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Phenibut, a GABAB agonist structurally similar to baclofen, is not approved for medical use in the USA but is available through internet suppliers for recreational use. Calls to poison control centers for phenibut have increased over the last 5 years, and there are many case reports of severe acute intoxications and withdrawals requiring hospitalization. This case report describes the autopsy and toxicology findings of a 26-year-old male found dead at home with phenibut containers on the scene. Autopsy findings and routine toxicology testing were generally unremarkable. Scene findings prompted the validation of a qualitative liquid chromatography--tandem mass spectrometry method that confirmed the presence of phenibut in blood and urine.

N-Hydroxysuccinimide esters of small molecules are widely used to modify biomolecules such as antibodies or proteins. Primary amine groups preferably react with the ester to form covalent amide bonds. Currently, protocols strongly recommend replacing the buffer reagent tris(hydroxymethyl)aminomethane, and it has even been proposed as a stop reagent. Here, we show that TRIS indeed does not interfere with biotinylation of biomolecules with NHS chemistry.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.

A toxicological investigation was conducted for alcohol-to-jet (ATJ) fuels intended as a 50:50 blend with petroleum-derived fuel Jet Propulsion (JP)-8. The ATJ synthetic paraffinic kerosene (SPK) fuel was produced by Gevo (Englewood CO) and derived either from biomass (bio) or non-biomass sources. All toxicity tests were performed with one or both ATJ fuels following addition of a standard additive package required for JP-8. The primary fuel, Gevo (bio) ATJ SPK produced from biomass-derived iso-butanol, exhibited the same dermal irritation potential in rabbits as JP-8; the non-biomass-derived fuel was less irritating. The Gevo (bio) fuel was non-clastogenic in micronucleus testing with rats and neither version was mutagenic in the bacterial reverse mutation assay. A 90-day study was performed with Gevo (bio) ATJ SPK by exposing male and female Fischer 344 rats to target concentrations of 0, 200, 700 or 2000 mg/m3 of fuel, 6 hr per day, 5 days a week for 69 exposure days and included neurobehavioral assays and reproductive health evaluations in the study design. Results were negative or limited to irritant effects in the respiratory system due to exposure to a vapor and aerosol mixture in the 2000 mg/m3 exposure group. Occupational exposure limits for JP-8 were proposed for these ATJ fuels since these fuels display similar or somewhat lower toxicity than JP-8. As both versions of the Gevo ATJ jet fuel were similar, handling of either fuel alone or in a blend with petroleum-derived JP-8 appears unlikely to increase human health risks for workers.

A new Pseudomonas putida strain (AQ8) was isolated from a decommissioned oil refinery's soil in Italy and characterized for its ability to degrade BTEX. The draft genome of the new strain was sequenced and annotated for genes that encode enzymes putatively involved in BTEX degradation and quorum sensing. The strain was transformed with a plasmid expressing lactonase, which cleaves the autoinducer quorum sensing signal molecule, the acyl-homoserine lactone, to obtain a quorum sensing minus strain. P. putida AQ8 depleted the 40% on average of all the components of the initial BTEX concentration in 36 h. The quorum sensing minus strain, in the same time interval, depleted only the 10% of the initial BTEX concentration. The role of quorum sensing in regulating the expression of the annotated benzene/toluene dioxygenase gene (benzA) and biphenyl/toluene/benzene dioxygenase (bphA) genes, which are involved in BTEX degradation, was studied by quantitative RT-real-time quantitative (q)PCR analysis. The qPCR data showed decreased levels of expression of the benzA and bphA genes in the quorum sensing minus strain. Our results showed, for the first time, quorum sensing modulation of the level of transcription of dioxygenase genes in the upper BTEX oxidation pathway.

Objective Smoking cue-(SC) elicited craving can lead to relapse in SC-vulnerable individuals. Thus, identifying treatments that target SC-elicited craving is a top research priority. Reduced drug cue neural activity is associated with recovery and is marked by a profile of greater tonic (resting) activation in executive control regions, and increased connectivity between executive and salience regions. Evidence suggests the GABA-B agonist baclofen can reduce drug cue-elicited neural activity, potentially through its actions on the resting brain. Based on the literature, we hypothesize that baclofen's effects in the resting brain can predict its effects during SC exposure. Methods In this longitudinal, double blind, placebo-controlled neuropharmacological study 43 non-abstinent, sated treatment-seeking cigarette smokers (63% male) participated in an fMRI resting-state scan and a SC-reactivity task prior to (T1) and 3 weeks following randomization (T2; baclofen: 80 mg/day; n = 21). Subjective craving reports were acquired before and after SC exposure to explicitly examine SC-induced craving. Results Whole-brain full-factorial analysis revealed a group-by-time interaction with greater resting brain activation of the right dorsolateral prefrontal cortex (dlPFC) at T2 in the baclofen group (BAC) (pFWEcorr = 0.02), which was associated with reduced neural responses to SCs in key cue-reactive brain regions; the anterior ventral insula and ventromedial prefrontal cortex (pFWEcorr ＜ 0.01). BAC, but not the placebo group reported decreased SC-elicited craving (p = 0.02). Conclusion Results suggest that baclofen mitigates the reward response to SCs through an increase in tonic activation of the dlPFC, an executive control region. Through these mechanisms, baclofen may offer SC-vulnerable smokers protection from SC-induced relapse.

Non-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure – HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL−1 or 107 CFU mL−1) in milk. For inoculum levels of 104 CFU mL−1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL−1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).

Polyaniline-chitosan (PANI-Cs) blends were prepared by in situ oxidative polymerization with acetic (CH 3 COOH) acid and hydrochloric (HCl) acid as dopants. Using Fourier transform infrared (FTIR) spectroscopy, the C N and C C stretching of quinoid and benzenoid of PANI in the PANI-Cs blends was confirmed at ranges 1528cm − 1 to 1571cm − 1 and 1490cm − 1 to 1493cm − 1 , respectively. PANI emeraldine state was stipulated in all samples and PANI-Cs HCl samples have the highest degree of oxidation which is 48.56 ± 0.23%. The ultraviolet-visible (UV–vis) spectroscopy identified the glucopyranose of Cs (282–308 nm), and π − π * transition of benzenoid (389–437 nm) and quinoid rings (551–554 nm) of PANI. The PANI-Cs blends yielded optical band gap values of 1.968 ± 0.014 to 2.009 ± 0.005 eV due to the presence of PANI. One-way ANOVA was also performed, and results showed the significant effect of using different dopants towards the degree of oxidation of the samples. Frequency dependent behavior was observed to the PANI-Cs samples with AC conductivity values around 1.2–4.1 Scm − 1 at 10 5 –10 7 Hz. The 10 2 Hz dielectric peak intensity of the samples varied due to influence of dopants on charge polarization. Lastly, PANI-Cs samples showed dominance of DC loss and relaxation at low frequencies 10–10 3 Hz.

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-β-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-β-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1., Alterations of the mucosal microbiota, including Lactobacillus bacteria, are associated with infections caused by the fungus Candida albicans. Here, MacAlpine et al. show that some Lactobacillus strains produce a small molecule that blocks C. albicans filamentation and biofilm formation, and thus virulence, through inhibition of a fungal kinase.

UV-C light is a non-thermal technology with potential application in food industries. The objective was to assess UV-C radiation effect (13.44 W/m2) on microbiological decontamination and some quality characteristics of Cantaloupe melon juice, during refrigerated storage. Juice was inoculated with Listeria innocua (non-pathogenic surrogate of L. monocytogenes) and Alicyclobacillus acidoterrestris spores (spoilage indicators) used as indicators of the UV-C treatment efficacy. Total mesophylls and yeasts and molds were also enumerated. Results demonstrated that 5 min of exposure allowed 3.7 ± 0.3 log10 cycles reduction of L. innocua, while for A. acidoterrestris, 20 min were required to decrease 4.7 ± 0.1 log10 cycles. At the end of refrigerated storage for 13 days, UV-C treated juices retained color, total phenolics content and antioxidant activity, and yeast and molds did not grow. Since UV-C process was effective on microbial inactivation and allowed juice quality maintenance, it can be considered as a promising alternative to thermal pasteurization.

A spore-forming bacterium, Alicyclobacillus acidoterrestris, is often responsible for fruit juices quality degradation. The objective was to study the influence of ultrasounds and combinations with temperature (thermosonication) on A. acidoterrestris spores inactivation in apple juices. Commercially available juice was artificially inoculated with the bacterium (∼105 CFU/mL). Sonication was carried out in an ultrasonic bath (35 kHz; 120–480 W) at room temperature and at 70, 80, 85, 90 and 95 °C for different times. Thermal treatments at the same temperatures were performed as a control. Sonication had no significant effect on A. acidoterrestris spores inactivation. However, when applied at 70 and 80 °C, it allowed 1 log-cycle more of inactivation when compared to thermal treatments and at the end of the processes. Ultrasounds at higher temperatures required approximately half of the treatment time to attain the same inactivation that occurred when the thermal processes were applied alone. In thermosonicated juices, spores decreased by 4.8, 4.7 and 5.5 log-cycles (at 85, 90 and 95 °C, respectively) after 90, 60 and 20 min. The Weibull model satisfactorily fitted inactivation data of thermosonicated juices. Thermosonication is efficient for A. acidoterrestris spores inactivation, with a drastic impact on spores’ loads when high temperatures are used.

SUMMARY Evasion of killing by immune cells is crucial for fungal survival in the host. For the human fungal pathogen Candida albicans, internalization by macrophages induces a transition from yeast to filaments that promotes macrophage death and fungal escape. Nutrient deprivation, alkaline pH, and oxidative stress have been implicated as triggers of intraphagosomal filamentation; however, the impact of other host-derived factors remained unknown. Here, we show that lysates prepared from macrophage-like cell lines and primary macrophages robustly induce C. albicans filamentation. Enzymatic treatment of lysate implicates a phosphorylated protein, and bioactivity-guided fractionation coupled to mass spectrometry identifies the immunomodulatory phosphoprotein PTMA as a candidate trigger of C. albicans filamentation. Immunoneutralization of PTMA within lysate abolishes its activity, strongly supporting PTMA as a filament-inducing component of macrophage lysate. Adding to the known repertoire of physical factors, this work implicates a host protein in the induction of C. albicans filamentation within immune cells., Graphical Abstract, In brief The human fungal pathogen Candida albicans filaments within host macrophages, enabling its escape. Case et al. demonstrate that lysates prepared from macrophage-like cell lines and primary macrophages induce C. albicans filamentation and implicate the immunomodulatory protein prothymosin alpha (PTMA) as a trigger of filamentation produced by host immune cells.

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+T cells in regulating immunological processes, we generated novel single-domain antibodies (nanobodies, Nbs) that specifically recognize human CD4. After in depth analysis of their binding properties, recognized epitopes, and effects on T cell proliferation, activation and cytokine release, we selected CD4 Nbs that did not interfere with crucial T cell processesin vitroand converted them into immune tracers for non-invasive molecular imaging.By optical imaging, we demonstrate the ability of a high-affinity CD4-Nb to specifically visualize CD4+cellsin vivousing a xenograft model. Furthermore, time-resolved immune positron emission tomography (immunoPET) of a human CD4 knock-in mouse model showed rapid accumulation of64Cu-radiolabeled CD4-Nb in CD4+T cell-rich tissues. We propose that the CD4 Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

Background Carob (Ceratonia siliqua L.) is an evergreen tree that belongs to the Leguminosae family and is typical of the Mediterranean basin. It is well known for its valuable locust bean gum obtained from carob seeds. However, the food industry can obtain different carob products from carob fruit after processing. Carob products are good sources of dietary fibre, sugars, and a range of bioactive compounds such as polyphenols and D-pinitol. Scope and approach Bioactive compounds present in carob fruit and its derived products help control many health problems such as diabetes, heart diseases, and gastrointestinal disorders due to their anti-hyperglycaemic, antioxidant, and anti-inflammatory activities. So, carob products have a great potential to be used as a functional food ingredient. Key findings and conclusions This article focuses on carob characteristics and processing, chemical composition, health benefits, and applications in food formulations to explore the potential of carob in developing a wide variety of health-beneficial food products.

Candida albicans is a leading human fungal pathogen, which can cause superficial infections or life-threatening systemic disease in immunocompromised individuals. The ability to transition between yeast and filamentous forms is a major virulence trait of C. albicans, and a key regulator of this morphogenetic transition is the molecular chaperone Hsp90. To explore the mechanisms governing C. albicans morphogenesis in response to Hsp90 inhibition, we performed a functional genomic screen using the gene replacement and conditional expression collection to identify mutants that are defective in filamentation in response to the Hsp90 inhibitor, geldanamycin. We found that transcriptional repression of genes involved in mitochondrial function blocked filamentous growth in response to the concentration of the Hsp90 inhibitor used in the screen, and this was attributable to increased resistance to the compound. Further exploration revealed that perturbation of mitochondrial function reduced susceptibility to two structurally distinct Hsp90 inhibitors, geldanamycin and radicicol, such that filamentous growth was restored in the mitochondrial mutants by increasing the compound concentration. Deletion of two representative mitochondrial genes, MSU1 and SHY1, enhanced cellular efflux and reduced susceptibility to diverse intracellularly acting compounds. Additionally, screening a C. albicans efflux pump gene deletion library implicated Yor1 in the efflux of geldanamycin and Cdr1, in the efflux of radicicol. Deletion of these transporter genes restored sensitivity to Hsp90 inhibitors in MSU1 and SHY1 homozygous deletion mutants, thereby enabling filamentation. Taken together, our findings suggest that mitochondrial dysregulation elevates cellular efflux and consequently reduces susceptibility to xenobiotics in C. albicans.

Patients with breast or ovarian cancer have not benefited from improved efficacy with pegylated liposomal doxorubicin relative to free drug, likely due to the limited extent of the enhanced permeability and retention (EPR) effect, further compromising drug bioavailability in the tumor. Herein it is hypothesized that targeting nucleolin overexpressed in tumor endothelial cells (readily accessible from the vascular compartment), besides cancer cells, with PEGASEMP (doxorubicin hydrochloride in a lipid-based pegylated nanoparticle functionalized with a 31-aminoacid peptide targeting nucleolin), lessens the dependence on high systemic exposures and EPR effect for successful tumor targeting. This strategy has resulted in improved intracellular tumor bioavailability of doxorubicin, at low systemic exposure, associated with a safe toxicological profile. Levels of cell surface nucleolin dictated the antitumor activity of PEGASEMP against nucleolin-overexpressing solid tumors of diverse histological origin, evidencing a significant growth inhibition of malignant mesothelioma over the standard of care. Those observations were paralleled by an impairment of the nucleolin-positive vasculature and downregulation of typically overexpressed genes. Patient stratification based on nucleolin mRNA expression correlated with prognosis and enabled identification of breast and mesothelioma tumors that may potentially benefit from PEGASEMP. Overall, a novel principle of drug delivery is presented with potential therapeutic impact across nucleolin-overexpressing human cancers. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request.

PCR-based deep sequencing of 16S rRNA gene allows for detection of a wide array of bacterial pathogens in culture-negative specimens. Ion 16S metagenomics kit and Ion Reporter metagenomics workflow (Ion 16S mNGS) provides an end-to-end solution with integrated workflow. Ninety-eight clinical samples with the diagnosis generated with 16S rRNA gene PCR/chain termination (Sanger) sequencing (16S CS) was used to assess the performance of Ion 16S mNGS. Compared to species level detection of 16S CS, the Ion 16S mNGS had 88% sensitivity and 76% specificity. When accounting for genus level of detection, the Ion 16S mNGS had 100% sensitivity. Notably, Ion 16S mNGS generated diagnosis in 13% of 16S CS and culture-negative samples. In addition, Ion 16S mNGS had the advantage of detecting more than 1 pathogen in 16S CS positive samples. We showed that the workflow had high reproducibility.

Multiple studies about tumor biology have revealed the determinant role of the tumor microenvironment in cancer progression, resulting from the dynamic interactions between tumor cells and surrounding stromal cells within the extracellular matrix. This malignant microenvironment highly impacts the efficacy of anticancer nanoparticles by displaying drug resistance mechanisms, as well as intrinsic physical and biochemical barriers, which hamper their intratumoral accumulation and biological activity.Currently, two-dimensional cell cultures are used as the initial screening method in vitro for testing cytotoxic nanocarriers. However, this fails to mimic the tumor heterogeneity, as well as the three-dimensional tumor architecture and pathophysiological barriers, leading to an inaccurate pharmacological evaluation.Biomimetic 3D in vitro tumor models, on the other hand, are emerging as promising tools for more accurately assessing nanoparticle activity, owing to their ability to recapitulate certain features of the tumor microenvironment and thus provide mechanistic insights into nanocarrier intratumoral penetration and diffusion rates.Notwithstanding, in vivo validation of nanomedicines remains irreplaceable at the preclinical stage, and a vast variety of more advanced in vivo tumor models is currently available. Such complex animal models (e.g., genetically engineered mice and patient-derived xenografts) are capable of better predicting nanocarrier clinical efficiency, as they closely resemble the heterogeneity of the human tumor microenvironment.Herein, the development of physiologically more relevant in vitro and in vivo tumor models for the preclinical evaluation of anticancer nanoparticles will be discussed, as well as the current limitations and future challenges in clinical translation.

Background Pseudomonas chlororaphis strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus Sclerotinia sclerotiorum. This bacterium secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. In order to be successful, a biocontrol agent must be able to persist in the environment and avoid the threat of grazing predators. The focus of the current study was to investigate whether PA23 is able to resist grazing by the protozoan predator Acanthamoeba castellanii (Ac) and to define the role of bacterial metabolites in the PA23-Ac interaction. Methods Ac was co-cultured with PA23 WT and a panel of derivative strains for a period of 15 days, and bacteria and amoebae were enumerated on days 1, 5, 10 and 15. Ac was subsequently incubated in the presence of purified PRN, PHZ, and KCN and viability was assessed at 24, 48 and 72 h. Chemotactic assays were conducted to assess whether PA23 compounds exhibit repellent or attractant properties towards Ac. Finally, PA23 grown in the presence and absence of amoebae was subject to phenotypic characterization and gene expression analyses. Results PRN, PHZ and HCN were found to contribute to PA23 toxicity towards Ac trophozoites, either by killing or inducing cyst formation. This is the first report of PHZ-mediated toxicity towards amoebae. In chemotaxis assays, amoebae preferentially migrated towards regulatory mutants devoid of extracellular metabolite production as well as a PRN mutant, indicating this antibiotic has repellent properties. Co-culturing of bacteria with amoebae led to elevated expression of the PA23 phzI/phzR quorum-sensing (QS) genes and phzA and prnA, which are under QS control. PHZ and PRN levels were similarly increased in Ac co-cultures, suggesting that PA23 can respond to predator cues and upregulate expression of toxins accordingly. Conclusions PA23 compounds including PRN, PHZ and HCN exhibited both toxic and repellent effects on Ac. Co-culturing of bacteria and amoebae lead to changes in bacterial gene expression and secondary metabolite production, suggesting that PA23 can sense the presence of these would-be predators and adjust its physiology in response.

Several scientific studies point fruits as rich sources of antioxidants but mainly focus on their edible part. However, fruits wastes are abundant sources of bioactive compounds and nutrients, which are considered to be health beneficial. The main purpose was to characterise juice, pulp, peel and seeds of Piel de Sapo melon, in terms of several physicochemical characteristics (soluble solids content, titratable acidity, pH, potassium, colour and water activity), some bioactive compounds (total phenolics, vitamin C, chlorophylls and total carotenoids) and total antioxidant activity. Juice, pulp, peel and seeds represent 47, 19, 27 and 5% of melon total weight, respectively. Peel and seeds stood out by their higher concentration of total phenolics compounds and antioxidant activity when compared to edible parts. The highest potassium concentration was found in seeds. Chlorophylls were only detected in peel, while carotenoids were not detected in any part of the melon analysed. Juice and pulp contributed to 69% of vitamin C amount of the whole fruit. However, its concentration in peel was equivalent to the ones observed in juice and pulp. These results pointed out the importance of fruit wastes valorisation and the development of strategies for their re-utilisation.

Computational redesign of chondroitinase ABC introduces 37 point mutations to improve enzyme stability and activity., Maintaining biocatalyst stability and activity is a critical challenge. Chondroitinase ABC (ChABC) has shown promise in central nervous system (CNS) regeneration, yet its therapeutic utility is severely limited by instability. We computationally reengineered ChABC by introducing 37, 55, and 92 amino acid changes using consensus design and forcefield-based optimization. All mutants were more stable than wild-type ChABC with increased aggregation temperatures between 4° and 8°C. Only ChABC with 37 mutations (ChABC-37) was more active and had a 6.5 times greater half-life than wild-type ChABC, increasing to 106 hours (4.4 days) from only 16.8 hours. ChABC-37, expressed as a fusion protein with Src homology 3 (ChABC-37-SH3), was active for 7 days when released from a hydrogel modified with SH3-binding peptides. This study demonstrates the broad opportunity to improve biocatalysts through computational engineering and sets the stage for future testing of this substantially improved protein in the treatment of debilitating CNS injuries.

Plant growth promoting bacteria (PGPB) are a growing subset of agricultural adjuncts which can be used to increase crop yield and plant productivity. Although substantial research has been conducted on the metabolites and active molecules secreted by PGPBs, relatively little is known about their effects on the global transcriptome of the host plant. The present study was carried out to investigate changes in the gene expression landscape of early vegetative Brassica napus following treatment with Pseudomonas chlororaphis PA23. This PGPB was isolated from the soybean rhizosphere and has been extensively studied as a biocontrol agent. However, little is known about its effects on plant growth and development. Using a combination of RNA-sequencing and physiological analyses, we identified increased abundance of mRNA transcripts associated with photosynthesis and phytohormone response. Phenotypically we observed increased photosynthetic rates and larger root and shoot systems in B. napus following P. chlororaphis PA23 treatment. Lastly, we identified auxin production by P. chlororaphis PA23 which likely contributes to changes in gene expression and the observed phenotypic differences in root and shoot structures. Together, the results of our study suggest that PA23 is a potent plant growth promoting agent with the potential for field applications as an agricultural adjunct.

The U.S. Air Force (USAF) has pursued development of alternative fuels to augment or replace petroleum-based jet fuels. Hydroprocessed esters and fatty acids (HEFA) renewable jet fuel is certified for use in commercial and USAF aircraft. HEFA feedstocks include camelina seed oil (

Fischer-Tropsch (FT) Synthetic Paraffinic Kerosene (SPK) jet fuel is a synthetic organic mixture intended to augment petroleum-derived JP-8 jet fuel use by the U.S. armed forces. The FT SPK testing program goal was to develop a comparative toxicity database with petroleum-derived jet fuels that may be used to calculate an occupational exposure limit (OEL). Toxicity investigations included the dermal irritation test (FT vs. JP-8 vs. 50:50 blend), 2 in vitro genotoxicity tests, acute inhalation study, short-term (2-week) inhalation range finder study with measurement of bone marrow micronuclei, 90-day inhalation toxicity, and sensory irritation assay. Dermal irritation was slight to moderate. All genotoxicity studies were negative. An acute inhalation study with F344 rats exposed at 2000 mg/m

The hypotensive and antihypertensive activities of the aqueous extract (AE) and butanolic fraction (ButF) isolated from Cecropia glaziovii Sneth have been demonstrated in previous studies in animal models. This study aimed to evaluate the molecular mechanism of action responsible for the vasodilatory effect of procyanidins, flavanols, and flavonoids found in C. glaziovii in endothelial cell culture. For this purpose, we analyzed the effect of procyanidin B2 and B3 compounds, catechin, epicatechin, orientin, isoorientin, and isovitexin in the mobilization of Ca2+ in rat endothelial cell cultures. Parallel associations with different antagonists were examined by considering the following in vivo hypotensive mechanisms: blockage of L-type calcium channels, action on β-2 adrenergic receptors, and vasodilation via the nitric oxide pathway. All measurements of calcium mobilization were carried out by using the fluorescence measurement methodology in a Flexstation M3 spectrophotometer. The results indicate that some of the compounds have mixed actions, acting through different calcium mobilization pathways. The mobilization induced by such compounds significantly decreased when they were incubated with their corresponding antagonists. Taken together, our data suggest that the beneficial effects seen with the popular use of Cecropia glaziovii Sneth in pathological conditions, such as systemic arterial hypertension, seem to be related to the plant's hypotensive effect, very probably promoted by the actions of flavonols, flavonoids, and procyanidins, by different pathways of calcium mobilization.

Background The relationship between antibiotic consumption and resistance is relatively well defined at the population/ecologic level. Increases in antimicrobial consumption correlate with increased antibiotic resistance for clinical and surveillance isolates. However, the impact of antimicrobial consumption on nosocomial transmission of resistant bacteria is less well defined. This study explores the association between antimicrobial consumption, hand hygiene, and horizontal resistant organism transmission. Methods A retrospective cohort pilot study was conducted. Vancomycin-resistant Enterococcus spp. (VRE) horizontal transmission events during a 2-year period were identified. VRE transmission events were defined as isolation of genetically similar VRE strain-types (determined using pulsed field gel electrophoresis) from patients who were temporally and geographically co-localized within our hospital. The Centers for Disease Control and Prevention Antimicrobial Use and Resistance Module was utilized to collect antibacterial consumption data of commonly utilized agents. Hand hygiene was quantified using floor-by-floor peer audit data. Regression techniques were employed to assess population-level relationships between study variables and transmission events. Results One hundred nineteen transmission events were identified. Hand hygiene estimates were homogeneous and did not correlate with VRE transmission rates. Stepwise-multivariate linear regression revealed that aztreonam consumption was associated with a lower rate of transmissions in the medical intensive care unit ( P =0.031), and carbapenem consumption was associated with a higher rate of VRE transmission events on one of two oncology floors ( P =0.033). Discussion/Conclusion Consumption of aztreonam and carbapenems was associated with VRE horizontal transmission rates. Further studies are necessary to identify other associations and elucidate the full clinical significance of this finding.

Summary This work aimed to develop an orange juice powder by spray drying with lactic acid bacteria (Lactobacillus plantarum 299v and Pediococcus acidilactici HA-6111-2), testing their survival both during drying and storage (room temperature and 4 °C). Initially, the best conditions for spray drying were chosen to allow the best survival of each LAB: (i) inlet air temperature of 120 °C and (ii) 0.5:2 ratio of the orange juice soluble solids and drying agent added (prebiotics: 10 DE maltodextrin or gum Arabic). Survival of LAB was not affected by drying process, and it was higher when cultures were stored at 4 °C. A slightly higher protection was conferred by 10 DE maltodextrin, in the case of L. plantarum and at 4 °C. Pediococcus acidilactici was more resistant during storage at 4 °C, with logarithmic reductions lower than 1 log-unit. It was demonstrated that it is possible to produce a functional nondairy product, orange juice powder supplemented with prebiotic compounds, containing viable LAB for at least 7 months, when stored at 4 °C.

Alicyclobacillus acidoterrestris is a spore-forming bacterium, which is responsible for quality degradation of fruit juices. Non-thermal treatments, such as ultraviolet-C radiation (UV-C) and ultrasound (US), are promising methods for microbial inactivation. The main objective was to study the influence of UV-C, ultrasound and combinations of both treatments on A. acidoterrestris spores inactivation in apple juices. A thermal treatment at 95 °C was used as control. Commercial juices artificially inoculated with the bacterium were exposed to the treatments. Results showed that ultrasound had the lowest impact on A. acidoterrestris inactivation. When UV-C radiation was applied, the number of spores decreased drastically after 8 min, being more effective than a 95 °C thermal treatment. In terms of final microbial loads, combination of US and UV-C did not result in synergetic effects. However, inactivation rates of the combined treatments were significantly different from the ones obtained when the treatments were applied individually.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the pathogenic fungus Sclerotinia sclerotiorum . This bacterium secretes the antibiotics pyrrolnitrin (PRN) and phenazine (PHZ), together with degradative enzymes and siderophores. Strain PA23 also produces hydrogen cyanide (HCN); however, the role of this compound in PA23 antifungal (AF) activity remains unknown. The aim of the current study was to characterize an hcn mutant and determine whether HCN contributes to biocontrol. Analysis of an hcn mutant revealed decreased AF activity both in vitro and in a newly established model of S. sclerotiorum root-rot infection in lettuce. When glycine (20 mM) and ferric chloride (100 µM) were included as media amendments, elevated AF activity was observed. Moreover glycine, but not FeCl 3, increased HCN production and hcnA-lacZ transcriptional activity in the wild type. As the metabolic precursor for HCN biosynthesis, glycine is expected to enhance HCN production at the post-transcriptional level. We postulated that glycine might be indirectly affecting HCN transcription through the PhzRI quorum-sensing (QS) system. Analysis of phzR-lacZ and phzI-lacZ activity together with autoinducer levels revealed upregulation by glycine. This compound also increased expression of the QS-controlled prnA and phzA and their endproducts but, for reasons unknown, FeCl 3 exerted a repressive effect. In a QS-deficient background the opposite occurred; FeCl 3 increased hcnA-lacZ expression whereas glycine had no effect. HCN is under control of QS and the global regulator ANR. We present a model depicting how these regulators, in combination with media amendments, impact PA23 hcn expression.

Ultraviolet-C radiation (UV-C) is widely used as an alternative strategy to control microorganism in food products. Alicyclobacillus acidoterrestris is a thermo-acidophilic, non-pathogenic and spore-forming bacterium, able to grow at low pH and high temperatures. It is a concern, particularly in apple juice thermal pasteurization, because it is responsible for quality degradation. The main objective of this work was to study the influence of UV-C radiation treatments with seven different intensities (0.34, 0.86, 2.59, 5.59, 8.45, 11.50 and 13.44 W/m2) on A. acidoterrestris inactivation in apple juices. Treatments were carried out in a camera (75 × 70 × 45 cm3) with four germicidal UV lamps with peak emission at 254 nm. Commercial juices were artificially inoculated with bacterium, with initial loads around 107 CFU/mL and then exposed to UV-C radiation and the treatment impact on microbial loads was assessed throughout exposure times. The UV transmittance at 254 nm of the juice was 58%. Results showed that the log-survival of A. acidoterrestris decreased linearly with treatment time, for all intensities tested. When the most severe intensity was used, the number of spores decreased drastically (around 5-log reduction) after 8 min of treatment. Overall it can be concluded that UV-C radiation is a promising treatment with a drastic impact on the loads of A. acidoterrestris in apple juices, especially when high intensities are used.

Contaminated food with Listeria monocytogenes is the predominant route of transmission of listeriosis to humans, a severe illness with a high mortality rate. Food processing environments can be colonized by persistent strains, repeatedly isolated for months or years. This study aimed to investigate the biofilm formation capacity of six strains of L. monocytogenes that persisted for long periods in two cheese processing plants in comparison to seven strains isolated sporadically from the same plants. Persistent strains produced more biofilm than non-persistent strains on stainless steel and silicon rubber surfaces; no significant differences were observed on polyvinyl chloride (PVC). In a polystyrene microtiter plate assay with crystal violet staining, no evidence was found that persistent strains have higher ability to form biofilm than non-persistent strains, and no correlation was identified between biofilm formation in the microtiter plate and on the three other surfaces tested.

We have developed a technology that facilitates the targeted delivery of glucocorticoids to arthritic joints thereby side-stepping systemic immune suppression while potentially improving efficacy. Our technology loads phototherapeutics inside of red blood cell (RBC) carriers. This ensures that the inactive, RBC-contained phototherapeutic drug is transported throughout the body and released from the RBC carrier only after triggered release by long-wavelength light. We have demonstrated that medications bound to vitamin B12 can be photo-released from RBCs using tissue penetrating, longwavelength red, far-red, and near infrared light in the collagen antibody-induced arthritis model.

The author E. M. Gonçalves gratefully acknowledges the support of Fundação para a Ciência e Tecnologia through the research units UIDP/04035/2020 (GeoBioTec). The author J. Pinheiro acknowledges financial support to Fundação para a Ciência e a Tecnologia (FCT), through the strategic project UIDB/04292/2020 granted to MARE, and the Integrated Program of SR&TD “SmartBioR—Smart Valorization of Endogenous Marine Biological Resources Under a Changing Climate” (Centro-01-0145-FEDER-000018) co-funded by Centro 2020 program, Portugal 2020, European Union through the European Regional Development Fund. Teresa R.S. Brandão also gratefully acknowledges the Post-Doctoral Grant (SFRH/ BPD/101179/2014) to FCT. This work was also supported by National Funds from FCT through project UID/Multi/50016/2013. In order to improve the overall quality of frozen carrots (Daucus carota L.), the degradation kinetics of several attributes were quantified during frozen storage under isothermal and nonisothermal conditions. The experimental results showed that the analyzed quality parameters were significantly affected by both tested frozen regimes. For both storage conditions, the degradation of color parameters and drip loss followed zero-order kinetics, and the texture was successfully described by a fractional kinetic model. A first-order kinetic model was adequate in describing total vitamin C decay under isothermal storage conditions. The storage temperature effect was adequately modeled by the Arrhenius law. The carrots shelf life under isothermal storage conditions of −18°C, using a threshold of 50% vitamin C content, will be of 118 days. Practical applications: The objective of this work was to evaluate the degradation kinetics of several quality attributes of carrots when subjected to various frozen storage temperature conditions that may occur along the distribution chain. The quality attributes degradation mechanisms are governed by chemical and/or physical changes that need to be systematically evaluated for further incorporation into product and process designs. The achieved results will be an insight to help manufacturers to predict and optimize products quality and determine its shelf life. info:eu-repo/semantics/publishedVersion

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of “phz-box” sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.

Pseudomonas chlororaphis PA23 is a biocontrol agent that, in addition to producing antifungal compounds, synthesizes polyhydroxyalkanoate (PHA) polymers as a carbon and energy sink. Quorum sensing (QS) and the anaerobic regulator (ANR) are required for PA23-mediated fungal suppression; however, the role of these regulators in PHA production is unknown. Strains lacking either QS or ANR accumulated less PHA polymers when propagated on Ramsay's minimal medium (RMM) with glucose or octanoate as the carbon source. In the acyl-homoserine lactone (AHL)-deficient background, all six of the genes in the pha locus (phaC1, phaC2, phaZ, phaD, phaF, phaI) showed reduced expression in RMM glucose, and all except phaC2 were repressed in RMM octanoate. Although changes in gene activity were observed in the anr mutant, they were less pronounced. Analysis of the promoter regions for QS- and ANR-binding consensus sequences revealed putative phzboxes upstream of phaZ and phaI, but no anr boxes were identified. Our findings indicate that altered pha gene expression likely contributes to the lower PHA accumulation in the QS- and ANR-deficient strains, which may be in part indirectly mediated. This study is the first to show that mcl-PHA production is under QS and ANR control.

A protein isolate (ERPI) was prepared from ethanol-treated rapeseed meal and used as a stabilizing agent in sunflower and rapeseed oil-in-water emulsions. The aim of the current study was to explore the influence of protein and oil concentrations on initial stability of sunflower and rapeseed oil-in-water emulsions by evaluating Gibbs free energy (ΔG) and particle size distribution. The 7-day dynamics of emulsion stability was investigated by turbidity measurement as well. A 3(2) factorial design was applied to assess the significance of oil (5%, 10% and 15% w/w) and ERPI protein (0.25%, 0.5% and 1.0% w/w) addition on stability of the emulsions. The results demonstrated that the increase of oil concentrations from 5 to 15% positively influenced the initial stability of sunflower and rapeseed oil-in-water emulsions. In both oil types, ERPI protein supplementation at all levels resulted in significant differences in the stability of 5% and 10% oil emulsions but did not alter the initial stability of the emulsions prepared with either 15% sunflower or rapeseed oil. With a few exceptions, there was a good agreement between Gibbs free energy data and microstructural profiles of the emulsions. Overall, emulsions with all sunflower oil concentrations and 1.0% ERPI protein exhibited better initial and a 7-day stability dynamics compared to all rapeseed oil-based emulsions. The study demonstrated the potential of ethanol-treated rapeseed meal protein isolate to serve as an emulsifying agent in sunflower and rapeseed oil containing emulsions.

This work evaluated the effect of the air-drying temperature and vacuum packaging on quality and nutritional compounds of dehydrated galega kale, previously steam blanched, during 5 months of storage. When compared with the vacuum packaged kale, the packaging without vacuum yielded improved nutritional features, in general. This approach combined with a drying temperature of 40°C resulted in retention percentages of 62, 38, 92, and 48% for vitamin C, total phenolic content, total antioxidant capacity, and chlorophylls, respectively. The acceptance of the dried product by the consumer was assessed through a focus group. The participants classified the product as a practical and convenient alternative to cook healthier dishes. The appearance of dried galega kale was described as being fragile and being with nice smell, natural colour, and flavour similar to the fresh product. In conclusion, the herein presented product was addressed as an innovation with multiple possible applications in several recipes.

Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis, and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3, and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites on C. albicans Hsp90, and substitutions at these residues compromised Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90-dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network, and reveals new targets for development of antifungal drugs. This article is protected by copyright. All rights reserved.

The objective of this work was to investigate the effect of stress conditions frequently encountered in food-associated environments on virulence-associated characteristics of eight strains of Listeria monocytogenes. Strains were grown at low (11 °C, cold stress) and optimal (37 °C) temperatures and in high NaCl concentrations (6% NaCl, 11 °C; cold-osmotic stress) and tested for their ability to invade the human intestinal epithelial Caco-2 cells. Results demonstrate that the correlation between exposure to cold stress and increased invasion phenotype is strain-dependent as strains investigated exhibited different behaviours, i.e. exposure to cold stress conditions resulted in a significant increase of invasion levels in five out of the eight strains tested, when compared to growth under optimal conditions. On the other hand, when these cold-adapted cells were subsequently submitted to high salt concentrations and low temperature, their enhanced ability to invade Caco-2 was lost. Surprisingly, saturated fatty acids (SFA) and branched chain fatty acids (BCFA) decreased when L. monocytogenes were exposed to stress conditions as opposed to what has been observed in other studies, therefore highlighting that further studies will need to deepen in the understanding of the lipid metabolism of these strains. The effect of stress conditions on the survival of three selected L. monocytogenes strains through an in vitro gastrointestinal (GI) tract digestion model was further investigated. The exposure to cold-osmotic stress increased the survival of one strain through the GI tract.

BP/MS is a new experimental proteomic platform for systematic study of native protein complexes and global protein-protein interaction networks based on deep biochemical purification of soluble protein extracts and mass spectrometry identification of coeluting proteins. Herein, we describe the application of this methodology to draft a high-confidence protein interaction network for Candida albicans.

Polyaniline⁻chitosan (PAni⁻Cs) composite films were synthesized using a solution casting method with varying PAni concentrations. Polyaniline powders used in the composite synthesis were polymerized using acetic acid as the dopant media. Raman spectroscopy revealed that the PAni powders synthesized using hydrochloric acid and acetic acid did not exhibit significant difference to the chemical features of PAni, implying that PAni was formed in varying concentrations of the dopant media. The presence of agglomerated particles on the surface of the Cs composite, which may have been due to the presence of PAni powders, was observed with scanning electron microscope⁻energy dispersive X-ray spectroscopy (SEM⁻EDX). Ultraviolet⁻visible (UV⁻Vis) spectroscopy further showed the interaction of PAni with Cs where the Cs characteristic peak shifted to a higher wavelength. Cell viability assay also revealed that the synthesized PAni⁻Cs composites were nontoxic and may be utilized for future biomedical applications.

Extensive loss of dopaminergic neurons and aggregation of the protein α-synuclein into ubiquitin-positive Lewy bodies represents a major neuropathological hallmark of Parkinson's disease (PD). At present, the generation of large nuclear-associated Lewy bodies from endogenous wild-type α-synuclein, translationally regulated under its own promoter in human cell culture models, requires costly and time-consuming protocols. Here, we demonstrate that fully differentiated human SH-SY5Y neuroblastoma cells grown in three-dimensional cell culture develop Lewy-body-like pathology upon exposure to exogenous α-synuclein species. In contrast to most cell- and rodent-based PD models, which exhibit multiple diffuse α-synuclein aggregates throughout the cytoplasm, a single large nuclear inclusion that is immunopositive for α-synuclein and ubiquitin is rapidly obtained in our model. This was achieved without the need for overexpression of α-synuclein or genetic modification of the cell line. However, phosphorylation of α-synuclein within these inclusions was not observed. The system described here provides an ideal tool to screen compounds to therapeutically intervene in Lewy body formation, and to investigate the mechanisms involved in disease progression in synucleinopathies., Editor's choice: This study describes a novel 3D cell culture model that recapitulates key features of neuropathology in synucleinopathies, including large, nuclear-associated α-synuclein and ubiquitin-positive inclusions.

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ, PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay&rsquo, s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz- (PA23-63) and phz- prn- (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz- prn- (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz- (PA23-63), prn- (PA23-8), and phz- prn- (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz- and prn- mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

Environments combining JP-8 jet fuel exposure with heightened ambient noise may accelerate hearing loss induced by noise. To reduce animal use and facilitate kinetic modeling of this military aviation fuel, tissue-specific parameters are required, including water, protein, and lipid content. However, tissues involved in hearing, including cochlea, brainstem, frontal, and temporal lobe, have not been characterized before. Therefore, water content was determined by lyophilization of rat auditory tissues and the protein of the freeze dried remainder was quantified using a bicinchoninic acid assay. Lipids were extracted from fresh-frozen rat auditory tissues and separated into neutral lipids, free fatty acids, neutral phospholipids, and acidic phospholipids using solid phase extraction. Phospholipid fractions were confirmed by 31 P nuclear magnetic resonance analysis showing distinct phospholipid profiles. Lipid content in reference tissues, such as kidney and adipose, confirmed literature values. For the first time, lipid content in the rat auditory pathway was determined showing that total lipid content was lowest in cochlea and highest in brainstem compared with frontal and temporal lobes. Auditory tissues displayed distinct lipid fraction profiles. The information on water, protein, and lipid composition is necessary to validate algorithms used in mathematical models and predict partitioning of chemicals of future interest into these tissues. This research may reduce the use of animals to measure partition coefficients for prospective physiological models.

Candida albicans hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2 is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3 co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae., eLife digest Billions of microorganisms live on, and in, the human body. Known as the human microbiome, most of these microscopic hitchhikers are harmless. But, for people with a compromised immune system, common species can sometimes cause disease. For example, the yeast Candida albicans, which colonises between 30 and 70% of the population, is normally harmless, but can switch to a disease-causing version that makes branching structures called hyphae. These hyphae grow fast, piercing and damaging the tissues around them. Immune cells called macrophages usually engulf invading microbes. These cells recognise sugars on the outside of C. albicans, and respond by wrapping their membranes around the yeast, drawing the microorganism in, and sealing it into closed structures called phagosomes. Then, the macrophages fill the phagosomes with acid, enzymes and destructive chemicals, which breaks the yeast down. Yet, C. albicans hyphae grow larger than macrophages, making them difficult to control. Maxson et al. have now tracked the immune response revealing how macrophages try to control large hyphae. The immune cells were quick to engulf C. albicans in its normal yeast form, but the response slowed down in the presence of hyphae. Electron microscopy revealed that the large structures were only partly taken in. Rather than form a closed phagosome, the macrophages made a cuff around the middle of the hypha, leaving the rest hanging out. The process starts with a receptor called CR3, which detects sugars on the outside of the hyphae. CR3 is a type of integrin, a molecule that sends signals from the surface to the inside of the immune cell. A network of filaments called actin assemble around the hypha, squeezing the membrane tight. The macrophage then deploys free radicals and other damaging chemicals inside the closed space. The seal is not perfect, and some molecules do leak out, but the effect slows the growth of the yeast. When a phagosome cannot engulf an invading microbe, a state that is referred to as being “frustrated”, the leaking of damaging chemicals can harm healthy tissues and lead to inflammation and disease. These findings reveal that macrophages do at least try to form a complete seal before releasing their cocktail of chemicals. Understanding how the immune system handles this situation could open the way for new treatments for C. albicans infections, and possibly similar diseases related to “frustrated engulfment” (such as asbestos exposure, where asbestos fibers are also too large to engulf). However, one next step will be to find out what happens to partly engulfed hyphae, and how this differs from the fate of fully engulfed yeast.

Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABAARs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [3H]-GABA release, induced by nicotinic receptor (nAChR) activation or K+ depolarization, and [3H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K+ solutions led to a concentration-dependent and Ca2+-dependent [3H]-GABA release in control and mdx synaptosomes. [3H]-GABA release induced by 10 μM nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K+-induced [3H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and β2-containing nAChRs were involved in nicotine-induced [3H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [3H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t1/2) and increased (44%) rate of [3H]-GABA uptake (Vmax) compared to controls. The apparent transporter affinity (Km) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABAAR in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD.

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