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Native amine dehydrogenases offer sustainable access to chiral amines, so the search for scaffolds capable of converting more diverse carbonyl compounds is required to reach the full potential of this alternative to conventional synthetic reductive aminations. Here we report a multidisciplinary strategy combining bioinformatics, chemoinformatics and biocatalysis to extensively screen billions of sequences in silico and to efficiently find native amine dehydrogenases features using computational approaches. In this way, we achieve a comprehensive overview of the initial native amine dehydrogenase family, extending it from 2,011 to 17,959 sequences, and identify native amine dehydrogenases with non-reported substrate spectra, including hindered carbonyls and ethyl ketones, and accepting methylamine and cyclopropylamine as amine donor. We also present preliminary model-based structural information to inform the design of potential (R)-selective amine dehydrogenases, as native amine dehydrogenases are mostly (S)-selective. This integrated strategy paves the way for expanding the resource of other enzyme families and in highlighting enzymes with original features., (© 2024. The Author(s).)

Biosourced and biodegradable plastics offer a promising solution to reduce environmental impacts of plastics for specific applications. Here, we report a novel bacterium named Alteromonas plasticoclasticus MED1 isolated from the marine plastisphere that forms biofilms on foils of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Experiments of degradation halo, plastic matrix weight loss, bacterial oxygen consumption and heterotrophic biosynthetic activity showed that the bacterial isolate MED1 is able to degrade PHBV and to use it as carbon and energy source. The likely entire metabolic pathway specifically expressed by this bacterium grown on PHBV matrices was shown by further genomic and transcriptomic analysis. In addition to a gene coding for a probable secreted depolymerase, a gene cluster was located that encodes characteristic enzymes involved in the complete depolymerization of PHBV, the transport of oligomers, and in the conversion of the monomers into intermediates of central carbon metabolism. The transcriptomic experiments showed the activation of the glyoxylate shunt during PHBV degradation, setting the isocitrate dehydrogenase activity as regulated branching point of the carbon flow entering the tricarboxylic acid cycle. Our study also shows the potential of exploring the natural plastisphere to discover new bacteria with promising metabolic capabilities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance., Significance: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4., (©2023 The Authors; Published by the American Association for Cancer Research.)

Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of several genes including protooncogene cellular myelocytomatosis (c-Myc). c-Myc is difficult to target directly by agents due to its disordered alpha helical protein structure and predominant nuclear localization. The epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. MT-1 is a bivalent BD inhibitor that is 100-fold more potent than the first-in-class BD inhibitor JQ1. MT-1 decreased cell viability and causes cell cycle arrest in G0/G1 phase in castration-sensitive and resistant PC cell lines in a dose-dependent fashion. The inhibition of c-Myc function by MT-1 was molecularly corroborated by the de-repression of Protein Kinase D1 (PrKD) and increased phosphorylation of PrKD substrate proteins: threonine 120, serine 11, and serine 216 amino acid residues in β-Catenin, snail, and cell division cycle 25c (CDC25c) proteins, respectively. The treatment of 3D cell cultures derived from three unique clinically annotated heavily pretreated patient-derived PC xenografts (PDX) mice models with increasing doses of MT-1 demonstrated the lowest IC 50 in tumors with c-Myc amplification and clinically resistant to Docetaxel, Cabazitaxel, Abiraterone, and Enzalutamide. An intraperitoneal injection of either MT-1 or in combination with 3jc48-3, an inhibitor of obligate heterodimerization with MYC-associated protein X (MAX), in mice implanted with orthotopic PC PDX, decreased tumor growth. This is the first pre-clinical study demonstrating potential utility of MT-1 in the treatment of PC with c-Myc dysregulation.

Evidence suggests that β-(2,6)-levan-type fructooligosaccharides (FOSs) possess higher prebiotic potential and selectivity than their β-(2,1)-inulin-type counterparts. The focus of the present work was to develop an enzymatic approach for the synthesis of levan-type FOSs, employing levanases (EC 3.2.1.65), specifically those performing endo-hydrolysis on levans. To identify new levanases, a selection of candidates was obtained via in silico exploration of the levanase family biodiversity through a sequence-driven approach. A collection of 113 candidates was screened according to their specific activities on low- and high-molecular-weight (MW) levan as well as thermal stability. The most active levanases were able to hydrolyze both types of levan with similar efficiency. This ultimately revealed 10 active, highly evolutionary distant and diverse candidate levanases, which demonstrated preferential hydrolysis of levan over inulin. The end-product profile differed significantly depending on levanase with levanbiose, levantriose, and levantetraose being the major FOSs. Among them, the catalytic properties of 5 selected potential new levanases (LEV9 from Belliella Baltica , LEV36 from Dyadobacter fermentans , LEV37 from Capnocytophaga ochracea , LEV79 from Vibrio natriegens , LEV91 from Paenarthrobacter aurescens ) were characterized, especially in terms of pH and temperature profiles, thermal stability, and kinetic parameters. The identification of these novel levanases is expected to contribute to the production of levan-type FOSs with properties surpassing those of commercial preparations.

In 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes., (© 2022 Wiley-VCH GmbH.)

There is substantial evidence that in addition to nicotine, other compounds found in tobacco smoke significantly influence smoking behavior. Further, recent years have seen an explosion in the availability of non-combusted products that deliver nicotine, such as e-cigarettes and "home-brew" vaping devices that are essentially unregulated. There are many thousands of compounds in tobacco smoke alone, and new products are constantly introducing new compounds. Uncovering which of these compounds are active, across multiple smoking-relevant subtypes of the nicotinic acetylcholine receptor (nAChR) that influence tobacco/nicotine addiction, requires a high-throughput screening (HTS) approach. Accordingly, we developed a panel of HTS-friendly cell-based assays, all performed in the same cellular background and using the same membrane potential dye readout, to measure the function of the α3β4-, α4β2-, and α6β2-nAChR subtypes. These subtypes have each been prominently and consistently associated with human smoking behavior. We validated our assays by performing pilot screening of an expanded set of the Prestwick FDA-approved drug library. The screens displayed excellent performance parameters, and moderate hit rates (mean of 1.2% across all three assays) were achieved when identifying antagonists (chosen since effects of endogenous antagonists on consumption of nicotine/tobacco products are under-studied). Validation rates using an orthogonal assay ( 86 Rb + efflux) averaged 73% across the three assays. The resulting panel of assays represents a valuable new platform with which to screen and identify nAChR subtype-selective compounds. This provides a resource for identifying smoking-related compounds in both combusted and non-combusted tobacco products, with potential relevance in the search for additional smoking-cessation therapies., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants' occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants' tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants' emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant's tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant's tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world., (© 2021. The Author(s).)

Despite the success of some nitrilases in industrial applications, there is a constant demand to broaden the catalog of these hydrolases, especially robust ones with high operational stability. By using the criteria of thermoresistance to screen a collection of candidate enzymes heterologously expressed in Escherichia coli , the enzyme Nit phym from the mesophilic organism Paraburkholderia phymatum was selected and further characterized. Its quick and efficient purification by heat treatment is of major interest for large-scale applications. The purified nitrilase displayed a high thermostability with 90% of remaining activity after 2 days at 30°C and a half-life of 18 h at 60°C, together with a broad pH range of 5.5-8.5. Its high resistance to various miscible cosolvents and tolerance to high substrate loadings enabled the quantitative conversion of 65.5 g⋅L -1 of 3-phenylpropionitrile into 3-phenylpropionic acid at 50°C in 8 h at low enzyme loadings of 0.5 g⋅L -1 , with an isolated yield of 90%. This study highlights that thermophilic organisms are not the only source of industrially relevant thermostable enzymes and extends the scope of efficient nitrilases for the hydrolysis of a wide range of nitriles, especially trans -cinnamonitrile, terephthalonitrile, cyanopyridines, and 3-phenylpropionitrile., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bessonnet, Mariage, Petit, Pellouin, Debard, Zaparucha, Vergne-Vaxelaire and de Berardinis.)

The cellular cytotoxicity of APY0201, a PIKfyve inhibitor, against multiple myeloma was initially identified in an unbiased in vitro chemical library screen. The activity of APY0201 was confirmed in all 25 cell lines tested and in 40% of 100 ex vivo patient-derived primary samples, with increased activity in primary samples harboring trisomies and lacking t(11;14). The broad anti-multiple myeloma activity of PIKfyve inhibitors was further demonstrated in confirmatory screens and showed the superior potency of APY0201 when compared to the PIKfyve inhibitors YM201636 and apilimod, with a mid-point half maximal effective concentration (EC 50 ) at nanomolar concentrations in, respectively, 65%, 40%, and 5% of the tested cell lines. Upregulation of genes in the lysosomal pathway and increased cellular vacuolization were observed in vitro following APY0201 treatment, although these cellular effects did not correlate well with responsiveness. We confirm that PIKfyve inhibition is associated with activation of the transcription factor EB, a master regulator of lysosomal biogenesis and autophagy. Furthermore, we established an assay measuring autophagy as a predictive marker of APY0201 sensitivity. Overall, these findings indicate promising activity of PIKfyve inhibitors secondary to disruption of autophagy in multiple myeloma and suggest a strategy to enrich for likely responders., (Copyright© 2020 Ferrata Storti Foundation.)

Seventy-six FDA-approved oncology drugs and emerging therapeutics were evaluated in 25 multiple myeloma (MM) and 15 non-Hodgkin's lymphoma cell lines and in 113 primary MM samples. Ex vivo drug sensitivities were mined for associations with clinical phenotype, cytogenetic, genetic mutation, and transcriptional profiles. In primary MM samples, proteasome inhibitors, dinaciclib, selinexor, venetoclax, auranofin, and histone deacetylating agents had the broadest cytotoxicity. Of interest, newly diagnosed patient samples were globally less sensitive especially to bromodomain inhibitors, inhibitors of receptor tyrosine kinases or non-receptor kinases, and DNA synthesis inhibitors. Clustering demonstrated six broad groupings of drug sensitivity linked with genomic biomarkers and clinical outcomes. For example, our findings mimic clinical observations of increased venetoclax responsiveness in t(11;14) patients but also identify an increased sensitivity profile in untreated patients, standard genetic risk, low plasma cell S-Phase, and in the absence of Gain(1q) and t(4;14). In contrast, increased ex vivo responsiveness to selinexor was associated with biomarkers of poor prognosis and later relapse patients. This "direct to drug" screening resource, paired with functional genomics, has the potential to successfully direct appropriate individualized therapeutic approaches in MM and to enrich clinical trials for likely responders.

Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo , we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as "SBI-183," suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1., (©2019 American Association for Cancer Research.)

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L -1 ·h -1 ) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L -1 ·h -1 )., (© 2019 Wiley Periodicals, Inc.)

Flavin-dependent halogenases (FHals) catalyse the halogenation of electron-rich substrates, mainly aromatics. Halogenated compounds have many applications, as pharmaceutical, agrochemicals or as starting materials for the synthesis of complex molecules. By exploring the sequenced bacterial diversity, we discovered and characterized XszenFHal, a novel FHal from Xenorhabdus szentirmaii, a symbiotic bacterium of entomopathogenic nematode. The substrate scope of XszenFHal was examined and revealed activities towards tryptophan, indole and indole derivatives, leading to the formation of the corresponding 5-chloro products. XszenFHal makes a valuable addition to the panel of flavin-dependent halogenases already discovered and enriches the potential for biotechnology applications by allowing access to 5-halogenated indole derivatives.

We explored a collection of 2-deoxyribose-5-phosphate aldolases (DERAs) from biodiversity for their nucleophile substrate promiscuity. The DERAs were screened using as nucleophiles propanone, propanal, cyclobutanone, cyclopentanone, dihydroxyacetone, and glycolaldehyde with l-glyceraldehyde-3-phosphate as an electrophile in aldol addition. A DERA from Arthrobacter chlorophenolicus (DERA Arthro ) efficiently allowed the synthesis of the corresponding aldol adducts in good yields, displaying complementarity in terms of configuration and substrate specificity with fructose-6-phosphate aldolase, the only previously known aldolase with a large nucleophile tolerance.

Background: Terpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established-mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes., Results: The potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule., Conclusion: IPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance.

Most of the "repressor, open reading frame, kinase" (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrK mitis ) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose of 1.5 10 6  s -1  M -1 and 2.7 10 5  s -1  M -1 , respectively, but also significant ones toward D-tagatose (3.5 10 2  s -1  M -1 ) and the unnatural monosaccharides D-altrose (1.1 10 4  s -1  M -1 ) and D-talose (3.4 10 2  s -1  M -1 ). Specific activities measured for all hexoses showed a high stereopreference for D- over L-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 °C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.

Trigonelline (TG; N- methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields ( Z )-2-(( N- methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into ( E )-2-(( N- methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available., Competing Interests: The authors declare no conflict of interest.

Dihydroxyacetone phosphate (DHAP)-dependent rhamnulose aldolases display an unprecedented versatility for ketones as electrophile substrates. We selected and characterized a rhamnulose aldolase from Bacteroides thetaiotaomicron (RhuABthet) to provide a proof of concept. DHAP was added as a nucleophile to several α-hydroxylated ketones used as electrophiles. This aldol addition was stereoselective and produced branched-chain monosaccharide adducts with a tertiary alcohol moiety. Several aldols were readily obtained in good to excellent yields (from 76 to 95 %). These results contradict the general view that aldehydes are the only electrophile substrates for DHAP-dependent aldolases and provide a new C-C bond-forming enzyme for stereoselective synthesis of tertiary alcohols., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transaminases are efficient tools for the stereoselective conversion of prochiral ketones into valuable chiral amines. Notably, the diversity of naturally occurring α-transaminases offers access to a wide range of L- and D-α-amino acids. We describe here two continuous colorimetric assays for the quantification of transamination activities between a keto acid and a standard donor substrate (L- or D-Glutamic acid or cysteine sulfinic acid). These assays are helpful for kinetic studies as well as for high-throughput screening of enzyme collections.

Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.

Efficient bi-enzymatic cascades combining aldolases and α-transaminases were designed for the synthesis of γ-hydroxy-α-amino acids. These recycling cascades provide high stereoselectivity, atom economy, and an equilibrium shift of the transamination. l-syn or anti-4-hydroxyglutamic acid and d-anti-4,5-dihydroxynorvaline were thus prepared in 83-95% yield in one step from simple substrates.

In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

Three regiodivergent Baeyer-Villiger mono-oxygenases (enantioselectively) oxidized a series of cyclic α,β-unsaturated ketones into (chiral) either enol-lactones or ene-lactones. An easy-to-use and efficient biocatalytic process based on a host-microorganism deprived of unwanted activities (knock-out mutant) was developed to enable the exclusive synthesis of unsaturated lactones.

A monitoring program of the pre-treatment susceptibility of clinical isolates of bacteria from diseased dogs and cats was active between the years 2002 and 2009. Susceptibility of each isolated strain to a panel of nine antibiotics (amoxicillin/clavulanic acid, ampicillin, penicillin, clindamycin, doxycycline, enrofloxacin, marbofloxacin, trimethoprim and trimethoprim/sulfamethoxazole) was assessed. The Minimum Inhibitory Concentration (MIC) of marbofloxacin was also determined by a standardized microdilution technique following CLSI recommendations. In total, 1857 bacterial strains were collected throughout Europe from cases of otitis, respiratory, urinary and dermatological infections. Although bacterial susceptibility varied for each of the antibiotics within the panel, patterns of susceptibility were similar to those described in the literature for comparable time periods and geographical areas. With a clinical resistance varying from 0 to 14.48% against the isolated strains, marbofloxacin susceptibility was very high and remains an effective antibiotic for the treatment of otitis, urinary, respiratory and dermatological infections in companion animals., (Copyright © 2014. Published by Elsevier Ltd.)

Millions of protein database entries are not assigned reliable functions, preventing the full understanding of chemical diversity in living organisms. Here, we describe an integrated strategy for the discovery of various enzymatic activities catalyzed within protein families of unknown or little known function. This approach relies on the definition of a generic reaction conserved within the family, high-throughput enzymatic screening on representatives, structural and modeling investigations and analysis of genomic and metabolic context. As a proof of principle, we investigated the DUF849 Pfam family and unearthed 14 potential new enzymatic activities, leading to the designation of these proteins as β-keto acid cleavage enzymes. We propose an in vivo role for four enzymatic activities and suggest key residues for guiding further functional annotation. Our results show that the functional diversity within a family may be largely underestimated. The extension of this strategy to other families will improve our knowledge of the enzymatic landscape.

Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr(176), but not Trp(231), resulted in the loss of TWEAK binding to Fn14 substantiating Tyr(176) as the anchoring residue. Importantly, mutation of TWEAK at Tyr(176) did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.

Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D(3) C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P < 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D(3) or 1alpha-hydroxyvitamin D(3), respectively, suggesting impaired C-25-hydroxylation of vitamin D(3). Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms.

Microbial communities in sewage sludge and green waste co-composting were investigated using culture-dependent methods and community level physiological profiles (CLPP) with Biolog Microplate. Different microbial groups characterized each stage of composting. Bacterial densities were high from beginning to end of composting, whereas actinomycete densities increased only after bio-oxidation phase i.e. after 40days. Fungal populations become particularly high during the last stage of decomposition. Cluster analyses of metabolic profiles revealed a similar separation between two groups of composts at 67days for bacteria and fungi. Principal component analysis (PCA) applied to bacterial and fungal CLPP data showed a chronological distribution of composts with two phases. The first one (before 67days), where the composts were characterized by the rapid decomposition of non-humic biodegradable organic matter, was significantly correlated to the decrease of C, C/N, organic matter (OM), fulvic acid (FA), respiration, cellulase, protease, phenoloxidase, alkaline and acid phosphatases activities. The second phase corresponding to the formation of polycondensed humic-like substances was significantly correlated to humic acid (HA) content, pH and HA/FA. The influent substrates selected on both factorial maps showed that microbial communities could adapt their metabolic capacities to the particular environment. The first phase seems to be focused on easily degradable substrate utilization whereas the maturation phase appears as multiple metabolisms, which induce the release of metabolites and their polymerization leading to humification processes., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background: Diatoms represent the predominant group of eukaryotic phytoplankton in the oceans and are responsible for around 20% of global photosynthesis. Two whole genome sequences are now available. Notwithstanding, our knowledge of diatom biology remains limited because only around half of their genes can be ascribed a function based onhomology-based methods. High throughput tools are needed, therefore, to associate functions with diatom-specific genes., Results: We have performed a systematic analysis of 130,000 ESTs derived from Phaeodactylum tricornutum cells grown in 16 different conditions. These include different sources of nitrogen, different concentrations of carbon dioxide, silicate and iron, and abiotic stresses such as low temperature and low salinity. Based on unbiased statistical methods, we have catalogued transcripts with similar expression profiles and identified transcripts differentially expressed in response to specific treatments. Functional annotation of these transcripts provides insights into expression patterns of genes involved in various metabolic and regulatory pathways and into the roles of novel genes with unknown functions. Specific growth conditions could be associated with enhanced gene diversity, known gene product functions, and over-representation of novel transcripts. Comparative analysis of data from the other sequenced diatom, Thalassiosira pseudonana, helped identify several unique diatom genes that are specifically regulated under particular conditions, thus facilitating studies of gene function, genome annotation and the molecular basis of species diversity., Conclusions: The digital gene expression database represents a new resource for identifying candidate diatom-specific genes involved in processes of major ecological relevance.

Background: Brown algae (Phaeophyceae) are phylogenetically distant from red and green algae and an important component of the coastal ecosystem. They have developed unique mechanisms that allow them to inhabit the intertidal zone, an environment with high levels of abiotic stress. Ectocarpus siliculosus is being established as a genetic and genomic model for the brown algal lineage, but little is known about its response to abiotic stress., Results: Here we examine the transcriptomic changes that occur during the short-term acclimation of E. siliculosus to three different abiotic stress conditions (hyposaline, hypersaline and oxidative stress). Our results show that almost 70% of the expressed genes are regulated in response to at least one of these stressors. Although there are several common elements with terrestrial plants, such as repression of growth-related genes, switching from primary production to protein and nutrient recycling processes, and induction of genes involved in vesicular trafficking, many of the stress-regulated genes are either not known to respond to stress in other organisms or are have been found exclusively in E. siliculosus., Conclusions: This first large-scale transcriptomic study of a brown alga demonstrates that, unlike terrestrial plants, E. siliculosus undergoes extensive reprogramming of its transcriptome during the acclimation to mild abiotic stress. We identify several new genes and pathways with a putative function in the stress response and thus pave the way for more detailed investigations of the mechanisms underlying the stress tolerance of brown algae.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes ( approximately 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.

Carboxyl (C)-terminal fragments of parathyroid hormone (PTH) oppose the calcemic, phosphaturic, and bone-resorbing effects of active hormone. To study the action of these fragments on 1,25(OH)(2)D (1,25-dihydroxyvitamin D) synthesis, we infused parathyroidectomized rats with human or rat active 1-34 or 1-84 PTH at doses selected to produce similar calcemic responses. Human active PTH influenced neither phosphate nor 1,25(OH)(2)D concentrations. However, active 1-34 rat PTH decreased phosphate to the same level as vehicle-treated rats and increased 1,25(OH)(2)D to very high levels, whereas active 1-84 PTH decreased phosphate but maintained 1,25(OH)(2)D. As the latter effect could have been due to C-terminal fragment generation during its metabolic breakdown, we infused a mixture of rat C-terminal fragments alone or with rat 1-34. The C-terminal fragments decreased 1,25(OH)(2)D and prevented hypocalcemic-induced 1,25(OH)(2)D synthesis. When infused with active rat 1-34, they lowered the 1,25(OH)(2)D level to that seen with intact rat 1-84. The C-terminal fragments did not influence either basal or rat 1-34- or 1-84-induced CYP27B1 mRNA levels, suggesting that their inhibitory effects on 1,25(OH)(2)D synthesis appears to be post-transcriptional.

Hypocalcemia secondary to vitamin D3 (D3) depletion (D-Ca-) perturbs extra- and intracellular calcium (Ca). To study the effect of cyclic nutritional changes in the D3 and calcium (Ca) repletion state, we investigated the lasting effects of calcium or D3 repletion on calcium and bone metabolism using a novel depletion-repletion-redepletion protocol. D-Ca- rats presenting osteomalacia without rickets and a significant impairment in whole body mineral content (BMC) accretion were repleted with either calcium alone [3% (Ca+3) or 0.5% (Ca+0.5)] or D3 and then switched back to the original D-Ca- diet. All repletion protocols, except Ca+0.5, normalized serum (S) Ca and parathyroid hormone (PTH) but Ca+3 exhibited growth retardation and hypophosphatemia. D3 normalized BMC in D-Ca- and healed osteomalacia while Ca+0.5 led to 50% normalization. In contrast, rickets with no BMC accretion was observed in Ca+3 most likely secondary to hypophosphatemia. Upon redepletion, S Ca rapidly decreased while S PTH and phosphate increased. D3 and Ca+0.5 survived the redepletion protocols but all Ca+3 died within 5 days upon sudden Ca withdrawal whereas progressive Ca redepletion significantly delayed the death rate. Data indicate that during the calcium redepletion period, correction of hypophosphatemia in Ca+3 allowed calcification of the enlarged growth plates thus resulting in an increased demand for calcium. It is postulated that this increased demand for calcium, in conjunction with low dietary calcium and the bone calcium reservoir incapacity to provide sufficient calcium to sustain S Ca, led to the observed acute hypocalcemia which was most likely the cause of death. This hypothesis is further supported by the observation that Ca+3 submitted to a progressive Ca deprivation exhibited a delay in death rate, a progressive involution of rickets and survival only upon return to the D-Ca- phenotype. Furthermore, in Ca+3, increasing dietary phosphate by 0.6% to achieve a Ca/P ratio similar to Ca+0.5 or D3 prevented the development of hypophosphatemia, slightly increased S Ca, significantly increased BMC, prevented the development of rickets and allowed 100% survival during rapid calcium withdrawal. Collectively, data clearly demonstrate the importance of the dietary Ca/P ratio to maintain S Ca/P at optimum concentrations for bone health.

How do we extrapolate the final position of hand trajectory that suddenly vanishes behind a wall? Studies showing maintenance of cortical activity after objects in motion disappear suggest that internal model of action may be recalled to reconstruct the missing part of the trajectory. Although supported by neurophysiological and brain imaging studies, behavioural evidence for this hypothesis is sparse. Further, in humans, it is unknown if the recall of internal model of action at motion observation can be tuned with kinematic features of movement. Here, we propose a novel experiment to address this question. Each stimulus consisted of a dot moving either upwards or downwards, and corresponding to vertical arm movements that were masked in the last part of the trajectory. The stimulus could either move according to biological and or non-biological kinematic laws of pointing tasks. We compared subjects' estimations of the stimulus vanishing or final positions after biological and after non-biological motion displays. Subjects systematically overestimated the vanishing and final position for the two directions (up and down) and the two kinematics displayed (biological and non-biological). However, estimation of the final position decreased in precision and increased in variability for movements that violated the kinematic laws of arm pointing task. The results suggest that motion inference does not rely only upon visual extrapolating mechanisms based on past visual trajectory information. We propose that motion estimation relies on internal models that contain specific kinematic details of vertical arm movement, which can be rapidly recalled during motion observation.

Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests approximately 900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.

Aims: The purpose of this multicentre clinical randomized controlled blinded prospective trial was to determine whether an inspiratory impedance threshold device (ITD), when used in combination with active compression-decompression (ACD) cardiopulmonary resuscitation (CPR), would improve survival rates in patients with out-of-hospital cardiac arrest., Methods and Results: Patients were randomized to receive either a sham (n = 200) or an active impedance threshold device (n = 200) during advanced cardiac life support performed with active compression-decompression cardiopulmonary resuscitation. The primary endpoint of this study was 24 h survival. The 24 h survival rates were 44/200 (22%) with the sham valve and 64/200 (32%) with the active valve (P = 0.02). The number of patients who had a return of spontaneous circulation (ROSC), intensive care unit (ICU) admission, and hospital discharge rates was 77 (39%), 57 (29%), and 8 (4%) in the sham valve group versus 96 (48%) (P = 0.05), 79 (40%) (P = 0.02), and 10 (5%) (P = 0.6) in the active valve group. Six out of ten survivors in the active valve group and 1/8 survivors in the sham group had normal neurological function at hospital discharge (P = 0.1)., Conclusion: The use of an impedance valve in patients receiving active compression-decompression cardiopulmonary resuscitation for out-of-hospital cardiac arrest significantly improved 24 h survival rates.

Chromosome 14 is one of five acrocentric chromosomes in the human genome. These chromosomes are characterized by a heterochromatic short arm that contains essentially ribosomal RNA genes, and a euchromatic long arm in which most, if not all, of the protein-coding genes are located. The finished sequence of human chromosome 14 comprises 87,410,661 base pairs, representing 100% of its euchromatic portion, in a single continuous segment covering the entire long arm with no gaps. Two loci of crucial importance for the immune system, as well as more than 60 disease genes, have been localized so far on chromosome 14. We identified 1,050 genes and gene fragments, and 393 pseudogenes. On the basis of comparisons with other vertebrate genomes, we estimate that more than 96% of the chromosome 14 genes have been annotated. From an analysis of the CpG island occurrences, we estimate that 70% of these annotated genes are complete at their 5' end.

CYP27A is considered the main vitamin D(3) (D(3))-25 hydroxylase in humans. Our purpose was to evaluate the effect of the D(3) nutritional and hormonal status on hepatic CYP27A mRNA, cellular distribution, transcription rate, and enzyme activity. Studies were carried out in normal and in D-depleted rats supplemented with D(3), 25OHD(3), or 1,25(OH)(2)D(3). CYP27A exhibited a significant gender difference and was observed throughout the hepatic acinus not only in hepatocytes but also in sinusoidal endothelial, stellate, and Kupffer cells. Neither D(3) nor 25OHD(3) influenced CYP27A mRNA levels. However, 1,25(OH)(2)D(3) repletion led to a 60% decrease in CYP27A mRNA, which was accompanied by a 46% decrease in mitochondrial D(3)-25 hydroxylase activity. The effect of 1,25(OH)(2)D(3) was mediated by a significant decrease in CYP27A transcription, whereas its mRNA half-life remained unchanged. Our data indicate that CYP27A is present in hepatic parenchymal and sinusoidal cells and that the gene transcript is not influenced by the D(3) nutritional status but is transcriptionally regulated by 1,25(OH)(2)D(3) exposure.

The phenomenological tradition stands opposed to contemporary cognitive science in this, that so far from being a mere thing in the physical world, a thing whose mind would contain some mental representation, the lived body is constituted in its sense of being for the subject by being used and actively appropriated by this self-same subject, as agent. Though not already generally acknowledged, there are important similarities between this conception and one, which emerges from the literature of brain cartography over the last twenty years, that of a functional morphogenetic neurodynamics, induced and modulated by experience in a relatively autonomous way, in relation both to the anatomic structures of the body, as well as the cytoarchitectonic frontiers of brain tissue. Taking advantage of the latter similarities, neuroscientific data can be employed to confirm the Husserlian theory of constitution against the attempts on the part of cognitive science to discredit it, and this whether we are talking about its later modification at the hands of Merleau-Ponty or the original version, which is that of a subjective (transcendental) auto-constitution of the lived body on the basis of the practico-kinaesthetic experience of <>.