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In this structure-activity relationship (SAR) study, we report the development of dual inhibitors with antiviral properties targeting the SARS-CoV-2 main protease (M pro ) and human cathepsin L (hCatL). The novel molecules differ in the aliphatic amino acids at the P2 site and the fluorine position on the phenyl ring at the P3 site. The identified dual inhibitors showed K i values within 1.61 and 10.72 μM against SARS-CoV-2 M pro ; meanwhile, K i values ranging from 0.004 to 0.701 μM toward hCatL were observed. A great interdependency between the nature of the side chain at the P2 site and the position of the fluorine atom was found. Three dual-targeting inhibitors exhibited antiviral activity in the low micromolar range with CC 50 values >100 μM. Docking simulations were executed to gain a deeper understanding of the SAR profile. The findings herein collected should be taken into consideration for the future development of dual SARS-CoV-2 M pro /hCatL inhibitors., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Despite several major achievements in the development of vaccines and antivirals, the fight against SARS-CoV-2 and the health problems accompanying COVID-19 are still ongoing. SARS-CoV-2 main protease (M pro ), an essential viral cysteine protease, is a crucial target for the development of antiviral agents. A virtual screening analysis of in-house cysteine protease inhibitors against SARS-CoV-2 M pro allowed us to identify two hits (i.e., 1 and 2) bearing a methyl vinyl ketone warhead. Starting from these compounds, we herein report the development of Michael acceptors targeting SARS-CoV-2 M pro , which differ from each other for the warhead and for the amino acids at the P2 site. The most promising vinyl methyl ketone-containing analogs showed sub-micromolar activity against the viral protease. SPR38, SPR39, and SPR41 were fully characterized, and additional inhibitory properties towards hCatL, which plays a key role in the virus entry into host cells, were observed. SPR39 and SPR41 exhibited single-digit micromolar EC 50 values in a SARS-CoV-2 infection model in cell culture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections., (© 2022. The Author(s).)

Human African Trypanosomiasis (HAT) is a neglected tropical disease widespread in sub-Saharan Africa. Rhodesain, a cysteine protease of Trypanosoma brucei rhodesiense , has been identified as a valid target for the development of anti-HAT agents. Herein, we report a series of urea-bond-containing Michael acceptors, which were demonstrated to be potent rhodesain inhibitors with K i values ranging from 0.15 to 2.51 nM, and five of them showed comparable k 2nd values to that of K11777, a potent antitrypanosomal agent. Moreover, most of the urea derivatives exhibited single-digit micromolar activity against the protozoa, and the presence of substituents at the P3 position appears to be essential for the antitrypanosomal effect. Replacement of Phe with Leu at the P2 site kept unchanged the inhibitory properties. Compound 7 (SPR7) showed the best compromise in terms of rhodesain inhibition, selectivity, and antiparasitic activity, thus representing a new lead compound for future SAR studies., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The SARS-CoV-2 epidemic is pressuring healthcare systems worldwide. Disease outcomes in certain subgroups of patients are still scarce, and data are needed. Therefore, we describe here the experience of four dialysis centers of the Brescia Renal COVID Task Force. During March 2020, within an overall population of 643 hemodialysis patients, SARS-CoV-2 RNA positivity was detected in 94 (15%). At disease diagnosis, 37 of the 94 (39%) patients (group 1) were managed on an outpatient basis, whereas the remaining 57 (61%) (group 2) required hospitalization. Choices regarding management strategy were made based on disease severity. In group 1, 41% received antivirals and 76% hydroxychloroquine. Eight percent died and 5% developed acute respiratory distress syndrome (ARDS). In group 2, 79% received antivirals and 77% hydroxychloroquine. Forty two percent died and 79% developed ARDS. Overall mortality rate for the entire cohort was 29%. History of ischemic cardiac disease, fever, older age (over age 70), and dyspnea at presentation were associated with the risk of developing ARDS, whereas fever, cough and a C-reactive protein higher than 50 mg/l at disease presentation were associated with the risk of death. Thus, in our population of hemodialysis patients with SARS-CoV-2 infection, we documented a wide range of disease severity. The risk of ARDS and death is significant for patients requiring hospital admission at disease diagnosis., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Aims: Atrial fibrillation incidence is increasing due to ageing population and electrical cardioversion (ECV) is overused because of atrial fibrillation recurrences. Study's aim was to evaluate value of novel three-dimensional echocardiographic-derived left atrial conduit (LAC) function quantification in predicting early atrial fibrillation recurrence after ECV., Methods: We included 106 patients [74 (64-78) years] who underwent ECV for persistent nonvalvular atrial fibrillation. For all clinical data and simultaneous left atrial and left ventricular (LV) three-dimensional full-volume data sets were available before ECV. We computed LAC as: [(LV maximum - LV minimum) - (left atrial maximum - left atrial minimum) volume], expressed as % LV stroke volume. Atrial fibrillation recurrence was checked with Holter monitoring., Results: One month after ECV 66 patients were in sinus rhythm and 40 experienced atrial fibrillation recurrence. Pre-ECV patients with atrial fibrillation recurrence showed higher LAC contribution to LV filling (P < 0.0001) and noninvasively estimated left atrial stiffness (P < 0.0001) compared with sinus rhythm patients. There were no other differences, neither in clinical characteristics nor in LV properties. At multivariate LAC (P < 0.001), left atrial stiffness (P = 0.002) and volume (P = 0.043) predicted early atrial fibrillation relapse, even when compared with other confounding factors. Receiver-operating characteristics area (ROC) analysis confirmed LAC as best atrial fibrillation recurrence predictor (0.84, P < 0.0001), cut-off value more than 54% exhibiting reasonable sensibility-specificity (76-75%)., Conclusion: Atrial fibrillation makes LV filling dependent on reciprocation between left atrial reservoir/conduit phases. Our data suggest that LAC larger contribution to filling in persistent atrial fibrillation patients reflects left atrial and LV diastolic dysfunction, which skews atrio-ventricular interaction that leads to atrial fibrillation perpetuation, making LAC a powerful atrial fibrillation recurrence predictor after ECV.

We have simplified the published procedure (5) for measuring sweat rates in individual human sweat glands. Sweat secretion rates were obtained from sweat drops secreted on the forearm by multiple individual glands. We computed a ratio between CFTR-dependent (by intradermal microinjection of a β adrenergic cocktail) and CFTR-independent (by methacoline as cholinergic stimulus) sweat secretion rates. We obtained a reproducible, approximately linear readout of CFTR function with measurements performed by two different independent teams. We considered three groups (CF subjects, CF carriers and non-CF controls, n=22 in each group); their mean ratios was respectively 0.000, 0.104 and 0.205 The average ratio of CF subjects was consistent with diagnosis in 3 additional cases clinically resembling CF. All groups were clearly discriminated, with sensibility and specificity ranging from 82% to 100%. A software was developed for detecting sweat droplets. This bioassay is suitabile for multicentre studies focusing on CFTR targeted therapies, controversial diagnosis and functional relevance of rare CFTR mutations., (Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Shwachman-Diamond syndrome (SDS) is an inherited disease caused by mutations of a gene encoding for SBDS protein. So far little is known about SBDS exact function. SDS patients present several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with increased risk of leukemic evolution. So far, the molecular mechanisms that underlie neutropenia, MDS and AML in SDS patients have been poorly investigated. STAT3 is a key regulator of several cellular processes including survival, differentiation and malignant transformation. Moreover, STAT3 has been reported to regulate neutrophil granulogenesis and to induce several kinds of leukemia and lymphoma. STAT3 activation is known to be regulated by mTOR, which in turn plays an important role in cellular growth and tumorigenesis. Here we show for the first time, to the best of our knowledge, that both EBV-immortalized B cells and primary leukocytes obtained from SDS patients present a constitutive hyper-activation of mTOR and STAT3 pathways. Interestingly, loss of SBDS expression is associated with this process. Importantly, rapamycin, a well-known mTOR inhibitor, is able to reduce STAT3 phosphorylation to basal levels in our experimental model. A novel therapeutic hypothesis targeting mTOR/STAT3 should represent a significant step forward into the SDS clinical practice.

Rationale: Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown., Objectives: We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses., Methods: We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study., Measurements and Main Results: We found that chemoattractant-induced activation of β1 and β2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of β1 and β2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient., Conclusions: Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type-selective leukocyte adhesion deficiency disease, providing new insights into CF pathogenesis.

Biodrying refers to the decomposition of organic municipal solid waste (MSW) under aerobic conditions. This process usually lasts 14 days and requires large amounts of air to be injected into the waste matrix. The efficiency of the biodrying process depends on several geotechnical parameters, including initial moisture content, initial organic content, bulk density, dry density, solid particle density, and porosity. To examine the potential influence of these parameters on the biodrying process, we analyzed 13 biodried MSW samples. The results revealed a strong positive linear relationship between the initial moisture content and the mass loss percentage. In the first three days of the biodrying process, the waste mass rapidly decreased; afterwards, the daily mass loss occurred at a less rapid, more constant rate. The established average mass removal ratio between the volatile solids and water was 1:6.38 with a standard deviation of 1.06. Dry and solid particle densities were preserved in all 13 experiments; thus, the corresponding void ratio remained unchanged. This finding suggests that the settlement and degradation of MSW that occur during the biodrying process did not significantly influence the airflow rate., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Despite the vast amount of water on Earth, only a small percent is suitable for consumption, and these resources are diminishing. Moreover, water resources are unevenly distributed, leading to significant disparities in access to drinking water between countries and populations. Increasing consumption and the expanding human population necessitate the development of novel wastewater treatment technologies and the use of water treatment byproducts in other areas, such as fertilisers. However, water treatment sludge often cannot be used to enhance crop production due to the presence of parasite eggs, particularly from roundworms (Ascaridae family), which are resistant to environmental factors and can pose a threat for several years. Legislation prohibits the use of sludge containing parasite eggs as fertiliser. In some cases, water may not contain parasite eggs but larvae, which require different detection methods. Additionally, the presence of eggs does not necessarily indicate danger since they may lose infectivity due to prolonged storage or exposure to chemical compounds in the sewage. This paper reviews European Union regulations on wastewater treatment, the selected parasitic diseases related to the presence of parasites in wastewater, the spectrum of detection methods, and highlights differences in viability and invasiveness, which is intended to draw attention to the need to determine both biological properties of parasites. [ABSTRACT FROM AUTHOR]

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. In a previous study, we screened for reduced virulence using Tn5 transposon mutants and identified a LysR-type transcriptional regulator (LTTR) as a potential virulence factor in Pcal. However, the role of LTTR in Pcal virulence has not been thoroughly investigated. In this study, we demonstrated that the Pcal NN14 mutant (with Tn5 insertion in the LTTR-encoding gene) showed reduced disease symptoms and bacterial populations in cabbage, indicating that LTTR contributes to Pcal virulence. RNA-seq analysis identified 39 LTTR-dependent genes. Genes associated with 13 of the type three secretion system (T3SS), two of flagellar apparatus, ABC transporters, and transcription factors were expressed at lower levels in the NN14 mutant compared to the wild type. Conversely, tssH and hcp, type six secretion system (T6SS)-related genes, showed higher expression in NN14. Furthermore, these differences in gene expression were observed in minimal medium, but not in nutrient-rich medium, suggesting that LTTR acts as a global regulator responsive to nutrient conditions. Additionally, LTTR activated the expression of T3SS-related genes during Pcal infection. We also demonstrated that NN14 showed a reduced ability to induce hypersensitive reaction (HR) cell death in non-host plants. Collectively, these results suggest that LTTR contributes to Pcal virulence by regulating T3SS in response to environmental changes. [ABSTRACT FROM AUTHOR]

Food waste needs to be processed immediately after disposal, because it causes leachate and unpleasant odors. The purpose of this study was to utilize food waste using a rotary kiln composter. Food waste was chopped and placed into a rotary kiln composter equipped with an aeration system (6 L/min), stirring mechanism, and remotecontrol functionality via mobile phone for automated operation. Further, a bulking agent was added with a ratio of food waste vs bulking agent of 70:30. The addition of microorganism mass was varied to speed up the composting process for 14 days. The results showed that the compost derived from food waste was stable within 3 days. The temperature of the food waste matrix reached 46 °C, and the pH value of the compost was close to neutral since day 4. In this study, no leachate was produced, either R1, R2, R3, or R4. The final total organic carbon (TOC) content was 12.12-15.22%, Total-N was 0.83-1.04%, phosphorus was 0.18%, and potassium was 1.05%. On the basis of the C / N value and the germination index (GI), the R2, R3, and R4 reactors produced mature and stable compost on the third day with C/N values < 20 and GI > 100%. This result is a good breakthrough because the rotary kiln composter can accelerate the composting process of food waste within 3 days. [ABSTRACT FROM AUTHOR]

Background: Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells., Methods: Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays., Results: We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTRinh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis., Conclusions: We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF., General Significance: Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)