Functional Characterization Of Genes Research Articles

Multiple Metabolic Enzymes Can Be Involved in Cross-Resistance to 4-Hydroxyphenylpyruvate-Dioxygenase-Inhibiting Herbicides in Wild Radish.

A wild radish population (R) has been recently confirmed to be cross-resistant to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides without previous exposure to these herbicides. This cross-resistance is endowed by enhanced metabolism. Our study identified one 2-oxoglutarate/Fe(II)-dependent dioxygenase gene (Rr2ODD1) and two P450 genes (RrCYP704C1 and RrCYP709B1), which were significantly more highly expressed in R versus susceptible (S) plants. Gene functional characterization using Arabidopsis transformation showed that overexpression of RrCYP709B1 conferred a modest level of resistance to mesotrione. Ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that tissue mesotrione levels in RrCYP709B1 transgenic Arabidopsis plants were significantly lower than that in the wild type. In addition, overexpression of Rr2ODD1 or RrCYP704C1 in Arabidopsis endowed resistance to tembotrione and isoxaflutole. Structural modeling indicated that mesotrione can bind to CYP709B1 and be easily hydroxylated to form 4-OH-mesotrione. Although each gene confers a modest level of resistance, overexpression of the multiple herbicide-metabolizing genes could contribute to HPPD-inhibiting herbicide resistance in this wild radish population.

Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

Generation and Characterization of a Transgenic Mouse That Specifically Expresses the Cre Recombinase in Spermatids.

Spermiogenesis is the step during which post-meiotic cells, called spermatids, undergo numerous morphological changes and differentiate into spermatozoa. Thousands of genes have been described to be expressed at this stage and could contribute to spermatid differentiation. Genetically-engineered mouse models using Cre/LoxP or CrispR/Cas9 are the favored approaches to characterize gene function and better understand the genetic basis of male infertility. In the present study, we produced a new spermatid-specific Cre transgenic mouse line, in which the improved iCre recombinase is expressed under the control of the acrosomal vesicle protein 1 gene promoter (Acrv1-iCre). We show that Cre protein expression is restricted to the testis and only detected in round spermatids of stage V to VIII seminiferous tubules. The Acrv1-iCre line can conditionally knockout a gene during spermiogenesis with a > 95% efficiency. Therefore, it could be useful to unravel the function of genes during the late stage of spermatogenesis, but it can also be used to produce an embryo with a paternally deleted allele without causing early spermatogenesis defects.

Transcriptional Regulation Technology for Gene Perturbation in Fission Yeast.

Isolation and introduction of genetic mutations is the primary approach to characterize gene functions in model yeasts. Although this approach has proven very powerful, it is not applicable to all genes in these organisms. For example, introducing defective mutations into essential genes causes lethality upon loss of function. To circumvent this difficulty, conditional and partial repression of target transcription is possible. While transcriptional regulation techniques, such as promoter replacement and 3' untranslated region (3'UTR) disruption, are available for yeast systems, CRISPR-Cas-based technologies have provided additional options. This review summarizes these gene perturbation technologies, including recent advances in methods based on CRISPR-Cas systems for Schizosaccharomyces pombe. We discuss how biological resources afforded by CRISPRi can promote fission yeast genetics.

Rapidly generating homozygous mutate zebrafish in F0 generation by technical integration of CRISPR/Cas9 and gynogenesis

Gene editing technique has been widely applied for gene function characterization. However, such an approach is often time-consuming to obtain homozygous mutant. In this article, through timing the earlier injection of embryos, the gene editing efficiency is significantly improved, and together with the technique of the UV inactivated sperm and heat shock treatment for blocking the first cleavage, the gene-edited homozygous zebrafish are successfully obtained in F0 generation, rather than in the third or more generations as reported in the existing results. It is concluded that genetic purification of artificial gynogenesis can play critical role in more efficiently preparing the gene-edited homozygous individuals in fish.

Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.

UltraSEQ, a Universal Bioinformatic Platform for Information-Based Clinical Metagenomics and Beyond.

Applied metagenomics is a powerful emerging capability enabling the untargeted detection of pathogens, and its application in clinical diagnostics promises to alleviate the limitations of current targeted assays. While metagenomics offers a hypothesis-free approach to identify any pathogen, including unculturable and potentially novel pathogens, its application in clinical diagnostics has so far been limited by workflow-specific requirements, computational constraints, and lengthy expert review requirements. To address these challenges, we developed UltraSEQ, a first-of-its-kind accurate and scalable metagenomic bioinformatic tool for potential clinical diagnostics and biosurveillance utility. Here, we present the results of the evaluation of our novel UltraSEQ pipeline using an in silico-synthesized metagenome, mock microbial community data sets, and publicly available clinical data sets from samples of different infection types, including both short-read and long-read sequencing data. Our results show that UltraSEQ successfully detected all expected species across the tree of life in the in silico sample and detected all 10 bacterial and fungal species in the mock microbial community data set. For clinical data sets, even without requiring data set-specific configuration setting changes, background sample subtraction, or prior sample information, UltraSEQ achieved an overall accuracy of 91%. Furthermore, as an initial demonstration with a limited patient sample set, we show UltraSEQ's ability to provide antibiotic resistance and virulence factor genotypes that are consistent with phenotypic results. Taken together, the above-described results demonstrate that the UltraSEQ platform offers a transformative approach for microbial and metagenomic sample characterization, employing a biologically informed detection logic, deep metadata, and a flexible system architecture for the classification and characterization of taxonomic origin, gene function, and user-defined functions, including disease-causing infections. IMPORTANCE Traditional clinical microbiology-based diagnostic tests rely on targeted methods that can detect only one to a few preselected organisms or slow, culture-based methods. Although widely used today, these methods have several limitations, resulting in rates of cases of an unknown etiology of infection of >50% for several disease types. Massive developments in sequencing technologies have made it possible to apply metagenomic methods to clinical diagnostics, but current offerings are limited to a specific disease type or sequencer workflow and/or require laboratory-specific controls. The limitations associated with current clinical metagenomic offerings result from the fact that the backend bioinformatic pipelines are optimized for the specific parameters described above, resulting in an excess of unmaintained, redundant, and niche tools that lack standardization and explainable outputs. In this paper, we demonstrate that UltraSEQ uses a novel, information-based approach that enables accurate, evidence-based predictions for diagnosis as well as the functional characterization of a sample.

Research on digital characterization and identification process model of functional genes for intelligent innovative design

The inheritance and variation of functional knowledge is an important way to achieve product innovation. Analogous biological genetic inheritance can solve the problems with the inheritance and variation of functional knowledge. However, the different expressions of functional knowledge in different domains lead designers to consuming a lot of time for functional knowledge characterization and identification, which reduces the efficiency of functional knowledge application. Therefore, in order to solve the problem of inefficient application of functional knowledge in the genetic process caused by irregular expression of functional knowledge, the paper proposes a digital characterization and identification process model of functional genes for product intelligent innovative design. The process model enables product intelligent innovative design through digital characterization of functional genes and rapid identification of functional genes using computer assistance. The process model consists of four following steps. First, extract functional knowledge from patents and perform preliminary functional gene characterization. Second, characterize functional genes digitally by using computerized digital coding techniques. Third, identify the required functional genes by using image similarity matching algorithm. It assists designers to quickly identify the functional genes required for the target system. Fourth, obtain the target product design scheme. By “transcribing” and “translating” the required functional genes and using TRIZ tools to solve the translated scheme, the product innovative design scheme is finally obtained. The feasibility and effectiveness of the model are verified by the intelligent innovative design of shared bicycle parking management device.

Transcriptome profiling reveals characteristics of hairy root and the role of AhGLK1 in response to drought stress and post-drought recovery in peanut

BackgroundHR (hairy root) has emerged as a valuable tissue for the rapid characterization of plant gene function and enzyme activity in vivo. AhGLK1 (Arachis hypogaea L. golden2-like 1) is known to play a role in post-drought recovery. However, it is unclear (a) whether HR has properties that are distinct from those of PR (primary root); and (b) which gene networks are regulated by AhGLK1 in response to drought stress and recovery in peanut.ResultsWe found that cells of the root tip cortex were larger in HR than in PR, while a total of 850 differentially expressed genes (DEGs) were identified in HR compared to PR. Eighty-eight of these DEGs, relating to chlorophyll and photosynthesis, were upregulated in HR. In addition, AhGLK1-OX (AhGLK1-overexpressing) HR showed a green phenotype, and had a higher relative water content than 35 S::eGFP (control) HR during drought stress. RNA-seq analysis showed that 74 DEGs involved both in the drought response and the post-drought recovery process were significantly enriched in the galactose metabolism pathway. GO terms enrichment analysis revealed that 59.19%, 29.79% and 17.02% of the DEGs mapped to the ‘biological process’ (BP), ‘molecular function’ (MF) and ‘cellular component’ (CC) domains, respectively. Furthermore, 20 DEGs involved in post-drought recovery were uniquely expressed in AhGLK1-OX HR and were significantly enriched in the porphyrin metabolism pathway. GO analysis showed that 42.42%, 30.30% and 27.28% of DEGs could be assigned to the BP, MF and CC domains, respectively. Transcription factors including bHLH and MYB family members may play a key role during drought stress and recovery.ConclusionOur data reveal that HR has some of the characteristics of leaves, indicating that HR is suitable for studying genes that are mainly expressed in leaves. The RNA-seq results are consistent with previous studies that show chlorophyll synthesis and photosynthesis to be critical for the role of AhGLK1 in improving post-drought recovery growth in peanut. These findings provide in-depth insights that will be of great utility for the exploration of candidate gene functions in relation to drought tolerance and/or post-drought recovery ability in peanut.

Tree Genetics: Molecular and Functional Characterization of Genes

Forests represent one of the most important ecosystems on Earth, covering approximately 30% of the total global land area [...]

Improving Agrobacterium tumefaciens-Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.).

In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T1 OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The Agrobacterium-mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

DCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.

Genome-wide identification of the MATE gene family and functional characterization of PbrMATE9 related to anthocyanin in pear

Plant multidrug and toxic compound extrusion (MATE) genes play an important role in the process of detoxification, plant morphogenesis, and anthocyanin accumulation. However, whether the MATE gene family functions in pear peel coloration is still unknown. To evaluate and identify the MATE gene family members which are involving in anthocyanin accumulation and coloration in pear. In this study, 85 MATE genes were identified in the reference pear genome of ‘Dangshansuli’ through genome-wide identification. Based on gene structure and phylogenetic tree analysis, the MATE family was divided into five subfamilies. RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) indicated that the expression patterns of PbrMATEs were tissue-specific. 28.24% (24) of PbrMATE genes were expressed in the fruits, and 44.71% (38) of PbrMATE genes were expressed in the leaves. Additionally, we found that the expression levels of PbrMATE9, PbrMATE26, PbrMATE50, and PbrMATE69 in debagged fruits with red peel were significantly higher than those in bagged fruits without red peel, according to our bagging/debagging treatment of ‘Mantianhong’. The expression pattern of PbrMATE9 was consistent with the variation trend in anthocyanin content, suggesting that it might play an important role in anthocyanin accumulation in response to light exposure. Subcellular localization showed that PbrMATE9 was a membrane protein. More strikingly, the transient overexpression of PbrMATE9 promoted anthocyanin accumulation in the peel of pear, and the expression of structural genes (PbrCHI, PbrANS, PbrDFR, and PbrUFGT) in the anthocyanin biosynthesis pathway also increased significantly. Through co-expression network analysis, the transcription factors were identified, such as WRKY, COL, GATA, and BBX, which might be involved in the regulation of PbrMATE9. The study has enriched the genetic resources and improved the understanding of the regulation network of anthocyanin accumulation in pear.

Systematic identification and characterization of the soybean (Glycine max) B-box transcription factor family

In plants, the B-box (BBX) transcription factors (TFs) are a subfamily of zinc-finger TFs that act to regulate diverse plant growth and development processes. The BBX TFs have been the subject of considerable attention, and are well characterized in diverse plant species, including in rice (Oryza sativa) and Arabidopsis thaliana, but less so in the economically important soybean (Glycine max). In this work, we systematically identified and characterized 57 soybean BBX genes (GmBBX1 to GmBBX57). These genes were mapped to all 20 soybean chromosomes and were divided into five clades with high intra-clade intron–exon similarity. The majority of GmBBX gene promoter cis-acting elements were responsive to light, abscisic acid, salicylic acid and methyl jasmonate, as well as a diverse array of other stimuli. Quantitative RT-PCR indicated that several GmBBX genes exhibited tissue-specific and phytohormone- and abiotic stress-responsiveness. The results of this study will be useful in the continued characterization of soybean BBX gene functions and provide new ideas for soybean breeding.

Screening and characterization of highly responsive genes related to the blast disease caused by Pyricularia oryzae in rice (Oryza sativa)

Blast, caused by Pyricularia oryzae, has been regarded as one of the most critical diseases in rice plants (Oryza sativa). Seeking a list of candidate genes is a major step for resistance breeding programs. In this study, at least 24 RNA-Seq datasets related to blast disease infection in rice plants have been obtained from databases. Among them, four microarray datasets obtained from blast-infected leaf samples in Indica rice varieties, including GSE122258, GSE126961, GSE78266 and GSE39635 were selected for further in silico analysis. Particularly, a total of 216 differentially expressed genes in blast-treated leaves were obtained by exploring four microarray atlas. Among them, at least 32 blast-responsive genes that exhibited similar expression patterns were selected for further bioinformatics analysis. Particularly, these differentially expressed genes encode important transcription factors and enzyme families. By using Expasy and Yloc tools, the physic-chemical properties of these proteins, including lengths, molecular weights, iso-electric points and grand average of hydropathy were greatly variable and the majority of these blast-responsive gene-encoding proteins was localized in the cytoplasm and nucleus. To sum up, this current study has provided fundamental knowledge about potential candidate differentially expressed genes for further functional characterization of genes aiming to control blast resistance in rice.

Genome-Wide Identification of Wheat KNOX Gene Family and Functional Characterization of TaKNOX14-D in Plants.

The KNOX genes play important roles in maintaining SAM and regulating the development of plant leaves. However, the TaKNOX genes in wheat are still not well understood, especially their role in abiotic stress. In this study, a total of 36 KNOX genes were identified, and we demonstrated the function of the TaKNOX14-D gene under mechanical injury and cold stress. Thirty-six TaKNOX genes were divided into two groups, and thirty-four TaKNOX genes were predicted to be located in the nucleus by Cell-PLoc. These genes contained five tandem duplications. Fifteen collinear gene pairs were exhibited in wheat and rice, one collinear gene pair was exhibited in wheat and Arabidopsis. The phylogenetic tree and motif analysis suggested that the TaKNOX gene appeared before C3 and C4 diverged. Gene structure showed that the numbers of exons and introns in TaKNOX gene are different. Wheat TaKNOX genes showed different expression patterns during the wheat growth phase, with seven TaKNOX genes being highly expressed in the whole growth period. These seven genes were also highly expressed in most tissues, and also responded to most abiotic stress. Eleven TaKNOX genes were up-regulated in the tillering node during the leaf regeneration period after mechanical damage. When treating the wheat with different hormones, the expression patterns of TaKNOX were changed, and results showed that ABA promoted TaKNOX expression and seven TaKNOX genes were up-regulated under cytokinin and auxin treatment. Overexpression of the TaKNOX14-D gene in Arabidopsis could increase the leaf size, plant height and seed size. This gene overexpression in Arabidopsis also increased the compensatory growth capacity after mechanical damage. Overexpression lines also showed high resistance to cold stress. This study provides a better understanding of the TaKNOX genes.

Evaluation of In Vitro Morphogenic Response of Triticum urartu, a Donor of Au Genome of Modern Wheat

Triticum urartu Thum. ex Gandil. is a wild diploid wheat species (2n = 2x = 14) that is an Au genome donor of modern polyploid cultivars of durum and bread wheat. In the last decade, this relict species has attracted breeders as donors of various agronomically important characteristics to broaden the genetic diversity of cultivated wheat. In addition, T. urartu can be considered as a model species for studying the evolution, biology and genomics of wheat without the cross-influence of homologous sub-genomes. Various genetic engineering technologies, including transgenesis and genome editing, may be applied to facilitate the functional characterization of genes located in A chromosomes. Such biotechnological techniques are still required for the efficient tissue culture systems to allow easy plant regeneration. The objective of our study was to assess the abilities of in vitro plant regeneration from zygotic immature embryo-derived tissues of spring and winter types of T. urartu. Three synthetic auxins, 2,4-D, Dicamba and Picloram, at four concentrations were studied to stimulate morphogenic responses in spring T. urartu. The induction medium supplemented with 4 mg·L−1 Dicamba stimulated the highest frequency of regenerable callus production (65.8%), promoting the generation of 5.7 plants. Although the presence of 2 mg·L−1 2,4-D was less effective in stimulating regenerable callus formation (53.2%) than Dicamba, it allowed the regeneration of more plants from one regenerable callus (9.3 plants). These two treatments also successfully initiated morphogenesis in winter assertions; however, their regenerative capacity was generally lower. The frequency of regenerable callus production was accession-dependent and fluctuated within 31.3 to 49.2%, with a formation of an average 2.2–5.8 plants per callus. The relatively simple and fast regeneration system described in this study could be further used as the basis for regenerating transgenic plants of T. urartu.

Full-length transcriptome analysis of two chemotype and functional characterization of genes related to sesquiterpene biosynthesis in Atractylodes lancea

Atractylodes lancea (Thunb.) DC. is an important medicinal plant mainly distributed in China. A. lancea is rich in volatile oils and has a significant effect on various diseases, including coronavirus disease 2019 (COVID-19). Based on the signature constituents of volatile oils, A. lancea is divided into two chemotypes: the Dabieshan and Maoshan chemotype. Gas chromatography-mass spectrometry (GC–MS) results revealed that the hinesol and β-eudesmol contents in the Dabieshan chemotype were higher than those in the Maoshan chemotype. Next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing technologies were combined to investigate the molecular mechanisms of sesquiterpenoid biosynthesis in A. lancea. A total of 42 differentially expressed genes (DEGs) for terpenoid biosynthesis were identified in the two chemotype groups, and nine full-length terpene synthase (TPS) genes were identified. Subcellular localization revealed that AlTPS1 and AlTPS2 proteins were localized in the nucleus and endoplasmic reticulum. They use FPP as a substrate to generate sesquiterpenoids. AlTPS1 catalyzes biosynthesis of elemol while AlTPS2 is observed to perform β-farnesene synthase activity. This study provides information for understanding the differences in the accumulation of terpenoids in two chemotypes of A. lancea and lays a foundation for further elucidation of the molecular mechanism of sesquiterpenoid biosynthesis.

Characterization of age-associated B cells in early drug-naïve rheumatoid arthritis patients.

Age-associated B cells (ABCs) are an immune cell subset linked to autoimmunity, infection and ageing, and whose pathophysiological importance was recently highlighted using single cell synovial tissue profiling. To elucidate their pathophysiological relevance, peripheral blood (PB) ABCs from early rheumatoid arthritis (eRA) patients naïve to disease-modifying anti-rheumatic drugs (DMARDs) were compared with their synovial fluid (SF) counterparts, and to PB ABCs from psoriatic arthritis patients and healthy controls. PB and SF B-cell subsets were phenotyped by multi-parameter flow cytometry, sorted and subjected to gene expression profiling (NanoString nCounter® Immunology V2 Panel) and functional characterization (stimulated cytokine measurements by immunoassay). PB ABCs of eRA patients, which are transcriptionally distinct from those of control cohorts, express chemokine receptors and adhesion molecules, such as CXCR3, that favour homing to inflammatory sites over lymphoid tissue. These cells are an activated, class-switched B-cell subset expressing high levels of HLA-DR, co-stimulatory molecules and T-bet. Their secretion profile includes IL-12p70 and IL-23 but low levels of IL-10. High surface expression of FcRL family members, including FcRL3, furthermore suggests a role for these cells in autoimmunity. Finally, and unlike in the periphery where they are rare, ABCs are the predominant B-cell subsets in SF. These observations indicate the predilection of ABCs for inflammatory tissue in RA, where their propensity for antigen presentation and pro-inflammatory phenotype may support autoimmune pathology. Their potential as a therapeutic target therefore warrants further study.

Functional genomics of the lactic acid bacterium Limosilactobacillus fermentum LAB-1: metabolic, probiotic and biotechnological perspectives

A genome-based systematic analysis was conducted to characterize the metabolic, probiotic, fitness, and safety properties of Limosilactobacillus fermentum LAB-1, a lactic acid bacterium demonstrating strong antimicrobial effects against clinical pathogens. Gene functional characterization revealed a large number of genes for carbohydrate metabolism and a heterofermentative system for carbon dissimilation. Genes for intact pyruvate oxidation, pentose phosphate, and PRPP biosynthetic pathways were identified. Substantial carbohydrate-active enzymes and transporters were also predicted. Metabolic reconstruction revealed complete sets of enzymes for arginine, lysine, methionine, threonine, proline, and ornithine biosynthesis. The bacterium harbors a diverse range of peptidases, and a large variety of peptide and amino acid uptake systems. It encodes restriction-modification and CRISPR-Cas systems for protection against phage infections and carries a wide spectrum of stress proteins for adaptation in the gut and industrial conditions. Genes related to the biosynthesis of B-group and K vitamins were identified allowing its application for novel bio-enriched food production. Other beneficial traits of probiotic and industrial importance such as production of flavor compounds, exopolysaccharide, acetoin, and butanediol were identified. Three antimicrobial peptides were predicted which showed >98% sequence-identity to experimentally validated bacteriocins. Negative traits such as transmissible antibiotic resistance, pathogenicity or virulence appeared to be absent suggesting the strain to be considered safe. The genome analysis will allow precisely targeted laboratory research and full exploitation of the probiotic potentials towards functional-food, biotechnology and health-related applications.