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Establishing a drug-screening platform is critical for the discovery of potential antiviral agents against SARS-CoV-2. In this study, we developed a platform based on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate SARS-CoV-2 infectivity, with the aim of evaluating potential antiviral agents for anti-SARS-CoV-2 activity and cardiotoxicity. Cultured myocytes of iPSC-CMs and immortalized human cardiomyocyte cell line (AC-16) were primarily characterized for the expression of cardiac markers and host receptors of SARS-CoV-2. An infectivity model for the wild-type SARS-CoV-2 strain was then established. Infection modeling involved inoculating cells with SARS-CoV-2 at varying multiplicities of infection (MOIs) and then quantifying infection using immunofluorescence and plaque assays. Only iPSC-CMs, not AC16 cells, expressed angiotensin-converting enzyme 2 (ACE-2), and quantitative assays confirmed the dose-dependent infection of iPSC-CMs by SARS-CoV-2, unlike the uninfectable AC16 cells lacking the expression of ACE2. Cytotoxicity was evaluated using MTT assays across a concentration range. An assessment of the plant-derived compound panduratin A (panA) showed cytotoxicity at higher doses (50% cytotoxic concentration (CC50) 10.09 μM) but promising antiviral activity against SARS-CoV-2 (50% inhibition concentration (IC50) 0.8–1.6 μM), suppressing infection at concentrations 10 times lower than its CC50. Plaque assays also showed decreased viral production following panA treatment. Overall, by modeling cardiac-specific infectivity, this iPSC-cardiomyocyte platform enables the reliable quantitative screening of compound cytotoxicity alongside antiviral efficacy. By combining disease pathogenesis and pharmacology, this system can facilitate the evaluation of potential novel therapeutics, such as panA, for drug discovery applications. [ABSTRACT FROM AUTHOR]

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most prevalent genetic diseases affecting the kidneys. A genetically specific mutation model is required to comprehend its pathophysiology and to develop a drug treatment. In this study, we successfully developed human induced pluripotent stem cells (hiPSCs) named MUi027-A from skin fibroblasts of a patient diagnosed with ADPKD and carrying the PKD1 frameshift mutation (c.7946_7947delCT). MUi027-A cells showed the same genetic fingerprints as the parental cells, including the presence of the PKD1 mutation. MUi027-A hiPSCs displayed embryonic stem cell-like characteristics with the capability of differentiating into the three germ layers. Upon directed differentiation, MUi027-A hiPSCs could be differentiated into tubular organoids with the expression of renal cell markers. Furthermore, we compared the efficiency of cyst formation in two human iPSC lines with different PKD1 mutations. When cyst formation was induced by either forskolin or blebbistatin, MUi027-A hiPSC-derived kidney organoids displayed higher frequencies of cyst formation when compared to organoids generated from an iPSC cell line with non-truncating PKD1 mutation genotype (c.5878C > T), suggesting the presence of physiological differences in the mechanism of cyst formation between different PKD1 mutants. Overall, we generated and characterized a novel human iPSC line with a specific PKD mutation and demonstrated its potential as a disease model to study the pathophysiology of genetic determinants in the development of ADPKD disease. [ABSTRACT FROM AUTHOR]

Although the TlyA hemolysin from Helicobacter pylori has been implicated as a potential virulence factor involved in mediating host cell colonization and hence disease progression, its structural determinants underlying the biological activity are still largely uncertain. In this study, an important role of the formation of a particular disulfide bond for functional oligomeric assembly of the H. pylori TlyA toxin was evidently elucidated. The 27-kDa TlyA recombinant protein was overexpressed in Escherichia coli , subsequently purified to near homogeneity by cation exchange chromatography, and proven to be hemolytically active against sheep erythrocytes. Additionally, TlyA-induced hemolytic activity was significantly diminished under conditions of disulfide bond reduction with a thiol-reducing agent, dithiothreitol. When the purified TlyA protein was subjected to modified SDS-PAGE under non-reducing conditions, the presence of an oligomeric state of this protein was clearly revealed by its apparent molecular mass of ∼48 kDa. Recombinant E. coli cells expressing TlyA also displayed contact-dependent hemolysis of erythrocytes, suggesting TlyA localization at the bacterial outer membrane and thus supporting the formation of disulfide-bonded TlyA. Homology-based modeling and in silico structural assembly analysis of TlyA signified potential intermolecular, rather than intramolecular, disulfide bonding through Cys124 and Cys128. Subsequently, single substitution of either of these Cys residues with Ser severely affected the oligomeric assembly of both TlyA mutants and hence abolished their hemolytic activity. Altogether, our present data provide pivotal evidence that the formation of intermolecular disulfide bonding between Cys124 and Cys128 plays a critical role in structural assembly of a biologically active-TlyA oligomer. • Hemolysis induced by H. pylori TlyA was drastically decreased under reducing conditions. • 27-kDa TlyA formed ∼48-kDa oligomers as analyzed by non-reducing semi-native SDS-PAGE. • Cys124−Cys128 intermolecular disulfide bonding was revealed through TlyA-dimers by in silico analysis. • TlyA-induced hemolysis and oligomeric assembly were affected by C124S or C128S single-mutation. [ABSTRACT FROM AUTHOR]

The membrane perturbing action of the VacA toxin from Helicobacter pylori is responsible for vacuole formation in intracellular compartments and the induction of apoptosis. The VacA toxin contains 2 major domains, p33 and p55, which are involved in receptor binding and membrane pore formation, respectively. Improved methodologies for VacA purification and assays are urgently needed for further detailed investigations on the mechanism of action of this significant virulence factor. We found that by fusing mouse DHFR with the N-terminus of the full-length (p88) VacA toxin, expression levels in recombinant E. coli were substantially increased when compared to the conventional (His)6-tagged protein. The DHFR-VacA fusion protein was active in sulforhodamine dye-release assays using liposomes at acidic pH in a concentration-dependent manner. Enzymatic activity of DHFR in the fusion protein was comparable to a commercial reference sample of purified DHFR; however, activity was insensitive to inhibition by methotrexate. Our findings suggest that the VacA p88 toxin with a modified N-terminus still maintains its capability for membrane insertion and that pH-dependent conformational changes occur during interaction of VacA with membranes. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is associated with severe and chronic diseases of the stomach and duodenum such as peptic ulcer, non-cardial adenocarcinoma and gastric lymphoma, making Helicobacter pylori the only bacterial pathogen which is known to cause cancer. The worldwide rate of incidence for these diseases is extremely high and it is estimated that about half of the world's population is infected with H. pylori . Among the bacterial virulence factors is the vacuolating cytotoxin A (VacA), which represents an important determinant of pathogenicity. Intensive characterization of VacA over the past years has provided insight into an ample variety of mechanisms contributing to host-pathogen interactions. The toxin is considered as an important target for ongoing research for several reasons: i) VacA displays unique features and structural properties and its mechanism of action is unrelated to any other known bacterial toxin; ii) the toxin is involved in disease progress and colonization by H. pylori of the stomach; iii) VacA is a potential and promising candidate for the inclusion as antigen in a vaccine directed against H. pylori and iv) the vacA gene is characterized by a high allelic diversity, and allelic variants contribute differently to the pathogenicity of H. pylori . Despite the accumulation of substantial data related to VacA over the past years, several aspects of VacA-related activity have been characterized only to a limited extent. The biologically most significant effect of VacA activity on host cells is the formation of membrane pores and the induction of vacuole formation. This review discusses recent findings and advances on structure-function relations of the H. pylori VacA toxin, in particular with a view to membrane channel formation, oligomerization, receptor binding and apoptosis. [ABSTRACT FROM AUTHOR]

Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10−3 ng and 5 × 10−6 ng of target copy per reaction. We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting. [ABSTRACT FROM AUTHOR]

Vacuolating cytotoxin A (VacA) is a highly polymorphic virulence protein produced by the human gastric pathogen Helicobacter pylori which can cause gastritis, peptic ulcer and gastric cancer. Here, we present an optimized protein preparation of the mature full-length VacA variants (m1-and m2-types) and their 33-kDa N-terminal and 55/59-kDa C-terminal domains as biologically active recombinant proteins fused with an N-terminal His (6) tag. All recombinant VacA constructs were over-expressed in Escherichia coli as insoluble inclusions which were soluble when phosphate buffer (pH 7.4) was supplemented with 5–6 M urea. Upon immobilized-Ni2+ affinity purification under 5-M urea denaturing conditions, homogenous products (>95% purity) of 55/59-kDa domains were consistently obtained while only ~80% purity of both mature VacA variants and the 33-kDa truncate was achieved, thus requiring additional purification by size-exclusion chromatography. After successive refolding via optimized stepwise dialysis, all refolded VacA proteins were proven to possess both cytotoxic and vacuolating activity against cultured human gastric epithelial cells albeit the activity observed for VacA-m2 was lower than the m1-type variant. Such an optimized protocol described herein was effective for production of high-purity recombinant VacA proteins in large amounts (~30–40 mg per liter culture) that would pave the way for further studies on sequence-structure and function relationships of different VacA variants. • High-purity preparation of functional VacA-m1/m2 variants fused with N-terminal His (6) tag was optimally devised. • Efficient solubilization of VacA protein inclusions was achieved in 5–6 M urea. • Urea-denatured VacA proteins were purified to homogeneity via IMAC or IMAC followed by gel filtration. • Denaturant removal by optimized stepwise dialysis yielded high recovery of refolded VacA proteins. • Refolded VacA-m1 proteins were more active than m2-type variant against cultured human gastric cells. [ABSTRACT FROM AUTHOR]