Multivariate optimization of a green procedure for determination of emerging polycyclic aromatic nitrogen heterocycles in PM2.5 from sites with different characteristics.

Emerging polycyclic aromatic nitrogen heterocycles (PANHs) contributes significantly to the health risk associated with inhaling polluted air. However, there is a lack of analytical methods with the needed performance to their determination. This study presents the optimization and validation for the first time of a green microscale extraction procedure for the determination of twenty-one PANHs, including carbazole, indole, and quinolone classes, in particulate matter (PM2.5) samples by gas chromatography-mass spectrometry. A simplex-centroid mixture design and full factorial design (23) were employed to optimize the following extraction parameters: type and volume of solvent, sample size, extraction time, and necessity of a cleanup step. Low limits of detection and quantification (LOD < 0.97 pg m-3 and LOQ < 3.24 pg m-3, respectively) were obtained in terms of matrix-matched calibration. The accuracy and precision of the method were adequate, with recoveries in three levels between 73 to 120% and intraday and interday relative standard deviations from 2.0 to 12.9% and 7.3 to 18.9%, respectively. The green character of the method was evaluated using the Analytical Greenness (AGREE) tool, where a score of 0.69 was obtained, indicating a great green procedure. The method was applied to PM2.5 samples collected from sites with different characteristics; the concentrations ranged from 69.3 pg m-3 (2-methylcarbazole) to 11,874 pg m-3 (carbazole) for individual PANHs and from 2306 to 24,530 pg m-3 for ∑21PANHs. Principal component analysis (PCA) and hierarchical clustering enabled discrimination of the sampling sites according to the PANHs concentrations. The score plots formed two distinct groups, one with samples containing higher concentrations of PANHs, corresponding to sites with a major influence from diesel emissions, and another group with minor PANH contents, corresponding to sites impacted by emissions from urban traffic and industrial activities.

Lethal and sublethal effects of an emulsion based on Pogostemon cablin (Lamiaceae) essential oil on the coffee berry borer, Hypothenemus hampei.

The global search for eco-friendly and human-safe pesticides has intensified, and research on essential oils (EOs) has expanded due to their remarkable insecticidal activities and apparent human-safe. Despite this, most of the literature focuses on short-term and simplified efforts to understand lethal effects, with only a few comprehensive studies addressing sublethal exposures. To fill this shortcoming, we explore the lethal and sublethal effects of Pogostemon cablin (Lamiaceae) EO and an EO-based emulsion (18%) using the coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae) as a model. First, we determine the toxicity of EO and EO-based emulsion using dose-mortality curves and lethal times. Second, we subjected adult females of H. hampei to sublethal doses to assess whether they affected their behavior, reproductive output, and histological features. Our findings reveal that patchoulol (43.05%), α-Guaiene (16.06%), and α-Bulnesene (13.69%) were the main components of the EO. Furthermore, the EO and its emulsion had similar toxicity, with dose-mortality curves and lethal times overlapping 95% confidence intervals. We also observed that sublethal exposure of females of H. hampei reduces reproduction and feeding, increases walking activity, and causes histopathological changes in the midgut. This study advances the knowledge of the lethal and sublethal effects of an eco-friendly substance on insects.Responsible Editor: Giovanni Benelli.

eSPC: an online data-analysis platform for molecular biophysics.

All biological processes rely on the formation of protein-ligand, protein-peptide and protein-protein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is critical for understanding basic cellular mechanisms. Many different technologies have been designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference, among others). Evaluation of the data from binding experiments and their fitting is an essential step towards the quantification of binding affinities. Here, user-friendly online tools to analyze biophysical data from steady-state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments are presented. The modules of the data-analysis platform (https://spc.embl-hamburg.de/) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (Kd) and thermal unfolding parameters such as melting temperatures (Tm).

Microscale extraction combined with gas chromatography/mass spectrometry for the simultaneous determination of polycyclic aromatic hydrocarbons and polycyclic aromatic sulfur heterocycles in marine sediments.

This paper describes a novel method based on an ultrasound-assisted extraction microscale device (UAE-MSD) for the rapid and simultaneous determination of polycyclic aromatic hydrocarbons (PAH) and polycyclic aromatic sulfur heterocycles (PASH) in marine sediments. Solvent extraction conditions were optimized by applying a simplex-centroid mixture design. Optimum conditions were used to validate and determine the concentrations of 17 PAH and 7 PASH. The best conditions were obtained by extracting sediments with 500 µL of DCM:MeOH (65:35, v:v) over 23 min of sonication. Analytes were determined by gas chromatography/mass spectrometry in selective ion monitoring (GC-MS/SIM). Matrix effects were evaluated, and matrix-matched calibration was used for quantitation. Analytical method validation was carried out using the certified reference material NIST SRM 1941b, as well as sediment spiked with PASH at three concentration levels. Recoveries ranged between 70.0 ± 3.5% and 119 ± 9.1% for PAH and 80.6 ± 10.4% and 120 ± 10% for PASH. Linearity (R2) was ≥0.99 for all compounds. Method detection limits ranged from 8.8 to 30.2 ng g-1, while limits of quantification ranged from 29.4 to 1011 ng g-1. UAE-MSD was applied to marine sediments exposed to different anthropogenic impacts collected in Todos os Santos Bay, Brazil. PAH concentrations ranged from

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Targeting the Oncogenic TBX2 Transcription Factor With Chromomycins.

The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic processes including the promotion of proliferation and bypass of senescence. Importantly, based on compelling biological evidences, TBX2 has been considered as a potential target for new anticancer therapies. There has therefore been a substantial interest to identify molecules with TBX2-modulatory activity, but no such substance has been found to date. Here, we adopt a targeted approach based on a reverse-affinity procedure to identify the ability of chromomycins A5 (CA5) and A6 (CA6) to interact with TBX2. Briefly, a TBX2-DNA-binding domain recombinant protein was N-terminally linked to a resin, which in turn, was incubated with either CA5 or CA6. After elution, bound material was analyzed by UPLC-MS and CA5 was recovered from TBX2-loaded resins. To confirm and quantify the affinity (KD) between the compounds and TBX2, microscale thermophoresis analysis was performed. CA5 and CA6 modified the thermophoretic behavior of TBX2, with a KD in micromolar range. To begin to understand whether these compounds exerted their anti-cancer activity through binding TBX2, we next analyzed their cytotoxicity in TBX2 expressing breast carcinoma, melanoma and rhabdomyosarcoma cells. The results show that CA5 was consistently more potent than CA6 in all tested cell lines with IC50 values in the nM range. Of the cancer cell types tested, the melanoma cells were most sensitive. The knockdown of TBX2 in 501mel melanoma cells increased their sensitivity to CA5 by up to 5 times. Furthermore, inducible expression of TBX2 in 501mel cells genetically engineered to express TBX2 in the presence of doxycycline, were less sensitive to CA5 than the control cells. Together, the data presented in this study suggest that, in addition to its already recognized DNA-binding properties, CA5 may be binding the transcription factor TBX2, and it can contribute to its cytotoxic activity.

Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae.

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (ß-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Targeted Metabolite Profiling-Based Identification of Antifungal 5-n-Alkylresorcinols Occurring in Different Cereals against Fusarium oxysporum.

A rapid and convenient biochemometrics-based analysis of several cereal-derived extracts was used to identify n-alkyl(enyl)resorcinols (AR) as antifungals against Fusarium oxysporum. Total AR content and liquid chromatography/mass spectrometry (LC-MS)-based profiles were recorded for each extract, in addition to their antifungal activity, to help integrate these chemical and biological datasets by orthogonal partial least squares regression. In this study, we developed and used a micro-scale amended medium (MSAM) assay to evaluate the in vitro mycelial growth inhibition at low amounts of extracts. Triticale husk-derived extracts had the highest AR content (662.1 µg olivetol equivalent/g dry extract), exhibiting >79% inhibition at the highest doses (10.0⁻1.0 µg/µL). Correlation of the chemical and antifungal datasets using supervised metabolite profiling revealed that 5-n-nonadecanylresorcinol, 5-n-heneicosylresorcinol, and 5-n-tricosyl-resorcinol were the most active ARs occurring in cereal products from Colombia. Hence, we propose the biochemometrics-based approach as a useful tool for identifying AR-like antifungals against F. oxysporum.

Microscale direct transesterification of microbial biomass with ethanol for screening of microorganisms by its fatty acid content

Abstract We present an improved method of direct transesterification suitable for the quantitative analysis of multiple dry samples for its fatty acid content, using a minimal amount of biomass and reactants. The method features an acid-catalyzed direct alcoholysis of microgram samples of dry biomass; the rationale behind the solvent and reagent proportions chosen is discussed. The method was validated using seven microbial strains with diverse lipid content (Saccharomyces cerevisiae, Saccharomyces boulardii, Candida tropicalis, Haematococcus pluvialis, Chlorella vulgaris, Spirulina platensis and Schizochytrium limacinum), and compared with a macroscale direct transesterification method, and with gravimetric analysis of lipids extracted with solvents. The microscale method showed a conversion of 98.06 ± 0.87% of the lipids, using approximately 3 mg of dry biomass, 1mL of 0.2M H2SO4 dissolved in anhydrous ethanol (the acid is the catalyzer and ethanol the reactant)). The mixture was maintained at 70 °C for 20 h with periodic mixing, and then extracted with 2mL n-heptane and analyzed by GC-FID. The lipid content was then calculated considering dilution and sample mass. This method is effective, reliable, and technically attractive for analytical and comparative purposes.

Looking for Novel Capsid Protein Multimerization Inhibitors of Feline Immunodeficiency Virus.

Feline immunodeficiency virus (FIV) is a member of the retroviridae family of viruses. It causes acquired immunodeficiency syndrome (AIDS) in worldwide domestic and non-domestic cats and is a cause of an important veterinary issue. The genome organization of FIV and the clinical characteristics of the disease caused by FIV are similar to human immunodeficiency virus (HIV). Both viruses infect T lymphocytes, monocytes, and macrophages, with a similar replication cycle in infected cells. Thus, the infection of cats with FIV is also a useful tool for the study and development of novel drugs and vaccines against HIV. Anti-retroviral drugs studied extensively with regards to HIV infection have targeted different steps of the virus replication cycle: (1) disruption of the interaction with host cell surface receptors and co-receptors; (2) inhibition of fusion of the virus and cell membranes; (3) blocking of the reverse transcription of viral genomic RNA; (4) interruption of nuclear translocation and integration of viral DNA into host genomes; (5) prevention of viral transcript processing and nuclear export; and (6) inhibition of virion assembly and maturation. Despite the great success of anti-retroviral therapy in slowing HIV progression in humans, a similar therapy has not been thoroughly investigated for FIV infection in cats, mostly because of the little structural information available for FIV proteins. The FIV capsid protein (CA) drives the assembly of the viral particle, which is a critical step in the viral replication cycle. During this step, the CA protein oligomerizes to form a protective coat that surrounds the viral genome. In this work, we perform a large-scale screening of four hundred molecules from our in-house library using an in vitro assembly assay of p24, combined with microscale thermophoresis, to estimate binding affinity. This screening led to the discovery of around four novel hits that inhibited capsid assembly in vitro. These may provide new antiviral drugs against FIV.

MECHANICAL CHARACTERIZATION OF CELLS AND TISSUES AT THE MICRO SCALE

Cell-substrate interactions are relevant for a number of biological and clinical applications e.g. to determine the effectiveness of medical implants. Cells are natural transducers that respond to and sense signals originating in their microenvironment. One important cell signaling mechanism is known as chemo-mechanical transduction. This refers to the use of external mechanical cues to initiate internal biochemical cellular processes and vice versa. One key factor to characterize and understand these interactions is the evaluation of the mechanical forces present at the cell-substrate interface. Recent advances in the micro and nanotechnology fields have allowed the development of new tools for the measurement of cellular and tissue forces. These tools have provided a means to study extremely low cellular and subcellular forces (pN-µN) as well as detailed small-scale tissue mechanics. This paper will review some of the most significant approaches to characterize the mechanical properties of cells and tissues at the micro-scale. Material properties, device fabrication, and design issues will be discussed.

Las interacciones célula-sustrato juegan un papel fundamental en gran número de aplicaciones biológicas y clínicas. Las células son transductores naturales que sensan y responden a señales en su entorno fisiológico. Uno de los mecanismos más importantes empleados en la caracterización de interacciones celulares es la transducción químico-mecánica, la cual se basa en la implementación de señales externas que se aplican a la célula con el fin de inducir diversos procesos bioquímicos al interior de ésta y viceversa. Los avances alcanzados en el campo de la micro y nanotecnología han permitido el desarrollo de nuevas herramientas para medir fuerzas a nivel celular o incluso sub-celular (pN-µN), y dilucidar la mecánica de los tejidos en la escala micrométrica. La presente revisión literaria describe algunos de los micro-dispositivos empleados actualmente para caracterizar las propiedades mecánicas de las células y tejidos en la micro-escala.