on different tumor cell lines. The present study was aimed to analyze and validate the biological effects of the most active compounds tested, in combination with antagomiRNA molecules targeting two miRNAs, miR-221-3p and miR-222-3p. The obtained results show that a "combination therapy", produced by combining the antagomiRNAs targeting miR-221-3p, miR-222-3p and the palladium allyl complex 4d, is very effective in inducing apoptosis, supporting the concept that the combination treatment of cancer cells with antagomiRNAs targeting a specific upregulated oncomiRNAs (in this study miR-221-3p and miR-222-3p) and metal-based compounds represents a promising therapeutic strategy to increase the efficacy of the antitumor protocol, reducing side effects at the same time.

The Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) Is under Post-Transcriptional Control of microRNAs: Analysis of the Effects of agomiRNAs Mimicking miR-145-5p, miR-101-3p, and miR-335-5p.

(1) Background: MicroRNAs are involved in the expression of the gene encoding the chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator); the objective of this short report is to study the effects of the treatment of bronchial epithelial Calu-3 cells with molecules mimicking the activity of pre-miR-145-5p, pre-miR-335-5p, and pre-miR-101-3p, and to discuss possible translational applications of these molecules in pre-clinical studies focusing on the development of protocols of possible interest in therapy; (2) Methods: CFTR mRNA was quantified by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The production of the CFTR protein was assessed by Western blotting; (3) Results: The treatment of Calu-3 cells with agomiR-145-5p caused the highest inhibition of CFTR mRNA accumulation and CFTR production; (4) Conclusions: The treatment of target cells with the agomiR pre-miR-145-5p should be considered when CFTR gene expression should be inhibited in pathological conditions, such as polycystic kidney disease (PKD), some types of cancer, cholera, and SARS-CoV-2 infection.

Production and Characterization of K562 Cellular Clones Hyper-Expressing the Gene Encoding α-Globin: Preliminary Analysis of Biomarkers Associated with Autophagy.

One of the most relevant pathophysiological hallmarks of ß-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the obtained clones seemed to trigger autophagy spontaneously, probably to overcome the accumulation/toxicity of the α-globin. We propose this new model system for the screening of pharmacological agents able to activate the full program of autophagy to reduce α-globin accumulation, but the model may be also suitable for new therapeutical approaches targeted at the reduction of the expression of the α-globin gene.

The rs368698783 (G>A) Polymorphism Affecting LYAR Binding to the Aγ-Globin Gene Is Associated with High Fetal Hemoglobin (HbF) in ß-Thalassemia Erythroid Precursor Cells Treated with HbF Inducers

The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5'-GGTTAT-3') is located within the 5'-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in ß-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify ß-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in ß-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of ß-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment.

Effects of Sirolimus treatment on patients with ß-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells.

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. ß-Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in ß-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4+ and CD8+ T cells, respectively. The main results of the present study are that treatment of ß-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4+ and CD8+ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that ß-Thalassemia patients are considered prone to immune deficiency.

Combined Treatment of Bronchial Epithelial Calu-3 Cells with Peptide Nucleic Acids Targeting miR-145-5p and miR-101-3p: Synergistic Enhancement of the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes for a chloride channel defective in Cystic Fibrosis (CF). Accordingly, upregulation of its expression might be relevant for the development of therapeutic protocols for CF. MicroRNAs are deeply involved in the CFTR regulation and their targeting with miRNA inhibitors (including those based on Peptide Nucleic Acids, PNAs)is associated with CFTR upregulation. Targeting of miR-145-5p, miR-101-3p, and miR-335-5p with antisense PNAs was found to be associated with CFTR upregulation. The main objective of this study was to verify whether combined treatments with the most active PNAs are associated with increased CFTR gene expression. The data obtained demonstrate that synergism of upregulation of CFTR production can be obtained by combined treatments of Calu-3 cells with antisense PNAs targeting CFTR-regulating microRNAs. In particular, highly effective combinations were found with PNAs targeting miR-145-5p and miR-101-3p. Content of mRNAs was analyzed by RT-qPCR, the CFTR production by Western blotting. Combined treatment with antagomiRNAs might lead to maximized upregulation of CFTR and should be considered in the development of protocols for CFTR activation in pathological conditions in which CFTR gene expression is lacking, such as Cystic Fibrosis.

Expression of γ-globin genes in ß-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin).

Introduction: ß-thalassemia is caused by autosomal mutations in the ß-globin gene, which induce the absence or low-level synthesis of ß-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with ß-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for ß-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the ß+/ß+ and ß+/ß0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from ß-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with ß-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies.

Synergistic Effects of A Combined Treatment of Glioblastoma U251 Cells with An Anti-miR-10b-5p Molecule and An AntiCancer Agent Based on 1-(3',4',5'-Trimethoxyphenyl)-2-Aryl-1H-Imidazole Scaffold.

(1) Background: In the development of new and more effective anticancer approaches, combined treatments appear of great interest. Combination therapy could be of importance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer of the central nervous system, with a median survival of 15 months. This study aimed to verify the activity on a glioblastoma cancer cell line of one of the most active compounds of a novel series of tubulin polymerization inhibitors based on the 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold, used in combination with a miRNA inhibitor molecule targeting the oncomiRNA miR-10b-5p. This microRNA was selected in consideration of the role of miR-10b-5p on the onset and progression of glioblastoma. (2) Methods: Apoptosis was analyzed by Annexin-V and Caspase 3/7 assays, efficacy of the anti-miR-10b-5p was assessed by determining the miR-10b-5p content by RT-qPCR. (3) Results: The results obtained show that a "combination therapy" performed by combining the use of an anti-miR-10b-5p and a 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole derivative is an encouraging strategy to boost the efficacy of anticancer therapies and at the same time to reduce side effects.

Teaching during COVID-19 pandemic in practical laboratory classes of applied biochemistry and pharmacology: A validated fast and simple protocol for detection of SARS-CoV-2 Spike sequences.

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.

Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis.

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.