Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.     

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.     

Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran

Fascioliasis is an important human and animal disease caused by Fasciola hepatica and Fasciola gigantica. In Iran, the distribution of these two species overlaps in most areas, including the northern human endemic province of Gilan where both fasciolids are simultaneously found in individual cattle and buffaloes. A phenotypic study of fasciolid adult flukes from naturally infected bovines from Gilan was carried out by means of an exhaustive morphometric analysis using traditional microscopic measurements and an allometric model. The Iranian fasciolids were compared to F. hepatica and F. gigantica standard populations, i.e. from geographical areas where both species do not co-exist (Bolivia and Burkina Faso, respectively). Although morphometric values somewhat overlapped, there were clear differences in allometric growth. The allometric function was adjusted to 25 pairs of variables. Results obtained revealed that Iranian F. hepatica-like specimens are larger than the F. hepatica standard and Iranian F. gigantica-like specimens are longer and narrower than the F. gigantica standard, but with smaller body area. Measurements which permit a specific differentiation in allopatric populations (distance between ventral sucker and posterior end of the body; ratio between body length and body width) overlap in the specimens from Gilan, thus proving the presence of intermediate forms. When compared to the standard populations, the different Iranian fasciolid morphs show greater differences in F. gigantica-like specimens than in F. hepatica-like specimens. This study shows that simple, traditional microscopic measurements may be sufficient for the morphometric characterisation of fasciolids, even in areas where intermediate forms are present.     

Increase of autophagy and attenuation of apoptosis by Salvigenin promote survival of SH-SY5Y cells following treatment with H2O2

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. Here, we tried to elucidate the possible neuroprotective effect of Salvigenin, a natural polyphenolic compound, on oxidative stress-induced apoptosis and autophagy in human neuroblastoma SH-SY5Y cells. We measured cell viability by MTT test and found that 25?μM is the best protective concentration of Salvigenin. GSH and SOD assays suggested that Salvigenin activates antioxidant factors. At the same time, measurement of ER stress-associated proteins including calpain and caspase-12 showed the ability of Salvigenin to decrease ER stress. We found that Salvigenin could decrease the apoptotic factors. Salvigenin inhibited H₂O₂-induced caspase-3 which is a hallmark of apoptosis in addition to reducing Bax\Bcl-2 ratio by 1.45 fold. Additionally, Salvigenin increased the levels of autophagic factors. Our results showed an increase in LC3-II/LC3-I ratio, Atg7, and Atg12 in the presence of 25?μM of Salvigenin by about 1.28, 1.25, and 1.54 folds, respectively, compared to H₂O₂-treated cells. So it seems that H₂O₂ cytotoxicity mainly results from apoptosis. Besides, Salvigenin helps cells to survive by inhibiting apoptosis and enhancing autophagy that opens a new horizon for the future experiments.     

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.     

Conquering the cytokine storm in COVID-19-induced ARDS using placenta-derived decidua stromal cells

Acute respiratory distress syndrome (ARDS) is the most common cause of death in COVID-19 patients. The cytokine storm is the main driver of the severity and magnitude of ARDS. Placenta-derived decidua stromal cells (DSCs) have a stronger immunosuppressive effect than other sources of mesenchymal stromal cells. Safety and efficacy study included 10 patients with a median age of 50 (range 14–68) years with COVID-19-induced ARDS. DSCs were administered 1–2 times at a dose of 1 × 10⁶/kg. End points were safety and efficacy by survival, oxygenation and effects on levels of cytokines. Oxygenation levels increased from a median of 80.5% (range 69–88) to 95% (range 78–99) (p = 0.012), and pulmonary infiltrates disappeared in all patients. Levels of IL-6 decreased from a median of 69.3 (range 35.0–253.4) to 11 (range 4.0–38.3) pg/ml (p = 0.018), and CRP decreased from 69 (range 5–169) to 6 (range 2–31) mg/ml (p = 0.028). Two patients died, one of a myocardial infarction and the other of multiple organ failure, diagnosed before the DSC therapy. The other patients recovered and left the intensive care unit (ICU) within a median of 6 (range 3–12) days. DSC therapy is safe and capable of improving oxygenation, decreasing inflammatory cytokine level and clearing pulmonary infiltrates in patients with COVID-19.