In vitro antiproliferative effects of Vatairea macrocarpa (Benth.) Ducke lectin on human tumor cell lines and in vivo evaluation of its toxicity in Drosophila melanogaster.

Tumor cells may develop alterations in glycosylation patterns during the initial phase of carcinogenesis. These alterations may be important therapeutic targets for lectins with antitumor action. This work aimed to evaluate the in vitro cytotoxicity of VML on tumor and non-tumor cells (concentration of 25 µg/mL and then microdiluted) and evaluate its in vivo toxicity at different concentrations (1.8, 3.5 and 7.0 µg/mL), using Drosophila melanogaster. Toxicity in D. melanogaster evaluated mortality rate, as well as oxidative stress markers (TBARS, iron levels, nitric oxide levels, protein and non-protein thiols). The cytotoxicity assay showed that VML had cytotoxic effect on leukemic lines HL-60 (IC50 = 3.5 µg/mL), KG1 (IC50 = 18.6 µg/mL) and K562 (102.0 µg/mL). In the toxicity assay, VML showed no reduction in survival at concentrations of 3.5 and 7.0 µg/mL and did not alter oxidative stress markers at any concentrations tested. Cytotoxicity of VML from HL-60, KG1 and K562 cells could arise from the interaction between the lectin and specific carbohydrates of tumor cells. In contrast, effective concentrations of VML against no-tumor cells human keratinocyte - HaCat and in the D. melanogaster model did not show toxicity, suggesting that VML is a promising molecule in vivo studies involving leukemic cells.

Modeling the influence of propionic acid concentration and pH on the kinetics of Salmonella Typhimurium.

Salmonella Typhimurium is a foodborne pathogen often found in the poultry production chain. Antibiotics have been used to reduce S. Typhimurium contamination in poultry aviaries and improve chicken growth. However, antibiotics were banned in several countries. Alternatively, organic acids, such as propionic acid (PA), can control pathogens. This study determined the PA minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and mathematically modeled S. Typhimurium growth/inactivation kinetics under the influence of PA at different pH values (4.5, 5.5, and 6.5) which are within the pH range of the chicken gastrointestinal tract. The PA MIC against S. Typhimurium was pH-dependent, resulting in 5.0, 3.5 and 9.0 mM undissociated PA at pH 4.5, 5.5, and 6.5, respectively. The Baranyi and Roberts and the Weibull model fit growth and inactivation data well, respectively. Secondary models were proposed. The validated model predicted 3-log reduction of S. Typhimurium in 3 h at 68.2 mM of undissociated PA and pH 4.5. The models presented a good capacity to describe the kinetics of S. Typhimurium subjected to PA, representing a useful tool to predict PA antibacterial action depending on the pH.

Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

Delirium During Critical Illness and Subsequent Change of Treatment in Patients With Cancer: A Mediation Analysis.

OBJECTIVES: To assess whether delirium during ICU stay is associated with subsequent change in treatment of cancer after discharge. DESIGN: Retrospective cohort study. SETTING: A 50-bed ICU in a dedicated cancer center. PATIENTS: Patients greater than or equal to 18 years old with a previous proposal of cancer treatment (chemotherapy, target therapy, hormone therapy, immunotherapy, radiotherapy, oncologic surgery, and bone marrow transplantation). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We considered delirium present if Confusion Assessment Method for the ICU was positive. We assessed the association between delirium and modification of the treatment after discharge. We also performed a mediation analysis to assess both the direct and indirect (i.e., mediated by the development of functional dependence after discharge) of delirium on modification of cancer treatment and whether the modification of cancer treatment was associated with mortality at 1 year. We included 1,134 patients, of whom, 189 (16.7%) had delirium. Delirium was associated with the change in cancer treatment (adjusted odds ratio [OR], 3.80; 95% CI, 2.72-5.35). The association between delirium in ICU and change of treatment was both direct and mediated by the development of functional dependence after discharge. The proportion of the total effect of delirium on change of treatment mediated by the development of functional dependence after discharge was 33.0% (95% CI, 21.7-46.0%). Change in treatment was associated with increased mortality at 1 year (adjusted OR, 2.68; 95% CI, 2.01-3.60). CONCLUSIONS: Patients who had delirium during ICU stay had a higher rate of modification of cancer treatment after discharge. The effect of delirium on change in cancer treatment was only partially mediated by the development of functional dependence after discharge. Change in cancer treatment was associated with increased 1-year mortality.

DVL, lectin from Dioclea violacea seeds, has multiples mechanisms of action against Candida spp via carbohydrate recognition domain.

Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.

Dioclea violacea lectin increases the effect of neomycin against multidrug-resistant strains and promotes the purification of the antibiotic in immobilized lectin column.

DVL is a Man/Glc-binding lectin from Dioclea violacea seeds that has the ability to interact with the antibiotic gentamicin. The present work aimed to evaluate whether the DVL has the ability to interact with neomycin via CRD and to examine the ability of this lectin to modulate the antibiotic effect of neomycin against multidrug-resistant strains (MDR). The hemagglutinating activity test revealed that neomycin inhibited the hemagglutinating activity of DVL with a minimum inhibitory concentration of 50 mM, indicating that the antibiotic interacts with DVL via the carbohydrate recognition domain (CRD). DVL immobilized on cyanogen bromide-activated Sepharose® 4B bound 41 % of the total neomycin applied to the column, indicating that the DVL-neomycin interaction is efficient for purification processes. Furthermore, the minimum inhibitory concentrations (MIC) obtained for DVL against all strains studied were not clinically relevant. However, when DVL was combined with neomycin, a significant increase in antibiotic activity was observed against S. aureus and P. aeruginosa. These results demonstrate the first report of lectin-neomycin interaction, indicating that immobilized DVL has the potential to isolate neomycin by affinity chromatography. Moreover, DVL increased the antibiotic activity of neomycin against MDR, suggesting that it is a potent adjuvant in the treatment of infectious diseases.

MiR-200c-3p expression may be associated with worsening of the clinical course of patients with COVID-19.

COVID-19 represents a public health emergency, whose mechanism of which is not fully understood. It is speculated that microRNAs may play a crucial role in host cells after infection by SARS-CoV-2. Thus, our study aimed to analyze the expression of miR-200c-3p in saliva samples from patients with COVID-19. One handred eleven samples from patients with COVID-19 were divided into 4 groups. Group I: 39 patients negative for Covid-19; Group II: 37 positive and symptomatic patients, with no indication of hospitalization; Group III: 21 patients with respiratory disorders (hospitalized); Group IV: 14 patients with severe conditions (oxygen therapy). The expression levels of miR-200c-3p were determined using qPCR. We found greater expression of miR-200c-3p in patients in group IV (p<0.0001), and also verified that patients aged ≥42 years had a higher expression of this miR (p=0.013). Logistic regression analysis revealed that the expression of miR-200c-3p and systemic arterial hypertension are factors independently associated with patients in group IV (p<0.0001). Our results suggest that miR-200c-3p is a predictor of severity independent of COVID-19 risk factors, which could represent a way of screening patients affected by SARS-CoV-2.

Noninvasive ventilation in critically ill very old patients with pneumonia: A multicenter retrospective cohort study.

BACKGROUND: Very old patients (≥ 80 years-old, VOP) are increasingly admitted to intensive care units (ICUs). Community-acquired pneumonia (CAP) is a common reason for admission and the best strategy of support for respiratory failure in this scenario is not fully known. We evaluated whether noninvasive ventilation (NIV) would be beneficial compared to invasive mechanical ventilation (IMV) regarding hospital mortality. METHODS: Multicenter cohort study of VOPs admitted with CAP in need of IMV or NIV to 11 Brazilian ICUs from 2009 through 2012. We used logistic regression models to evaluate the association between the initial ventilatory strategy (NIV vs. IMV) and hospital mortality adjusting for confounding factors. We evaluated effect modification with interaction terms in pre-specified sub-groups. RESULTS: Of 369 VOPs admitted for CAP with respiratory failure, 232 (63%) received NIV and 137 (37%) received IMV as initial ventilatory strategy. IMV patients were sicker at baseline (median SOFA 8 vs. 4). Hospital mortality was 114/232 (49%) for NIV and 90/137 (66%) for IMV. For the comparison NIV vs. IMV (reference), the crude odds ratio (OR) was 0.50 (95% CI, 0.33-0.78, p = 0.002). This association was largely confounded by antecedent characteristics and non-respiratory SOFA (adjOR = 0.70, 95% CI, 0.41-1.20, p = 0.196). The fully adjusted model, additionally including Pao2/Fio2 ratio, pH and Paco2, yielded an adjOR of 0.81 (95% CI, 0.46-1.41, p = 0.452). There was no strong evidence of effect modification among relevant subgroups, such as Pao2/Fio2 ratio ≤ 150 (p = 0.30), acute respiratory acidosis (p = 0.42) and non-respiratory SOFA ≥ 4 (p = 0.53). CONCLUSIONS: NIV was not associated with lower hospital mortality when compared to IMV in critically ill VOP admitted with CAP, but there was no strong signal of harm from its use. The main confounders of this association were both the severity of respiratory dysfunction and of extra-respiratory organ failures.

Characteristics and outcomes of patients with COVID-19 admitted to the ICU in a university hospital in São Paulo, Brazil - study protocol.

OBJECTIVES: We designed a cohort study to describe characteristics and outcomes of patients with coronavirus disease (COVID-19) admitted to the intensive care unit (ICU) in the largest public hospital in Sao Paulo, Brazil, as Latin America becomes the epicenter of the pandemic. METHODS: This is the protocol for a study being conducted at an academic hospital in Brazil with 300 adult ICU beds dedicated to COVID-19 patients. We will include adult patients admitted to the ICU with suspected or confirmed COVID-19 during the study period. The main outcome is ICU survival at 28 days. Data will be collected prospectively and retrospectively by trained investigators from the hospital's electronic medical records, using an electronic data capture tool. We will collect data on demographics, comorbidities, severity of disease, and laboratorial test results at admission. Information on the need for advanced life support and ventilator parameters will be collected during ICU stay. Patients will be followed up for 28 days in the ICU and 60 days in the hospital. We will plot Kaplan-Meier curves to estimate ICU and hospital survival and perform survival analysis using the Cox proportional hazards model to identify the main risk factors for mortality. ClinicalTrials.gov: NCT04378582. RESULTS: We expect to include a large sample of patients with COVID-19 admitted to the ICU and to be able to provide data on admission characteristics, use of advanced life support, ICU survival at 28 days, and hospital survival at 60 days. CONCLUSIONS: This study will provide epidemiological data about critically ill patients with COVID-19 in Brazil, which could inform health policy and resource allocation in low- and middle-income countries.

Crystal structure and specific location of a germin-like protein with proteolytic activity from Thevetia peruviana.

Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical ß-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.

Machaerium acutifolium lectin alters membrane structure and induces ROS production in Candida parapsilosis.

Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.

Spectrum crispness sensory scale correlation with instrumental acoustic high-sampling rate and mechanical analyses.

Mechanical and acoustical instrumental tests can help to predict and compare the sensory crispness of food products. This study proposed a method to correlate crispness sensory analyses to instrumental parameters using the standard products of the Spectrum Crispness Sensory Scale. An acoustic system was developed with a high-sampling rate for characterizing food crispness. Force-displacement and acoustic signals were measured during penetration and compression tests of the products in the Spectrum Scale. A band-pass filter suppressed the texture analyzer's engine and gearbox noise. High correlations were obtained between acoustical and sensory parameters in both tests; however, the penetration test better differentiated the products. The high-sampling rate acoustical measurement system was capable of discriminate crispy products, although the trained sensory panel was still more sensitive to small differences.

Characterization of Three Osmotin-Like Proteins from Plumeria rubra and Prospection for Adiponectin Peptidomimetics.

BACKGROUND: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet. OBJECTIVE AND METHODS: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2). RESULTS: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide. CONCLUSION: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis.

The Galactose-Binding Lectin Isolated from Vatairea macrocarpa Seeds Enhances the Effect of Antibiotics Against Staphylococcus aureus-Resistant Strain.

The use of natural products together with standard antimicrobial drugs has recently received more attention as a strategy to combat infectious diseases caused by multidrug-resistant (MDR) microorganisms. This study aimed to evaluate the capacity of a galactose-binding lectin from Vatairea macrocarpa seeds (VML) to modulate antibiotic activity against standard and MDR Staphylococcus aureus and Escherichia coli bacterial strains. The minimum inhibitory concentration (MIC) obtained for VML against all strains was not clinically relevant (MIC ≥ 1024 µg/mL). However, when VML was combined with the antibacterial drugs gentamicin, norfloxacin and penicillin, a significant increase in antibiotic activity was observed against S. aureus, whereas the combination of VML and norfloxacin presented decreased and, hence, antagonistic antibiotic activity against E. coli. By its inhibition of hemagglutinating activity, gentamicin (MIC = 50 mM) revealed its interaction with the carbohydrate-binding site (CBS) of VML. Using molecular docking, it was found that gentamicin interacts with residues that constitute the CBS of VML with a score of - 120.79 MDS. It is this interaction between the antibiotic and the lectin's CBS that may be responsible for the enhanced activity of gentamicin in S. aureus. Thus, our results suggest that the VML can be an effective modulating agent against S. aureus. This is the first study to report the effect of lectins as modulators of bacterial sensitivity, and as such, the outcome of this study could lay the groundwork for future research involving the use of lectins and conventional antibiotics against such infectious diseases such as community-acquired methicillin-resistant S. aureus (MRSA).

Dioclea violacea lectin modulates the gentamicin activity against multi-resistant strains and induces nefroprotection during antibiotic exposure.

Gentamicin is an aminoglycoside antibiotic used to treat infections of various origins. In the last few decades, the constant use of gentamicin has resulted in increased bacterial resistance and nephrotoxicity in some cases. In this study, we examined the ability of Dioclea violacea lectin (DVL) in modulate the antimicrobial activity of gentamicin and reduce the nephrotoxicity induced by this drug. The minimum inhibitory concentration (MIC) obtained for DVL against all strains studied was not clinically relevant (MIC ≥ 1024 µg/mL). However, when DVL was combined with gentamicin, a significant increase in antibiotic action was observed against Staphylococcus aureus and Escherichia coli. DVL also reduced antibiotic tolerance in S. aureus during 10 days of continuous treatment. In addition, DVL presented a nephroprotective effect, reducing sodium excretion, N-Gal expression and urinary protein, that are important markers of glomerular and tubular injuries. Taken together, studies of inhibition of hemagglutinating activity, fluorescence spectroscopy and molecular docking revealed that gentamicin can interact with DVL via the carbohydrate recognition domain (CRD), suggesting that the results obtained in this study may be directly related to the interaction of DVL-gentamicin and with the ability of the lectin to interact with glycans present in the cells of the peritoneum.

Ultrasound-Guided Percutaneous Dilational Tracheostomy: A Systematic Review of Randomized Controlled Trials and Meta-Analysis.

INTRODUCTION: Percutaneous dilational tracheostomy (PDT) is a common and increasingly used procedure in the intensive care unit (ICU). It is usually performed with bronchoscopy guidance. Ultrasound has emerged as a useful tool in order to assist PDT, potentially improving its success rate and reducing procedural-related complications. OBJECTIVE: To investigate whether the ultrasound-guided PDT is equivalent or superior to the bronchoscopy-guided or anatomical landmarks-guided PDT with regard to procedural-related and clinical complications. METHODS: A systematic review of randomized clinical trials was conducted comparing an ultrasound-guided PDT to the control groups (either a bronchoscopy-guided PDT or an anatomical landmark-guided PDT) in patients undergoing a PDT in the ICU. The primary outcome was the incidence of major procedural-related and clinical complication rates. The secondary outcome was the incidence of minor complication rates. Random-effect meta-analyzes were used to pool the results. RESULTS: Four studies fulfilled the inclusion criteria and they were analyzed. The studies included 588 participants. There were no differences in the major complication rates between the patients who were assigned to the ultrasound-guided PDT when compared to the control groups (pooled risk ratio [RR]: 0.48; 95% confidence interval [CI]: 0.13-1.71, I2 = 0%). The minor complication rates were not different between the groups, but they had a high heterogeneity (pooled RR: 0.49; 95% CI 0.16-1.50; I2 = 85%). The sensitivity analyzes that only included the randomized controlled trials that used a landmark-guided PDT as the control group showed lower rates of minor complications in the ultrasound-guided PDT group (pooled RR: 0.55; 95% CI: 0.31-0.98, I2 = 0%). CONCLUSION: The ultrasound-guided PDT seems to be safe and it is comparable to the bronchoscopy-guided PDT regarding the major and minor procedural-related or clinical complications. It also seems to reduce the minor complications when compared to the anatomical landmark-guided PDT.