Simple optical nanomotion method for single-bacterium viability and antibiotic response testing.

Antibiotic resistance is nowadays a major public health issue. Rapid antimicrobial susceptibility tests (AST) are one of the options to fight this deadly threat. Performing AST with single-cell sensitivity that is rapid, cheap, and widely accessible, is challenging. Recent studies demonstrated that monitoring bacterial nanomotion by using atomic force microscopy (AFM) upon exposure to antibiotics constitutes a rapid and highly efficient AST. Here, we present a nanomotion detection method based on optical microscopy for testing bacterial viability. This novel technique only requires a very basic microfluidic analysis chamber, and an optical microscope equipped with a camera or a mobile phone. No attachment of the microorganisms is needed, nor are specific bacterial stains or markers. This single-cell technique was successfully tested to obtain AST for motile, nonmotile, gram-positive, and gram-negative bacteria. The simplicity and efficiency of the method make it a game-changer in the field of rapid AST.

Oncolytic DNX-2401 Virus for Pediatric Diffuse Intrinsic Pontine Glioma.

BACKGROUND: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking. METHODS: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses. RESULTS: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×1010 (the first 4 patients) or 5×1010 (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire. CONCLUSIONS: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.).

An Optical Fiber-Based Nanomotion Sensor for Rapid Antibiotic and Antifungal Susceptibility Tests.

The emergence of antibiotic and antifungal resistant microorganisms represents nowadays a major public health issue that might push humanity into a post-antibiotic/antifungal era. One of the approaches to avoid such a catastrophe is to advance rapid antibiotic and antifungal susceptibility tests. In this study, we present a compact, optical fiber-based nanomotion sensor to achieve this goal by monitoring the dynamic nanoscale oscillation of a cantilever related to microorganism viability. High detection sensitivity was achieved that was attributed to the flexible two-photon polymerized cantilever with a spring constant of 0.3 N/m. This nanomotion device showed an excellent performance in the susceptibility tests of Escherichia coli and Candida albicans with a fast response in a time frame of minutes. As a proof-of-concept, with the simplicity of use and the potential of parallelization, our innovative sensor is anticipated to be an interesting candidate for future rapid antibiotic and antifungal susceptibility tests and other biomedical applications.

Adolescents and youths' opinions about the factors associated with cannabis use: a qualitative study based on the I-Change model.

BACKGROUND: To learn about the experiences and opinions of adolescent non-consumers and regular cannabis users about cannabis use and the factors that determine its use, using the I-Change explanatory model as a basis. METHODS: Qualitative methodology with a content analysis was used. Focus groups were conducted with adolescents who were non-regular cannabis users (those who had not tried cannabis or had only experimented with it before) and semi-structured interviews were conducted with adolescent and young adult in recovery who were in a detoxification program. A deductive analysis of the audio-recorded and transcribed interviews was performed, using the domains of the I-Change Model as a reference. RESULTS: Personal problems, social problems or family problems can lead to cannabis use. There was a lack of knowledge and low risk perception about consumption of this drug. There are other factors that influence consumption, the perception of advantages, such as the feeling of freedom and the influence of the peer group. The consumption of this substance in girls is changing, becoming more and more equal to that of boys. The family has an important role to play in preventing drug use. CONCLUSION: Knowledge of these factors is of vital importance as a prior step to the development of efficient intervention measures adjusted to the needs identified and the characteristics of the population.

Performance evaluation of Elecsys SARS-CoV-2 Antigen immunoassay for diagnostic of COVID-19.

One of the challenges for control and prevention of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the early diagnostic at the point of care. Several tests based on qualitative antigen detection have been developed; one of these is Elecsys SARS-CoV-2 Antigen immunoassay (Roche Diagnostics). In total, 523 nasopharyngeal swabs were randomly selected with the aims to evaluate sensitivity, specificity, cross-reactivity, positive and negative predictive value (PPV, NPV), and agreement of Elecsys SARS-CoV-2 Antigen immunoassay using reverse transcription-polymerase chain reaction (RT-PCR) STAT-NAT® coronavirus disease-2019 as reference test. Cross-reactivity was estimated using samples positive by RT-PCR to other respiratory viruses (influenza virus, parainfluenza virus, rhinovirus, coronavirus OC43, and HKU1). The overall sensitivity of Elecsys SARS-CoV-2 Antigen was 89.72% (288/321); specificity was 90.59% (183/202); and cross-reactivity to other respiratory viruses were not detected. Elecsys SARS-CoV-2 Antigen immunoassay showed a high sensitivity in samples with cycle threshold value <30, which ranged from 92.81% to 95.40%, independently of symptoms. PPV and NPV were 93.81% and 84.72%, respectively. The κ coefficient was 0.79 (95% confidence interval: 0.73-0.84), showing substantial agreement between both tests. The results suggest Elecsys SARS-CoV-2 Antigen immunoassay could be used as an alternative to RT-PCR testing, or in complement with it, to identify infectious individuals and reduce SARS-CoV-2 transmission.

Rhodococcus as Biofactories for Microbial Oil Production.

Bacteria belonging to the Rhodococcus genus are frequent components of microbial communities in diverse natural environments. Some rhodococcal species exhibit the outstanding ability to produce significant amounts of triacylglycerols (TAG) (>20% of cellular dry weight) in the presence of an excess of the carbon source and limitation of the nitrogen source. For this reason, they can be considered as oleaginous microorganisms. As occurs as well in eukaryotic single-cell oil (SCO) producers, these bacteria possess specific physiological properties and molecular mechanisms that differentiate them from other microorganisms unable to synthesize TAG. In this review, we summarized several of the well-characterized molecular mechanisms that enable oleaginous rhodococci to produce significant amounts of SCO. Furthermore, we highlighted the ability of these microorganisms to degrade a wide range of carbon sources coupled to lipogenesis. The qualitative and quantitative oil production by rhodococci from diverse industrial wastes has also been included. Finally, we summarized the genetic and metabolic approaches applied to oleaginous rhodococci to improve SCO production. This review provides a comprehensive and integrating vision on the potential of oleaginous rhodococci to be considered as microbial biofactories for microbial oil production.

Nano-Motion Analysis for Rapid and Label Free Assessing of Cancer Cell Sensitivity to Chemotherapeutics.

Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method's applicability.

Interferon gamma, TGF-ß1 and RANTES expression in upper airway samples from SARS-CoV-2 infected patients.

Upper respiratory tract is the primary site of SARS-CoV-2 replication. Releasing of pro and anti-inflammatory mediators plays an important role in the immunopathogenesis of Coronavirus Disease 2019 (COVID-19). The aim of this study was to evaluate the early inflammatory response in upper airway by measuring of IFN-γ, TGF-ß1 and RANTES at mRNA level. Forty five SARS-CoV-2 infected patients were enrolled, whose were divided in two groups: asymptomatic and symptomatic. Twenty healthy persons, SARS-CoV-2 negative were included as controls. Higher IFN-γ expression was detected in SARS-CoV-2 infected patients in comparison with controls (p = 0.0393). IFN-γ expression was increased in symptomatic patients (p = 0.0405). TGF-ß1 and RANTES expressions were lower in SARS-CoV-2 infected patients than controls (p < 0.0001; p = 0.0011, respectively). A significant correlation between IFN-γ and TGF-ß1 was observed in SARS-CoV-2 asymptomatic patients (r = +0.61, p = 0.0014). The findings suggest that imbalance between IFN-γ and TGF-ß1 expression could be an impact in clinical expression of SARS-CoV-2 infection.

A perspective view on the nanomotion detection of living organisms and its features.

The insurgence of newly arising, rapidly developing health threats, such as drug-resistant bacteria and cancers, is one of the most urgent public-health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro-fabricated cantilever and by monitoring its displacements with an atomic force microscope-based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life-threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.

ICOS Costimulation at the Tumor Site in Combination with CTLA-4 Blockade Therapy Elicits Strong Tumor Immunity.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) blockade therapy is able to induce long-lasting antitumor responses in a fraction of cancer patients. Nonetheless, there is still room for improvement in the quest for new therapeutic combinations. ICOS costimulation has been underscored as a possible target to include with CTLA-4 blocking treatment. Herein, we describe an ICOS agonistic aptamer that potentiates T cell activation and induces stronger antitumor responses when locally injected at the tumor site in combination with anti-CTLA-4 antibody in different tumor models. Furthermore, ICOS agonistic aptamer was engineered as a bi-specific tumor-targeting aptamer to reach any disseminated tumor lesions after systemic injection. Treatment with the bi-specific aptamer in combination with CTLA-4 blockade showed strong antitumor immunity, even in a melanoma tumor model where CTLA-4 treatment alone did not display any significant therapeutic benefit. Thus, this work provides strong support for the development of combinatorial therapies involving anti-CTLA-4 blockade and ICOS agonist tumor-targeting agents.

A structure-based approach towards the identification of novel antichagasic compounds: Trypanosoma cruzi carbonic anhydrase inhibitors.

Trypanosoma cruzi carbonic anhydrase (TcCA) has recently emerged as an interesting target for the design of new compounds to treat Chagas disease. In this study we report the results of a structure-based virtual screening campaign to identify novel and selective TcCA inhibitors. The combination of properly validated computational methodologies such as comparative modelling, molecular dynamics and docking simulations allowed us to find high potency hits, with KI values in the nanomolar range. The compounds also showed trypanocidal effects against T. cruzi epimastigotes and trypomastigotes. All the candidates are selective for inhibiting TcCA over the human isoform CA II, which is encouraging in terms of possible therapeutic safety and efficacy.

Exploratory study on the presence of bisphenol A and bis(2-ethylhexyl) phthalate in the Santa Catarina River in Monterrey, N.L., Mexico.

The current problem related to the supply of drinking water for the metropolitan area of Monterrey, which comprises of five million inhabitants, and the important role played by the El Cuchillo Dam, found downstream of the Santa Catarina River basin as a surface source of drinking water for the city, renders this river an interesting site for assessment. This work evaluates the degree to which the surface water and the subsoil of the river are affected by emerging organic pollutants due to their existence, even at low concentrations, representing a toxic risk enhanced by the absence of stricter standards for regulating these substances. Based on fieldwork, three discharge points that could affect the quality of the surface water were selected: two points on the river stream, and three groundwater wells. Gas chromatography results showed the presence of BisPhenol A (BPA) and bis(2-EthylHexyl) Phthalate (DEHP) in both the surface water and subsoil at different seasons of the year. The highest concentration levels in the samples taken from both types of water were 0.9 and 60 µg L-1 for BPA and DEHP, respectively. Results of this research did not reveal the level at which the aquifer is affected by these substances.

Insights into the Metabolism of Oleaginous Rhodococcus spp.

Some species belonging to the Rhodococcus genus, such as Rhodococcus opacus, R. jostii, and R. wratislaviensis, are known to be oleaginous microorganisms, since they are able to accumulate triacylglycerols (TAG) at more than 20% of their weight (dry weight). Oleaginous rhodococci are promising microbial cell factories for the production of lipids to be used as fuels and chemicals. Cells could be engineered to create strains capable of producing high quantities of oils from industrial wastes and a variety of high-value lipids. The comprehensive understanding of carbon metabolism and its regulation will contribute to the design of a reliable process for bacterial oil production. Bacterial oleagenicity requires an integral configuration of metabolism and regulatory processes rather than the sole existence of an efficient lipid biosynthesis pathway. In recent years, several studies have been focused on basic aspects of TAG biosynthesis and accumulation using R. opacus PD630 and R. jostii RHA1 strains as models of oleaginous bacteria. The combination of results obtained in these studies allows us to propose a metabolic landscape for oleaginous rhodococci. In this context, this article provides a comprehensive and integrative view of different metabolic and regulatory attributes and innovations that explain the extraordinary ability of these bacteria to synthesize and accumulate TAG. We hope that the accessibility to such information in an integrated way will help researchers to rationally select new targets for further studies in the field.

Nanomotion Detection Method for Testing Antibiotic Resistance and Susceptibility of Slow-Growing Bacteria.

Infectious diseases are caused by pathogenic microorganisms and are often severe. Time to fully characterize an infectious agent after sampling and to find the right antibiotic and dose are important factors in the overall success of a patient's treatment. Previous results suggest that a nanomotion detection method could be a convenient tool for reducing antibiotic sensitivity characterization time to several hours. Here, the application of the method for slow-growing bacteria is demonstrated, taking Bordetella pertussis strains as a model. A low-cost nanomotion device is able to characterize B. pertussis sensitivity against specific antibiotics within several hours, instead of days, as it is still the case with conventional growth-based techniques. It can discriminate between resistant and susceptible B. pertussis strains, based on the changes of the sensor's signal before and after the antibiotic addition. Furthermore, minimum inhibitory and bactericidal concentrations of clinically applied antibiotics are compared using both techniques and the suggested similarity is discussed.

Stereoselective Synthesis of Highly Substituted Tetrahydropyrans through an Evans Aldol-Prins Strategy.

A direct and general method for the synthesis of naturally occurring 2,3,4,5,6-pentasubstituted tetrahydropyrans has been developed, employing ß,γ-unsaturated N-acyl oxazolidin-2-ones as key starting materials. The combination of the Evans aldol addition and the Prins cyclization allowed the diastereoselective and efficient generation of the desired oxacycles in two fashions: a one-pot Evans aldol-Prins protocol, in which five new σ bonds and five contiguous stereocenters were straightforwardly generated, and a two-step version, which additionally permitted the isolation of ß,γ-unsaturated alcohol precursors bearing an N-acyl oxazolidin-2-one in the α position. From these alcohols were also obtained halogenated pentasubstituted tetrahydropyrans as well as 2,3,4,5-tetrasubstituted tetrahydrofurans, shedding light on the mechanism of the process. Computational studies were consistent with the experimental findings, and this innovative Evans aldol-Prins strategy was performed for the preparation of a battery of more than 30 densely substituted tetrahydropyrans, unprecedentedly fused to a 1,3-oxazinane-2,4-dione ring, both in a racemic fashion and in an enantiomeric fashion. These novel molecules were successfully submitted to several transformations to permit simple access to a variety of differently functionalized tetrahydropyrans. Most of these unique molecules were evaluated for their antimicrobial activity against Gram-positive and Gram-negative bacteria and the yeast Candida albicans, and some structure-activity relationships were established.

Searching for New Leads To Treat Epilepsy: Target-Based Virtual Screening for the Discovery of Anticonvulsant Agents.

The purpose of this investigation is to contribute to the development of new anticonvulsant drugs to treat patients with refractory epilepsy. We applied a virtual screening protocol that involved the search into molecular databases of new compounds and known drugs to find small molecules that interact with the open conformation of the Nav1.2 pore. As the 3D structure of human Nav1.2 is not available, we first assembled 3D models of the target, in closed and open conformations. After the virtual screening, the resulting candidates were submitted to a second virtual filter, to find compounds with better chances of being effective for the treatment of P-glycoprotein (P-gp) mediated resistant epilepsy. Again, we built a model of the 3D structure of human P-gp, and we validated the docking methodology selected to propose the best candidates, which were experimentally tested on Nav1.2 channels by patch clamp techniques and in vivo by the maximal electroshock seizure (MES) test. Patch clamp studies allowed us to corroborate that our candidates, drugs used for the treatment of other pathologies like Ciprofloxacin, Losartan, and Valsartan, exhibit inhibitory effects on Nav1.2 channel activity. Additionally, a compound synthesized in our lab, N, N'-diphenethylsulfamide, interacts with the target and also triggers significant Na1.2 channel inhibitory action. Finally, in vivo studies confirmed the anticonvulsant action of Valsartan, Ciprofloxacin, and N, N'-diphenethylsulfamide.

Rhodococcus bacteria as a promising source of oils from olive mill wastes.

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2-2.7 g/l) and lipids (77-83% of CDW, 1.8-2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1-1.6 g/l of cell biomass and 7.1-14.0% of CDW, 0.1-0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.

The effect of treatment for sleep apnoea on determinants of blood pressure control.

Our aim was to assess the effect of continuous positive airway pressure (CPAP) on the nocturnal evolution of peripheral chemosensitivity, renin-angiotensin-aldosterone system activity, sympathetic tone and endothelial biomarkers in obstructive sleep apnoea (OSA) patients with isolated nocturnal hypertension (INH) or day-night sustained hypertension (D-NSH).In a crossover randomised trial, 32 OSA patients newly diagnosed with hypertension and without antihypertensive treatment were randomly assigned to 12 weeks of CPAP or sham CPAP. Peripheral chemosensitivity was evaluated before and after sleep using the hypoxic withdrawal test (%ΔVI).At baseline, D-NSH patients showed higher %ΔVI before sleep and higher levels of aldosterone and diurnal catecholamines. CPAP only reduced the nocturnal increase of %ΔVI in INH patients (6.9%, 95% CI 1.0-12.8%; p=0.026). CPAP-induced change from baseline in %ΔVI after sleep was 7.5% (95% CI 2.6-12.2%, p=0.005) in the INH group and 5.7% (95% CI 2.2-9.3%, p=0.004) in the D-NSH group. In contrast, %ΔVI before sleep only decreased with CPAP in the D-NSH patients (3.0%, 95% CI 0.5-5.6%; p=0.023).In conclusion, CPAP reduces the nocturnal increase of peripheral chemosensitivity experienced by INH patients and corrects the high daytime sensitivity of patients with D-NSH. Differences in response to CPAP between these patients can help better understand the mechanisms of perpetuation of hypertension in sleep apnoea.

Role of TGF-ß in metastatic colon cancer: it is finally time for targeted therapy.

Colorectal cancer (CRC) is one of the most frequent tumor types in Western countries. Approximately 20 % of patients show metastasis at the time of diagnosis, with the liver being one of the most affected organs. Transforming growth factor-beta (TGF-ß) plays a regulatory role not only in the physiology of the normal colon but also in the development of CRC and its metastatic process. In this review, we analyze the molecular mechanisms leading to TGF-ß dysregulation in tumor and stroma cells and the modification of the microenvironment that fosters CRC metastasis. Recent genomic studies have identified a CRC subtype with a mesenchymal and aggressive phenotype having TGF-ß as a hub gene of this signature. Consistent with these findings, the inhibition of TGF-ß signaling has been shown to impair experimental CRC metastasis to the liver. Based on these and other results conducted in various tumor types, the pharmaceutical industry has developed a variety of strategies to target TGF-ß. We provide up-to-date information of these therapies, which are currently in preclinical or clinical trials.