Comparative genomic analysis of Pseudomonas aeruginosa strains susceptible and resistant to carbapenems and aztreonam isolated from patients with healthcare-associated infections in a Mexican hospital.

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that causes different infections on immunocompromised patients. Within PA accessory genome, differences in virulence, antibiotic resistance and biofilm formation have been described between strains, leading to the emergence of multidrug-resistant strains. The genome sequences of 17 strains isolated from patients with healthcare-associated infections in a Mexican hospital were genomically and phylogenetically analyzed and antibiotic resistance genes, virulence genes, and biofilm formation genes were detected. Fifteen of the 17 strains were resistant to at least two of the carbapenems meropenem, imipenem, and the monobactam aztreonam. The antibiotic resistance (mexA, mexB, and oprM) and the biofilm formation (pslA and pslD) genes were detected in all strains. Differences were found between strains in accessory genome size. The strains had different sequence types, and seven strains had sequence types associated with global high risk epidemic PA clones. All strains were represented in two groups among PA global strains. In the 17 strains, horizontally acquired resistance genes to aminoglycosides and beta-lactams were found, mainly, and between 230 and 240 genes that encode virulence factors. The strains under study were variable in terms of their accessory genome, antibiotic resistance, and virulence genes. With these characteristics, we provide information about the genomic diversity of clinically relevant PA strains.

Effect of Routine Invasive vs Conservative Strategy in Older Adults With Frailty and Non-ST-Segment Elevation Acute Myocardial Infarction: A Randomized Clinical Trial.

Importance: To our knowledge, no randomized clinical trial has compared the invasive and conservative strategies in frail, older patients with non-ST-segment elevation acute myocardial infarction (NSTEMI). Objective: To compare outcomes of invasive and conservative strategies in frail, older patients with NSTEMI at 1 year. Design, Setting, and Participants: This multicenter randomized clinical trial was conducted at 13 Spanish hospitals between July 7, 2017, and January 9, 2021, and included 167 older adult (≥70 years) patients with frailty (Clinical Frailty Scale score ≥4) and NSTEMI. Data analysis was performed from April 2022 to June 2022. Interventions: Patients were randomized to routine invasive (coronary angiography and revascularization if feasible; n = 84) or conservative (medical treatment with coronary angiography for recurrent ischemia; n = 83) strategy. Main Outcomes and Measures: The primary end point was the number of days alive and out of the hospital (DAOH) from discharge to 1 year. The coprimary end point was the composite of cardiac death, reinfarction, or postdischarge revascularization. Results: The study was prematurely stopped due to the COVID-19 pandemic when 95% of the calculated sample size had been enrolled. Among the 167 patients included, the mean (SD) age was 86 (5) years, and mean (SD) Clinical Frailty Scale score was 5 (1). While not statistically different, DAOH were about 1 month (28 days; 95% CI, -7 to 62) greater for patients managed conservatively (312 days; 95% CI, 289 to 335) vs patients managed invasively (284 days; 95% CI, 255 to 311; P = .12). A sensitivity analysis stratified by sex did not show differences. In addition, we found no differences in all-cause mortality (hazard ratio, 1.45; 95% CI, 0.74-2.85; P = .28). There was a 28-day shorter survival in the invasive vs conservatively managed group (95% CI, -63 to 7 days; restricted mean survival time analysis). Noncardiac reasons accounted for 56% of the readmissions. There were no differences in the number of readmissions or days spent in the hospital after discharge between groups. Neither were there differences in the coprimary end point of ischemic cardiac events (subdistribution hazard ratio, 0.92; 95% CI, 0.54-1.57; P = .78). Conclusions and Relevance: In this randomized clinical trial of NSTEMI in frail older patients, there was no benefit to a routine invasive strategy in DAOH during the first year. Based on these findings, a policy of medical management and watchful observation is recommended for older patients with frailty and NSTEMI. Trial Registration: ClinicalTrials.gov Identifier: NCT03208153.

Current knowledge in the use of bacteriophages to combat infections caused by Pseudomonas aeruginosa in cystic fibrosis.

Pseudomonas aeruginosa (PA) is considered the first causal agent of morbidity and mortality in people with cystic fibrosis (CF) disease. Multi-resistant strains have emerged due to prolonged treatment with specific antibiotics, so new alternatives have been sought for their control. In this context, there is a renewed interest in therapies based on bacteriophages (phages) supported by several studies suggesting that therapy based on lytic phages and biofilm degraders may be promising for the treatment of lung infections in CF patients. However, there is little clinical data about phage studies in CF and the effectiveness and safety in patients with this disease has not been clear. Therefore, studies regarding on phage characterization, selection, and evaluation in vitro and in vivo models will provide reliable information for designing effective cocktails, either using mixed phages or in combination with antibiotics, making a great progress in clinical research. Hence, this review focuses on the most relevant and recent findings on the activity of lytic phages against PA strains isolated from CF patients and hospital environments, and discusses perspectives on the use of phage therapy on the treatment of PA in CF patients.

Use of Cocktail of Bacteriophage for Salmonella Typhimurium Control in Chicken Meat.

Foodborne diseases are extremely relevant and constitute an area of alert for public health authorities due to the high impact and number of people affected each year. The food industry has implemented microbiological control plans that ensure the quality and safety of its products; however, due to the high prevalence of foodborne diseases, the industry requires new microbiological control systems. One of the main causative agents of diseases transmitted by poultry meat is the bacterium Salmonella enterica. Disinfectants, antibiotics, and vaccines are used to control this pathogen. However, they have not been efficient in the total elimination of these bacteria, with numerous outbreaks caused by this bacterium observed today, in addition to the increase in antibiotic-resistant bacteria. The search for new technologies to reduce microbial contamination in the poultry industry continues to be a necessity and the use of lytic bacteriophages is one of the new solutions. In this study, 20 bacteriophages were isolated for Salmonella spp. obtained from natural environments and cocktails composed of five of them were designed, where three belonged to the Siphoviridae family and two to the Microviridae family. This cocktail was tested on chicken meat infected with Salmonella Typhimurium at 10 °C, where it was found that this cocktail was capable of decreasing 1.4 logarithmic units at 48 h compared to the control.

Incidence and Predictors of Progression in Asymptomatic Patients With Stable Heart Failure.

Data from previous heart failure (HF) trials suggest that patients with mild symptoms (NYHA II) actually have a poor clinical outcome. However, these studies did not assess clinical stability and rarely included patients in NYHA I. We sought to determine the incidence of short-term clinical progression in supposedly stable HF patients in NYHA I. In addition, we aimed to investigate the predictive value of widely available electrocardiographic and echocardiographic parameters for short-term disease progression. This is a retrospective study including 153 consecutive patients with HF with reduced and mid-range ejection fraction (HFrEF: LVEF<40%; HFmrEF: LVEF 40-49%) in NYHA I with no history of decompensation within the previous 6 months. All patients underwent comprehensive baseline echocardiographic and electrocardiographic assessment. The primary endpoint was the composite of cardiovascular death, hospitalization and need for intensification of HF treatment within a 12 month follow-up period. The cumulative incidence of HF progression was 17.8%, with a median time to event of 193 days. Death and hospitalization due to HF accounted for three-quarters of the events. QRS duration ≥120ms and mitral regurgitation grade >1 showed to be significant predictors of HF progression (HR: 8.92, p<0.001; and HR: 4.10, p<0.001, respectively). Patients without these risk factors had a low incidence of clinical events (3.8%). In conclusion, almost one in five supposedly stable HF patients in NYHA I experience clinical progression in short-term follow-up. Simple electrocardiographic and echocardiographic predictors may be useful for risk stratification and could help to improve individual HF patient management and outcomes.

Antioxidant effect of resveratrol in single red blood cells measured by thermal fluctuation spectroscopy.

The human red blood cell (RBC) membrane has significant elastic capabilities which can be described measuring typical membrane edge fluctuations and mechanical properties by optical techniques. The RBC elastic properties can be affected by changes in the surrounding media. In an attempt to elucidate the molecular mechanisms of the interaction of resveratrol with the red cell membrane and of its antioxidant capacity the changes in mechanical properties of the RBC membrane were analyzed. These studies were carried out through measurements of RBC membrane fluctuations in the presence of the oxidant agent HClO using thermal fluctuation spectroscopy (TFS). The observed results showed that the elastic capabilities of RBC changed with low concentration of hypochlorous acid but without morphological changes. However, in the presence of resveratrol the deformation and decrease of elastic capabilities induced by HClO on RBC decreased. These in vitro results demonstrated the protective effect of RV against the detrimental effects triggered by HClO upon human erythrocytes.

Estrategia invasiva frente a conservadora en pacientes frágiles con IAMSEST. Diseño del ensayo clínico MOSCA-FRAIL / Invasive versus conservative strategy in frail patients with NSTEMI: the MOSCA-FRAIL clinical trial study design

Introducción y objetivos: Aunque las guías de práctica clínica recomiendan una estrategia invasiva para el infarto agudo de miocardio sin elevación del segmento ST (IAMSEST), en la práctica clínica esta estrategia se infrautiliza en ancianos frágiles. Además estos enfermos habitualmente quedan excluidos de los ensayos clínicos, por lo que la evidencia es escasa. Nuestra hipótesis es que una estrategia invasiva para el anciano con fragilidad y IAMSEST mejorará el pronóstico. Métodos: Se trata de un estudio prospectivo, multicéntrico y aleatorizado que compara una estrategia invasiva frente a una conservadora en ancianos frágiles con IAMSEST. Los criterios de inclusión son: IAMSEST, edad ≥ 70 años y fragilidad definida por al menos 4 criterios de la escala Clinical Frailty Scale. Se aleatorizará a los participantes a una estrategia invasiva (coronariografía y revascularización si se considera anatómicamente indicada) o conservadora (tratamiento médico y coronariografía solo en caso de inestabilidad clínica persistente). El objetivo principal será el número de días vivo fuera del hospital durante el primer año. El objetivo coprincipal será el tiempo hasta la presentación de muerte cardiovascular, reinfarto agudo de miocardio o revascularización tras el alta. El tamaño de la muestra estimado es de 178 pacientes (89 por grupo), asumiendo un incremento del 20% en la proporción de días vivo fuera del hospital con la estrategia invasiva. Resultados: Los resultados del estudio aportarán información novedosa para el tratamiento del anciano frágil con IAMSEST. Conclusiones: Nuestra hipótesis es que una estrategia invasiva mejorará el pronóstico de los pacientes ancianos frágiles con IAMSEST. Si esta hipótesis se confirmara, la situación de fragilidad no debería disuadir al cardiólogo de indicar un tratamiento invasivo. Ensayo registrado en ClinicalTrials.gov (Identificador: NCT03208153)

Introduction and objectives: Although clinical guidelines recommend invasive management in non-ST-segment elevation myocardial infarction (NSTEMI), this strategy is underused in frail elderly patients in the real world. Furthermore, these patients are underrepresented in clinical trials and therefore the evidence is scarce. Our hypothesis is that an invasive strategy will improve prognosis in elderly frail patients with NSTEMI. Methods: This will be a prospective, multicenter, randomized trial, in which the conservative and invasive strategies will be compared in patients meeting all of the following inclusion criteria: NSTEMI diagnosis, age ≥ 70 years, and frailty defined by a category ≥ 4 in the Clinical Frailty Scale. Participants will be randomized to an invasive (coronary angiogram and revascularization if anatomically amenable) or conservative (medical treatment and coronary angiogram only if persistent clinical instability) strategy. The primary endpoint will be the number of days alive out of hospital during the first year. The coprimary endpoint will be the time until the first cardiac event (cardiac death, reinfarction or postdischarge revascularization). We estimate a sample size of 178 patients (89 per arm), considering an increase of 20% in the proportion of days alive out of hospital with the invasive management. Results: The results of this study will add important knowledge to inform the management of frail elderly patients hospitalized with NSTEMI. Conclusions: We hypothesize that the invasive strategy will improve outcomes in frail elderly patients with NSTEMI. If this is confirmed, frailty status should not dissuade physicians from implementing an invasive management strategy. Clinical trial registration: URL: http://www.clinicaltrials.gov .Identifier: NCT03208153

Invasive Versus Conservative Strategy in Frail Patients With NSTEMI: The MOSCA-FRAIL Clinical Trial Study Design.

INTRODUCTION AND OBJECTIVES: Although clinical guidelines recommend invasive management in non-ST-segment elevation myocardial infarction (NSTEMI), this strategy is underused in frail elderly patients in the real world. Furthermore, these patients are underrepresented in clinical trials and therefore the evidence is scarce. Our hypothesis is that an invasive strategy will improve prognosis in elderly frail patients with NSTEMI. METHODS: This will be a prospective, multicenter, randomized trial, in which the conservative and invasive strategies will be compared in patients meeting all of the following inclusion criteria: NSTEMI diagnosis, age ≥ 70 years, and frailty defined by a category ≥ 4 in the Clinical Frailty Scale. Participants will be randomized to an invasive (coronary angiogram and revascularization if anatomically amenable) or conservative (medical treatment and coronary angiogram only if persistent clinical instability) strategy. The primary endpoint will be the number of days alive out of hospital during the first year. The coprimary endpoint will be the time until the first cardiac event (cardiac death, reinfarction or postdischarge revascularization). We estimate a sample size of 178 patients (89 per arm), considering an increase of 20% in the proportion of days alive out of hospital with the invasive management. RESULTS: The results of this study will add important knowledge to inform the management of frail elderly patients hospitalized with NSTEMI. CONCLUSIONS: We hypothesize that the invasive strategy will improve outcomes in frail elderly patients with NSTEMI. If this is confirmed, frailty status should not dissuade physicians from implementing an invasive management strategy. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov.Identifier: NCT03208153.

Freezing of gait in Parkinson's disease: functional neuroimaging studies of the frontal lobe.

OBJECTIVE: The objective of the study is to study a group of patients with Parkinson's disease (PD) with and without freezing of gait (FOG) and analyze neuropsychological differences, especially regarding executive functions, according to their performance in a set of tests, and potential anomalies in functional positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro-d-glucose integrated with computed tomography (18F-FDG PET/CT) neuroimaging results of the frontal lobe. METHODS: The study recruited 23 patients. We performed an 18F-FDG PET/CT scan for 17 patients (9 with FOG and 8 without FOG) and 6 controls. Frontal functions were evaluated in all the patients during their 'on' situation. RESULTS: In FDG PET studies, PD patients differed from controls in that they presented hypometabolic areas in the parietal and bilateral occipital regions, as well as in the bilateral frontal region, especially on the right side. Compared to patients without FOG, patients with FOG displayed more pronounced frontal and predominantly right-sided hypometabolism. Both groups of patients displayed a poorer performance than the control population in the frontal cognitive tests. A greater executive disfunction was found in patients with FOG. CONCLUSIONS: Our study revealed greater hypometabolism in FDG PET studies, predominantly in right-sided-specific motor regions of the frontal lobe in FOG patients and greater frontal disfunction in neuropsychological tests in PD patients with FOG. These data suggest that FOG may be related to functional impairment of the circuits connecting the frontal lobe and the basal ganglia.

In vitro effects of benzimidazole/thioether-copper complexes with antitumor activity on human erythrocytes.

Two cytotoxic copper(II) complexes with N-H and N-methylated benzimidazole-derived ligands (Cu-L1 and Cu-L1Me; L1=bis(2-methylbenzimidazolyl)(2-methylthioethyl)amine, L1Me=bis(1-methyl-2-methylbenzimidazolyl)(2-methylthioethyl)amine) were synthesized and exposed to human erythrocytes and molecular models of its membrane. The latter were bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of lipids present in the external and internal moieties of the human red cell membrane, respectively. Scanning electron microscopy (SEM) of erythrocytes incubated with solutions of both Cu(II) complexes showed that they induced morphological changes to the normal cells to echinocytes, and hemolysis at higher concentrations. Real-time observation of the dose-dependent effects of the complexes on live erythrocytes by defocusing microscopy (DM) confirmed SEM results. The formation of echinocytes implied that complex molecules inserted into the outer moiety of the red cell membrane. X-ray diffraction studies on DMPC and DMPE showed that none of these complexes interacted with DMPE and only Cu-L1 interacted with DMPC. This difference was explained by the fact that Cu-L1Me complex is more voluminous than Cu-L1 because it has two additional methyl groups; on the other hand, DMPC molecule has three methyl groups in its bulky terminal amino end. Thus, by steric hindrance Cu-L1Me molecules cannot intercalate into DMPC bilayer, which besides is present in the gel phase. These results, together with the increased antiproliferative capacity of the N-methylated complex Cu-L1Me over that of Cu-L1 are rationalized mainly based on its higher lipophilicity.

Effects of Thimerosal on Lipid Bilayers and Human Erythrocytes: An In Vitro Study.

Thimerosal (THI, ethyl-mercury thiosalicylate) is added to vaccines as a preservative; as a consequence, infants may have been exposed to bolus doses of Hg that collectively added up to nominally 200 µg Hg during the first 6 months of life. While several studies report an association between THI-containing vaccines and neurological disorders, other studies do not support the causal relation between THI and autism. With the purpose to understand the molecular mechanisms of the toxic effect of THI it was assayed on human red cells and in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids found in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of THI to interact with DMPC and DMPE was determined by X-ray diffraction and differential scanning calorimetry, whereas intact human erythrocytes were observed by optical, defocusing and scanning electron microscopy. The experimental findings of this study demonstrated that THI interacted in a concentration-dependent manner with DMPC and DMPE bilayers, and in vitro interacted with erythrocytes inducing morphological changes. However, concentrations were considerable higher than those present in vaccines.

Antioxidant Capacity of Gallic Acid in vitro Assayed on Human Erythrocytes.

Gallic acid (GA) is a polyphenol present in many plants. This study was aimed to investigate the molecular interaction of GA with the human erythrocyte membrane and to determine its antioxidant capacity. The molecular interaction with the membrane of human red cells and the antioxidant property was assayed on both human red cells and molecular models of its membrane. Observations by optical, scanning electron, and defocusing microscopy demonstrated that GA is capable to convert red cells from their normal biconcave shape to crenated echinocytes. This result indicates that GA molecules are positioned in the outer monolayer of the red cell membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were selected as classes of phospholipids found in the outer and inner monolayers of the red cell membrane, respectively. X-ray diffraction and differential scanning calorimetry showed that GA was preferentially bound to DMPC bilayers. Experiments related to the antioxidant capacity of GA indicated that this compound offsets HClO oxidative capacity on DMPE bilayers. In addition, optical, scanning, defocusing microscopy, and hemolysis assays confirmed the protective capacity of GA against HClO deleterious effects on human red cells. As a conclusion, GA would be capable to block the access of oxidants into the lipid bilayer, and thus avoid their access into red cells.

Structural and functional effects of benzimidazole/thioether-copper complexes with antitumor activity on cell membranes and molecular models.

Two cytotoxic copper(II) complexes with N-H and N-methylated benzimidazole-derived ligands (Cu-L(2) and Cu-L(2Me)) were synthesized and made to interact with human erythrocytes and molecular models of their plasmatic membranes. The latter consisted in lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), lipids of the types present in the outer and inner monolayers of the human erythrocyte membrane, respectively. Initial assessment of the interaction of the complexes with DMPC and DMPE consisted of X-ray diffraction studies, which showed preferential interactions with the former. Scanning electron microscopy (SEM) of erythrocytes incubated with solutions of the Cu(II) complexes evidenced deformation of the cells to stomatocytes and knizocytes by Cu-L(2) and Cu-L(2Me) due to interactions with the inner and outer leaflets of the cell membranes, respectively. This was further confirmed by real-time observation of the dose-dependent effects of the complexes on live erythrocytes by defocusing microscopy (DM). The combined observations, including the increased antiproliferative activity of the N-methylated complex Cu-L(2Me) over that of Cu-L(2) is rationalized based on the higher lipophilicity of the former. This property would facilitate passive diffusion of Cu-L(2Me) through the cell membrane, particularly in the initial stages when the DMPC-rich outer leaflet is involved. In contrast, the benzimidazole N-H groups of Cu-L(2) may participate in hydrogen bonding with DMPE polar groups; this result is consistent with the formation of stomatocyte induced by the latter complex.

Morphological Effects and Antioxidant Capacity of Solanum crispum (Natre) In Vitro Assayed on Human Erythrocytes.

In order to gain insight into the molecular mechanism of the antioxidant properties of Solanum crispum, aqueous extracts of its leaves were assayed on human erythrocytes and molecular models of its membrane. Phenolics and alkaloids were detected by HPLC-MS. Scanning electron and defocusing microscopy showed that S. crispum changed erythrocytes from the normal shape to echinocytes. These results imply that molecules present in the aqueous extracts were located in the outer monolayer of the erythrocyte membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction showed that S. crispum preferentially interacted with DMPC bilayers. Experiments regarding its antioxidant properties showed that S. crispum neutralized the oxidative capacity of HClO on DMPE bilayers; defocusing microscopy and hemolysis assays demonstrated the protective effect of S. crispum against the oxidant effects of HClO on human erythrocytes.

Morphological Effects Induced In Vitro by Propranolol on Human Erythrocytes.

Despite the extended use and well-documented information, there are insufficient reports concerning the effects of propranolol on the structure and functions of cell membranes, particularly those of human erythrocytes. Aimed to better understand the molecular mechanisms of its interactions with cell membranes, human erythrocyte and molecular models of the red cell membrane were utilized. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of propranolol to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction. Moreover, we took advantage of the capability of differential scanning calorimetry to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from propranolol interaction with DMPC and DMPE multilamellar vesicles. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy. Results indicated that propranolol induced morphological changes to human erythrocytes and interacted in a concentration-dependent manner with phospholipid bilayer.

Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes.

Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures.

Effects of sodium metavanadate on in vitro neuroblastoma and red blood cells.

Toxicity of vanadium on cells is one of the less studied effects. This prompted us to study the structural effects induced on neuroblastoma and erythrocytes by vanadium (V) sodium metavanadate. This salt was incubated with mice cholinergic neuroblastoma cells and intact human erythrocytes. To learn whether metavanadate interacts with membrane lipid bilayers it was incubated with bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). These are phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Exposure of neuroblastoma cells to metavanadate showed significant decreases in cell viability as well as in cell number correlating with inhibition of aconitase activity. In scanning electron microscopy (SEM) and defocusing microscopy (DM) it was observed that induced on erythrocytes the formation of echinocytes. However, no effects were obtained when metavanadate was made to interact with DMPC and DMPE multibilayers and liposomes, assays performed by X-ray diffraction and differential scanning calorimetry (DSC), respectively. These results imply that the effects of metavanadate on erythrocytes are through interactions with proteins located in the membrane outer moiety, and could still involve other minor lipid components as well. Also, partly unsaturated lipids could interact differently the fully saturated chains in the model systems.

Real-time study of shape and thermal fluctuations in the echinocyte transformation of human erythrocytes using defocusing microscopy.

We present a real-time method to measure the amplitude of thermal fluctuations in biological membranes by means of a new treatment of the defocusing microscopy (DM) optical technique. This approach was also applied to study the deformation of human erythrocytes to its echinocyte structure. This was carried out by making three-dimensional shape reconstructions of the cell and measuring the thermal fluctuations of its membrane, as the cell is exposed to the anti-inflammatory drug naproxen and as it recovers its original shape, when it is subsequently cleansed of the drug. The results showed biomechanical changes in the membrane even at low naproxen concentration (0.2 mM). Also, we found that when the cell recovered its original shape, the membrane properties were different compared to the nondrugged initial erythrocyte, indicating that the drug administration-recovery process is not completely reversible.