Earthquake forecasting in Italy, before and after Umbria-Marche seismic sequence 1997. A review of the earthquake occurrence modeling at different spatio-temporal-magnitude scales.

The main goal of this work is to review the scientific researches carried out before and after the Umbria-Marche sequence related to the earthquake forecasting/prediction in Italy. In particular, I focus the attention on models that aim addressing three main practical questions: was (is) Umbria-Marche a region with high probability of occurrence of a destructive earthquake? Was a precursory activity recorded before the mainshock(s)? What was our capability to model the spatio-temporal-magnitude evolution of that seismic sequence? The models are reviewed pointing out what we have learned after the Umbria-Marche earthquakes, in terms of physical understanding of earthquake occurrence process, and of improving our capability to forecast earthquakes and to track in real-time seismic sequences

Structure-function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity

Regulation of the phosphate starvation response in Corynebacterium glutamicum by the PhoRS two-component system

In bacteria, the recognition of environmental changes and the necessary adaptations to these changes is in many cases accomplished by two-component signal transduction systems. They are composed of a usually membrane-bound sensor kinase and a response regulator. In response to specific environmental signals, the sensor kinase controls the phosphorylation state of the response regulator and thus its activity. With few exceptions, response regulators function as transcriptional regulators. Within the 3.3 Mb-genome of the gram-positive bacterium Corynebacterium glutamicum genes for 13 two-component systems were identified. By directed mutagenesis, a set of 12 strains was constructed each lacking the genes for one sensor kinase and its response regulator. In one case, only the sensor kinase, but not the response regulator could be deleted. In this work it was tested, whether one of the two-component systems of C. glutamicum is involved in the phosphate starvation response. To this end, the mutants mentioned above were cultivated under phosphate excess and phosphate limitation. One of the mutants grew poorer than the wild type under phosphate limitation, but not under phosphate excess. The growth defect could be abolished by expression of the missing genes from a plasmid. These results indicate a function of the deleted two-component system in the phosphate starvation response and therefore the corresponding genes were named phoS (phosphate sensor kinase) and phoR (phosphate response regulator). With the aim to identify the target genes of response regulator PhoR, DNA microarray analyses were performed. In a first set of experiments, the transcriptom of the $\Delta$phoRS mutant before and after a shift from phosphate excess to phosphate starvation was compared. Genes known to be induced by phosphate starvation in the wild type were not induced in the mutant, explaining the impaired growth of the $\Delta$phoRS mutant under phosphate limitation. The pstSCAB genes encoding a high-affinity ABC transporter for phosphate were still partially induced in the $\Delta$phoRS mutant under phosphate limitation, indicating the existence of an additional, currently unknown regulation of the pst operon. In a second set of experiments, the transcriptom of the $\Delta$phoRS mutant was compared to that of the wild type. Under phosphate excess, 27 genes showed a lower and 15 genes a higher mRNA level in the mutant compared to the wild type. Under phosphate-limited conditions, all of the known P$_{i}$ starvation-inducible genes (pstSCAB, ugpAEBC, glpQ, phoH, nucH and ushA) showed lower mRNA levels in the deletion mutant, sustaining the previous DNA microarray data. Interestingly, the mRNA level of the pitA gene encoding a low-affinity P$_{i}$ transporter decreased after a shift from P$_{i}$ excess to P$_{i}$ limitation in the wild type, but not in the $\Delta$phoRS mutant. By primer extension analysis of pstS, ugpA and phoR, the DNA microarray data were confirmed and the transcriptional start sites of these genes were determined. In summary, the data indicated that the PhoS-PhoR two-component system is responsible for the activation of many phosphate starvation genes, but also for repression of the pitA gene. In order to demonstrate the phosphorylation reactions characteristic for two-component signal transduction systems, the sensor kinase PhoS and the response regulator PhoR were both modified with a carboxyterminal histidine tag, overproduced in Escherichia coli and subsequently purified by affinity chromatography. Solubilization and purification of the integral membrane protein PhoS were performed with the detergent N,Ndimethyldodecylamine-N-oxide (LDAO). Both the autokinase activity of solubilized PhoS and the phosphoryl transfer from phosphorylated PhoS to PhoR were demonstrated with the isolated proteins. The binding of the response regulator PhoR to the promoters of pstSCAB, ugpAEBC, phoRS and pitA was analysed by gel shift analyses. With the entire PhoR protein it was not possible to show binding, irrespective of the conditions applied. However, a derivative of PhoR in which the aminoterminal 125 amino acid residues were deleted and replaced by a histidine tag did bind to the four promoter regions mentioned above, but not to the negative control promoters clpP1P2 and clpC. Additionally, the PhoR binding site within the pst promoter was analysed by DNase I footprinting, which indicated a protected region localized 170 to 205 bps upstream of pstS transcriptional start site

Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression.

Toll like receptors (TLRs) are important pattern recognition receptors that can detect pathogen and danger associated molecular patterns to initiate an innate immune response. TLR1 and 2 heterodimerize at the plasma membrane upon binding to triacylated lipopeptides from bacterial cell walls, or to the synthetic ligand Pam3CSK4. TLR1/2 dimers interact with adaptor molecules TIRAP and MyD88 to initiate a signalling cascade that leads to activation of key transcription factors, including NF-kB. Despite TLRs being extensively studied over the last two decades, the real-time kinetics of ligand binding and receptor activation remains largely unexplored. We aimed to study the kinetics of TLR activation and recruitment of adaptors, using TLR1/2 dimer interactions with adaptors MyD88 and TIRAP. Bioluminescence resonance energy transfer (BRET) allows detection of real-time protein-protein interactions in living cells, and was applied to study adaptor recruitment to TLRs. Energy transfer showed interactions between TLR2 and TIRAP, and between TLR2 and MyD88 only in the presence of TIRAP. Quantitative BRET and confocal microscopy confirmed that TIRAP is necessary for MyD88 interaction with TLR2. Furthermore, constitutive proximity between the proteins in the absence of Pam3CSK4 stimulation was observed with BRET, and was not abrogated with lowered protein expression, changes in protein tagging strategies, or use of the brighter NanoLuc luciferase. However, co-immunoprecipitation studies did not demonstrate constitutive interaction between these proteins, suggesting that the interaction observed with BRET likely represents artefacts of protein overexpression. Thus, caution should be taken when utilizing protein overexpression in BRET studies and in investigations of the TLR pathway

Activated CD8+CD38+ Cells Are Associated With Worse Clinical Outcome in Hospitalized COVID-19 Patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that spread around the world during the past 2 years, has infected more than 260 million people worldwide and has imposed an important burden on the healthcare system. Several risk factors associated with unfavorable outcome were identified, including elderly age, selected comorbidities, immune suppression as well as laboratory markers. The role of immune system in the pathophysiology of SARS-CoV-2 infection is indisputable: while an appropriate function of the immune system is important for a rapid clearance of the virus, progression to the severe and critical phases of the disease is related to an exaggerated immune response associated with a cytokine storm. We analyzed differences and longitudinal changes in selected immune parameters in 823 adult COVID-19 patients hospitalized in the Martin University Hospital, Martin, Slovakia. Examined parameters included the differential blood cell counts, various parameters of cellular and humoral immunity (serum concentration of immunoglobulins, C4 and C3), lymphocyte subsets (CD3+, CD4+, CD8+, CD19+, NK cells, CD4+CD45RO+), expression of activation (HLA-DR, CD38) and inhibition markers (CD159/NKG2A). Besides already known changes in the differential blood cell counts and basic lymphocyte subsets, we found significantly higher proportion of CD8+CD38+ cells and significantly lower proportion of CD8+NKG2A+ and NK NKG2A+ cells on admission in non-survivors, compared to survivors; recovery in survivors was associated with a significant increase in the expression of HLA-DR and with a significant decrease of the proportion of CD8+CD38+cells. Furthermore, patients with fatal outcome had significantly lower concentrations of C3 and IgM on admission. However, none of the examined parameters had sufficient sensitivity or specificity to be considered a biomarker of fatal outcome. Understanding the dynamic changes in immune profile of COVID-19 patients may help us to better understand the pathophysiology of the disease, potentially improve management of hospitalized patients and enable proper timing and selection of immunomodulator drugs

Immune Profile in Patients With COVID-19 : Lymphocytes Exhaustion Markers in Relationship to Clinical Outcome

The velocity of the COVID-19 pandemic spread and the variable severity of the disease course has forced scientists to search for potential predictors of the disease outcome. We examined various immune parameters including the markers of immune cells exhaustion and activation in 21 patients with COVID-19 disease hospitalised in our hospital during the first wave of the COVID-19 pandemic in Slovakia. The results showed significant progressive lymphopenia and depletion of lymphocyte subsets (CD3+, CD4+, CD8+ and CD19+) in correlation to the disease severity. Clinical recovery was associated with significant increase in CD3+ and CD3+CD4+ T-cells. Most of our patients had eosinopenia on admission, although no significant differences were seen among groups with different disease severity. Non-survivors, when compared to survivors, had significantly increased expression of PD-1 on CD4+ and CD8+ cells, but no significant difference in Tim-3 expression was observed, what suggests possible reversibility of immune paralysis in the most severe group of patients. During recovery, the expression of Tim-3 on both CD3+CD4+ and CD3+CD8+ cells significantly decreased. Moreover, patients with fatal outcome had significantly higher proportion of CD38+CD8+ cells and lower proportion of CD38+HLA-DR+CD8+ cells on admission. Clinical recovery was associated with significant decrease of proportion of CD38+CD8+ cells. The highest AUC values within univariate and multivariate logistic regression were achieved for expression of CD38 on CD8+ cells and expression of PD1 on CD4+ cells alone or combined, what suggests, that these parameters could be used as potential biomarkers of poor outcome. The assessment of immune markers could help in predicting outcome and disease severity in COVID-19 patients. Our observations suggest, that apart from the degree of depletion of total lymphocytes and lymphocytes subsets, increased expression of CD38 on CD3+CD8+ cells alone or combined with increased expression of PD-1 on CD3+CD4+ cells, should be regarded as a risk factor of an unfavourable outcome in COVID-19 patients. Increased expression of PD-1 in the absence of an increased expression of Tim-3 on CD3+CD4+ and CD3+CD8+ cells suggests potential reversibility of ongoing immune paralysis in patients with the most severe course of COVID-19

A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types

The anti-fibrotic actions of relaxin are mediated through a NO-sGC-cGMP-dependent pathway in renal myofibroblasts<i> in vitro </i>and enhanced by the NO donor, diethylamine NONOate

INTRODUCTION: The anti-fibrotic hormone, relaxin, has been inferred to disrupt TGF-beta1/Smad2 phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly involved in the anti-fibrotic actions of relaxin using a selective NO scavenger and sGC inhibitor, and comparing and combining relaxin’s effects with that of an NO donor. METHODS AND RESULTS: Primary renal cortical myofibroblasts isolated from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8nM), the NO donor, diethylamine NONOate (DEA/NO; 0.5-5uM) or the combined effects of RLX (16.8nM) and DEA/NO (5uM) over 72 hours. The effects of RLX (16.8nM) and DEA/NO (5uM) were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100uM) or sGC inhibitor, ODQ (5uM) over 72 hours. Furthermore, the effects of RLX (30nM), DEA/NO (5uM) and RLX (30nM)+DEA/NO (5uM) on cGMP levels were directly measured, in the presence or absence of ODQ (5uM). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2 and α-smooth muscle actin (α-SMA; a measure myofibroblast differentiation) (cell layer) were assessed by gelatin zymography and Western blotting, respectively. At the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9 levels by 25-33%, while inhibiting pSmad2 and α-SMA expression by up to 50% (all p<0.05 vs untreated and vehicle-treated cells). However, 5uM of DEA/NO was required to produce the effects seen with 16.8nM of RLX over 72 hours. The anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and ODQ (both p<0.01 vs RLX alone or DEA/NO alone), but were significantly enhanced when added in combination (all p<0.05 vs RLX alone). Additionally, the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all increased cGMP levels by 12-16-fold over basal levels; all p<0.01 vs vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all p<0.05 vs the respective treatments alone). CONCLUSIONS: These findings confirmed that RLX mediates its TGF-beta1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO