Peak inspiratory pressure lacks accuracy to estimate plateau pressure in infants with severe obstructive lower airway disease.

PURPOSE: In children with acute respiratory distress syndrome receiving mechanical ventilation, the peak inspiratory pressure (PIP) is close to plateau pressure (PPLAT) when inspiratory flow approaches zero. We aimed to evaluate the reliability of PIP to estimate PPLAT in infants with severe respiratory viral infection (SRVI), characterized by increased airway resistance, and the accuracy of an equational model to estimates PPLAT (ePPLAT) based on PIP. METHODS: This was a retrospective observational study including mechanically ventilated children (1 to 24 month old) with SRVI, whose respiratory mechanics measurements were performed to evaluate PIP and PPLAT. The measured PPLAT was compared with the result of the equation: ePPLAT = PIP - [5.067 - (0.858 × static compliance) - (0.018 × inspiratory resistance) - (0.390 × pressure above positive-end expiratory pressure) + (4.989 × inspiratory time)]. RESULTS: Thirty-seven patients were included, with a median age of 3 (2-5) months. They presented a high inspiratory and expiratory resistance (136 ± 43 and 168 ± 66 cmH2O/L/s, respectively) and a moderate reduction in static compliance: 0.75 ± 0.3 mL/kg/cmH2O. PIP overestimated PPLAT (33 ± 3 and 26 ± 5 cmH2O, p = 0.01), with a mean difference of 7.3 ± 4 cmH2O. Moreover, the Bland-Altman analysis demonstrated a mean difference between PPLAT and ePPLAT of 1.0 ± 4.0 cmH2O, with 95% limits of agreement of -6.9 and 8.8. CONCLUSIONS: A significant difference between PIP and PPLAT was observed in infants with SRVI. The equation model was inaccurate for estimating PPLAT based on PIP. Any estimation of PPLAT from PIP needs to consider the resistance component of the respiratory system.

Comparative molecular profiling of multidrug-resistant Pseudomonas aeruginosa identifies novel mutations in regional clinical isolates from South India.

Objectives: We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. Methods: We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Results: Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (blaNDM-1) within our sample. Conclusions: This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.

Revisiting the orbital complications of acute rhinosinusitis

Abstract Objective: The present study revisited three classification systems of orbital complications of acute rhinosinusitis (ARS) (Chandler, Mortimore & Wormald, and Velasco e Cruz & Anselmo-Lima) and observed which of them presented the best clinical applicability. Methods: Clinical data and CT scan findings of patients with orbital infection were retrospectively collected. To compare the three classification systems, we revised and graded all CT images accordingly, and divided the patients into four groups: Eyelid cellulitis (EC), orbital cellulitis (OC), subperiosteal abscess (SA), and orbital abscess (OA). The groups were compared regarding the presence of sinus opacification, the need for hospitalization and/or surgical treatment, and the presence of further complications/sequelae. Results: 143 patients were included. The median number of sinuses involved in patients in the OC, SA, and OA groups was 2.0. ARS was rarely associated with signs of EC (present in both Chandler's and Mortimore & Wormald's classifications. The hospitalization rate was significantly lower in the EC group compared to the other three groups. Surgery was performed in all cases in the OA group, in 58.1% in the SA group, 19.4% in the OC group, and 12.5% in the EC group (p-value < 0.0001 ). Complications were present at higher rates in the OA group compared to the other three groups. Conclusions: ARS was rarely associated with Eyelid Cellulitis. The stratification in the other three groups showed to be clinically relevant. Velasco e Cruz & Anselmo-Lima's classification system proved valid, simple, and effective for categorizing orbital complications of ARS. Level of evidence: 3.

Revisiting the orbital complications of acute rhinosinusitis.

OBJECTIVE: The present study revisited three classification systems of orbital complications of acute rhinosinusitis (ARS) (Chandler, Mortimore & Wormald, and Velasco e Cruz & Anselmo-Lima) and observed which of them presented the best clinical applicability. METHODS: Clinical data and CT scan findings of patients with orbital infection were retrospectively collected. To compare the three classification systems, we revised and graded all CT images accordingly, and divided the patients into four groups: Eyelid cellulitis (EC), orbital cellulitis (OC), subperiosteal abscess (SA), and orbital abscess (OA). The groups were compared regarding the presence of sinus opacification, the need for hospitalization and/or surgical treatment, and the presence of further complications/sequelae. RESULTS: 143 patients were included. The median number of sinuses involved in patients in the OC, SA, and OA groups was 2.0. ARS was rarely associated with signs of EC (present in both Chandler's and Mortimore & Wormald's classifications. The hospitalization rate was significantly lower in the EC group compared to the other three groups. Surgery was performed in all cases in the OA group, in 58.1% in the SA group, 19.4% in the OC group, and 12.5% in the EC group (p-value < 0.0001). Complications were present at higher rates in the OA group compared to the other three groups. CONCLUSIONS: ARS was rarely associated with Eyelid Cellulitis. The stratification in the other three groups showed to be clinically relevant. Velasco e Cruz & Anselmo-Lima's classification system proved valid, simple, and effective for categorizing orbital complications of ARS.

Small molecule targeting of transcription-replication conflict for selective chemotherapy.

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.

Guaranteed approximation error estimation of neural networks and model modification.

Approximation error is a key measure in the process of model validation and verification for neural networks. In this paper, the problems of guaranteed error estimation of neural networks and applications to assured system modeling and assured neural network compression are addressed. First, a concept called guaranteed error estimation of feedforward neural networks is proposed, which intends to provide the worst-case approximation error of a trained neural network with respect to a compact input set essentially containing an infinite number of values. Given different prior information about the original system, two approaches including Lipschitz constant analysis and set-valued reachability analysis methods are developed to efficiently compute upper-bounds of approximation errors. Based on the guaranteed approximation error estimation framework, an optimization for obtaining parameter values from data set is proposed. A robotic arm and neural network compression examples are presented to illustrate the effectiveness of our approach.

A better understanding of air quality resulting from the effects of the 2020 pandemic in a city in the equatorial region (Fortaleza, Brazil).

The year 2020 was atypical due to the pandemic caused by the SARS-CoV-2 virus (COVID-19), providing a unique opportunity to understand changes in air quality due to the reduction in urban activity. Therefore, the aim of the present study was to perform an integrated evaluation on the influence of the effects of the 2020 pandemic on air quality in the city of Fortaleza, investigating levels of PM2.5, PM10, NO2, NO, SO2, CO, and O3, corresponding health risks, as well as the influence of meteorological variables and urban activity. In all phases analyzed, significant reductions were found in NOx, NO, NO2, and CO. A considerable reduction in PM2.5 and PM10 was found in the early phases, with an increase in the later phases. These findings are explained by the nearly 50% reduction in vehicular traffic and the consequent reduction in fossil fuel emissions, mainly in the partial lockdown and total lockdown periods, as well as reductions in commercial (stores/shops) and industrial activities. The variation in O3 was initially non-significant, followed by a considerable increase in the last three phases analyzed; this increase was influenced by changes in temperature and the incidence of sunlight. SO2 concentrations increased in the period studied, demonstrating that the vehicular fleet, local commerce, and other activities are not the predominant sources of this compound. Estimated health risks were reduced by half during the lockdown period, especially for non-smokers, followed by a drastic increase in the last three phases. The planetary boundary layer was positively correlated with O3 and PM10 and negatively correlated with NOx, NO2, and NO, indicating its influence on the distribution of pollutants in the lower atmosphere and, consequently, air quality.

Novel Peptide Therapeutic Approaches for Cancer Treatment.

Peptides are increasingly being developed for use as therapeutics to treat many ailments, including cancer. Therapeutic peptides have the advantages of target specificity and low toxicity. The anticancer effects of a peptide can be the direct result of the peptide binding its intended target, or the peptide may be conjugated to a chemotherapy drug or radionuclide and used to target the agent to cancer cells. Peptides can be targeted to proteins on the cell surface, where the peptide-protein interaction can initiate internalization of the complex, or the peptide can be designed to directly cross the cell membrane. Peptides can induce cell death by numerous mechanisms including membrane disruption and subsequent necrosis, apoptosis, tumor angiogenesis inhibition, immune regulation, disruption of cell signaling pathways, cell cycle regulation, DNA repair pathways, or cell death pathways. Although using peptides as therapeutics has many advantages, peptides have the disadvantage of being easily degraded by proteases once administered and, depending on the mode of administration, often have difficulty being adsorbed into the blood stream. In this review, we discuss strategies recently developed to overcome these obstacles of peptide delivery and bioavailability. In addition, we present many examples of peptides developed to fight cancer.

Product inhibition kinetics determinations - Substrate interaction affinity and enzymatic kinetics using one quantitative FRET assay.

Product inhibition is a common phenomenon during enzyme-catalyzed reactions. Almost all product molecules of an enzyme reaction should have some structural similarities to the substrate, and can thus still have affinities to the active site of the enzyme as product inhibitor. Currently, the characterizations of product inhibition are generally carried out by different methods to determine product binding affinity to the enzyme and the enzyme kinetics parameters, and then these parameters are combined to determine product inhibition. However, due to different sensitivity and variations, kinetics parameters determined from different methods are often not compatible, resulting in not accurate measurement. Here, we report a novel method that determines the two different classes of kinetics parameters, IC50 and Ki(or KD), Kcat and KM, using one single assay method-quantitative FRET(qFRET) assay for characterizing the product inhibition of pre-SUMO1's maturation by its protease SENP1. One method to determine all kinetics parameters provides, for the first time, not only a convenient method to determine all kinetics parameters, but more importantly, a novel approach to combine different measurements with mutually compatible results and errors.

Design, Synthesis, and Structural Characterization of Lysine Covalent BH3 Peptides Targeting Mcl-1.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Airway Resistance and Respiratory Compliance in Children with Acute Viral Bronchiolitis Requiring Mechanical Ventilation Support.

BACKGROUND: Acute viral bronchiolitis (AVB) is a very frequent disease that affects the lower airways of young children increasing the inspiratory and expiratory resistance in variable degree as well as reducing the pulmonary compliance. It would be desirable to know whether these variables are associated with the outcome. OBJECTIVES: To evaluate the respiratory mechanics in infants with AVB requiring mechanical ventilation (MV) support and to evaluate if respiratory mechanics predict outcomes in children with AVB supported on MV. To evaluate the respiratory mechanics in infants with AVB submitted to MV. MATERIALS AND METHODS: A prospective observational study was conducted in two pediatric intensive care units (PICUs) between February 2016 and March 2017. Included were infants (1 month to 1 year old) admitted with AVB and requiring MV for >48 hours. Auto-PEEP, dynamic compliance (Cdyn), static compliance (Cstat), expiratory resistance (ExRes), and inspiratory resistance (InRes) were evaluated once daily on the second and third day of MV. RESULTS: A total of 64 infants (median age of 2.8 months and a mean weight of 4.8 ± 1.7 kg) were evaluated. A mean positive inspiratory pressure (PIP) of 31.5 ± 5.2 cmH2O, positive end-expiratory pressure (PEEP) of 5.5 ± 1.4 cmH2O, resulting in a mean airway pressure (MAP) of 12.5 ± 2.2 cmH2O and delta pressure of 22.5 ± 4.4 cmH2O without difference between the two hospitals. Measurements of respiratory mechanics showed high values of InRes and ExRes (median 142 [IQ25-75 106-180] cmH2O/L/s and 158 [IQ25-75 130-195.3] cmH2O/L/s, respectively), accompanied by decreased Cdyn and Cstat (0.46 ± 0.19 and 0.81 ± 0.25 mL/kg/cmH2O, respectively). None of the variables was associated with mortality, length of MV, or length of PICU stay. CONCLUSION: Infants with AVB requiring MV support present very high InRes and ExRes values. These findings might be the reason for the aggressive ventilatory parameters, especially PIP, required to ventilate this group of children with lower airway obstruction. CLINICAL SIGNIFICANCE: Monitoring respiratory mechanics could represent a useful tool to guide the ventilatory strategy to be adopted in patients with AVB. HOW TO CITE THIS ARTICLE: Andreolio C, Piva JP, Bruno F, da Rocha TS, Garcia PCR. Airway Resistance and Respiratory Compliance in Children with Acute Viral Bronchiolitis Requiring Mechanical Ventilation Support. Indian J Crit Care Med 2021;25(1):88-93.

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast.

RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.

Human ARMT1 structure and substrate specificity indicates that it is a DUF89 family damage-control phosphatase.

Metabolite damage control is a critical but poorly defined aspect of cellular biochemistry, which likely involves many of the so far functionally uncharacterized protein domain (domains of unknown function; DUFs). We have determined the crystal structure of the human DUF89 protein product of the C6ORF211 gene to 1.85 Å. The crystal structure shows that the protein contains a core α-ß-α fold with an active site-bound metal ion and α-helical bundle N-terminal cap, which are both conserved features of subfamily III DUF89 domains. The biochemical activities of the human protein are conserved with those of a previously characterized budding yeast homolog, where an in vitro phosphatase activity is supported by divalent cations that include Co2+, Ni2+, Mn2+ or Mg2+. Full steady-state kinetics parameters of human DUF89 using a standard PNPP phosphatase assay revealed a six times higher catalytic efficiency in presence of Co2+ compared to Mg2+. The human enzyme targets a number of phosphate substrates similar to the budding yeast homolog, while it lacks a previously indicated methyltransferase activity. The highest activity on substrate was observed with fructose-1-phosphate, a potent glycating agent, and thus human DUF89 phosphatase activity may also play a role in limiting the buildup of phospho-glycan species and their related damaged metabolites.

N-locking stabilization of covalent helical peptides: Application to Bfl-1 antagonists.

Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N-lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N-locked covalent BH3 peptides that were designed to selectively target the anti-apoptotic protein Bfl-1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl-1 and cellular activity. The crystal structure of the complex between such N-locked covalent peptide and Bfl-1 provided insights on the geometry of the N-locking strategy and of the covalent bond between the agent and Bfl-1.

Profiling of behavioral effects evoked by ketamine and the role of 5HT2 and D2 receptors in ketamine-induced locomotor sensitization in mice.

Ketamine has addictive potential, a troublesome fact due to its promising use as a therapeutic drug. An important phenomenon associated with drug addiction is behavioral sensitization, usually characterized as augmented locomotion. However, other behaviors may also be susceptible to sensitization, and/or interfere with locomotor activity. Thus, this study drew a comprehensive behavioral 'profiling' in an animal model of repeated administration of ketamine. Adult Swiss mice received single daily ketamine injections (30 or 50 mg/Kg, i.p.), which were followed by open field testing for 7 days (acquisition period, ACQ). A ketamine challenge (sensitization test, ST) was carried out after a 5-day withdrawal. Locomotion, rearing, grooming, rotation and falling were assessed during ACQ and ST. All behaviors were affected from the first ACQ day onwards, with no indication of competition between locomotion and the other behaviors. Only locomotion in response to 30 mg/Kg of ketamine both escalated during ACQ and expressed increased levels at ST, evidencing development and expression of locomotor sensitization. Considering the involvement of serotonin 5HT(2) and dopamine D(2) receptors on addiction mechanisms, we further tested the involvement of these receptors in ketamine-induced sensitization. Ketanserin (5HT2 antagonist, 3 mg/Kg, s.c.) prevented ketamine-evoked development of locomotor sensitization. However, ketanserin pretreatment during ACQ failed to inhibit its expression during ST. Raclopride (D2 antagonist, 0.5 mg/Kg, s.c.) evoked less robust reductions in locomotion but prevented the development of ketamine-evoked sensitization. Pretreatment during ACQ further inhibited the expression of sensitization during ST. These results indicate that a partial overlap in serotonergic and dopaminergic mechanisms underlies ketamine-induced locomotor sensitization.

Oxyresveratrol drives caspase-independent apoptosis-like cell death in MDA-MB-231 breast cancer cells through the induction of ROS.

Earlier studies from our laboratory have demonstrated that Oxyresveratrol (OXY), a hydroxyl-substituted stilbene, exhibits potent inhibition of human melanoma cell proliferation. The present study defines a cytotoxic effect of OXY on the highly chemo-resistant, triple-negative human breast cancer cell line MDA-MB-231. OXY-mediated cell death resulted in accumulation of cells at the sub-G1 phase of the cell cycle, induced chromatin condensation, DNA fragmentation, phosphatidylserine externalization and PARP cleavage, indicative of apoptosis. Interestingly, morphology and cell viability studies with the pan-caspase inhibitor, QVD-OPH revealed that OXY-induced cell death was caspase-independent. Docking studies also showed that OXY can bind to the S1 site of caspase-3, and could also exert an inhibitory effect on this executioner caspase. The immunoblot analysis demonstrating the absence of caspase cleavage during cell death further confirmed these findings. OXY was also observed to induce the production of reactive oxygen species, which caused the depolarization of the mitochondrial membrane resulting in translocation of Apoptosis Inducing Factor (AIF) into the nucleus. Pretreatment of the cells with N-Acetyl Cysteine antioxidant prevented cell death resulting from OXY treatment. Thus, OXY initiates ROS-mediated, apoptosis-like cell death, involving mitochondrial membrane depolarization, translocation of AIF into the nucleus, and DNA fragmentation, resulting in caspase-independent cell death in MDA-MB-231 cells. The cytotoxicity manifested by OXY was also observed in 3D cell culture models and primary cells, thereby providing a basis for the utilization of OXY as a novel template for the future design of anticancer therapeutics.

Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy.

We have recently investigated the reactivity of aryl-fluorosulfates as warheads to form covalent adducts with Lys, Tyr, and His residues. However, the rate of reaction of aryl-fluorosulfates seemed relatively slow, putting into question their effectiveness to form covalent adducts in cell. Unlike the previously reported agents that targeted a relatively remote Lys residue with respect to the target's binding site, the current agents were designed to more directly juxtapose an aryl-fluorosulfate with a Lys residue that is located within the binding pocket of the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP). We found that such new agents can effectively and rapidly form a covalent adduct with XIAP-BIR3 in vitro and in cell, approaching the rate of reaction, cellular permeability, and stability that are similar to what attained by acrylamides when targeting Cys residues. Our studies further validate aryl-fluorosulfates as valuable Lys-targeting electrophiles, for the design of inhibitors of both enzymes and protein-protein interactions.

Covalent Inhibitors of Protein-Protein Interactions Targeting Lysine, Tyrosine, or Histidine Residues.

We have recently reported a series of Lys-covalent agents targeting the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP) using a benzamide-sulfonyl fluoride warhead. Using XIAP as a model system, we further investigated a variety of additional warheads that can be easily incorporated into binding peptides and analyzed their ability to form covalent adducts with lysine and other amino acids, including tyrosine, histidine, serine, and threonine, using biochemical and biophysical assays. Moreover, we tested aqueous, plasma stability, cell permeability, and cellular efficacy of the most effective agents. These studies identified aryl-fluoro sulfates as likely the most suitable electrophiles to effectively form covalent adducts with Lys, Tyr, and His residues, given that these agents were cell permeable and stable in aqueous buffer and in plasma. Our studies contain a number of general findings that open new possible avenues for the design of potent covalent protein-protein interaction antagonists.

Recent insights into natural product inhibitors of matrix metalloproteinases.

Members of the matrix metalloproteinase (MMP) family have biological functions that are central to human health and disease, and MMP inhibitors have been investigated for the treatment of cardiovascular disease, cancer and neurodegenerative disorders. The outcomes of initial clinical trials with the first generation of MMP inhibitors proved disappointing. However, our growing understanding of the complexities of the MMP function in disease, and an increased understanding of MMP protein architecture and control of activity now provide new opportunities and avenues to develop MMP-focused therapies. Natural products that affect MMP activities have been of strong interest as templates for drug discovery, and for their use as chemical tools to help delineate the roles of MMPs that still remain to be defined. Herein, we highlight the most recent discoveries of structurally diverse natural product inhibitors to these proteases.