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The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3 and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19. [ABSTRACT FROM AUTHOR]

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pHdependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity. [ABSTRACT FROM AUTHOR]

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 mM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19. [ABSTRACT FROM AUTHOR]

3C-like protease (3CLpro) processes and liberates functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19. It has been suggested that 3CLpro is catalytically active as a dimer, making the dimerization interface a target for antiviral development. Guided by structural analysis, here we introduced single amino acid substitutions at nine residues at three key sites of the dimer interface to assess their impact on dimerization and activity. We show that at site 1, alanine substitution of S1 or E166 increased by twofold or reduced relative activity, respectively. At site 2, alanine substitution of S10 or E14 eliminated activity, whereas K12A exhibited (60% relative activity. At site 3, alanine substitution of R4, E290, or Q299 eliminated activity, whereas S139A exhibited 46% relative activity. We further found that the oligomerization states of the dimer interface mutants varied; the inactive mutants R4A, R4Q, S10A/C, E14A/ D/Q/S, E290A, and Q299A/E were present as dimers, demonstrating that dimerization is not an indication of catalytically active 3CLpro. In addition, present mostly as monomers, K12A displayed residual activity, which could be attributed to the conspicuous amount of dimer present. Finally, differential scanning calorimetry did not reveal a direct relationship between the thermodynamic stability of mutants with oligomerization or catalytic activity. These results provide insights on two allosteric sites, R4/E290 and S10/E14, that may promote the design of antiviral compounds that target the dimer interface rather than the active site of severe acute respiratory syndrome coronavirus 2 3CLpro. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is responsible for coronavirus disease 2019 (COVID‐19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID‐19 are yet to be devised. It is crucial that antivirals that interfere with the SARS‐CoV‐2 life cycle be identified and developed. 3‐Chymotrypsin‐like protease (3CLpro) is an attractive antiviral drug target against SARS‐CoV‐2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro‐catalyzed reaction. While DMSO (5%‐20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide‐binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID‐19. [ABSTRACT FROM AUTHOR]

Background: Approximately 5% of COVID-19 patients develop respiratory failure and need ventilatory support, yet little is known about the impact of mechanical ventilation strategy in COVID-19. Our objective was to describe baseline characteristics, ventilatory parameters, and outcomes of critically ill patients in the largest referral center for COVID-19 in Sao Paulo, Brazil, during the first surge of the pandemic. Methods: This cohort included COVID-19 patients admitted to the intensive care units (ICUs) of an academic hospital with 94 ICU beds, a number expanded to 300 during the pandemic as part of a state preparedness plan. Data included demographics, advanced life support therapies, and ventilator parameters. The main outcome was 28-day survival. We used a multivariate Cox model to test the association between protective ventilation and survival, adjusting for PF ratio, pH, compliance, and PEEP. Results: We included 1503 patients from March 30 to June 30, 2020. The mean age was 60 ± 15 years, and 59% were male. During 28-day follow-up, 1180 (79%) patients needed invasive ventilation and 666 (44%) died. For the 984 patients who were receiving mechanical ventilation in the first 24 h of ICU stay, mean tidal volume was 6.5 ± 1.3 mL/kg of ideal body weight, plateau pressure was 24 ± 5 cmH2O, respiratory system compliance was 31.9 (24.4–40.9) mL/cmH2O, and 82% of patients were ventilated with protective ventilation. Noninvasive ventilation was used in 21% of patients, and prone, in 36%. Compliance was associated with survival and did not show a bimodal pattern that would support the presence of two phenotypes. In the multivariable model, protective ventilation (aHR 0.73 [95%CI 0.57–0.94]), adjusted for PF ratio, compliance, PEEP, and arterial pH, was independently associated with survival. Conclusions: During the peak of the epidemic in Sao Paulo, critically ill patients with COVID-19 often required mechanical ventilation and mortality was high. Our findings revealed an association between mechanical ventilation strategy and mortality, highlighting the importance of protective ventilation for patients with COVID-19. [ABSTRACT FROM AUTHOR]

Hexokinase (HK) catalyzes the first step in glucose metabolism, making it an exciting target for the inhibition of tumor initiation and progression due to their elevated glucose metabolism. The upregulation of hexokinase-2 (HK2) in many cancers and its limited expression in normal tissues make it a particularly attractive target for the selective inhibition of cancer growth and the eradication of tumors with limited side effects. The design of such safe and effective anticancer therapeutics requires the development of HK2-specific inhibitors that will not interfere with other HK isozymes. As HK2 is unique among HKs in having a catalytically active N-terminal domain (NTD), we have focused our attention on this region. We previously found that NTD activity is affected by the size of the linker helix-α13 that connects the N- and C-terminal domains of HK2. Three nonactive site residues (D447, S449, and K451) at the beginning of the linker helix-α13 have been found to regulate the NTD activity of HK2. Mutation of these residues led to increased dynamics, as shown via hydrogen deuterium exchange analysis and molecular dynamic simulations. D447A contributed the most to the enhanced dynamics of the NTD, with reduced calorimetric enthalpy of HK2. Similar residues exist in the C-terminal domain (CTD) but are unnecessary for HK1 and HK2 activity. Thus, we postulate these residues serve as a regulatory site for HK2 and may provide new directions for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of HK2. [ABSTRACT FROM AUTHOR]

The authors discuss a study by G. D. Perkins and colleagues, published within the issue which reported the Randomized Evaluation of COVID-19 Therapy-Respiratory Support (RECOVERY-RS) platform trial that assessed whether noninvasive ventilation or high flow nasal oxygen (HFNO) was more effective than conventional oxygen therapy for hospitalized patients with COVID-19. Topics include key finding from the trial and assumption on noninvasive ventilation based on the available evidence.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease 2019 (COVID-19). An appealing antiviral drug target is the coronavirus 3C-like protease (3CLpro) that is responsible for the processing of the viral polyproteins and liberation of functional proteins essential for the maturation and infectivity of the virus. In this study, multiple thermal analytical techniques have been implemented to acquire the thermodynamic parameters of 3CLpro at different buffer conditions. 3CLpro exhibited relatively high thermodynamic stabilities over a wide pH range; however, the protease was found to be less stable in the presence of salts. Divalent metal cations reduced the thermodynamic stability of 3CLpro more than monovalent cations; however, altering the ionic strength of the buffer solution did not alter the stability of 3CLpro. Furthermore, the most stable thermal kinetic stability of 3CLpro was recorded at pH 7.5, with the highest enthalpy of activation calculated from the slope of Eyring plot. The biochemical and biophysical properties of 3CLpro explored here may improve the solubility and stability of 3CLpro for optimum conditions for the setup of an enzymatic assay for the screening of inhibitors to be used as lead candidates in the discovery of drugs and design of antiviral therapeutics against COVID-19. [ABSTRACT FROM AUTHOR]

To assess epidemiology and management practices of Latin America Pediatric Rheumatologists (LAPR) about childhood-onset systemic lupus erythematosus (cSLE). A cross-sectional study was performed in 288 LAPR PANLAR members based on online survey about cSLE practices. The response rate of web-based survey by LAPR was 170/288(59%) and the majority worked in university hospitals (63%). The ACR and/or SLICC classification criteria (99%) and disease activity tools (97%) were almost universally used by LAPR, whereas damage index (70%) and CHAQ (58%) instruments were less frequently used. Laboratory exams, diagnostic imaging, and biopsies were generally available (> 75%), however low availability for densitometry (66%). Drug access was excellent for the most common prescribed medications (> 75%), except for belimumab (11%). Emerging mosquito-borne diseases were also reported: dengue (20%), chikungunya (11%), and Zika (8%). Groups were further divided in two, according to the median number of cSLE patients followed by LAPR in the last year: groups A and B (≥ 25 and < 25, respectively). Frequencies of condom in combination with other contraceptive methods were significantly higher in group A than B (p = 0.01). The frequencies of reported pregnancy (p < 0.001) and non-adherence to therapy were significantly higher in group A (p = 0.023). Alcohol intake (p = 0.004) and illicit drug use (p = 0.007) were also reported more frequently by LAPR of group A in at least one cSLE patient. This first large web-based survey demonstrated an overall excellent access for diagnosis and therapy by LAPR, probably related to their high rate of practices in tertiary care of university hospitals. Adherence to therapy, pregnancy, and substance abuse was identified as major challenges in this population, particularly in larger centers. [ABSTRACT FROM AUTHOR]

The high proliferation rate of tumor cells demands high energy and metabolites that are sustained by a high glycolytic flux known as the ‘Warburg effect’. The activation and further metabolism of glucose is initiated by hexokinase, a focal point of metabolic regulation. The human hexokinase 2 (HK2) is overexpressed in all aggressive tumors and predominantly found on the outer mitochondrial membrane, where interactions through its N-terminus initiates and maintains tumorigenesis. Here, we report the structure of HK2 in complex with glucose and glucose-6-phosphate (G6P). Structural and biochemical characterization of the mitochondrial conformation reveals higher conformational stability and slow protein unfolding rate (ku) compared with the cytosolic conformation. Despite the active site similarity of all human hexokinases, the N-domain of HK2 is catalytically active but not in hexokinase 1 and 3. Helix-α13 that protrudes out of the N-domain to link it to the C-domain of HK2 is found to be important in maintaining the catalytic activity of the N-half. In addition, the N-domain of HK2 regulates the stability of the whole enzyme in contrast with the C-domain. Glucose binding enhanced the stability of the wild-type (WT) enzyme and the single mutant D657A of the C-domain, but it did not increase the stability of the D209A mutant of the N-domain. The interaction of HK2 with the mitochondria through its N-half is proposed to facilitate higher stability on the mitochondria. The identification of structural and biochemical differences between HK2 and other human hexokinase isozymes could potentially be used in the development of new anticancer therapies. [ABSTRACT FROM AUTHOR]

We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity. [ABSTRACT FROM AUTHOR]

Fluorescence quenching of lipid-boundpyrene was used to assessthe binding of cytochrome c(cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM)POPC/DOPE/TOCL, with the conditions that it did ordid not contain oxidized phosphatidylcholine molecules, i.e.,1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine(PazePC), or a mixture of two hydroperoxide isomers derivedfrom POPC (POPCOX). The binding isotherms reveal two dissociationconstants, KD1and KD2, representing, respectively, thelow- and high-affinity states of the membrane. These dissociationconstants probably are due to the lipid reorganization promoted bycyt c, as observed in giant unilamellar vesiclesthat contain fluorescent cardiolipin (CL). The presence of PazePC,which has a nonreactive carboxylic group, increased the KD1and KD2values 1.2- and 4.5-fold, respectively. The presence of POPCOXwhich has a reactive peroxide group, decreased the KD1value 1.5-fold, increased the KD2value 10-fold, and significantlyreduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt cincubatedwith liposomes containing POPCox demonstrated a mass increase correspondingto the formation of nonenal adducts as hydrophobic anchors. Electronicabsorption spectroscopy, circular dichroism, and magnetic circulardichroism demonstrated that all of the lipids studied promoted changesin the cyt ccoordination sphere. Therefore,in the presence of CL, the oxidation of zwitterionic lipids also promoteschanges in the cyt cstructure and in the affinityfor lipid bilayers. [ABSTRACT FROM AUTHOR]

Green propolis is produced by Apis mellifera honeybees using Baccharis dracunculifolia D.C. (Asteraceae) as substrate. This Southern Brazilian native plant and green propolis have been used in traditional medicine to treat gastric diseases, inflammation and liver disorders. Investigate the effects of baccharin (Bac) or p -coumaric acid (pCA) isolated from B. dracunculifolia D.C. (Asteraceae) over the inflammation induced by lipopolysaccharide (LPS) in vivo. Inflammation was induced by LPS injection into air-pouches in mice, which were subsequently treated with Bac or pCA. Lavage fluid was collected from air pouches for the quantification of cellular influx via microscopy, and quantification of inflammatory mediators via colorimetric methods, ELISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). LPS-induced inflammation increased cellular influx and increased the levels of parameters related to vascular permeability and edema formation, such as nitric oxide (NO) and protein extravasation. Moreover, LPS increased the levels of cytokines and eicosanoids in the air-pouches. Importantly, both Bac and pCA suppressed the infiltration of neutrophils, production of NO and protein extravasation. Notably, the compounds promote differential regulation of cytokine and eicosanoid production. Our results suggest that Bac from green propolis directly affects inflammation by inhibiting the production of cytokines and eicosanoids, while pCA may exert direct, but also indirect effects on inflammation by stimulating the production of regulatory effectors such as interkeukin-10 in vivo. [Display omitted] • Green propolis' compounds exhibit broad anti-inflammatory activities. • Bac or pCA suppress neutrophil recruitment and promotes vascular integrity. • Both compounds inhibit inflammatory mediators, but pCA also induces IL-10. [ABSTRACT FROM AUTHOR]

The article presents a study which compares the function of a bilevel ventilator with the noninvasive-positive pressure ventilation (NPPV) modes to nine different intensive care unit (ICU) ventilators in the presence of leaks. It states that the ventilators were set at 12 cm water pressure support and 5 cm water positive end-expiratory pressure. Furthermore, it shows that only the Vision and Servo I ventilators require no adjustments as they adapted to increasing leaks.

High phosphate intake is known to aggravate renal osteodystrophy along various pathogenetic pathways. Recent studies have raised the possibility that dysregulation of the osteocyte Wnt/β-catenin signaling pathway is also involved in chronic kidney disease (CKD)-related bone disease. We investigated the role of dietary phosphate and its possible interaction with this pathway in an experimental model of adynamic bone disease (ABD) in association with CKD and hypoparathyroidism. Partial nephrectomy (Nx) and total parathyroidectomy (PTx) were performed in male Wistar rats. Control rats with normal kidney and parathyroid function underwent sham operations. Rats were divided into three groups and underwent pair-feeding for 8 weeks with diets containing either 0.6% or 1.2% phosphate: sham 0.6%, Nx+PTx 0.6%, and Nx+PTx 1.2%. In the two Nx+PTx groups, serum creatinine increased and blood ionized calcium decreased compared with sham control group. They also presented hyperphosphatemia and reduced serum parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) levels. Fractional urinary excretion of phosphate increased in Nx+PTx 1.2% rats despite lower PTH and FGF23 levels than in sham group. These biochemical changes were accompanied by a decrease in bone formation rates. The Nx+PTx 1.2% group had lower bone volume (BV/TV), higher osteoblast and osteocyte apoptosis, and higher SOST and Dickkopf-1 gene expression than the Nx+PTx 0.6% group. Nx+PTx 0.6% rat had very low serum sclerostin levels, and Nx+PTx 1.2% had intermediate sclerostin levels compared with sham group. Finally, there was a negative correlation between BV/TV and serum sclerostin. These results suggest that high dietary phosphate intake decreases bone volume in an experimental model of CKD-ABD, possibly via changes in SOST expression through a PTH-independent mechanism. These findings could have relevance for the clinical setting of CKD-ABD in patients who low turnover bone disease might be attenuated by optimal control of phosphate intake and/or absorption. [ABSTRACT FROM AUTHOR]

The peptidolytic enzyme THIMET-oligopeptidase (TOP) is able to act as a reducing agent in the peroxidase cycle of myoglobin (Mb) and horseradish peroxidase (HRP). The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme. EPR (electron paramagnetic resonance) analysis using DBNBS spin trapping revealed that TOP also prevented the formation of tryptophanyl radical in Mb challenged by H2O2. The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging. These findings are discussed towards a possible occurrence of these reactions in cells. [ABSTRACT FROM AUTHOR]

Simple Summary: In the different livestock production systems, forage is the main feed resource. However, the availability and quality of the forage fluctuate throughout the year due to variable environmental conditions, such as temperature, humidity, location, or lack of rainfall. In semiarid regions, this fact is even more critical. The option for forage plants adapted to the semiarid climate, such as cactus cladodes, becomes indispensable for the sustainability of the systems. Nonetheless, it is necessary to combine the cactus with high-fiber-content feeds (silage, hay, and agroindustry residues, among others) to increase fiber contents in the diet to promote ideal rumen conditions. Based on the knowledge that cactus cladodes (Opuntia spp. and Nopalea spp.) are one of the most viable crops in semiarid regions, the association with a by-product rich in NDF proves to be a more feasible alternative in terms of price and availability, with the producer making the final decision. We aimed to evaluate the effect of the cactus cladodes Nopalea cochenillifera (L). Salm-Dyck. (NUB) and cactus cladodes Opuntia stricta (Haw.) Haw. (OUB), both combined with sugarcane bagasse (SB) plus urea, Tifton hay (TH), corn silage (CS), and sorghum silage (SS) plus urea on nutrient intake and digestibility, ruminal dynamics, and parameters. Five male sheep, fistulated in the rumen, were assigned in a 5 × 5 Latin square design. The NUB provided a higher intake of dry matter (DM) and any nutrients than SS. TH provided larger pools of DM and iNDF. The OUB and CS provided a higher DM degradation. CS provided a higher NDF degradation rate. OUB provided a lower ruminal pH. Depending on the collection time, the lowest pH value was estimated at 3.79 h after the morning feeding. There was an interaction between treatments and collection time on VFA concentrations. Due to the high degradation rate, greater energy intake, less change in rumen pH, greater volatile fatty acid production, and feasibility, we recommend using cactus associated with sugarcane bagasse plus urea in sheep diets. [ABSTRACT FROM AUTHOR]

The proprotein convertases (PCs) are calcium-dependent proteases responsible for processing precursor proteins into their active forms in eukariotes. The PC1/3 is a pivotal enzyme of this family that participates in the proteolytic maturation of prohormones and neuropeptides inside the regulated secretory pathway. In this paper we demonstrate that mouse proprotein convertase 1/3 (mPC1/3) has a lag phase of activation by substrates that can be interpreted as a hysteretic behavior of the enzyme for their hydrolysis. This is an unprecedented observation in peptidases, but is frequent in regulatory enzymes with physiological relevance. The lag phase of mPC1/3 is dependent on substrate, calcium concentration and pH. This hysteretic behavior may have implications in the physiological processes in which PC1/3 participates and could be considered an additional control step in the peptide hormone maturation processes as for instance in the transformation of proinsulin to insulin. [ABSTRACT FROM AUTHOR]

Objectives: To measure the impact of clusters of double triggering on clinical outcomes.Design: Prospective cohort study.Setting: Respiratory ICU in Brazil.Patients: Adult patients under recent mechanical ventilation and with expectation of mechanical ventilation for more than 24 hours after enrollment.Interventions: None.Measurements and Main Results: We used a dedicated software to analyze ventilator waveforms throughout the entire period of mechanical ventilation and detect double triggering. We defined a cluster of double triggering as a period of time containing at least six double triggering events in a 3-minute period. Patients were followed until hospital discharge. We addressed the association between the presence and the duration of clusters with clinical outcomes. A total of 103 patients were enrolled in the study and 90 (87%) had at least one cluster of double triggering. The median number of clusters per patient was 19 (interquartile range, 6-41), with a median duration of 8 minutes (6-12 min). Compared with patients who had no clusters, patients with at least one cluster had longer duration of mechanical ventilation (7 d [4-11 d] vs 2 d [2-3 d]) and ICU length of stay (9 d [7-16 d] vs 13 d [2-8 d]). Thirty-three patients had high cumulative duration of clusters of double triggering (≥ 12 hr), and it was associated with longer duration of mechanical ventilation, fewer ventilator-free days, and longer ICU length of stay. Adjusted by duration of mechanical ventilation and severity of illness, high cumulative duration of clusters was associated with shorter survival at 28 days (hazard ratio, 2.09 d; 95% CI, 1.04-4.19 d).Conclusions: Clusters of double triggering are common and were associated with worse clinical outcomes. Patients who had a high cumulative duration of clusters had fewer ventilator-free days, longer duration of mechanical ventilation, longer ICU length of stay, and shorter survival than patients with low cumulative duration of cluster. [ABSTRACT FROM AUTHOR]

Since 2003, we have seen the emergence of novel viruses, such as SARS-CoV-1, MERS, ZIKA, swine flu virus H1N1, Marburg, Monkeypox, Ebola, and SARS-CoV-2, but none of them gained pandemic proportions similar to SARS-CoV-2. This could be attributed to unique viral traits, allowing its rapid global dissemination following its emergence in October 2019 in Wuhan, China, which appears to be primarily driven by the emergence of highly transmissible and virulent variants that also associate, in some cases, with severe disease and considerable mortality caused by fatal pneumonia, acute respiratory distress syndrome (ARDS) in infected individuals. Mechanistically, several factors are involved in viral pathogenesis, and epigenetic alterations take the front seat in host-virus interactions. The molecular basis of all viral infections, including SARS-CoV-2, tightly hinges on the transitory silencing of the host gene machinery via epigenetic modulation. SARS-CoV-2 also hijacks and subdues the host gene machinery, leading to epigenetic modulation of the critical host elements responsible for antiviral immunity. Epigenomics is a powerful, unexplored avenue that can provide a profound understanding of virus-host interactions and lead to the development of epigenome-based therapies and vaccines to counter viruses. This review discusses current developments in SARS-CoV-2 variation and its role in epigenetic modulation in infected hosts. This review provides an overview, especially in the context of emerging viral strains, their recombinants, and their possible roles in the epigenetic exploitation of host defense and viral pathogenesis. It provides insights into host-virus interactions at the molecular, genomic, and immunological levels and sheds light on the future of epigenomics-based therapies for SARS-CoV-2 infection. • SARS-CoV-2 manipulates the host gene machinery through epigenetic alterations and modulatesl critical host elements responsible for antiviral immunity. • Unique viral traits, including recombination among variants, have driven the rapid global dissemination of SARS-CoV-2, and these traits could be associated with severe disease, and mortality. • Conceptually, the potential of epigenome-based therapies for treating SARS-CoV-2 infection is a likely possibility. [ABSTRACT FROM AUTHOR]

Background: Protective mechanical ventilation is recommended for patients with acute respiratory distress syndrome (ARDS), but it usually requires controlled ventilation and sedation. Using neurally adjusted ventilatory assist (NAVA) or pressure support ventilation (PSV) could have additional benefits, including the use of lower sedative doses, improved patient–ventilator interaction and shortened duration of mechanical ventilation. We designed a pilot study to assess the feasibility of keeping tidal volume (VT) at protective levels with NAVA and PSV in patients with ARDS. Methods: We conducted a prospective randomized crossover trial in five ICUs from a university hospital in Brazil and included patients with ARDS transitioning from controlled ventilation to partial ventilatory support. NAVA and PSV were applied in random order, for 15 min each, followed by 3 h in NAVA. Flow, peak airway pressure (Paw) and electrical activity of the diaphragm (EAdi) were captured from the ventilator, and a software (Matlab, Mathworks, USA), automatically detected inspiratory efforts and calculated respiratory rate (RR) and VT. Asynchrony events detection was based on waveform analysis. Results: We randomized 20 patients, but the protocol was interrupted for five (25%) patients for whom we were unable to maintain VT below 6.5 mL/kg in PSV due to strong inspiratory efforts and for one patient for whom we could not detect EAdi signal. For the 14 patients who completed the protocol, VT was 5.8 ± 1.1 mL/kg for NAVA and 5.6 ± 1.0 mL/kg for PSV (p = 0.455) and there were no differences in RR (24 ± 7 for NAVA and 23 ± 7 for PSV, p = 0.661). Paw was greater in NAVA (21 ± 3 cmH2O) than in PSV (19 ± 3 cmH2O, p = 0.001). Most patients were under continuous sedation during the study. NAVA reduced triggering delay compared to PSV (p = 0.020) and the median asynchrony Index was 0.7% (0–2.7) in PSV and 0% (0–2.2) in NAVA (p = 0.6835). Conclusions: It was feasible to keep VT in protective levels with NAVA and PSV for 75% of the patients. NAVA resulted in similar VT, RR and Paw compared to PSV. Our findings suggest that partial ventilatory assistance with NAVA and PSV is feasible as a protective ventilation strategy in selected ARDS patients under continuous sedation. Trial registration ClinicalTrials.gov (NCT01519258). Registered 26 January 2012, https://clinicaltrials.gov/ct2/show/NCT01519258 [ABSTRACT FROM AUTHOR]

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum . Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor ( K i = 0.4 μM) and an in vitro antimalarial compound as potent as bestatin (IC 50 = 18 μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn 2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC 50 = 82 μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria. [ABSTRACT FROM AUTHOR]

Objectives: In 2012, a new acute respiratory distress syndrome definition was proposed for adult patients. It was later validated for infants and toddlers. Our objective was to evaluate the prevalence, outcomes, and risk factors associated with acute respiratory distress syndrome in children up to 15 years according to the Berlin definition. Design: A prospective, multicenter observational study from March to September 2013. Setting: Seventy-seven PICU beds in eight centers: two private hospitals and six public academic hospitals in Brazil. Patients: All children aged 1 month to 15 years admitted to the participating PICUs in the study period. Interventions: None. Measurements and Main Results: All children admitted to the PICUs were daily evaluated for the presence of acute respiratory distress syndrome according to the American-European Consensus Conference and Berlin definitions. Of the 562 patients included, acute respiratory distress syndrome developed in 57 patients (10%) and 58 patients (10.3%) according to the Berlin definition and the American-European Consensus Conference definition, respectively. Among patients with acute respiratory distress syndrome according to the Berlin definition, nine patients (16%) were mild, 21 (37%) were moderate, and 27 (47%) were severe. Compared with patients without acute respiratory distress syndrome, patients with acute respiratory distress syndrome had significantly higher severity scores, longer PICU and hospital length of stay, longer duration of mechanical ventilation, and higher mortality (p < 0.001). The presence of two or more comorbidities and admission for medical reasons were associated with development of acute respiratory distress syndrome. Comparisons across the three the Berlin categories showed significant differences in the number of ventilator-free days (21, 20, and 5 d, p = 0.001) and mortality for severe acute respiratory distress syndrome (41%) in comparison with mild (0) and moderate (15%) acute respiratory distress syndrome(p = 0.02). No differences in PICU or hospital stay were observed across the groups. Conclusions: The Berlin definition can identify a subgroup of patients with distinctly worse outcomes, as shown by the increased mortality and reduced number of ventilator-free days in pediatric patients with severe acute respiratory distress syndrome. [ABSTRACT FROM AUTHOR]

Abstract: Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136–142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10μM focusing on the molecular mechanism involved. PTZ promoted a dose–response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg2+ and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the PTP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since PTZ give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that PTZ bury in the inner mitochondrial membrane and the chemically generated PTZ cation radicals modify specific thiol groups that in the presence of Ca2+ result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MPT induction and may have implications for the cell death induced by PTZ. [Copyright &y& Elsevier]

Background: Noninvasive positive-pressure ventilation (NPPV) modes are currently available on bilevel and ICU ventilators. However, little data comparing the performance of the NPPV modes on these ventilators are available.Methods: In an experimental bench study, the ability of nine ICU ventilators to function in the presence of leaks was compared with a bilevel ventilator using the IngMar ASL5000 lung simulator (IngMar Medical; Pittsburgh, PA) set at a compliance of 60 mL/cm H(2)O, an inspiratory resistance of 10 cm H(2)O/L/s, an expiratory resistance of 20 cm H(2)O/ L/s, and a respiratory rate of 15 breaths/min. All of the ventilators were set at 12 cm H(2)O pressure support and 5 cm H(2)O positive end-expiratory pressure. The data were collected at baseline and at three customized leaks.Main Results: At baseline, all of the ventilators were able to deliver adequate tidal volumes, to maintain airway pressure, and to synchronize with the simulator, without missed efforts or auto-triggering. As the leak was increased, all of the ventilators (except the Vision [Respironics; Murrysville, PA] and Servo I [Maquet; Solna, Sweden]) needed adjustment of sensitivity or cycling criteria to maintain adequate ventilation, and some transitioned to backup ventilation. Significant differences in triggering and cycling were observed between the Servo I and the Vision ventilators.Conclusions: The Vision and Servo I were the only ventilators that required no adjustments as they adapted to increasing leaks. There were differences in performance between these two ventilators, although the clinical significance of these differences is unclear. Clinicians should be aware that in the presence of leaks, most ICU ventilators require adjustments to maintain an adequate tidal volume. [ABSTRACT FROM AUTHOR]

Abstract: Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved in oligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, and two are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalian immunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterial recombinant EP24.15 with oxidized glutathione concentration as low as 10 μM. The in vitro S-glutathionylation of EP24.15 was responsible for its oxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showed increased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also show that EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the protein oligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization by interprotein thiol–disulfide exchange. [Copyright &y& Elsevier]