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CTGrepeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of thisCTGexprepeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined byRNA sequencingand alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4,CYP2J2,DMD,MBNL3,CACNA1H,ROCK2,ACTB) or their association with splicing (SMN2,GCFC2,MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT,TNNT2,TTN,TPM1,SYNE1,CACNA1A,MTMR1,NEBL,TPM1),cellular signaling(NCOR2,CLIP1,LRRFIP2,CLASP1,CAMK2G), and other DM1-related genes (i.e.,NUMA1,MBNL2,LDB3) in addition to the disease-causingDMPKgene itself. Subsequent validation using a selected gene subset, includingMBNL1,MBNL2,INSR,ADD3, andCRTC2, further confirmed correction of the spliceopathy followingCTGexprepeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of theCTGexprepeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1.

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action—they form binary complexes with FKBP through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mTOR and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles that are named rapafucins using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin., Graphical abstract

Magnesium-sensitive transient receptor potential melastatin (TRPM) ion channels, TRPM6 and TRPM7, are present in several organs, but their roles in the heart remain unclear. Therefore, here, we studied the expression patterns of TRPM6 and TRPM7 in normal and diseased myocardium. Cardiac atrial tissue and cardiomyocytes were obtained from healthy pigs and undiseased human hearts as well as from hearts of patients with ischemic heart disease (IHD) or atrial fibrillation (AF). Immunofluorescence and ELISA were used to detect TRP proteins. TRPM6 and TRPM7 immunofluorescence signals, localized at/near the cell surface or intracellularly, were detected in pig and human atrial tissues. The TRP channel modulators carvacrol (CAR, 100 µM) or 2-aminoethoxydiphenyl borate (2-APB, 500 µM) decreased the TRPM7 signal, but enhanced that of TRPM6. At a higher concentration (2 mM), 2-APB enhanced the signals of both proteins. TRPM6 and TRPM7 immunofluorescence signals and protein concentrations were increased in atrial cells and tissues from IHD or AF patients. TRPM6 and TRPM7 proteins were both detected in cardiac atrial tissue, with relatively similar subcellular localization, but distinctive drug sensitivity profiles. Their upregulated expression in IHD and AF suggests a possible role of the channels in cardiac atrial disease. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:23 ispartof: location:Switzerland status: published

Due to the presence of a transmembrane domain, the subcellular mobility plan of membrane-bound or membrane-tethered transcription factors (MB-TFs) differs from that of their cytosolic counterparts. The MB-TFs are mostly locked in (sub)cellular membranes, until they are released by a proteolytic cleavage event or when the transmembrane domain (TMD) is omitted from the transcript due to alternative splicing. Here, we review the current knowledge on the proteolytic activation mechanisms of MB-TFs in plants, with a particular focus on regulated intramembrane proteolysis (RIP), and discuss the analogy with the proteolytic cleavage of MB-TFs in animal systems. We present a comprehensive inventory of all known and predicted MB-TFs in the model plant Arabidopsis thaliana and examine their experimentally determined or anticipated subcellular localizations and membrane topologies. We predict proteolytically activated MB-TFs by the mapping of protease recognition sequences and structural features that facilitate RIP in and around the TMD, based on data from metazoan intramembrane proteases. Finally, the MB-TF functions in plant responses to environmental stresses and in plant development are considered and novel functions for still uncharacterized MB-TFs are forecasted by means of a regulatory network-based approach.

Reading fluency is based on the automatic visual recognition of words. As a manifestation of the automatic processing of words, an automatic deviance detection of visual word stimuli can be observed in the early stages of visual recognition. To clarify whether this phenomenon occurs with Japanese kanji compounds—since their lexicality is related to semantic association—we investigated the brain response by utilizing three types of deviants: differences in font type, lexically correct or incorrect Japanese kanji compound words and pseudo-kanji characters modified from correct and incorrect compounds. We employed magnetoencephalography (MEG) to evaluate the spatiotemporal profiles of the related brain regions. The study included 22 adult native Japanese speakers (16 females). The abovementioned three kinds of stimuli containing 20% deviants were presented during the MEG measurement. Activity in the occipital pole region of the brain was observed upon the detection of font-type deviance within 250 ms of stimulus onset. Although no significant activity upon detecting lexically correct/incorrect kanji compounds or pseudo-kanji character deviations was observed, the activity in the posterior transverse region of the collateral sulcus (pCoS)—which is a fusiform neighboring area—was larger when detecting lexically correct kanji compounds than when detecting pseudo-kanji characters. Taken together, these results support the notion that the automatic detection of deviance in kanji compounds may be limited to a low-level feature, such as the stimulus stroke thickness.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of $$\upalpha$$ α -helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/.

Insecticide resistant pests become increasingly difficult to control in current day agriculture. Because of environmental and health concerns, the insecticide portfolio to combat agricultural pests is gradually decreasing. It is therefore crucial to make rational decisions on insecticide use to assure effective resistance management. However, resistance monitoring programs that inform on pest susceptibility and resistance are not yet common practice in agriculture. Molecular markers of resistance that are turned into convenient diagnostic tools are urgently needed and will only increase in importance. This review investigates which factors determine the strength, diagnostic value, and success of a diagnostic marker, and in which cases recent technical advances might provide new opportunities for decision making in an operational meaningful way.

Saccharolobus spindle-shaped virus 1 (SSV1) was one of the first viruses identified in the archaeal kingdom. Originally isolated from a Japanese species of Saccharolobus back in 1984, it has been extensively used as a model system for genomic, transcriptomic, and proteomic studies, as well as to unveil the molecular mechanisms governing the host–virus interaction. The purpose of this mini review is to supply a compendium of four decades of research on the SSV1 virus.

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models. ispartof: TOXINS vol:13 issue:12 ispartof: location:Switzerland status: published

Nucleotide sugar (NS) dehydratases play a central role in the biosynthesis of deoxy and amino sugars, which are involved in a variety of biological functions in all domains of life. Bacteria are true masters of deoxy sugar biosynthesis as they can produce a wide range of highly specialized monosaccharides. Indeed, deoxy and amino sugars play important roles in the virulence of gram-positive and gram-negative pathogenic species and are additionally involved in the biosynthesis of diverse macrolide antibiotics. The biosynthesis of deoxy sugars relies on the activity of NS dehydratases, which can be subdivided into three groups based on their structure and reaction mechanism. The best-characterized NS dehydratases are the 4,6-dehydratases that, together with the 5,6-dehydratases, belong to the NS-short-chain dehydrogenase/reductase superfamily. The other two groups are the less abundant 2,3-dehydratases that belong to the Nudix hydrolase superfamily and 3-dehydratases, which are related to aspartame aminotransferases. 4,6-Dehydratases catalyze the first step in all deoxy sugar biosynthesis pathways, converting nucleoside diphosphate hexoses to nucleoside diphosphate-4-keto-6-deoxy hexoses, which in turn are further deoxygenated by the 2,3- and 3-dehydratases to form dideoxy and trideoxy sugars. In this review, we give an overview of the NS dehydratases focusing on the comparison of their structure and reaction mechanisms, thereby highlighting common features, and investigating differences between closely related members of the same superfamilies.

Early Access; International audience; A growing world population as well as the need to enhance sustainability and health create challenges for crop breeding. To address these challenges, not only quantitative but also qualitative improvements are needed, especially regarding the macro- and micronutrient composition and content. In this review, we describe different examples of how the nutritional quality of crops and the bioavailability of individual nutrients can be optimised. We focus on increasing protein content, the use of alternative protein crops and improving protein functionality. Furthermore, approaches to enhance the content of vitamins and minerals as well as healthy specialised metabolites and long-chain polyunsaturated fatty acids are considered. In addition, methods to reduce antinutrients and toxins are presented. These approaches could help to decrease the 'hidden hunger' caused by micronutrient deficiencies. Furthermore, a more diverse crop range with improved nutritional profile could help to shift to healthier and more sustainable plant-based diets.

To feed successfully, ticks must bypass or suppress the host's defense mechanisms, particularly the immune system. To accomplish this, ticks secrete specialized immunomodulatory proteins into their saliva, just like many other blood-sucking parasites. However, the strategy of ticks is rather unique compared to their counterparts. Ticks' tendency for gene duplication has led to a diverse arsenal of dozens of closely related proteins from several classes to modulate the immune system's response. Among these are chemokine-binding proteins, complement pathways inhibitors, ion channels modulators, and numerous poorly characterized proteins whose functions are yet to be uncovered. Studying tick immunomodulatory proteins would not only help to elucidate tick-host relationships but would also provide a rich pool of potential candidates for the development of immunomodulatory intervention drugs and potentially new vaccines. In the present review, we will attempt to summarize novel findings on the salivary immunomodulatory proteins of ticks, focusing on biomolecular targets, structure-activity relationships, and the perspective of their development into therapeutics.

Cytochrome P450 mediated metabolism is a well-known mechanism of insecticide resistance. However, to what extent qualitative or quantitative changes are responsible for increased metabolism, is not well understood. Increased expression of P450 genes is most often reported, but the underlying regulatory mechanisms remain widely unclear. In this study, we investigate CYP392A16, a P450 from the polyphagous and major agricultural pest Tetranychus urticae. High expression levels of CYP392A16 and in vitro metabolism assays have previously associated this P450 with abamectin resistance. Here, we show that CYP392A16 is primarily localized in the midgut epithelial cells, as indicated by immunofluorescence analysis, a finding also supported by a comparison between feeding and contact toxicity bioassays. Silencing via RNAi of CYP392A16 in a highly resistant T. urticae population reduced insecticide resistance levels from 3400- to 1900- fold, compared to the susceptible reference strain. Marker-assisted backcrossing, using a single nucleotide polymorphism (SNP) found in the CYP392A16 allele from the resistant population, was subsequently performed to create congenic lines bearing this gene in a susceptible genetic background. Toxicity assays indicated that the allele derived from the resistant strain confers 3.6-fold abamectin resistance compared to the lines with susceptible genetic background. CYP392A16 is over-expressed at the same levels in these lines, pointing to cis-regulation of gene expression. In support of that, functional analysis of the putative promoter region from the resistant and susceptible parental strains revealed a higher reporter gene expression, confirming the presence of cis-acting regulatory mechanisms.

To feed successfully, ticks must bypass or suppress the host’s defense mechanisms, particularly the immune system. To accomplish this, ticks secrete specialized immunomodulatory proteins into their saliva, just like many other blood-sucking parasites. However, the strategy of ticks is rather unique compared to their counterparts. Ticks’ tendency for gene duplication has led to a diverse arsenal of dozens of closely related proteins from several classes to modulate the immune system’s response. Among these are chemokine-binding proteins, complement pathways inhibitors, ion channels modulators, and numerous poorly characterized proteins whose functions are yet to be uncovered. Studying tick immunomodulatory proteins would not only help to elucidate tick-host relationships but would also provide a rich pool of potential candidates for the development of immunomodulatory intervention drugs and potentially new vaccines. In the present review, we will attempt to summarize novel findings on the salivary immunomodulatory proteins of ticks, focusing on biomolecular targets, structure-activity relationships, and the perspective of their development into therapeutics.

Objectives To determine the prevalence of Melanocortin-4 Receptor (MC4R) mutations in a cohort of children and adolescents with overweight or obesity and to determine whether treatment responses differed between carriers and noncarriers. Methods Using target region capture sequencing, an MC4R mutation screen was performed in 1261 Danish children and adolescents enrolled at a tertiary multidisciplinary childhood obesity treatment center. Measurements of anthropometrics, blood pressure, fasting blood biochemistry including lipid and hormone levels, and dual-energy X-ray absorptiometry were performed at baseline and throughout treatment. Results Of 1209 children and adolescents that met all criteria to be included in the described analyses, 30 (2.5%) carried damaging or unresolved MC4R mutations. At baseline, mutation carriers exhibited higher concentrations of plasma thyroid-stimulating hormone (p = 0.003), and lower concentrations of plasma thyroxine (p = 0.010) compared to noncarriers. After a median of 1 year of treatment (range 0.5–4.0 years), body mass index (BMI) standard deviation score (SDS) was reduced in noncarriers but not in carriers, and this difference in treatment response was statistically significant (p = 0.005). Furthermore, HDL cholesterol was reduced in carriers, a response significantly different from that of noncarriers (p = 0.017). Conclusion Among Danish children and adolescents with overweight or obesity entering a tertiary lifestyle intervention, 2.5% carried damaging or unresolved MC4R mutations. In contrast to noncarriers, carriers of damaging or unresolved MC4R mutations failed to reduce their BMI SDS during obesity treatment, indicating a need for personalized treatment based on the MC4R genotype.

In 2019, the Nosology Committee of the International Skeletal Dysplasia Society provided an updated version of the Nosology and Classification of Genetic Skeletal Disorders. This is a reference list of recognized diseases in humans and their causal genes published to help clinician diagnosis and scientific research advances. Complementary to mammalian models, zebrafish has emerged as an interesting species to evaluate chemical treatments against these human skeletal disorders. Due to its versatility and the low cost of experiments, more than 80 models are currently available. In this article, we review the state-of-art of this "aquarium to bedside" approach describing the models according to the list provided by the Nosology Committee. With this, we intend to stimulate research in the appropriate direction to efficiently meet the actual needs of clinicians under the scope of the Nosology Committee. Yes

Background Tartary buckwheat (Fagopyrum tataricum) is an edible cereal crop whose sprouts have been marketed and commercialized for their higher levels of anti-oxidants, including rutin and anthocyanin. UDP-glucose flavonoid glycosyltransferases (UFGTs) play an important role in the biosynthesis of flavonoids in plants. So far, few studies are available on UFGT genes that may play a role in tartary buckwheat flavonoids biosynthesis. Here, we report on the identification and functional characterization of seven UFGTs from tartary buckwheat that are potentially involved in flavonoid biosynthesis (and have varying effects on plant growth and development when overexpressed in Arabidopsis thaliana.) Results Phylogenetic analysis indicated that the potential function of the seven FtUFGT proteins, FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, could be divided into three Arabidopsis thaliana functional subgroups that are involved in flavonoid biosynthesis of and anthocyanin accumulation. A significant positive correlation between FtUFGT8 and FtUFGT15 expression and anthocyanin accumulation capacity was observed in the tartary buckwheat seedlings after cold stress. Overexpression in Arabidopsis thaliana showed that FtUFGT8, FtUFGT15, and FtUFGT41 significantly increased the anthocyanin content in transgenic plants. Unexpectedly, overexpression of FtUFGT6, while not leading to enhanced anthocyanin accumulation, significantly enhanced the growth yield of transgenic plants. When wild-type plants have only cotyledons, most of the transgenic plants of FtUFGT6 had grown true leaves. Moreover, the growth speed of the oxFtUFGT6 transgenic plant root was also significantly faster than that of the wild type. At later growth, FtUFGT6 transgenic plants showed larger leaves, earlier twitching times and more tillers than wild type, whereas FtUFGT15 showed opposite results. Conclusions Seven FtUFGTs were isolated from tartary buckwheat. FtUFGT8, FtUFGT15, and FtUFGT41 can significantly increase the accumulation of total anthocyanins in transgenic plants. Furthermore, overexpression of FtUFGT6 increased the overall yield of Arabidopsis transgenic plants at all growth stages. However, FtUFGT15 shows the opposite trend at later growth stage and delays the growth speed of plants. These results suggested that the biological function of FtUFGT genes in tartary buckwheat is diverse. Electronic supplementary material The online version of this article (10.1186/s12870-019-1955-z) contains supplementary material, which is available to authorized users.

The Baltic Sea is a shallow basin of brackish water in which the spatial salinity gradient is one of the most important factors contributing to species distribution. The Baltic Sea is infamous for its annual cyanobacterial blooms comprised of Nodularia spumigena, Aphanizomenon spp., and Dolichospermum spp. that cause harm, especially for recreational users. To broaden our knowledge of the cyanobacterial adaptation strategies for brackish water environments, we sequenced the entire genome of Dolichospermum sp. UHCC 0315, a species occurring not only in freshwater environments but also in brackish water. Comparative genomics analyses revealed a close association with Dolichospermum sp. UHCC 0090 isolated from a lake in Finland. The genome closure of Dolichospermum sp. UHCC 0315 unraveled a mixture of two subtypes in the original culture, and subtypes exhibited distinct buoyancy phenotypes. Salinity less than 3 g L−1 NaCl enabled proper growth of Dolichospermum sp. UHCC 0315, whereas growth was arrested at moderate salinity (6 g L−1 NaCl). The concentrations of toxins, microcystins, increased at moderate salinity, whereas RNA sequencing data implied that Dolichospermum remodeled its primary metabolism in unfavorable high salinity. Based on our results, the predicted salinity decrease in the Baltic Sea may favor toxic blooms of Dolichospermum spp.

Active brown adipose tissue (BAT) has, since it rediscovery in adult humans in 2009, received much attention for its ability to increase energy expenditure when activated. By means of mitochondrial uncoupling activity BAT'S main function is to produce heat instead of storing energy such as in white adipose tissue (WAT). Therefore, BAT is considered a new potential target to treat obesity and the metabolic syndrome. However, the contribution of this thermogenic tissue is still a matter of debate among researchers. The aim of this review is to give an overview of the differences between classical brown adipocytes and inducible beige adipocytes in humans, and the potential activators of BAT in humans. Furthermore newly described genetic markers for identification of these two types of brown adipocytes are examined. Finally, the potential of the current measurement techniques, and the contribution of BAT activity to whole body energy expenditure are discussed.

Active brown adipose tissue (BAT) has, since it rediscovery in adult humans in 2009, received much attention for its ability to increase energy expenditure when activated. By means of mitochondrial uncoupling activity BAT'S main function is to produce heat instead of storing energy such as in white adipose tissue (WAT). Therefore, BAT is considered a new potential target to treat obesity and the metabolic syndrome. However, the contribution of this thermogenic tissue is still a matter of debate among researchers.The aim of this review is to give an overview of the differences between classical brown adipocytes and inducible beige adipocytes in humans, and the potential activators of BAT in humans. Furthermore newly described genetic markers for identification of these two types of brown adipocytes are examined. Finally, the potential of the current measurement techniques, and the contribution of BAT activity to whole body energy expenditure are discussed.

The endometrium, lining the uterine lumen, is highly essential for human reproduction. Its exceptional remodeling plasticity, including the transformation process to welcome and nest the embryo, is not well understood. Lack of representative and reliable study models allowing the molecular and cellular mechanisms underlying endometrium development and biology to be deciphered is an important hurdle to progress in the field. Recently, powerful organoid models have been developed that not only recapitulate endometrial biology such as the menstrual cycle, but also faithfully reproduce diseases of the endometrium such as endometriosis. Moreover, single-cell profiling endeavors of the endometrium in health and disease, and of derived organoids, start to provide deeper insight into cellular complexity and expression specificities, and in resulting tissue processes. This granular portrayal will not only help in understanding endometrium biology and disease, but also in pinning down the tissue's stem cells, at present not yet conclusively defined. Here, we provide a general overview of endometrium development and biology, and the efforts of modeling both the healthy tissue, as well as its key diseased form of endometriosis. The future of modeling and deciphering this key tissue, hidden inside the womb, looks bright. ispartof: JOURNAL OF PERSONALIZED MEDICINE vol:12 issue:7 ispartof: location:Switzerland status: published

Plants are a primary food source and can form the basis for renewable energy resources. The final size of their organs is by far the most important trait to consider when seeking increased plant productivity. Being multicellular organisms, plant organ size is mainly determined by the coordination between cell proliferation and cell expansion. The protease DA1 limits the duration of cell proliferation and thereby restricts final organ size. Since its initial identification as a negative regulator of organ growth, various transcriptional regulators of DA1, but also interacting proteins, have been identified. These interactors include cleavage substrates of DA1, and also proteins that modulate the activity of DA1 through post-translational modifications, such as ubiquitination, deubiquitination, and phosphorylation. In addition, many players in the DA1 pathway display conserved phenotypes in other dicot and even monocot species. In this review, we provide a timely overview of the complex, but intriguing, molecular mechanisms that fine-tune the activity of DA1 and therefore final organ size. Moreover, we lay out a roadmap to identify and characterize substrates of proteases and frame the substrate cleavage events in their biological context.

23 p.-5 fig.-2 tab., Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site-and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named rel(LS20), is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the Rel(LS20) shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research., Work in the Meijer lab was funded by the Spanish government through grant Bio2013- 41489-P of the Ministry of Economy and Competitiveness, and through grant Bio2016- 77883-C2-1-P of the Ministry of Economy, Industry and Competitiveness; the former grant also funded AMA and CGC. The Spanish government also supported DRB, JRLO, and CA.DRB was funded by grant Bio2016-77883-C2-2-P of the Ministry of Economy, Industry and Competitiveness, and JRLO and CA were supported by grant BFU2014-52070-C2-2-P of the Ministry of Economy and Competitiveness to CA.LJW's work was supported by Wellcome Trust grant WT098374AIA to Jeff Errington.

Overproduction of reactive oxygen species (ROS) in plant cells under environmental stress cause oxidative stress, which is one of major factors limiting the plant productivity. To prevent this limitation by stress conditions, a possible strategy is to strengthen the antioxidant defense of plants by gene manipulation of antioxidant enzymes and low molecular-weight (LMW) antioxidants. LMW antioxidants are important for the plant protection to environmental stress and also have nutritional merits for humans as antiaging and antidisease molecules. Sweetpotato [Ipomoea batatas (L.) Lam] as a nutritional food crop has many advantages in terms of the cultivation on the global marginal lands. Thus sweetpotato is considered as an emerging multifunctional food crop ensuring the food and nutrition security in the face of climate crisis, since it contains high levels of LMW antioxidants, minerals, and dietary fiber. This review describes the current status and prospects of metabolic engineering of two lipophilic antioxidants, carotenoids and tocopherols, in transgenic sweetpotato. In addition, the potentiality of an orange (Or) gene involved in enhanced tolerance to various abiotic stresses by high accumulation of carotenoids is introduced in detail. The rational metabolic engineering of LMW antioxidants can be applicable to all plant species to cope with oxidative stress in face of climate change and nutritional problems in aging society., National Research Foundation (NRF) of Korea - Korean Government (MSIT) [2020R1A2C1004560]; Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program [KGM5372113], This research is supported by The National Research Foundation (NRF) of Korea funded by the Korean Government (MSIT) (2020R1A2C1004560) and The Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5372113).

Plants accumulate silicon in their tissues as amorphous silica. The form of silicon taken up by plants is silicic acid, a neutral molecule that passes through membrane channels with water. After seminal work on rice identified an aquaporin that appeared to mediate the passage of silicic acid, several papers followed and classified similar channels (referred to as “transporters”) in a number of plant species. These channels have been described as essential for silicon uptake and specific for the metalloid. Herein, we critically review the published data on the characterisation of one channel in particular, Lsi1, and identify possible caveats in results and limitations in methods used. Our analysis does not support the suggestion that the identified channels are specific for silicic acid. Computational analyses of the size of the Lsi1 pore additionally suggest that it may not play a significant role in mediating the movement of silicic acid in planta. We suggest that to avoid further confusion, channels currently implicated in the transport of silicic acid in planta are not referred to as silicon-specific transporters. Future research including the use of molecular dynamics simulations will enable the unequivocal identification of channels involved in silicon transport in plants.

Introduction: Proteinuria contributes to progression of renal damage, partly by complement activation on proximal tubular epithelial cells. By pattern recognition, properdin has shown to bind to heparan sulfate proteoglycans on tubular epithelium and can initiate the alternative complement pathway (AP). Properdin however, also binds to C3b(Bb) and properdin binding to tubular cells might be influenced by the presence of C3b(Bb) on tubular cells and/or by variability in properdin proteinsin vitro. In this study we carefully evaluated the specificity of the properdin - heparan sulfate interaction and whether this interaction could be exploited in order to block alternative complement activation.Methods: Binding of various properdin preparations to proximal tubular epithelial cells (PTEC) and subsequent AP activation was determined in the presence or absence of C3 inhibitor Compstatin and properdin inhibitor Salp20. Heparan sulfate proteoglycan dependency of the pattern recognition of properdin was evaluated on PTEC knocked down for syndecan-1 by shRNA technology. Solid phase binding assays were used to evaluate the effectivity of heparin(oids) and recombinant Salp20 to block the pattern recognition of properdin.Results: Binding of serum-derived and recombinant properdin preparations to PTECs could be dose-dependently inhibited (P heparin(oid) > C3b.Discussion: In this study we showed that all properdin preparations recognize heparan sulfate/syndecan-1 on PTECs with and without Compstatin C3 blocking conditions. In contrast to Compstatin, recombinant Salp20 prevents heparan sulfate pattern recognition by properdin on PTECs. Both complement inhibitors prevented properdin-mediated C3 activation. Binding of properdin to C3b could also be blocked by heparin(oids) and recombinant Salp20. This work indicates that properdin serves as a docking station for AP activation on PTECs and a Salp20 analog or heparinoids may be viable inhibitors in properdin mediated AP activation.

The sulfur-containing amino acids methionine and cysteine play an important role in food industry. These amino acids are used to confer a sulfur smell or meat-related aroma to food products. Besides their use as food additives, methionine and cysteine participate in flavor formation in dairy fermentations. For instance, the characteristic aroma of Cheddar cheeses is derived from methionine. Therefore, bacterial strains with the ability to overproduce and secrete these amino acids are relevant for the food industry. In addition, the quantification of these compounds in food matrices is a laborious task that involves sample preparation and specific analytical methods such as high-performance liquid chromatography. The ability of bacteria to naturally sense metabolites has successfully been exploited to develop biosensors. The presence of a specific metabolite is sensed by the biosensors, and it is subsequently translated into the expression of one or more reporter genes. In this study we aim to develop biosensors to detect methionine and cysteine, which are produced and secreted by wild-type Lactococcus lactis strains. We employed two strategies to create L. lactis biosensors, the first one is based on the methionine auxotrophy of this bacterium and the second strategy is based on a cysteine-responsive promoter. The characterization of the biosensors showed their specific response to the presence of these amino acids. Subsequently, we applied the methionine biosensor to quantify the presence of methionine in bacterial supernatants of wild-type L. lactis that naturally secretes methionine to benchmark the performance of our biosensors. The methionine biosensor responded linearly to the amounts of methionine present in the bacterial supernatants, i.e., the increases in the biosensor cell densities were proportional to the amounts of methionine present in the supernatants. The biosensors developed in this study tackle the limitations of amino acid quantification and the selection of strains with secretion of amino acids. These biosensors may eventually be used for screening of engineered strains to increase methionine and cysteine production, and may facilitate the detection of these amino acids in complex food matrices.

Made available in DSpace on 2020-12-12T02:09:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Plants produce a vast array of defense compounds to protect themselves from pathogen attack or herbivore predation. Saponins are a specific class of defense compounds comprising bioactive glycosides with a steroidal or triterpenoid aglycone backbone. The model legume Medicago truncatula synthesizes two types of saponins, hemolytic saponins and nonhemolytic soyasaponins, which accumulate as specific blends in different plant organs. Here, we report the identification of the seed-specific transcription factor TRITERPENE SAPONIN ACTIVATION REGULATOR3 (TSAR3), which controls hemolytic saponin biosynthesis in developing M. truncatula seeds. Analysis of genes that are coexpressed with TSAR3 in transcriptome data sets from developing M. truncatula seeds led to the identification of CYP88A13, a cytochrome P450 that catalyzes the C-16a hydroxylation of medicagenic acid toward zanhic acid, the final oxidation step of the hemolytic saponin biosynthesis branch in M. truncatula. In addition, two uridine diphosphate glycosyltransferases, UGT73F18 and UGT73F19, which glucosylate hemolytic sapogenins at the C-3 position, were identified. The genes encoding the identified biosynthetic enzymes are present in clusters of duplicated genes in the M. truncatula genome. This appears to be a common theme among saponin biosynthesis genes, especially glycosyltransferases, and may be the driving force of the metabolic evolution of saponins. Ghent University Department of Plant Biotechnology and Bioinformatics VIB Center for Plant Systems Biology Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Institut de Recherche en Horticulture et Semences-Unités Mixtes de Recherche Université d’Angers INRAE Institut Agro, SFR 4207 QuaSaV VIB Metabolomics Core Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP)

The expression of short chain fatty acid receptors FFA2 and FFA3 in pancreatic islets raised interest in using them as drug targets for treating hyperglycemia in humans. This study aims to examine the efficacy of synthetic FFA2- and FFA3-ligands to modulate glucose-stimulated insulin secretion (GSIS) in human pseudoislets which display intact glucose responsiveness. The FFA2-agonists 4-CMTB and TUG-1375 inhibited GSIS, an effect reversed by the FFA2-antagonist CATPB. GSIS itself was not augmented by CATPB. The FFA3-agonists FHQC and 1-MCPC did not affect GSIS in human pseudoislets. For further drug evaluation we used mouse islets. The CATPB-sensitive inhibitory effect of 100 µM 4-CMTB on GSIS was recapitulated. The inhibition was partially sensitive to the Gi/o-protein inhibitor pertussis toxin. A previously described FFA2-dependent increase of GSIS was observed with lower concentrations of 4-CMTB (10 and 30 µM). The stimulatory effect of 4-CMTB on secretion was prevented by the Gq-protein inhibitor FR900359. As in human pseudoislets, in mouse islets relative mRNA levels were FFAR2 > FFAR3 and FFA3-agonists did not affect GSIS. The FFA3-agonists, however, inhibited GSIS in a pertussis toxin-sensitive manner in INS-1E cells and this correlated with relative mRNA levels of Ffar3 > > Ffar2. Thus, in humans, when FFA2-activation impedes GSIS, FFA2-antagonism may reduce glycemia.

ObjectiveHuman brown adipose tissue (BAT) activity decreases with age and obesity. In addition to uncoupling protein 1 (UCP1), several genetic markers of BAT in humans have been published. However, the link between human BAT activity and genetic markers has been inadequately explored. MethodsWhite adipose tissue (WAT) and BAT biopsies were obtained from 16 patients undergoing deep neck surgery. In vitro differentiated adipocytes were used to measure norepinephrine-stimulated mitochondrial uncoupling as a measure of in vitro BAT activity. Gene expression was determined in adipose tissue biopsies. ResultsNorepinephrine increased in vitro BAT activity in adipocytes derived from human BAT, and this increase was abolished by propranolol. Furthermore, in vitro BAT activity showed a negative correlation to age and BMI. UCP1 messenger RNA (mRNA) expression showed a positive correlation to in vitro BAT activity, while zinc finger protein of cerebellum 1 (ZIC1) mRNA showed a negative correlation to in vitro BAT activity. In human BAT biopsies, UCP1 mRNA showed negative correlations to age and BMI, while ZIC1 mRNA showed positive correlations to age and BMI. ConclusionsDifferentiated adipocytes derived from human BAT maintain intrinsic characteristics of the donor. High ZIC1 mRNA does not necessarily reflect high BAT activity.

Spirulina is rich in various antioxidants and nutraceuticals and it has proven to be effective in the treatment of various pathological conditions. This study explores the antioxidant effect of fermented and non-fermented Spirulina extracts on the proteome level using the yeast Saccharomyces cerevisiae as a model organism. Yeast cells were treated with fermented Spirulina water extract (SV), non-fermented Spirulina water extract (NFV), fermented Spirulina ethanol extract (SE), and non-fermented Spirulina ethanol extract (NFE). Cell lysates were prepared, and label-free quantitative proteome analysis was performed. In SV, when compared to NFV samples, the levels of most differentially expressed proteins were upregulated. Alternatively, SE compared to NFE samples showed a significant downregulation for the majority of the analyzed proteins involved in different cellular processes. Additionally, a higher downregulation of stress response related proteins was observed in SE compared to NFE samples, while their abundance in SV samples increased compared to NFV. This study provided a global view, on a proteome level, of how cells cope with exogenous antioxidants and remodel their cellular processes to maintain metabolic and redox balance. Furthermore, it combined for the first time the analysis of different extract effect, including the contribution of lactic acid fermentation to the cell activity.

The genus Pseudomonas hosts an extensive genetic diversity and is one of the largest genera among Gram-negative bacteria. Type strains of Pseudomonas are well known to represent only a small fraction of this diversity and the number of available Pseudomonas genome sequences is increasing rapidly. Consequently, new Pseudomonas species are regularly reported and the number of species within the genus is constantly evolving. In this study, whole genome sequencing enabled us to define 43 new Pseudomonas species and provide an update of the Pseudomonas evolutionary and taxonomic relationships. Phylogenies based on the rpoD gene and whole genome sequences, including, respectively, 316 and 313 type strains of Pseudomonas, revealed sixteen groups of Pseudomonas and, together with the distribution of cyclic lipopeptide biosynthesis gene clusters, enabled the partitioning of the P. putida group into fifteen subgroups. Pairwise average nucleotide identities were calculated between type strains and a selection of 60 genomes of non-type strains of Pseudomonas. Forty-one strains were incorrectly assigned at the species level and among these, 19 strains were shown to represent an additional 13 new Pseudomonas species that remain to be formally classified. This work pinpoints the importance of correct taxonomic assignment and phylogenetic classification in order to perform integrative studies linking genetic diversity, lifestyle, and metabolic potential of Pseudomonas spp.

The pregnane X receptor (PXR) is a nuclear receptor belonging to the NR1I sub-family and a known master regulator of xenobiotic metabolism. New roles have been recently proposed in mammals through its activation by vitamin K (VK) such as regulation of glucose metabolism, bone homeostasis, reproduction, neuronal development and cognitive capacities. In marine fish species little is known about PXR and its potential roles. Here, expression patterns of pxr transcripts and conservation of protein domains were determined in the Senegalese sole (Solea senegalensis), a marine flatfish model species in aquatic ecotoxicology. In addition to a full coding sequence transcript (sspxrl), two variants lacking DNA and/or ligand binding domains (sspxr2 and sspxr3) were also identified. The expression of sspxrl during early development and in adult tissues was ubiquitous, but highest levels were observed in liver, intestine and skin. Expression was also detected by in situ hybridization in chondrocytes and cells from the granular and inner nuclear layers in three month old fish. Finally, sspxrl expression was shown to be differentially regulated under physiological conditions related with fasting, VK and warfarin metabolism. The present work provides new and basic knowledge regarding pxr sequence and expression patterns in a marine flatfish species to unveil the potential impact of xenobiotics on marine fish physiology, and will allow a better and more ecosystemic environmental risk assessment of different pollutants over the marine environments with the development of reporter assays using PXR sequences from evolutionary distantly marine species (such as vertebrate and invertebrate marine species). (C) 2017 Elsevier Ltd. All rights reserved. Fundo Europeu de Desenvolvimento Regional (FEDER) Programa Operacional through research project-Universidade do Algarve (KLING project) [PROMAR 31-03-05-FEP0073] European Commission (ERDF-COMPETE) [PEst-C/MAR/LA0015/2011]

The master regulator, DnrI of Streptomyces peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.

// Africa Millan-Ucles 1,* , Susana Zuluaga 1,* , Miriam Marques 2 , Jesus Vallejo-Diaz 1 , Lorena Sanz 1 , Ariel E. Cariaga-Martinez 1 , Francisco X. Real 2,3 and Ana C. Carrera 1 1 Department of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Universidad Autonoma de Madrid, Cantoblanco, Madrid, Spain 2 Centro Nacional de Investigaciones Oncologicas, Melchor Fernandez Almagro 3, Madrid, Spain 3 Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain * These authors have contributted equally to this work Correspondence to: Ana C. Carrera, email: // Keywords : PI3Kbeta, PTEN, urothelial carcinoma, bladder cancer, siRNA Received : October 15, 2016 Accepted : October 25, 2016 Published : November 16, 2016 Abstract Alterations in phosphatidylinositol 3-kinase (PI3K) and in PTEN (phosphatase and tensin homolog), the negative regulator of the PI3K pathway, are found in nearly half of human tumors. As PI3Kβ, the main isoform activated in PTEN- mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kβ kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kβ depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN -mutant tumors more efficiently than PI3Kβ inhibition. A small proportion of these tumors was resistant to PI3Kβ downregulation; we analyzed what determined resistance in these cases. Using add-back experiments, we show that both PTEN mutation and low E-cadherin expression are necessary for PI3Kβ dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kβ associated with N-cadherin and that PIK3CB depletion selectively disrupted N-cadherin cell adhesions in PTEN -mutant bladder carcinoma. These results support the use of PIK3CB interfering RNA as a therapeutic approach for high-risk bladder cancers that show E-cadherin loss and express mutant PTEN .

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p

Beneficial Pseudomonas spp. produce an array of antimicrobial secondary metabolites such as cyclic lipopeptides (CLPs). We investigated the capacity of CLP-producing Pseudomonas strains and their crude CLP extracts to control rice blast caused by Magnaporthe oryzae, both in a direct manner and via induced systemic resistance (ISR). In planta biocontrol assays showed that lokisin-, white line inducing principle (WLIP)-, entolysin- and N3-producing strains successfully induced resistance to M. oryzae VT5M1. Furthermore, crude extracts of lokisin, WLIP and entolysin gave similar ISR results when tested in planta. In contrast, a xantholysin-producing strain and crude extracts of N3, xantholysin and orfamide did not induce resistance against the rice blast disease. The role of WLIP in triggering ISR was further confirmed by using WLIP-deficient mutants. The severity of rice blast disease was significantly reduced when M. oryzae spores were pre-treated with crude extracts of N3, lokisin, WLIP, entolysin or orfamide prior to inoculation. In vitro microscopic assays further revealed the capacity of crude N3, lokisin, WLIP, entolysin, xantholysin and orfamide to significantly inhibit appressoria formation by M. oryzae. In addition, the lokisin and WLIP biosynthetic gene clusters in the producing strains are described. In short, our study demonstrates the biological activity of structurally diverse CLPs in the control of the rice blast disease caused by M. oryzae. Furthermore, we provide insight into the non-ribosomal peptide synthetase genes encoding the WLIP and lokisin biosynthetic machineries. ispartof: FRONTIERS IN PLANT SCIENCE vol:10 ispartof: location:Switzerland status: published