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Digested sludge samples from domestic wastewater treatment plants located in Northern Italy were tested as far as the presence of viruses (enteric viruses and coliphages), bacteria (faecal coliforms, salmonella) and helminth eggs is concerned. Heavy metals were also analysed. Escherichia coli bacteriophages and faecal coliforms were isolated from all samples, while salmonellae and helminth eggs were isolated only from four and three out of 27 total samples, respectively. The 66% of sludge samples, 46 and 82% of aerobic and anaerobic digested sludges respectively, showed the presence of enteric viruses (enteroviruses and reoviruses). The virus concentrations ranged from 0.6 to 123 MPNCU/g. Results of this study suggest that significant concentrations of pathogens such as enteric viruses can be present in digested sludge, which showed compliance with Italian legislation. As suggested by other authors, there is the need for surveillance and reference indications both in EU (European Union) and Italian regulations concerning the use of sludge in agriculture. [ABSTRACT FROM AUTHOR]

The removal of toluene from an experimental gas-stream was studied in an industrial biofilter filled with poplar wood bark. Toluene degradation, approximately 85% through the operating period, resulted in low levels of toluene in the off-gas effluent. For a toluene load of 6.7 g m-3 h-1 the elimination capacity of the biofilter was found to be 6.0 g m-3 h-1. Toluene removal was due to biodegradative activity of microorganisms in the filter bed; the most probable number counts of toluene degraders increased from 2.4 x 102 to 6.4 x 107 MPN/g dry packing material in about seven months of air-toluene supply. The degradative capacity of a Burkholderia (Pseudomonas) cepacia strain, isolated from the biofilter material, as an example of the effectiveness of microbial toluene removal was tested in batch culture. The microorganism degraded completely 250 ppm of toluene supplied as sole carbon source in 24 hours. The high performance demonstrated for a long periodand the mechanical and physico-chemical stability of the biofilter favour its use in industrial full-scale off-gas control. [ABSTRACT FROM AUTHOR]

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.

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.)

We describe the long-term follow-up of a patient affected by chorea mollis, a rare variant of Sydenham's chorea of which there are very few reports in the literature. Our patient, a previously healthy 8-year-old boy developed progressive clumsiness, gait disturbance, generalised hypotonia and muscle weakness, choreic movements of the limbs and behavioural disturbances. Following the diagnosis of chorea mollis, the patient received prophylaxis (monthly injections of benzathine benzyl penicillin). Within a few weeks, his clinical conditions worsened and he became bedridden and incapable of standing and walking without assistance. The choreic movements were successfully treated with sodium valproate. Independent walking was achieved 14 months after the onset of the disease. At a 4-year follow-up, the patient showed a full neurological and psychiatric recovery. The clinical course observed in our patient shows that chorea mollis may not only have a dramatic course, but also have a good long-term prognosis.

The process of biodrying could be a good solution for municipal solid waste management, allowing the production of fuel with an interesting energy content. Previous work (Adani, F., Baido, D., Calcaterra, E., Genevini, P.L., 2002. The influence of biomass temperature on biostabilization-biodrying of municipal solid waste. Bioresource Technology 83 (3), 173-179) has indicated that appropriate management of the processing parameters (air-flow rate and biomass temperatures) could achieve biomass drying in very short times (8-9 days). However, the data of that work also evidenced that if the conditions do not consider pile turning, and the air-flow is always from one direction, temperature gradients arise within the biomass, resulting in a lack of homogeneity in the moisture and energy content of the final product. Therefore, a new laboratory study was conducted on municipal solid waste biodrying-biostabilization in an effort to obtain homogeneous final products. Our proposal to solve this lack of homogeneity is to periodically invert the air-flow direction. Thus, in line with a previous study, two trials, A and B, were carried out, dividing the biomass into three layers to study temperature and moisture gradients throughout the process, and a third trial (C) simulating air-flow inversion at regular intervals was introduced. The results suggest that the daily inversion of air-flow eliminates marked temperature differences and leads to a homogeneous final product.

The volatile organic carbon (VOC) and odours emitted during the aerobic biological processing of municipal solid waste (MSW) was studied in a pilot-scale reactor. VOCs were detected by different techniques on solid waste samples and the outlet air stream, before and after a biofilter. Organic compounds (alpha-pinene, beta-myrcene, D-limonene) were also measured in condensate water and leachate from the process. Results showed uniformity in the composition of the air in the solid waste samples, air sampled during the process and condensed water, indicating a matrix-derived origin of these compounds. Leachates, however, contained substances with a quite different molecular structure from the compounds identified in the gaseous fraction. Most of the substances in the gaseous effluent had a hydrocarbon-like structure, mainly terpenoids. The odour produced and detected through olfactometry agreed with GC-MS analyses. This was true above all for terpenes.

A laboratory study was carried out to obtain data on the influence of biomass temperature on biostabilization-biodrying of municipal solid waste (initial moisture content of 410 g kg wet weight (w.w.)(-1)). Three trials were carried out at three different biomass temperatures, obtained by airflow rate control (A = 70 degrees C, B = 60 degrees C and C = 45 degrees C). Biodegradation and biodrying were inversely correlated: fast biodrying produced low biological stability and vice versa. The product obtained from process A was characterized by the highest degradation coefficient (166 g kg TS0(-1); TS0(-1) = initial total solid content) and lowest water loss (409 g kg W0(-1); W0 = initial water content). Due to the high reduction of easily degradable volatile solid content and preservation of water, process A produced the highest biological stability (dynamic respiration index, DRI = 141 mg O2 kg VS(-1); VS = volatile solids) but the lowest energy content (EC = 10,351 kJ kg w.w.(-1)). Conversely, process C which showed the highest water elimination (667 g kg W0(-1)), and lowest degradation rate (18 g kg TS0(-1)) was optimal for refuse-derived fuel (RDF) production having the highest energy content (EC = 14,056 kJ kg w.w.(-1)). Nevertheless, the low biological stability reached, due to preservation of degradable volatile solids, at the end of the process (DRI = 1055 mg O2 kg VS(-1)), indicated that the RDF should be used immediately, without storage. Trial B showed substantial agreement between low moisture content (losses of 665 g kg W0(-1)), high energy content (EC = 13,558 kJ kg w.w.(-1)) and good biological stability (DRI = 166 mg O2 kg VS(-1)), so that, in this case, the product could be used immediately for RDF or stored with minimum pollutant impact (odors, leaches and biogas production).

High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. It is an inhibitor of sulfhydryl proteases and has procoagulant properties. It is also a source of the vasoactive peptide bradykinin (BK). It has been previously shown that HK binds to human umbilical vein endothelial cells (HUVEC) in culture. We have further characterized that interaction herein. Immunohistochemical experiments have indicated that when freshly obtained umbilical vein segments were treated with HK, washed, and probed with anti-HK antibodies, HK was localized on the endothelium. We next determined whether HUVEC-bound HK can be cleaved by plasma kallikrein to release BK. Cultured HUVEC were incubated with unlabeled HK for varying times, washed, and the kinetics of BK release by plasma kallikrein were assayed by radioimmunoassay. Results indicated that kallikrein released BK from HUVEC in proportion to the initial amount of bound HK. No release of BK occurred in the absence of kallikrein. Also, there was no BK release upon kallikrein treatment of the HUVEC not treated with exogenous HK. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of HUVEC-bound 125I-HK indicated that addition of kallikrein resulted in cleavage of HK, thus corroborating the BK release experiments. Comparison of cleavage patterns has also indicated that cell-bound HK is slightly less susceptible to digestion by kallikrein than free HK. Therefore, our data suggest that human HK can bind to vascular endothelium in situ and that plasma kallikrein can recognize endothelial-bound HK as a substrate and liberate the vasoactive peptide BK.