Biologically synthesized silver nanoparticles as potent antibacterial effective against multidrug-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most worrisome infectious bacteria due to its intrinsic and acquired resistance against several antibiotics and the recalcitrance of its infections; hence, the development of novel antimicrobials effective against multidrug-resistant P. aeruginosa is mandatory. In this work, silver nanoparticles obtained by green synthesis using a leaf extract and fungi were tested against a battery of clinical strains from cystic fibrosis, pneumonia and burnt patients, some of them with multidrug resistance. Both nanoparticles showed a potent antibacterial effect, causing severe damage to the cell wall, membrane and DNA, and inducing the production of reactive oxygen species. Moreover, the nanoparticles derived from fungi showed synergistic antibacterial effects with the antibiotics meropenem and levofloxacin for some clinical strains and both kinds of nanoparticles were nontoxic for larvae of the moth Galleria mellonella, encouraging further research for their implementation in the treatment of P. aeruginosa infections.

Antibacterial properties of phenothiazine derivatives against multidrug-resistant Acinetobacter baumannii strains.

AIM: As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial compounds is necessary. One strategy is to generate treatment alternatives by drug repurposing. METHODS AND RESULTS: In this work, phenotypic microarrays were used for the screening of miscellaneous compounds against the growth and biofilm formation of Acinetobacter baumannii, an important emergent multidrug-resistant opportunistic pathogen. The results showed that the phenothiazine derivatives, such as promethazine, trifluoperazine, thioridazine, and chlorpromazine, inhibited the growth of antibiotic-sensitive and multidrug-resistant strains (showing minimal inhibitory concentrations ranging from 0·05 to 0·6 g l-1 and minimal bactericidal concentrations ranging from 0·1 to 2·5 g l-1 ). All phenothiazine derivatives were active against biofilm cells (with minimal biofilm eradication concentrations ranging from 0·5 to >3 g l-1 ). Chlorpromazine promoted reactive oxigen species (ROS) production, and cell membrane and DNA damage. Chlorpromazine showed synergy with antibiotics such as ceftazidime, meropenem, and colistin and was an effective treatment for experimentally infected Galleria mellonella when combined with ceftazidime. CONCLUSIONS: It was demonstrated that phenothiazine derivatives, especially chlorpromazine, are drugs with attractive antibacterial properties against nosocomial MDR strains of A. baumannii, by generating ROS and cell membrane and DNA damage. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study indicates that repurposing phenothiazine derivatives for treating recalcitrant infections by A. baumannii could be promising.

Bacteria cultured from the gut of Meccus pallidipennis (Hemiptera: Reduviidae), a triatomine species endemic to Mexico.

The study of intestinal microbiota in vector insects like triatomines is paramount in parasitology because many parasitic species inhabit the vector's gut. Although knowledge on the gut microbiota in various vectors of the parasitic flagellate Trypanosoma cruzi has grown, research efforts have focused on South American triatomines. This study reports the isolation of bacterial microbiota in the anterior and posterior gut of Meccus pallidipennis (a triatomine species endemic to Mexico) by culture, as well as its identification by phenotypic and biochemical tests and its quantification by counting colony-forming units. The study was performed on fifth-instar nymph and adult specimens of M. pallidipennis, either laboratory-bred or collected in the field and either infected or not with T. cruzi. Overall, 17 bacterial species were identified, with the genera Bacillus and Staphylococcus being the most prevalent regardless of the origin of the insects. No differences were observed in the number of bacterial species in the gut of laboratory-bred and field-collected insects, neither with respect to life stage or infection status. In general, the Shannon-Weaver diversity index was higher in non-infected insects than in infected ones. Further studies using non-culture methods are required to determine whether bacterial species diversity is modified by laboratory breeding.

Assessing virus infection probability in an office setting using stochastic simulation.

Viral infections are an occupational health concern for office workers and employers. The objectives of this study were to estimate rotavirus, rhinovirus, and influenza A virus infection risks in an office setting and quantify infection risk reductions for two hygiene interventions. In the first intervention, research staff used an ethanol-based spray disinfectant to clean high-touch non-porous surfaces in a shared office space. The second intervention included surface disinfection and also provided workers with alcohol-based hand sanitizer gel and hand sanitizing wipes to promote hand hygiene. Expected changes in surface concentrations due to these interventions were calculated. Human exposure and dose were simulated using a validated, steady-state model incorporated into a Monte Carlo framework. Stochastic inputs representing human behavior, pathogen transfer efficiency, and pathogen fate were utilized, in addition to a mixed distribution that accounted for surface concentrations above and below a limit of detection. Dose-response curves were then used to estimate infection risk. Estimates of percent risk reduction using mean values from baseline and surface disinfection simulations for rotavirus, rhinovirus, and influenza A infection risk were 14.5%, 16.1%, and 32.9%, respectively. For interventions with both surface disinfection and the promotion of personal hand hygiene, reductions based on mean values of infection risk were 58.9%, 60.8%, and 87.8%, respectively. This study demonstrated that surface disinfection and the use of personal hand hygiene products can help decrease virus infection risk in communal offices. Additionally, a variance-based sensitivity analysis revealed a greater relative importance of surface concentrations, assumptions of relevant exposure routes, and inputs representing human behavior in estimating risk reductions.

Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments.

Pathogens that infect the gastrointestinal and respiratory tracts are subjected to intense pressure due to the environmental conditions of the surroundings. This pressure has led to the development of mechanisms of bacterial tolerance or persistence which enable microorganisms to survive in these locations. In this review, we analyze the general stress response (RpoS mediated), reactive oxygen species (ROS) tolerance, energy metabolism, drug efflux pumps, SOS response, quorum sensing (QS) bacterial communication, (p)ppGpp signaling, and toxin-antitoxin (TA) systems of pathogens, such as Escherichia coli, Salmonella spp., Vibrio spp., Helicobacter spp., Campylobacter jejuni, Enterococcus spp., Shigella spp., Yersinia spp., and Clostridium difficile, all of which inhabit the gastrointestinal tract. The following respiratory tract pathogens are also considered: Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cenocepacia, and Mycobacterium tuberculosis Knowledge of the molecular mechanisms regulating the bacterial tolerance and persistence phenotypes is essential in the fight against multiresistant pathogens, as it will enable the identification of new targets for developing innovative anti-infective treatments.

Correction to: Ouabain Accelerates Collective Cell Migration Through a cSrc and ERK1/2 Sensitive Metalloproteinase Activity.

The original version of the article unfortunately contained an error in the author group. Dr. Isabel Larré was not submitted and published in the original version.

Ouabain Accelerates Collective Cell Migration Through a cSrc and ERK1/2 Sensitive Metalloproteinase Activity.

Studies made in the Madin-Darby canine kidney (MDCK) epithelial cell line showed that ouabain regulates cell adhesion and cell-adhesion-related biological processes, such as migration. Here, we demonstrated that 10 nM ouabain accelerates collective cell migration and heals wounds in cultured MDCK cell monolayers. Ouabain-induced acceleration of cell migration depends on activation of the cSrc-ERK1/2 signaling cascade, as it was inhibited by the kinase inhibitors PP2 and PD98059. Activation of the cSrc-ERK1/2 signaling cascade increased expression and activation of the extracellular matrix metalloproteinase-2 (MMP-2). Inhibition of MMP activity using the generic inhibitor GM6001 or the potent iMMP-2 inhibitor prevented the accelerative effect of ouabain. Likewise, Focal Adhesion Kinase (FAK) inhibition with the transfection of dominant negative peptide FRNK impaired the effect of ouabain. These results suggest that ouabain binding to the Na+,K+-ATPase accelerates collective migration of MDCK cells through activation of the cSrc-ERK1/2-FAK signaling cascade and promoting secretion and MMP activity.

Dynamical age differences among coeval star clusters as revealed by blue stragglers.

Globular star clusters that formed at the same cosmic time may have evolved rather differently from the dynamical point of view (because that evolution depends on the internal environment) through a variety of processes that tend progressively to segregate stars more massive than the average towards the cluster centre. Therefore clusters with the same chronological age may have reached quite different stages of their dynamical history (that is, they may have different 'dynamical ages'). Blue straggler stars have masses greater than those at the turn-off point on the main sequence and therefore must be the result of either a collision or a mass-transfer event. Because they are among the most massive and luminous objects in old clusters, they can be used as test particles with which to probe dynamical evolution. Here we report that globular clusters can be grouped into a few distinct families on the basis of the radial distribution of blue stragglers. This grouping corresponds well to an effective ranking of the dynamical stage reached by stellar systems, thereby permitting a direct measure of the cluster dynamical age purely from observed properties.

Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.

Inhibition of quorum-sensing-dependent virulence factors and biofilm formation of clinical and environmental Pseudomonas aeruginosa strains by ZnO nanoparticles.

UNLABELLED: Quorum quenching decreases Pseudomonas aeruginosa virulence factors and biofilm formation, alleviating infections in animal models. Nevertheless, it is usually performed in laboratory strains such as PAO1 and PA14, and studies involving clinical or environmental isolates are scarce. In this work, the effects of ZnO nanoparticles, a potent quorum and virulence quencher for the PAO1 strain, were tested in six clinical strains from cystic fibrosis patients, a furanone C-30 resistant clinical strain from urine, two PA14 gallium resistant mutants, a PA14 C-30 resistant mutant and four environmental isolates. ZnO nanoparticles effectively decreased elastase, pyocyanin, and biofilm formation for most of the strains; regardless their origin or their resistance against the canonical quorum quencher C-30 or the novel antimicrobial gallium. The data indicate ZnO nanoparticles may have a broad spectrum for the quorum quenching of relevant strains and that may be an alternative to treat Ps. aeruginosa recalcitrant infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence inhibition by quorum quenchers in Pseudomonas aeruginosa is usually tested in laboratory strains and studies of their effects in relevant clinical and environmental strains are scarce. This study is significant as the effects of ZnO nanoparticles in QS-dependent virulence factor production were tested in six clinical strains from cystic fibrosis patients, a C-30 resistant clinical strain from urine, two PA14 gallium resistant mutants, a PA14 C-30 resistant mutant, and four environmental isolates. ZnO nanoparticles decreased elastase, pyocyanin, and biofilms for most of the strains; indicating they have broad spectrum and may be an alternative to treat Ps. aeruginosa infections.

Outcomes of splenectomy in patients with common variable immunodeficiency (CVID): a survey of 45 patients.

Splenectomy has been used in patients with common variable immunodeficiency disorders (CVID), mainly in the context of refractory autoimmune cytopenia and suspected lymphoma, but there are understandable concerns about the potential of compounding an existing immunodeficiency. With increasing use of rituximab as an alternative treatment for refractory autoimmune cytopenia, the role of splenectomy in CVID needs to be re-examined. This retrospective study provides the largest cohesive data set to date describing the outcome of splenectomy in 45 CVID patients in the past 40 years. Splenectomy proved to be an effective long-term treatment in 75% of CVID patients with autoimmune cytopenia, even in some cases when rituximab had failed. Splenectomy does not worsen mortality in CVID and adequate immunoglobulin replacement therapy appears to play a protective role in overwhelming post-splenectomy infections. Future trials comparing the effectiveness and safety of rituximab and splenectomy are needed to provide clearer guidance on the second-line management of autoimmune cytopenia in CVID.

Computational NMR coupling constants: shifting and scaling factors for evaluating 1JCH.

Optimized shifting and/or scaling factors for calculating one-bond carbon-hydrogen spin-spin coupling constants have been determined for 35 combinations of representative functionals (PBE, B3LYP, B3P86, B97-2 and M06-L) and basis sets (TZVP, HIII-su3, EPR-III, aug-cc-pVTZ-J, ccJ-pVDZ, ccJ-pVTZ, ccJ-pVQZ, pcJ-2 and pcJ-3) using 68 organic molecular systems with 88 (1)JCH couplings including different types of hybridized carbon atoms. Density functional theory assessment for the determination of (1)JCH coupling constants is examined, comparing the computed and experimental values. The use of shifting constants for obtaining the calculated coupling improves substantially the results, and most models become qualitatively similar. Thus, for the whole set of couplings and for all approaches excluding those using the M06 functional, the root-mean-square deviations lie between 4.7 and 16.4 Hz and are reduced to 4-6.5 Hz when shifting constants are considered. Alternatively, when a specific rovibrational contribution of 5 Hz is subtracted from the experimental values, good results are obtained with PBE, B3P86 and B97-2 functionals in combination with HIII-su3, aug-cc-pVTZ-J and pcJ-2 basis sets.

Anti-SpCas9 IgY Polyclonal Antibodies Production for CRISPR Research Use.

The CRISPR/Cas adaptative immune system has been harnessed as an RNA-guided, programmable genome editing tool, allowing for diverse biotechnological applications. The implementation of the system relies on the ability to detect the Cas9 protein in biological samples. This task is facilitated by employing antibodies, which exhibit several advantageous features and applications in the context of tropical neglected diseases. This study reports a one-month immunization scheme with the Cas9 protein fromStreptococcus pyogenes to produce IgY polyclonal antibodies (anti-SpCas9), which can be rapidly isolated by combining yolk de-lipidation with protein salting out using pectin and ammonium sulfate, respectively. Immunodetection assays indicate that the antibodies are highly sensitive, specific, and useful for detecting the SpCas9 protein in promastigotes ofLeishmania braziliensisexpressing exogenous SpCas9. Thus, the simple method for producing anti-SpCas9 IgY antibodies will accelerate CRISPR/Cas-based studies in Leishmania spp. This approach serves as a valuable research tool in this parasite model and holds the potential for wide application in various other biological samples, promoting the implementation of the system. In fact, a bioinformatics approach based on the identification of antigenic determinants in the SpCas9 protein suggests the possibility of using the anti-SpCas9 IgY antibodies in applications such as Prime and Base editing.

The Na+-K+-ATPase as self-adhesion molecule and hormone receptor.

Thanks to the homeostasis of the internal milieu, metazoan cells can enormously simplify their housekeeping efforts and engage instead in differentiation and multiple forms of organization (tissues, organs, systems) that enable them to produce an astonishing diversity of mammals. The stability of the internal milieu despite drastic variations of the external environment (air, fresh or seawater, gastrointestinal fluids, glomerular filtrate, bile) is due to transporting epithelia that can adjust their specific permeability to H(2)O, H(+), Na(+), K(+), Ca(2+), and Cl(-) over several orders of magnitude and exchange substances with the outer milieu with exquisite precision. This exchange is due to the polarized expression of membrane proteins, among them Na(+)-K(+)-ATPase, an oligomeric enzyme that uses chemical energy from ATP molecules to translocate ions across the plasma membrane of epithelial cells. Na(+)-K(+)-ATPase presents two types of asymmetries: the arrangement of its subunits, and its expression in one pole of the epithelial cell ("polarity"). In most epithelia, polarity consists of the expression of Na(+)-K(+)-ATPase towards the intercellular space and arises in part from the interaction of the extracellular segment of the ß-subunit with another ß-subunit present in a Na(+)-K(+)-ATPase molecule expressed by a neighboring cell. In addition to enabling the Na(+)-K(+)-ATPase to transport ions and water vectorially, this position exposes its receptors to ouabain and analogous cardiotonic steroids, which are present in the internal milieu because these were secreted by endocrine cells.

Incidence of anaphylaxis in the city of Alcorcon (Spain): a population-based study.

BACKGROUND: Relatively few studies have examined the incidence of anaphylaxis in the general population. OBJECTIVE: To report the incidence of anaphylaxis among the general population of the city of Alcorcon, Spain, using various public health care databases. METHODS: Episodes of anaphylaxis were recovered using validated alphanumeric strings in different fields of electronic clinical records used in the different public health settings in the city of Alcorcon (primary care, Emergency Department, hospitalized patients and Allergy Outpatient Clinic). Patients with anaphylaxis were tracked across the different clinical settings in Alcorcon. RESULTS: The incidence of anaphylaxis in Alcorcon was 103.37 episodes per 100 000 person-years (total standardized incidence rate of 112.2). There was a peak of 313.58 episodes in the 0-4 years age group and a different distribution of incidence rates (although non-significant) among different age groups between male patients and female patients. In most age groups, incidence tended to be higher for female patients aged over 10 years. Patients were attended at two or more levels in 76.78% of episodes, and a new evaluation was often made at a primary care centre (71.43%), Allergy Outpatient Clinic (75.6%), or both after the episode (58.93%). CONCLUSION AND CLINICAL RELEVANCE: This study revealed a higher rate of anaphylaxis than that in previous studies, although this incidence rate is probably lower than the real incidence rate. Studies exploring potential methodological, genetic and environmental factors accounting for these higher rates of anaphylaxis are required.

Bond Fukui functions as descriptor of the electron density reorganization in π conjugated systems.

The bond Fukui function is introduced and tested as a new reactivity index capable of predicting the evolution of bond breaking and formation processes during an organic reaction involving π conjugated systems. As an illustration, we examine many cases where substituted ethylenes and dienes may respond to different reagents to yield cycloaddition, Michael addition, and other reactions at double bonds.

Solvent effects in ionic liquids: empirical linear energy-density relationships.

Multiparameter linear energy-density relationships to model solvent effects in room temperature ionic liquids (RTILs) are introduced and tested. The model incorporates two solvent dependent and two specific solute-solvent parameters represented by a set of electronic indexes derived from the conceptual density functional theory. The specific solute-solvent interactions are described in terms of the electronic chemical potential for proton migration between the anion or cation and the transition state structure of a specific reaction. These indexes provide a quantitative estimation of the hydrogen bond (HB) acceptor basicity and the hydrogen bond donor acidity of the ionic solvent, respectively. A sound quantitative scale of HB strength is thereby obtained. The solvent dependent contributions are described by the global electrophilicity of the cation and nucleophilicity of the anion forming the ionic liquid. The model is illustrated for the kinetics of cycloaddition of cyclopentadiene towards acrolein. In general, cation HB acidity outweighs the remaining parameters for this reaction.

Tocilizumab therapy reduced intensive care unit admissions and/or mortality in COVID-19 patients.

INTRODUCTION: No therapy has yet proven effective in COVID-19. Tocilizumab (TCZ) in patients with severe COVID-19 could be an effective treatment. METHOD: We conducted a retrospective case-control study in the Nord Franche-Comté Hospital, France. We compared the outcome of patients treated with TCZ and patients without TCZ considering a combined primary endpoint: death and/or ICU admissions. RESULTS: Patients with TCZ (n=20) had a higher Charlson comorbidity index (5.3 [±2.4] vs 3.4 [±2.6], P=0.014), presented with more severe forms (higher level of oxygen therapy at 13L/min vs 6L/min, P<0.001), and had poorer biological findings (severe lymphopenia: 676/mm3 vs 914/mm3, P=0.037 and higher CRP level: 158mg/L vs 105mg/L, P=0.017) than patients without TCZ (n=25). However, death and/or ICU admissions were higher in patients without TCZ than in the TCZ group (72% vs 25%, P=0.002). CONCLUSION: Despite the small sample size and retrospective nature of the work, this result strongly suggests that TCZ may reduce the number of ICU admissions and/or mortality in patients with severe SARS-CoV-2 pneumonia.

Control of tight junctional sealing: roles of epidermal growth factor and prostaglandin E2.

Epithelia can adjust the permeability of the paracellular permeation route by regulating the degree of sealing of the tight junction. This is reflected by a transepithelial electrical resistance (TER) ranging from a few tenths to several thousand ohms times square centimeters, depending on the difference in composition between the fluid in the lumen and the interstitial fluid. Although teleologically sound, such correlation requires a physiological explanation. We have previously shown that urine extracts from different animal species increase the TER of Madin-Darby canine kidney (MDCK) monolayers and that these effects are mediated by epidermal growth factor (EGF) contained in the flowing intratubular fluid that eventually reaches the urine. This increase in TER is accompanied by an enhanced expression of claudin-4 (cln-4) and a decrement of cln-2. These changes are transient, peaking at approximately 16 h and returning to control values in approximately 24 h. In the present work we investigated how EGF provokes this transient response, and we found that the activation of extracellular-regulated kinases 1/2 (ERK1/2) by EGF is essential to increase TER and cln-4 content, but it does not appear to participate in cln-2 downregulation. On the other hand, prostaglandin synthesis, stimulated by EGF, functions as a negative feedback, turning off the signal initiated by EGF. Thus, PGE(2) blocks ERK1/2 by a mechanism that involves the G alpha(s) protein, adenylyl cyclase as well as protein kinase A in MDCK cells. In summary, the permeability of a given segment of the nephron depends on the expression of different claudin types, which may be modulated by EGF and prostaglandins.