Waste cooking oil and molasses for the sustainable production of extracellular lipase by Saitozyma flava.

Organic waste valorization is one of the principal goals of the circular economy. Bioprocesses offer a promising approach to achieve this goal by employing microorganisms to convert organic feedstocks into high value products through their metabolic activities. In this study, a fermentation process for yeast cultivation and extracellular lipase production was developed by utilizing food waste. Lipases are versatile enzymes that can be applied in a wide range of industrial fields, from detergent, leather, and biodiesel production to food and beverage manufacturing. Among several oleaginous yeast species screened, Saitozyma flava was found to exhibit the highest secreted lipase activity on pNP-butyrate, pNP-caproate, and pNP-caprylate. The production medium was composed of molasses, a by-product of the sugar industry, which provided nutrients for yeast biomass formation. At the same time, waste cooking oil was employed to induce and enhance extracellular lipase production. After 48 h of process, 20 g/L of yeast biomass and 150 mU/mgdw of lipase activity were achieved, with a productivity of 3 mU/mgdw /h. The purified lipase from S. flava showed optimal performances at temperature 28°C and pH 8.0, exhibiting a specific activity of 62 U/mg when using p-NPC as substrate.

Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis.

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5-3 h) with high conversions (95-99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50-mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.

Structural insights into the desymmetrization of bulky 1,2-dicarbonyls through enzymatic monoreduction.

Benzil reductases are dehydrogenases preferentially active on aromatic 1,2-diketones, but the reasons for this peculiar substrate recognition have not yet been clarified. The benzil reductase (KRED1-Pglu) from the non-conventional yeast Pichia glucozyma showed excellent activity and stereoselectivity in the monoreduction of space-demanding aromatic 1,2-dicarbonyls, making this enzyme attractive as biocatalyst in organic chemistry. Structural insights into the stereoselective monoreduction of 1,2-diketones catalyzed by KRED1-Pglu were investigated starting from its 1.77 Å resolution crystal structure, followed by QM and classical calculations; this study allowed for the identification and characterization of the KRED1-Pglu reactive site. Once identified the recognition elements involved in the stereoselective desymmetrization of bulky 1,2-dicarbonyls mediated by KRED1-Pglu, a mechanism was proposed together with an in silico prediction of substrates reactivity.

Cloning the putative gene of vinyl phenol reductase of Dekkera bruxellensis in Saccharomyces cerevisiae.

Vinylphenol reductase of Dekkera bruxellensis, the characteristic enzyme liable for "Brett" sensory modification of wine, has been recently recognized to belong to the short chain dehydrogenases/reductases family. Indeed, a preliminary biochemical characterisation has conferred to the purified protein a dual significance acting as superoxide dismutase and as a NADH-dependent reductase. The present study aimed for providing a certain identification of the enzyme by cloning the VPR gene in S. cerevisiae, a species not producing ethyl phenols. Transformed clones of S. cerevisiae resulted capable of expressing a biologically active form of the heterologous protein, proving its role in the conversion of 4-vinyl guaiacol to 4-ethyl guaiacol. A VPR specific protein activity of 9 ± 0.6 mU/mg was found in crude extracts of S. cerevisiae recombinant strain. This result was confirmed in activity trials carried out with the protein purified from transformant cells of S. cerevisiae by a his-tag purification approach; in particular, VPR-enriched fractions showed a specific activity of 1.83 ± 0.03 U/mg at pH 6.0. Furthermore, in agreement with literature, the purified protein behaves like a SOD, with a calculated specific activity of approximatively 3.41 U/mg. The comparative genetic analysis of the partial VPR gene sequences from 17 different D. bruxellesis strains suggested that the observed polymorphism (2.3%) and the allelic heterozygosity state of the gene do not justify the well described strain-dependent character in producing volatile phenols of this species. Actually, no correlation exists between genotype membership of the analysed strains and their capability to release off-flavours. This work adds valuable knowledge to the study of D. bruxellensis wine spoilage and prepare the ground for interesting future industrial applications.

Coronary calcium screening with dual-source CT: reliability of ungated, high-pitch chest CT in comparison with dedicated calcium-scoring CT.

PURPOSE: To investigate the reliability of ungated, high-pitch dual-source CT for coronary artery calcium (CAC) screening. MATERIALS AND METHODS: One hundred and eighty-five smokers underwent a dual-source CT examination with acquisition of two sets of images during the same session: (a) ungated, high-pitch and high-temporal resolution acquisition over the entire thorax (i.e., chest CT); (b) prospectively ECG-triggered acquisition over the cardiac cavities (i.e., cardiac CT). RESULTS: Sensitivity and specificity of chest CT for detecting positive CAC scores were 96.4 % and 100 %, respectively. There was excellent inter-technique agreement for determining the quantitative CAC score (ICC = 0.986). The mean difference between the two techniques was 11.27, representing 1.81 % of the average of the two techniques. The inter-technique agreement for categorizing patients into the four ranks of severity was excellent (weighted kappa = 0.95; 95 % CI 0.93-0.98). The inter-technique differences for quantitative CAC scores did not correlate with BMI (r = 0.05, p = 0.575) or heart rate (r = -0.06, p = 0.95); 87.2 % of them were explained by differences at the level of the right coronary artery (RCA: 0.8718; LAD: 0.1008; LCx: 0.0139; LM: 0.0136). CONCLUSION: Ungated, high-pitch dual-source CT is a reliable imaging mode for CAC screening in the conditions of routine chest CT examinations. KEY POINTS: • CAC is an independent risk factor for major cardiac events. • ECG-gated techniques are the reference standard for calcium scoring. • Great interest is directed toward calcium scoring on non-gated chest CT examinations. • Reliable calcium scoring can be obtained with dual-source CT in a high-pitch mode.

Stereoelectronic effects in the reaction of aromatic substrates catalysed by Halomonas elongata transaminase and its mutants.

A transaminase from Halomonas elongata and four mutants generated by an in silico-based design were recombinantly produced in E. coli, purified and applied to the amination of mono-substituted aromatic carbonyl-derivatives. While benzaldehyde derivatives were excellent substrates, only NO2-acetophenones were transformed into the (S)-amine with a high enantioselectivity. The different behaviour of wild-type and mutated transaminases was assessed by in silico substrate binding mode studies.

Enzymatic reduction of acetophenone derivatives with a benzil reductase from Pichia glucozyma (KRED1-Pglu): electronic and steric effects on activity and enantioselectivity.

A recombinant ketoreductase from Pichia glucozyma (KRED1-Pglu) was used for the enantioselective reduction of various mono-substituted acetophenones. Reaction rates of meta- and para-derivatives were consistent with the electronic effects described by σ-Hammett coefficients; on the other hand, enantioselectivity was determined by an opposite orientation of the substrate in the binding pocket. Reduction of ortho-derivatives occurred only with substrates bearing substituents with low steric impact (i.e., F and CN). Reactivity was controlled by stereoelectronic features (C[double bond, length as m-dash]O length and charge, shape of LUMO frontier molecular orbitals), which can be theoretically calculated.

Stereoselective reduction of aromatic ketones by a new ketoreductase from Pichia glucozyma.

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED1-Pglu was conducted; in contrast to most other known ketoreductases, KRED1-Pglu prefers space-demanding substrates, which are often converted with high stereoselectivity. A molecular modelling study was carried out for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrates. As a result, a new useful catalyst was identified, enabling the enantioselective preparation of different aromatic alcohols and hydroxyketones.

Radiological and clinical features of cerebral sinovenous thrombosis in newborns and older children.

BACKGROUND AND PURPOSE: Cerebral sinovenous thrombosis (CSVT) represents an increasingly recognized cause of pediatric stroke. Our purpose was to assess gender and age differences in the etiology, clinical presentation, and imaging features of CSVT in neonates and older children. METHODS: Subjects aged newborn to 18 years diagnosed with CSVT at the Lille university hospital between 2011 and 2014 were included. RESULTS: Eleven neonates and 16 non-neonates constituted the study population. The incidence of CSVT was significantly higher in male newborns. Clinical presentation did not vary significantly between the groups. Risk factors were age-dependent, with acute systemic illnesses significantly predominating in neonates (54%), whereas local infections, prothrombotic conditions, and trauma were more common in older children (36, 27, and 27% respectively). No predisposing factor could be identified in 36% of the neonates as compared to less than 5% of the non-neonates. Thrombosis of the deep venous structures was documented in 73% of the neonates whereas involvement of the superficial sinuses was significantly more frequent in the non-neonates group. Venous infarctions and extraparenchymal hemorrhages were significantly more frequent in the neonates group. CONCLUSION: Male patients are at higher risk for CSVT than females. In neonates, involvement of the deep venous structures is significantly more common. Brain parenchymal and extraparenchymal changes occur more frequently in this age group than in older children.

Comparison of four software packages for CT lung volumetry in healthy individuals.

OBJECTIVES: To compare CT lung volumetry (CTLV) measurements provided by different software packages, and to provide normative data for lung densitometric measurements in healthy individuals. METHODS: This retrospective study included 51 chest CTs of 17 volunteers (eight men and nine women; mean age, 30 ± 6 years), who underwent spirometrically monitored CT at total lung capacity (TLC), functional residual capacity (FRC), and mean inspiratory capacity (MIC). Volumetric differences assessed by four commercial software packages were compared with analysis of variance (ANOVA) for repeated measurements and benchmarked against the threshold for acceptable variability between spirometric measurements. Mean lung density (MLD) and parenchymal heterogeneity (MLD-SD) were also compared with ANOVA. RESULTS: Volumetric differences ranged from 12 to 213 ml (0.20 % to 6.45 %). Although 16/18 comparisons (among four software packages at TLC, MIC, and FRC) were statistically significant (P < 0.001 to P = 0.004), only 3/18 comparisons, one at MIC and two at FRC, exceeded the spirometry variability threshold. MLD and MLD-SD significantly increased with decreasing volumes, and were significantly larger in lower compared to upper lobes (P < 0.001). CONCLUSIONS: Lung volumetric differences provided by different software packages are small. These differences should not be interpreted based on statistical significance alone, but together with absolute volumetric differences. KEY POINTS: • Volumetric differences, assessed by different CTLV software, are small but statistically significant. • Volumetric differences are smaller at TLC than at MIC and FRC. • Volumetric differences rarely exceed spirometric repeatability thresholds at MIC and FRC. • Differences between CTLV measurements should be interpreted based on comparison of absolute differences. • MLD increases with decreasing volumes, and is larger in lower compared to upper lobes.

Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates.

Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase.

New insights into glycopeptide antibiotic binding to cell wall precursors using SPR and NMR spectroscopy.

Glycopeptide antibiotics, such as vancomycin and teicoplanin, are used to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. They inhibit bacterial cell wall biosynthesis by binding to the D-Ala-D-Ala C-terminus of peptidoglycan precursors. Vancomycin-resistant bacteria replace the dipeptide with the D-Ala-D-Lac depsipeptide, thus reducing the binding affinity of the antibiotics with their molecular targets. Herein, studies of the interaction of teicoplanin, teicoplanin-like A40926, and of their semisynthetic derivatives (mideplanin, MDL63,246, dalbavancin) with peptide analogues of cell-wall precursors by NMR spectroscopy and surface plasmon resonance (SPR) are reported. NMR spectroscopy revealed the existence of two different complexes in solution, when the different glycopeptides interact with Ac2KdAlaDAlaOH. Despite the NMR experimental conditions, which are different from those employed for the SPR measurements, the NMR spectroscopy results parallel those deduced in the chip with respect to the drastic binding difference existing between the D-Ala and the D-Lac terminating analogues, confirming that all these antibiotics share the same primary molecular mechanism of action and resistance. Kinetic analysis of the interaction between the glycopeptide antibiotics and immobilized AcKdAlaDAlaOH by SPR suggest a dimerization process that was not observed by NMR spectroscopy in DMSO solution. Moreover, in SPR, all glycopeptides with a hydrophobic acyl chain present stronger binding with a hydrophobic surface than vancomycin, indicating that additional interactions through the employed surface are involved. In conclusion, SPR provides a tool to differentiate between vancomycin and other glycopeptides, and the calculated binding affinities at the surface seem to be more relevant to in vitro antimicrobial activity than the estimations from NMR spectroscopy analysis.

Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes.

BACKGROUND: Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes. RESULTS: Recombinant S. cerevisiae BY4741∆Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, α-methyl-trans-cinnamaldehyde, and trans-ß-methyl-ß-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity. CONCLUSIONS: The OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes.

Thoracic applications of dual energy.

Computed tomography (CT) is the core imaging modality for the evaluation of thoracic disorders. With the recently developed dual-energy CT (DECT) technique, the clinical utility of CT in the management of pulmonary diseases can be expanded. The most actively investigated principle of dual energy is material decomposition based on attenuation differences at different energy levels. This technique provides two key insights into lung physiology, that is, regional perfusion and ventilation. This functional information is obtained in addition to morphologic information because high-resolution thoracic anatomy is entirely preserved on dual-energy thoracic CT. The second major possibility offered by DECT is virtual monochromatic imaging that represents a new option for standard chest CT in daily routine. In this review, imaging principles and clinical applications of dual-energy thoracic CT are described. Knowledge of the applications of DECT may lead to wider use of this technique in the field of respiratory disorders.

First-pass perfusion of non-small-cell lung cancer (NSCLC) with 64-detector-row CT: a study of technique repeatability and intra- and interobserver variability.

PURPOSE: This study was done to prospectively assess the repeatability and intra- and interobserver variability of first-pass perfusion with 64-detector-row computed tomography (CT) in non-small-cell lung cancer (NSCLC) with a maximum diameter of up to 8 cm. MATERIALS AND METHODS: Twelve patients with NSCLC underwent 64-detector-row first-pass CT perfusion (CTP) of the whole tumour. Two different techniques were used according to lesion size (cine mode; sequential mode). After 24 h, each study was repeated to assess repeatability. Lesion blood volume (BV), blood flow (BF), mean transit time (MTT) and peak enhancement intensity (PEI) were automatically calculated by two chest radiologists in two different reading sessions. Intra- and interobserver variability was also assessed. RESULTS: The first-pass CTP technique was repeatable and precise with within-subject coefficient of variation (WCV) of 9.3, 16.4, 11.2 and 14.9 %, respectively, for BV, BF, MTT and PEI. High intra- and interobserver agreement was demonstrated for each perfusion parameter, with Cronbach's α coefficients and intraclass correlation coefficients ranging from 0.99 to 1. Precision of measurements was slightly better for intraobserver analysis with WCV ranging between 1.05 and 3.03 %. CONCLUSIONS: Non-small-cell lung cancer first-pass perfusion performed with 64-detector-row CT showed good repeatability and high intra- and interobserver agreement for all perfusion parameters and may be considered a reliable and robust tool for assessing tumour vascularisation.

Enviromental endocrine disruptor risks in the central nervous system: Neurotoxic effects of PFOS and glyphosate.

Endocrine disruptors (EDs) pose significant risks to human and environmental health, with potential implications for neurotoxicity. This study investigates the synergistic neurotoxic effects of perfluorooctane sulfonate (PFOS) and glyphosate (GLY), two ubiquitous EDs, using SHSY5Y neuronal and C6 astrocytic cell lines. While individual exposures to PFOS and glyphosate at non-toxic concentrations did not induce significant changes, their combination resulted in a marked increase in oxidative stress and neuroinflammatory responses. Specifically, the co-exposure led to elevated levels of interleukin-6, tumor necrosis factor alpha, and interferon gamma, along with reduced interleukin-10 expression, indicative of heightened neuroinflammatory processes. These findings underscore the importance of considering the synergistic interactions of EDs in assessing neurotoxic risks and highlight the urgent need for further research to mitigate the adverse effects of these compounds on neurological health.

Effect of Pesticide Vinclozolin Toxicity Exposure on Cardiac Oxidative Stress and Myocardial Damage.

(1) Background: Vinclozolin is a popular fungicide used in fruit, ornamental plants, and vegetable crops. It has recently been seen that prolonged exposure to VZN can cause human or animal health damage to various organs, but little is known to date about its cardiovascular effects. In this study, we addressed the chronic effects of VZN on the myocardium and the enzymes involved in the cardiovascular function. (2) Methods: The animals were divided into four groups: group 1 served as the control, group 2 received 1 mg/kg of VZN by gavage, group 3 received 30 mg/kg of VZN by gavage, and group 4 received 100 mg/kg of VZN by gavage, for 30 days. (3) Results: Results showed that 100 mg/kg VZN markedly increased the plasma concentration of cardiac markers (CK-MB, cTnT, ANP, BNP). Moreover, compared to the control group, VZN treatment decreased the activity of SOD, CAT, and GPx, and downregulated the mRNA expression levels of Nrf2. Furthermore, collagen deposition was amplified owing to 100 mg/kg VZN cardiotoxicity. This harmful effect was confirmed by a histological study using hematoxylin and eosin (H&E) and Masson's trichrome staining. (4) Conclusion: Overall, our results proved the cardiotoxicity caused by chronic exposure to VZN.

Chemoenzymatic deacylation of ramoplanin.

The chemoenzymatic deacylation of ramoplanin A2 is described for the first time: ramoplanin A2 was Boc-protected and hydrogenated to Boc-protected tetrahydroramoplanin, which was subsequently deacylated using an acylase from Actinoplanes utahensis NRRL 12052. The chemoenzymatic process proceeded with 80% overall yield, which favourably compares with the previously described chemical deacylation.

Characterization, genome analysis and genetic tractability studies of a new nanocellulose producing Komagataeibacter intermedius isolate.

Bacterial nanocellulose (BC) is a highly versatile biopolymer currently pursued as a material of choice in varied themes of biomedical and material science research fields. With the aim to extend the biotechnological applications, the genetic tractability of the BC producers within the Komagataeibacter genus and its potential as an alternative host chassis in synthetic biology have been extensively studied. However, such studies have been largely focused on the model Komagataeibacter spp. Here, we present a novel K. intermedius strain capable of utilizing glucose, and glycerol sources for biomass and BC synthesis. Genome assembly identified one bacterial cellulose synthetase (bcs) operon containing the complete gene set encoding the BC biogenesis machinery (bcsI) and three additional copies (bcsII-IV). Investigations on the genetic tractability confirmed plasmid transformation, propagation of vectors with pBBR1 and p15A origin of replications and constitutive and inducible induction of recombinant protein in K. intermedius ENS15. This study provides the first report on the genetic tractability of K. intermedius, serving as starting point towards future genetic engineering of this strain.

Enzymatic continuous-flow preparation of nature-inspired phenolic esters as antiradical and antimicrobial agents.

A collection of nature-inspired lipophilic phenolic esters have been prepared by an enzymatic synthesis under flow conditions, using the immobilized lipase B from Candida antarctica (Novozyme 435®) as a catalyst in cyclopentyl methyl ether (CPME), a non-conventional and green solvent. Their antimicrobial activity against four selected bacterial strains together with their efficiency as radical scavengers were evaluated. The obtained compounds were characterized by enhanced lipophilicity in comparison with the parent non-esterified compounds, which increased the possibility of their use as additives in the food industry.