Pseudanabaena galeata CCNP1313 : biological activity and peptides production

Even cyanobacteria from ecosystems of low biodiversity, such as the Baltic Sea, can constitute a rich source of bioactive metabolites. Potent toxins, enzyme inhibitors, and anticancer and antifungal agents were detected in both bloom-forming species and less commonly occurring cyanobacteria. In previous work on the Baltic Pseudanabaena galeata CCNP1313, the induction of apoptosis in the breast cancer cell line MCF-7 was documented. Here, the activity of the strain was further explored using human dermal fibroblasts, African green monkey kidney, cancer cell lines (T47D, HCT-8, and A549(ACE2/TMPRSS2)) and viruses (SARS-CoV-2, HCoV-OC43, and WNV). In the tests, extracts, chromatographic fractions, and the main components of the P. galeata CCNP1313 fractions were used. The LC-MS/MS analyses of the tested samples led to the detection of forty-five peptides. For fourteen of the new peptides, putative structures were proposed based on MS/MS spectra. Although the complex samples (i.e., extracts and chromatographic fractions) showed potent cytotoxic and antiviral activities, the effects of the isolated compounds were minor. The study confirmed the significance of P. galeata CCNP1313 as a source of metabolites with potent activity. It also illustrated the difficulties in assigning the observed biological effects to specific metabolites, especially when they are produced in minute amounts

SARS-CoV-2 infects an <I>in vitro</I> model of the human developing pancreas through endocytosis

Recent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic β-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors in vitro. Moreover, we investigated mechanisms by which SARS-CoV-2 enters pancreatic cells, and found that ACE2 mediates the entry, while NRP1 and TMPRSS2 do not. Surprisingly, we found that in pancreatic progenitors, SARS-CoV-2 enters cells via cathepsin-dependent endocytosis, which is a different route than in respiratory tract. Therefore, pancreatic spheroids might serve as a model to study candidate drugs for endocytosis-mediated viral entry inhibition and to investigate whether SARS-CoV-2 infection may affect pancreas development, possibly causing lifelong health consequences

Generation of organotypic multicellular spheres by magnetic levitation : model for the study of human hematopoietic stem cells microenvironment

Q4Q3Background and Objective: The characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mesenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC. Methods and Results: Human bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, LonzaⓇ) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm3 and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells. Conclusions: We developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.https://orcid.org/0000-0002-9152-5552https://orcid.org/0000-0003-3075-9854https://orcid.org/0000-0002-0084-0339https://orcid.org/0000-0003-1881-9367N/

Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks. © 2021 The Author

3D multicellular spheroid for the study of human hematopoietic stem cells : synergistic effect between oxygen levels, mesenchymal stromal cells and endothelial cells

Q3Q3Introduction: The human bone marrow microenvironment is composed of biological, chemical and physical factors that act in a synergistic way to modulate hematopoietic stem cell biology, such as mesenchymal stromal cells (MSCs), endothelial cells (ECs) and low oxygen levels; however, it is difficult to mimic this human microenvironment in vitro. Methods: In this work, we developed 3D multicellular spheroid (3D-MS) for the study of human hematopoietic stem cells (HSCs) with some components of perivascular niche. HSCs were isolated from umbilical cord blood, MSCs were isolated from human bone marrow and a microvasculature EC line (CC-2811, Lonza®) was used. For the formation of a 3D structure, a magnetic levitation culture system was used. Cultures were maintained in 21%, 3% and 1% O2 for 15 days. Culture volume, sphericity index and cell viability were determined. Also, human HSC proliferation, phenotype and production of reactive oxygen species were evaluated. Results: After 15 days, 3D-MS exhibited viability greater than 80%. Histology results showed structures without necrotic centers, and higher cellular proliferation with 3% O2. An increase in the expression of the CD34 antigen and other hematopoietic antigens were observed to 1% O2 with MSCs plus ECs and low ROS levels. Conclusion: These findings suggest that 3D-MS formed by MSCs, ECs and HSCs exposed to low concentrations of oxygen (1–3% O2) modulate human HSC behavior and mimics some features of the perivascular niche, which could reduce the use of animal models and deepen the relationship between the microenvironment of HSC and human hematological diseases development.https://orcid.org/ 0000-0002-9152-5552Revista Internacional - IndexadaS

The interplay between the airway epithelium and tissue macrophages during the SARS-CoV-2 infection

The first line of antiviral immune response in the lungs is secured by the innate immunity. Several cell types take part in this process, but airway macrophages (AMs) are among the most relevant ones. The AMs can phagocyte infected cells and activate the immune response through antigen presentation and cytokine release. However, the precise role of macrophages in the course of SARS-CoV-2 infection is still largely unknown. In this study, we aimed to evaluate the role of AMs during the SARS-CoV-2 infection using a co-culture of fully differentiated primary human airway epithelium (HAE) and human monocyte-derived macrophages (hMDMs). Our results confirmed abortive SARS-CoV-2 infection in hMDMs, and their inability to transfer the virus to epithelial cells. However, we demonstrated a striking delay in viral replication in the HAEs when hMDMs were added apically after the epithelial infection, but not when added before the inoculation or on the basolateral side of the culture. Moreover, SARS-CoV-2 inhibition by hMDMs seems to be driven by cell-to-cell contact and not by cytokine production. Together, our results show, for the first time, that the recruitment of macrophages may play an important role during the SARS-CoV-2 infection, limiting the virus replication and its spread

DataSheet_1_The interplay between the airway epithelium and tissue macrophages during the SARS-CoV-2 infection.pdf

The first line of antiviral immune response in the lungs is secured by the innate immunity. Several cell types take part in this process, but airway macrophages (AMs) are among the most relevant ones. The AMs can phagocyte infected cells and activate the immune response through antigen presentation and cytokine release. However, the precise role of macrophages in the course of SARS-CoV-2 infection is still largely unknown. In this study, we aimed to evaluate the role of AMs during the SARS-CoV-2 infection using a co-culture of fully differentiated primary human airway epithelium (HAE) and human monocyte-derived macrophages (hMDMs). Our results confirmed abortive SARS-CoV-2 infection in hMDMs, and their inability to transfer the virus to epithelial cells. However, we demonstrated a striking delay in viral replication in the HAEs when hMDMs were added apically after the epithelial infection, but not when added before the inoculation or on the basolateral side of the culture. Moreover, SARS-CoV-2 inhibition by hMDMs seems to be driven by cell-to-cell contact and not by cytokine production. Together, our results show, for the first time, that the recruitment of macrophages may play an important role during the SARS-CoV-2 infection, limiting the virus replication and its spread.</p