All these processes highlight the complex relationship between ROS and NO metabolism which needs to be evaluated from a broad perspective.Reactive oxygen species (ROS) and nitric oxide (NO) are produced in all aerobic life forms under both physiological and adverse conditions. Unregulated ROS/NO generation causes nitro-oxidative damage, which has a detrimental impact on the function of essential macromolecules. ROS/NO production is also involved in signaling processes as secondary messengers in plant cells under physiological conditions. ROS/NO generation takes place in different subcellular compartments including chloroplasts, mitochondria, peroxisomes, vacuoles, and a diverse range of plant membranes. This compartmentalization has been identified as an additional cellular strategy for regulating these molecules. This assessment of subcellular ROS/NO metabolisms includes the following processes: ROS/NO generation in different plant cell sites; ROS interactions with other signaling molecules, such as mitogen-activated protein kinases (MAPKs), phosphatase, calcium (Ca Reactive oxygen species (ROS) are redox molecules mainly produced in aerobic organisms, with some of the most important being photosynthetic cells, in which ROS production needs to be continually regulated to prevent undesirable functional collateral damage. ROS, which are involved in signaling processes in subcellular compartments and cells, as well as in mechanisms of defense against biotic and abiotic stresses, are also essential for cell survival [Molecular oxygen , superoxide anion (O2•−), hydroxyl radical (•OH), and singlet oxygen (1O2), are metabolically closely associated with another family of molecules, called reactive nitrogen species (RNS), including peroxynitrite (ONOO−) and S-nitrosoglutathione (GSNO), derived from the nitric oxide (•NO) radical. Though generated under normal physiological conditions, these two molecular families are prone to uncontrolled overproduction under stressful circumstances, causing cellular nitro-oxidative damage which can hamper cell survival [In higher plants, the principal subcellular ROS production sites include chloroplasts, mitochondria, cytosols, vacuoles, peroxisomes, plasma membranes, apoplasts, and cell walls . ROS, susurvival ,5. Figursurvival ,7. 2+) is also actively involved in intra- and extra-cellular signaling networks [2+ and ROS signaling interactions are considered to be bidirectional, as Ca2+ is necessary for ROS production, while ROS is primarily required for the regulation of cellular Ca2+ [l-Arg-dependent NO synthase-like activity [ROS interact with many other signaling molecules which influence a broad spectrum of responses. More advanced transcriptomic techniques have identified important stress-responsive signaling molecules, such as mitogen-activated protein kinase (MAPK)and phosphatase, involved in ROS metabolism . Plant Mnetworks . Ca2+ anlar Ca2+ ,12. Strelar Ca2+ . The irolar Ca2+ . Furtherlar Ca2+ . The prelar Ca2+ ,17,18. Uactivity  and a niactivity , respect2. ROS are produced from oxygen under physiological conditions in response to abiotic and biotic factors, which eventuallyreducesO2 to water containing physiologically important inter medium molecules such as H2O2, O2•−and •OH [2•−, which is dismutated either chemically or enzymatically to H2O2 and then converted to •OH via a Haber-Weiss reaction triggered by iron ions (Fe2+/Fe3+) [2•−pool of less than 0.5%, the protonated superoxide radical HO2•, also called a hydroperoxyl or perhydroxyl radical, is comparatively more toxic [1O2, another toxic ROS species, is an electronically excited O2 generated in chloroplasts [2O2 and NO. For example, H2O2 acts as a retrograde signal from chloroplasts and mitochondria to the nucleus and triggers an appropriated response [Figure 2, respectively.Living organisms survive in an oxidizing environment where aerobic metabolism is supported by O−and •OH . Attenua2+/Fe3+)   and plastoquinone (PQ) level in photosystem PSII and then dismutated to H2O2 (ROS production in chloroplasts is directly related to photosynthetic light reactions in the thylakoid membrane. O to H2O2 .+ to enable ferredoxin-NADP reductase (FNR) to produce NADPH at PSI. Under stress conditions, the electron transfer chain (ETC) is overloaded, with part of the electron directed to O2, causing O2•− to be generated at PSI (Mehler reaction). O2•− is dismutated to H2O2 either spontaneously or by superoxide dismutase (SOD) in the thylakoid membrane. It is worth pointing out that the highly efficient SOD-catalyzed dismutation reaction occurs at a diffusion-limited rate of ∼2 × 109 M−1s−1, or ∼104 times the rate constant for spontaneous dismutation, and H2O2 is then rapidly detoxified by ascorbate peroxidase (APX). The electron flow from H2O at PSII to H2O at PSI is called the water-water cycle [2•− and H2O2 to suppress the production of •OH radicals, whose interaction with target molecules is thus prevented, leading to photoinhibition. This outcome is particularly important under stress conditions, such as high temperatures, drought and salinity. Depending on the plant species, several chloroplastic SOD isozymes, including Fe-SODs and Cu/Zn-SODs, bind to the thylakoid membrane located close to PSI [Under physiological conditions, the electron transport chain (ETC) from excited photosystem centers of the thylakoidal membrane is directed to NADPer cycle . This efe to PSI ,25. Simie to PSI . 1O2, whose formation is unique to the chloroplast, is generated in the thylakoid membrane, specifically through the transfer of energy from P680, the primary electron donor at PSII, to ground O2 [round O2 . S-nitrosation, which affect chloroplast function [S-nitrosated proteins identified include Rubisco, glyceraldehyde-3-phosphate dehydrogenase, dehydroascorbate reductase (DHAR), peroxiredoxin II E, fructose-1,6-bisphosphatase, phosphoribulokinase, and FNR [NO, which has been detected in chloroplasts, is generated by L-arginine- and NADPH-dependent NO synthase activity . It stimfunction . The nitfunction . The S-n and FNR ,32,33. A2•− overproduction, which is regulated by stromal and thylakoid-bound SODs, is dismutated to H2O2 and then regulated by the APX cycle as part of the water-water cycle. When H2O2 content, estimated at roughly 0.5µM under normal conditions, is exposed to differential stress, it increases to 1–15 µM. Thus, regulation of H2O2, which triggers retrograde signaling from chloroplasts to the nucleus, especially under stress conditions, is of great importance in preventing oxidative damage [2O2, mainly the ascorbate-glutathione (ASC-GSH) cycle, peroxiredoxins (Prxs), and thioredoxins (Trxs).As mentioned above, Oe damage . The chlThe ASC-GSH cycle is comprised of four enzymes: ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR). These enzymes require the presence of non-enzymatic antioxidants ascorbate (ASC) and glutathione (GSH) , which s2O2, alkyl hydroperoxide (ROOH), and ONOO−, present on various peroxide substrates. With a catalytic efficiency in the range of 105–108 M−1 s−1 greatly depending on thiol regeneration, Prxs contain one or two essential cysteines in conserved sequences and can be classified into four main groups in higher plants: 1-Cys Prxs, containing one conserved cysteine; 2-Cys Prxs; Prxs Q; and Prxs type II, containing two catalytic cysteines [Prx and Trx systems are involved in a complex redox homeostasis network aimed at protecting the chloroplast against oxidative damage ,37. Prxsysteines . Arabidoysteines . Trxs which are small thiol-disulphide proteins with 14 kDa, catalyze reversible disulphide bond formation, which facilitates the functional regulation of many proteins. Arabidopsis encloses twenty Trx isoforms, six of which  are present in chloroplasts . Chlorop1O2 scavenging chiefly occurs through reactions with biomolecules such as tocopherols [1O2-mediated chloroplast retrograde signaling [1O2 and O2•− have a short life-time and perform signaling functions due to their involvement in other signaling components such as proteins EXECUTER 1 and 2 [1O2 in nuclear gene modulation. The 1O2-producing Arabidopsis flu mutant is totally defenseless against paraquat stress and enhanced chloroplast APX expression, suggesting an interaction between 1O2 and H2O2 signals [1O2 [opherols , carotenopherols , and memopherols . β-carotignaling . 1O2 and 1 and 2 , which p signals . Xanthop signals . Vitaminals [1O2  to form als [1O2 , while tals [1O2 . 2O2 synthesized in the chloroplast in response to light reactions is transferred to nuclei without stromule formation when the chloroplast is adjacent to the nucleus [2O2 synthesis when the chloroplast near the nucleus is exposed to 405 nm laser light has been found to lead to a transfer of H2O2 to the nucleus [2O2-dependent oxidative PTMs. Inactivated PAP acts as a secondary messenger and assists in the transfer of information from the chloroplast to the nucleus [Stromules are tubular plastid extensions  whose sy nucleus . An in s nucleus . Further nucleus .2 under abiotic stress conditions with a concomitant production of O2•−, which is dismutated to H2O2 by MnSOD and quenched by APXs or Prxs. Unlike chloroplasts, mitochondria have only one Prx called Prx II F, while mitochondria have two Trxs (Trxo1 and Trxo2) which can activate alternative oxidase (AOX) [2O2 reacts with reduced Fe2+ and Cu+ to produce highly toxic •OH through a Fenton reaction. It is worth noting that the alternative oxidase (AOX) enzyme provides another route to the cytochrome pathway which facilitates its transfer to O2 to form H2O and dissipates energy in the form of heat to prevent the formation of ROS [2 absorbed by the mitochondrial membrane is reduced to O2•−. Mitochondria contain a respiratory electron transport chain (ETC) comprised of four key complexes: NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV), in addition to two mobile electron transporters, cytochrome c, and ubiquinone . The resse (AOX) . To funcn of ROS  especialn of ROS . Approxi2, reduces ROS production and counters changes in redox homeostasis. This reaction mixture is transduced to two endoplasmic reticulum (ER) transcription factors (TFs), Arabidopsis NAC domain-containing protein 13 (ANACO13) and ANACO17. These TFs are then translocalized to the nucleus in response to stress signals [2O2 production under heat and oxidative stress conditions [l-galactono-1,4-lactone dehydrogenase  which oxidizes l-galactono-lactone to ascorbic acid [Under stress conditions, the active form of the AOX homodimer can be reduced to the oxidized inactive form of the AOX monomer, which disrupts the mitochondrial ETC, resulting in enhanced gene-encoding AOX expression and other target such as mitochondrial dysfunctional stimulon (MDS) gene. Enhancement of AOX activity directs free electrons to O signals . However signals . Meanwhi signals . In maiz signals . The intnditions . In planbic acid  and suppbic acid . Mitochondria generate NO, which is produced by chemical reduction from nitrate and not by enzymatic activity . NO counMitochondria also generate extensions called matrixules which appear to play an, as of yet, little understood role in the ROS signaling mechanism . Plant peroxisomes are single membrane-bound organelles characterized by an adaptable and active nitro-oxidative metabolism ,71,72,732+ and calmodulin. Peroxisomal NO is necessary for various physiological functions such as pollen tube growth, leaf senescence, auxin-induced root organogenesis, and abiotic stress responses such as salinity, cadmium, and lead. Peroxisomes contain ONOO− and GSNO, while peroxisomal proteins, such as catalase, MDAR, GOX, hydroxypyruvate reductase, and malate dehydrogenase, prone to tyrosine nitration and S-nitrosation, have also been identified. Like chloroplasts and mitochondria, peroxisomes appear to generate peroxules under ROS stress conditions [NO was first identified in plant peroxisome organelles, in which a NOS like activity oxidizes L-arginine to NO using NADPH as a donor requiring Canditions . 2O2 generating •OH via the hydroxylic cycle, while proteomic analysis has revealed that RBOH is localized in the tonoplast region [2O2 is diffused through the tonoplast either directly or via aquaporins in vacuoles [•OH, a reaction which is triggered by enhanced phenolics, GSH and ascorbate present in vacuoles, suggesting that phenolics and fructans are synergistically active [Vacuoles, which contain abundant antioxidants and play a multifunctional role in plant metabolism, are among the largest redox homeostasis maintenance sites in response to elevated levels of ROS under abiotic stress conditions . Vacuolat region . Cellulavacuoles , which avacuoles . Enhancevacuoles  and drouvacuoles  conditiovacuoles . Solublevacuoles . Fructany active . Finallyy active . 2+-binding EF-hand motifs, indicating that RBOH activity is regulated by Ca2+ [2+, phosphorylation, phosphatidic acid, and NO, are also involved such as Ca2+-dependent andCa2+-CaM-dependent protein kinases, as well as a S-nitrosation mechanism [The plasma membrane is an important center of ROS production due to the presence of membrane-bound NADPH oxidase, also known as the respiratory burst oxidase homolog (RBOH). RBOH can transfer free electrons from its intracellular region to molecular oxygen via the apoplast through the plasma membrane . The com by Ca2+ ,88. In aechanism . Arabidopsis thaliana, nine in rice (Oryza sativa), eight in tomato (Solanum lycopersicum) and sweet orange (Citrus sinensis) and seven in common grapevine (Vitis vinifera). RBOH activity is involved in many plant processes including cell growth, plant development, stomatal closure, pollen tube growth, abiotic stress, symbiotic interactions, and pathogen responses [Although their number varies according to the plant species, RBOH genes belong to a multigene family composed often members in esponses . It is aesponses  and stonesponses . RBOH’s S-nitrosation [The apoplast, linking the environment and cells, is the plant cell region outside the plasma membrane. Apoplastic spaces are more complex than one would expect due to the presence of various components involved in ROS metabolism. These components, which have been found in the apoplast of different plant species, include APX, GR, guaiacol-dependent peroxidase, SOD isozymes, GSH, diamine oxidase (DAO), and polyamine oxidase , in addirosation . In many cases, apoplastic ROS metabolism is crucial for processes, such as axillary bud outgrowth , signal crk mutant plants [ROS signaling in apoplastic spaces involves local signals including: (i) sensing mechanisms directly via oxidative PTMs and (ii) sensing mechanisms indirectly via oxidation of metabolites present in apoplastic regions . Apoplast plants . A set ot plants . Anothert plants ,104. GRIt plants . 2O2 is simultaneously produced. The H2O2 produced plays a pivotal role in plant biotic and abiotic stress [Trxs, which are involved in regulating redox status under various environmental cues via thiol-disulfide interchange reactions, are also present in the apoplast . Secretic stress  and devec stress .2•− production, thus activating adaptive plant responses to abiotic stress conditions, particularly hyperthermia and drought [•OH production in vivo and in vitro has been observed in various plant cell walls [2•− and H2O2 generation in maize root cell walls has been confirmed by histochemical analysis. O2•−and •OH react with the methyl group of polysaturated fatty acids to form conjugated dienes, hydroperoxides, and peroxy radicals, which, in turn, react with metal ions such as Fe2+, while the lipid radicals formed initiate a further chain reaction [Cell walls are another major location for the accumulation of ROS , whose p drought . Diamine drought . •OH proll walls ,116, whireaction . S-nitrosoglutathione (GSNO) promotes cell wall remodeling and induces root hair formation in Arabidopsis [NO also affects cell wall metabolism  and indubidopsis . 2+. Biological responses to ROS depend on the type of ROS, signal magnitude, formation site and crosstalk with other signaling molecules such as MAPK, NO, and Ca2O2 mediates the activation of several components in the MAPK cascade by, for example, inactivating MAPK repressors, although, ROS have been observed to respond when this cascade is manipulated [The MAPK cascade is conserved in eukaryotes which initiate the signaling process when MAPK kinase kinase (MAPKKK) activates MAPK kinase (MAPKK) which, in turn, activates MAPKs. The relationship between the MAPK cascade and ROS has been described in various studies; exogenous Hipulated . The casRBOH gene, as well as WRKY transcription factors, both of which, they suggest, are phosphorylated by MAPK, thus revealing further crosstalk between MAPK phosphorylation and RBOH. MAPK cascades are considerably involved in retrograde signaling from chloroplast to the nucleus of the stressed plant cells . This ne2+ homeostasis is strictly regulated given the importance of Ca2+ as a second messenger which remains local or is transmitted throughout the whole cell in milliseconds to seconds. This regulation involves Ca2+ pumps, channels, cation exchangers, and Ca2+-binding proteins, as the Ca2+ cation regulates multiple cellular activities. Moreover, crosstalk between Ca2+ and other signaling molecules, including ROS and NO, which affects gene expression and Ca2+-dependent protein kinases, takes place under biotic and abiotic stress conditions [Cellular Canditions . 2+ signaling pathways can be both antagonistic and synergistic, depending on the ROS species, target protein type, duration, dosage, and tissue type. The formation and removal of ROS and their interaction with Ca2+ signaling are complex mechanisms which provide an excellent model to explore the molecular players involved in the interplay between ROS and other signaling cascades. An enhanced metabolic rate can lead to higher levels of O2, which increases the leakage of respiratory ETC and ROS concentrations. ROS generation in mitochondria is directly associated with the metabolic rate, and Ca2+ accumulation reduces the level of ROS generated by complexes I and III under normal conditions, while increasing ROS formation when these complexes are suppressed by pharmacological agents. Ca2+ may stimulate three dimensional (3-D) conformational alterations in respiratory ETC complexes, which further induces ROS production in mitochondria [2+ uptake reduces ROS formation. When membrane potential is in a depolarized range in the presence of ATP synthesis, ROS production is triggered which may be dependent on Ca2+ load. With a high Ca2+load in mitochondria, ROS generation independently enhances the mitochondrial metabolic rate [2+ on ROS production and eradication depends on subcellular compartments and cell tissue. CaM, a Ca2+binding protein with approximately 16 kDa, modulates antioxidative enzyme activity and is associated with ROS homeostasis. The suppressed or activated status of redox regulation may alter the activity, transcript level, open channel and trafficking [2+ signals are important for fully functioning cells involved in the discharge of Ca2+ from its storage site and the entry of Ca2+ through plasma membrane channels. The two main categories of channel proteins associated with receptor-triggered Ca2+ entry signals are transient receptor potential and store-operated Ca2+ channels [Mutual crosstalk between ROS and Cachondria . When melic rate . The ovefficking . Receptochannels . 2+ needs to be present in crosstalk with H2O2 for development purposes and in response to a diverse range of abiotic stresses [2+ together with CaM and NADPH redox power are strictly required [With regard to free-radical NO generation, Castresses . At the required .2O2 concentrations. Oxidant-dependent sulfhydryl GSH:GSSG cysteine conversion plays a key role in stress management by regulating redox homeostasis and also controls the function of various transcription factors [2O2 signaling and receptor-like kinase activation play a significant role in regulating redox-sensitive gene expression [2O2 signaling and accumulation also play an important role in activating enzymatic antioxidant-encoding gene expression and can be effectively used to regulate stress-responsive gene activity to withstand harsh environmental conditions. Furthermore, O2•− has specific targets including WRKY46, WRKY28, and WRKY22. Redox-sensitive genes include glutathione peroxidase (GPX), catalase (CAT), SOD2, quinone reductase, heme oxygenase-1, ferritin, GR, thioredoxin reductase, thioredoxin, metallothionin, cycloxygenase-2, and γ-glutamylcysteine synthase which is required for GSH synthesis [Redox balance in stressed cells is maintained by the ASC-GSH cycle which plays important role in regulating H factors . In addi factors , while Hpression . H2O2 siynthesis . 2+-ATPase, collagenase, and SRC tyrosine kinases. However, ROS not only regulate pre-existing protein activity but also induce the expression of certain genes that control the gene expression patterns of other signal transduction networks. Interest in how ROS and NO regulate gene expression under stress conditions and in identifying the TFs involved has been increasing. Available information indicates that highly complex signaling networks involvingH2O2 and NO, as well as phytohormones such as ABA, JA, and SA, are implicated in signaling cascades. Recently, up to sixty H2O2-responsive TFs involved in stresses such as cold, drought, heat, high light, and pathogens, were identified [S-nitrosothiols via a redox-dependent upregulation of TFs such as the ethylene-responsive TFs ERF1, ERF2 and myeloblast 1 (MYB1), as well as other stress response genes [Arabidopsis thaliana exposed to GSNO, RNAseq analysis reveals overexpression of TF groups including five members of the WRKY gene family involved in pathogen and abiotic stress responses, four members of the MYBTF family, heat stress TFs, and the transcription factor-binding TF GT-1 which binds to the cis-acting element BoxII [In eukaryotic cells, TFs are regulated by complex redox-sensitive systems, consisting of many systematically ordered elements, including GTPase, phosphatase, and lipase, as well as phospholipid-dependent and MAP kinases. ROS-regulated TFs contain reactive cysteine thiol groups whose redox status is affected such as Caentified . On the se genes . In the nt BoxII . Recentlnt BoxII  identifiIn eukaryotic cells, the IRE-IRP system consists of iron responsive elements (IREs) and iron regulatory proteins (IRPs). Iron (Fe) is an important nutrient which plays a significant role in cellular metabolism and morphogenesis. It acts as a co-factor in major enzymes such as ribonucleotide reductase, cytochrome P450, and leghemoglobin, as well as those involved in ROS metabolism including cytochrome c oxidase (COX), xanthine oxido-reductase, catalase, APX, Fe-SOD, and peroxidase . HoweverIRP1 and IRP2, which interact with IRE mRNA in the 3ʹ and 5ʹ untranslated regions (UTRs), are necessary for Fe homeostasis. Transferrin receptor-1 (TFR1) regulates Fe movements in cells and ferritin, as well as cellular Fe storage . The fatIRT1 gene is highly expressed in iron-deficient root cells, resulting in elevated levels of iron absorption which improves growth and development under iron-starved conditions. IRTI is involved in the absorption of other minerals, including Zn, Mn, Ca, Co, and Ni [It is crucial to monitor soil mineral absorption by root cells for plant growth and development purposes . Any def, and Ni , and in , and Ni . 3+ ions are reduced to Fe2+ ions by ferric chelate reductase enzyme FRO2 and then transported through the rhizodermal cell membrane by IRT1 [Arabidopsis plants, IRT1 is sequestered and gradually degraded by ubiquitination and phosphorylation, a process triggered by IRT1 binding to metal ions other than Fe [2O2 induced by RBOH1, with a concomitant boost in lignin biosynthesis and low phenolic secretion, which blocks apoplastic Fe mobilization efficiency [Plant root secretion of Fe-mobilizing phenolic compounds facilitates Fe detoxification phenomena . Fe3+ io by IRT1 . On the  by IRT1 . In Arab than Fe . IRT1 pr than Fe . Despite than Fe , IRT1 ub than Fe . Nitrateficiency . NO also preserves Fe homeostasis through crosstalk with ethylene and auxin phytohormones, ferritin and frataxin and through its ability to form nitrosyl iron complexes . New eleAs outlined above, ROS and NO play a crucial role in signaling mechanisms, which modulate plant growth and development, transpiration, hormonal regulation, germination, stomatal gaseous exchange, flowering, fruit ripening, defense responses, and programmed cell death (PCD) .S-nitrosation, nitration, and metal nitrosylation, in order to regulate gene expression and its interaction with phytohormones [Although the endogenous sources of enzymatic NO in higher plants remain a subject of debate such as it was mentioned previously, its presence and function in plant cells are clear . NO perfhormones .S-nitrosation and nitration are the most studied NO PTMs [2) to susceptible amino acids, with tyrosine (Tyr) being the most studied. In both cases, the PTM positively or negatively affects protein function but could also have no functional impact. In higher plants,  NO PTMs ,167. WhiS-nitrosation [S-nitrosation, which inhibits O2•−-generating RBOHD in Arabidopsis [2O2, are differentially affected by nitration and S-nitrosation. Nitration inhibits, while S-nitrosation triggers an increase in APX activity [S-nitrosation inhibit MDAR activity, GR function remains unaffected by either PTM [2O2, alkyl hydroperoxide (ROOH), and peroxynitrite (ONOO−), which are present in almost all cellular compartments [S-nitrosation [2•− and NO generation by RBOH and NOS-like activity, respectively. These NADP-DHs are differentially modulated by nitration/S-nitrosation: chloroplast FNR is inhibited by nitration, NADP-ICDH and NADP-ME are inhibited by both nitration and S-nitrosation [S-nitrosoglutathione reductase (GSNOR), involved in physiological and stress processes [S-nitrosation cellular capacity, is inhibited by S-nitrosation [Over the last ten years, significant advances have been made in the regulation by NO of ROS metabolism in plants. rosation . On the bidopsis , does nobidopsis . Differeactivity . Althougther PTM . As mentartments . In Arabrosation . NADPH-grosation , while Grosation . Remarkarocesses , which rrosation .Although NO regulates the expression of specific genes , high-th2+, cyclic GMP, and various plant hormones [NO, produced in plants to combat pathogen attacks, is directly involved in defense-related signaling pathways via secondary messenger protein kinases, cyclic AMP, signaling lipids, Cahormones . It alsohormones . S-nitrosation and tyrosine nitration, with non-expressor of pathogenesis-related gene 1 (NPR1) and AtRBOHD providing new insights into the role of NO in plant defenses. Many distinctive functions have been studied with regard to ROS-NO interactions, leading to the generation of related molecules such as NOx, ONOO−, N2O3, and NO2. They also act as oxidizing molecules on different proteins involved in NO-ROS signaling, resulting in structural and functional alterations in these proteins [Functional molecular analysis of NO-responsive genes in defense pathways has been used to identify encoding proteins for secondary metabolism and pathogen-related proteins . Skelly proteins .LcRBOH, LcMC-1-like and LcPirin genes, which play a role in ROS- and NO-mediated senescence and leaf abscission in litchi plants [2 metabolism in nodules. In a study of inoculated M. truncatula plants treated with an RBOH oxidase activity inhibitor, the NO scavenger clearly showed overlapping signaling pathways mediated by these molecules [MtNR1, Mtns-Hb1, and MtNiR genes have been observed to be upregulated in M. truncatula roots as a result of mediation by H2O2 of enzymatic transcriptional regulation to maintain NO homeostasis [Arabidopsis during plant pathogenesis, which leads to higher levels of NO production and affects RBOHD activity via S-nitrosation [Yang et al.  have elui plants . NO and i plants . Interaci plants . In addii plants ; this poolecules . Gene exolecules . Howevereostasis . Similarrosation . For ins2S), serotonin and melatonin. Finally, the complex relationships involved in cellular ROS/RNS metabolism need to be looked at from a broad perspective and we particularly hope that this assessment provides non-specialist researchers and academics alike with a clear understanding of this field of research. ROS/RNS, a fundamental feature of plant cell metabolism, increase nitro-oxidative damage when overproduced and prevent regulation by the corresponding antioxidant systems.


Ganoderma boninense that assembled as a single circular double-stranded DNA (dsDNA) of 86,549 bp with a G+C content of 26.81%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNA subunits, 4 hypothetical proteins, and 5 homing endonucleases.Here, we report the complete sequence of the mitochondrial (mt) genome of  Ganoderma boninense that assembled as a single circular double-stranded DNA (dsDNA) of 86,549 bp with a G+C content of 26.81%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNA subunits, 4 hypothetical proteins, and 5 homing endonucleases.Here, we report the complete sequence of the mitochondrial (mt) genome of  Elaeis guineensis Jacq) is the world’s most efficient oil-producing crop. Indonesia and Malaysia are major producers, but both are facing an economically important disease called basal stem rot disease that is caused by a basidiomycete fungus, Ganoderma boninense , apocytochrome b (cob), NADH dehydrogenase subunits 1 to 6 , ATP synthase subunits 6 to 9 , and ribosomal protein (rps3). All tRNA- and rRNA-encoding genes  and 4 hypothetical proteins were found on the positive strand and were clockwise oriented, except for trnW-CCA, which was found in the negative strand and was counterclockwise oriented. In addition, 5 homing endonucleases with LAGLIDADG motif patterns were identified.The PRJNA421251. In addition, all raw reads to build scaffold 204 were also deposited under the SRA run number SRR8351970.This mt genome shotgun project has been deposited at the NCBI database under the BioProject accession number


Staphylococcus aureus strain USA300 is believed to have originated from an ancestral methicillin-susceptible strain, although the details of that evolution remain unknown. To help understand the emergence of this highly successful strain, we sequenced the genomes of two methicillin-susceptible Staphylococcus aureus clinical strains that are very closely related to USA300.Predominant community-associated methicillin-resistant  Staphylococcus aureus strain USA300 is believed to have originated from an ancestral methicillin-susceptible strain, although the details of that evolution remain unknown. To help understand the emergence of this highly successful strain, we sequenced the genomes of two methicillin-susceptible Staphylococcus aureus clinical strains that are very closely related to USA300.Predominant community-associated methicillin-resistant  Staphylococcus aureus (MRSA) clones are still poorly understood, although it is hypothesized that they repeatedly arose from epidemic methicillin-susceptible S. aureus (MSSA) strains through acquisition of the mecA gene via horizontal transfer  RSII sequencing technology, with one single-molecule real-time (SMRT) cell. Contig assembly was done using the RS Hierarchical Genome Assembly Process (HGAP) protocol version 2.3.0.140936.p5 –17, with–N50 value of 2,823,074 bp. The estimated genome coverage was 368×, and the GC content was 32.82%. On the assembled chromosome of 2,795,888 bp, 2,924 genes were identified, of which 2,842 were coding sequences (CDS), 82 were RNA genes, and 76 were pseudogenes. Four contigs were assembled for MSSA strain H489 from 94,559 raw reads covering 1,215,818,661 sequenced bases, with an N50 value of 2,761,569 bp. The estimated genome coverage was 392×, and the GC content was 32.79%. On the chromosome of 2,757,748 bp, 2,874 genes were identified, of which 2,789 were CDS, 85 were RNA genes, and 94 were pseudogenes.Two contigs were assembled for MSSA strain C3948 from 85,037 raw reads, covering 1,142,835,600 sequenced bases, with an A complete analysis is under way to look at the major genetic components in these MSSA isolates and compare them with those found in MRSA USA300 to help shed light on the evolutionary path of the highly successful USA300 strain.CP020957 (C3948) and CP020959 (H489), with SRA accession numbers SRX5551895 (C3948) and SRX5552203 (H489).The chromosomal genome sequences have been deposited at GenBank under the accession numbers


Escherichia coli strain BL21 (DE3) in biotechnological processes makes the study of phage receptors in this strain particularly important. Here, eight phages infecting E. coli BL21 (DE3) via different receptors were isolated and subsequently identified as members of the genera T4virus, Js98virus, Felix01virus, T1virus, and Rtpvirus. Phage receptors were identified by whole-genome sequencing of phage-resistant E. coli strains and sequence comparison with wild-type BL21 (DE3). Results showed that the receptors for the isolated phages, designated vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoM_IME281, vB_EcoM_IME338, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341, and vB_EcoS_IME347 were FhuA, FepA, OmpF, lipopolysaccharide, Tsx, OmpA, FadL, and YncD, respectively. A polyvalent phage-resistant BL21 (DE3)-derived strain, designated PR8, was then identified by screening with a phage cocktail consisting of the eight phages. Strain PR8 is resistant to 23 of 32 tested phages including Myoviridae and Siphoviridae phages. Strains BL21 (DE3) and PR8 showed similar expression levels of enhanced green fluorescent protein. Thus, PR8 may be used as a phage resistant strain for fermentation processes. The findings of this study contribute significantly to our knowledge of phage-host interactions and may help prevent phage contamination in fermentation.Bacteria-based biotechnology processes are constantly under threat from bacteriophage infection, with phage contamination being a non-neglectable problem for microbial fermentation. The essence of this problem is the complex co-evolutionary relationship between phages and bacteria. The development of phage control strategies requires further knowledge about phage-host interactions, while the widespread use of  Bacteriophage, which were first discovered by Twort in 1915 and confirmed by d’Hérelle in 1917, are a highly diverse group of viruses that are ten times more abundant than their bacterial hosts in most environments . As alteMyoviridae phage Me1 and Siphoviridae phage Gifsy-1  was specifically designed for the overexpression of recombinant proteins. Understanding phage-host interactions, particularly the phage receptors of strain BL21 (DE3), is essential for the development of next-generation anti-phage strategies (E. coli strain derived from BL21 (DE3). We confirmed that FhuA, FepA, OmpF, LPS, Tsx, OmpA, FadL, and YncD can be used as receptors by BL21 (DE3)-infecting phages. Together, our findings provide data support for phage-host interaction studies and will aid in the control of phage contamination of E. coli BL21 (DE3).rategies . Howeverrategies . Here, pE. coli strain BL21  was stored at −70°C in 25% (v/v) glycerol. Plasmids pKDsg-ack, pCas9cr4, and pET-28a were stored at −20°C. All bacterial strains were cultured in Luria-Bertani (LB) broth . Medium was supplemented with agar (1.5%), soft agar (0.75%), 100 mg/L ampicillin (Amp), 50 mg/L spectinomycin (Spec), 50 mg/L kanamycin (Kana), or 100 μg/L anhydrotetracycline (aTc) when necessary.Bacteria and plasmids used in this study are listed in E. coli strain BL21 (DE3) were isolated from sewage samples collected from the State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China. The sewage samples were centrifuged at 13,000 × g for 10 min and then filtered through a 0.22-μm pore size filter . Thereafter, 4 mL of the filtrate were mixed with 200 μL of log-phase BL21 (DE3) cells [optical density at 600 nm (OD600) = 0.6] and inoculated into 2 mL of 3 × LB medium before being incubated at 37°C, 220 rpm until clarification. The resulting culture suspension was filtered as above and the phages were cultured using the double-layer agar plate method as described previously  and phage suspensions  obtained in the above procedure were mixed with soft agar and then poured onto the surface of LB-agar plates. The plates were incubated at 37°C for 12–18 h or until colonies were produced on the plaques. Single bacterial colonies were obtained by streaking, and their sensitivity to phage infection was examined using a spotting assay and a double-layer agar plate method (Log-phase BL21 (DE3) suspension  to achieve a MOI of 10, 1, 0.1, 0.01, 0.001, or 0.0001, and then incubated at 37°C, 220 rpm for 4 h. Culture supernatant was then filtered through a 0.22-μm filter, and the titer of the phage in the supernatant was measured using a double-layer agar plate method. Three replicates were conducted for determination. The MOI resulting in the highest phage titer was considered the optimal MOI of the phage.The optimal MOI and one-step growth curves for isolated phages were determined using methods described previously . Briefly7 CFU/mL) at the optimal MOI and incubated at 37°C for 5 min. The culture was then centrifuged at 12,000 × g for 1 min and the supernatant discarded. The pellet was then washed twice with LB medium and re-suspended in 20 mL of LB medium. The moment when the pellet was re-suspended in medium was defined as time zero. Then, the resulting culture was transferred to a shaker and incubated at 37°C, 220 rpm for 1.5 h. Three duplicate samples (200 μL) were collected every 10 min to determine the phage titer at different time points. Three replicates were conducted for determination. The one-step growth curve was obtained by plotting phage titer against time. The burst size was calculated by dividing the plateau phage titer by the initial phage titer.The one-step growth curve of a phage reflects dynamic changes in the number of particles during phage replication. To obtain a one-step growth curve for each of the isolated phages, phage suspension was added to 20 mL of log-phase BL21 (DE3) culture . Then, high-throughput sequencing of the DNA was performed on an Illumina MiSeq instrument . Genomes were assembled from filtered high-quality reads using the assembly algorithm Newbler version 3.0 with default parameters. Open reading frame prediction and genome annotation were carried out using the RAST1 tool and NCBI nucleotide collection (non-redundant nr database) BLASTp alignment, respectively. A phylogenetic tree was constructed using 75 terminase large subunit protein sequences of different phages from the Ounavirinae, Tevenvirinae, and Tunavirinae subfamilies in the International Committee on Taxonomy of Viruses virus taxonomy current release. The alignment of phage terminase large subunit sequences was carried out using ClustalW in MEGA 6 software, and then phylogenetic analysis was performed using the maximum likelihood method based on the JTT matrix-based model with 1000 bootstrap replicates.Phage genomic DNA was extracted using a modified phenol-chloroform extraction protocol . A 2 × 3E. coli strains and phage-sensitive strain BL21 (DE3) using a High Pure PCR Template Preparation Kit . Then, a 2 × 300 nt paired-end DNA library was constructed using the Illumina NEB Next® UltraTM II DNA library preparation kit and high-throughput sequencing was performed using Illumina MiSeq according to the manufacturer’s instructions. Sequence reads were mapped against the BL21 (DE3) reference genome (GenBank Accession No. CP001509.3) using CLC Genomics Workbench 9.0, followed by basic variant detection and gene deletion searches.Genomic DNA was extracted from phage-resistant 8 CFU/mL) bacterial culture (1 mL) was centrifuged at 12,000 × g for 1 min and the pellet resuspended in phosphate-buffered saline (0.9 mL). A 0.1 mL aliquot of phage solution (∼106 PFU/mL) was added to the cell suspension and the culture mixture was incubated at 37°C for 5 min to allow adsorption. In controls, LB medium was used instead of a cell suspension. Cultures were then centrifuged at 12,000 × g for 1 min and the titer of free phage in the supernatant was determined using the double-layer agar method. Three replicates were conducted for determination. The adsorption rate is estimated by k = (tC − tP)/P0/N/t, where k is the estimated adsorption rate; tP and P0 is the phage concentration at time t and 0, respectively; tC is the phage concentration in control group at the time t; N is the bacterial concentration.The sensitivity of bacterial strains to phages was examined by the double-layer agar plate method and adsorption assays. Phage-sensitive strains were selected to determine their phage adsorption capacity. Briefly, log-phase (10Escherichia coli BL21 (DE3) tonB, fhuA, fepA, ompF, waaG, tsx, ompA, and fadL deletion mutants were constructed using the Scarless Cas9 Assisted Recombineering (no-SCAR) system  expressed under the control of tetP as well as exo, bet, and gam genes, which constitute the λ-Red system, under the control of the arabinose-inducible promoter, araBP  system , 2017. Tr, ParaB . Cas9 ta2 or by manual selection of 20-bp fragments of DNA at the 3′- end of the target gene PAM region. To incorporate this 20-bp sequence into pKDsg-xxx, a ligation-independent cloning technique known as circular polymerase extension cloning (CPEC) was performed  by electroporation and selected using Amp and Spec, respectively. Transformed plasmids were verified by PCR. Next, BL21 (DE3) transformants containing pCas9cr4 and pKDsg-xxx were cultured to log phase for preparation of competent cells. Cells were incubated with 50 mM  L-arabinose for 20–30 min before preparation of competent cells to induce expression of the λ-Red recombination system in pKDsg-xxx. Finally, the target oligonucleotide was electro transformed into the recombinant competent cells, which were then plated on LB-agar containing 100 mg/L Amp, 50 mg/L Spec, and 100 μg/L aTc, and incubated at 30°C for 12 h.Deletion mutant strains were then constructed as follows. First, plasmids pCas9cr4 and pKDsg-xxx were sequentially transformed into Single colonies of deletion mutant strains were genotyped by PCR and Sanger sequencing. The phage sensitivity of the deletion mutant strains was then characterized via double-layer agar plate assays and adsorption assays, as described above.tonB, fhuA, fepA, ompF, waaG, tsx, ompA, and fadL were separately amplified using the CPEC technique and cloned into pET-28a to generate complementation plasmids. The complementation plasmids, designated pET-28a-xxx, were then transformed into their corresponding deletion mutations by electroporation to obtain complemented strains. The phage sensitivity of the complementation strains was confirmed by double-layer agar plate assays and adsorption assays, as described above.Complementation strains were also generated to determine whether wild-type copies of the deleted genes could restore phage sensitivity to the deletion mutants. E. coli BL21 (DE3) via different receptors were mixed to produce a phage cocktail. BL21 (DE3) mutant strains were then screened against the phage cocktail using the methods described above. Spotting assays and double-layer agar plate assays were used to identify the susceptibilities of the phage-resistant strains to the phage cocktail and to each of the phages individually. A polyvalent phage-resistant strain showing resistance to all the phages in the cocktail was selected for further analysis and was designated strain PR8.Eight phages isolated in this study that infect E. coli BL21 (DE3) were collected in our laboratory. The phage sensitivity of strain PR8 was evaluated by a double-layer agar plate method. Bacterial growth curves were generated for the BL21 (DE3) and PR8 strains to identify any differences in bacterial counts between the two strains during propagation. Briefly, overnight cultures were used to inoculate fresh LB medium at a ratio of 1:100 (v/v). The cultures were then incubated at 37°C, 220 rpm for 12 h. Three replicate samples (1 mL each) were collected every hour to determine the OD600 of the cultures. Three replicates were conducted for determination.A total of 32 phages isolated from China that can infect egfp was cloned into pET-28a, generating recombinant plasmid pET-28a-egfp, which was then transformed into PR8 and BL21 (DE3) electrocompetent cells, respectively. Recombinant strains containing pET-28a-egfp were separately cultured overnight and then used to inoculate fresh LB medium at a ratio of 1:100 (v/v). Cultures were incubated at 37°C with shaking to OD600 = 0.6 before the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.8 mM to induce the expression of EGFP. Three parallel 200 μL samples were taken in 96-well plates every hour and the fluorescence values were determined using a Multi-Mode Microplate Detection Platform SpectraMax® i3 . A strain that did not carry the expression vector was used as the control. Three replicates were conducted for determination. Fluorescence value = experimental group fluorescence value - control group fluorescence value.In addition, to identify differences in protein expression between polyvalent phage-resistant strain PR8 and wild-type strain BL21 (DE3), enhanced green fluorescent protein EGFP was selected as an indicator protein. Briefly, In this study, eight phages were sequentially separated from sewage . Phages 10 PFU/mL, indicating that they are highly infectious toward E. coli BL21 (DE3).Multiplicity of infection analysis assays showed that the optimal MOIs for phages IME18, IME253, IME281, IME338, IME339, IME340, IME341, and IME347 were 0.01, 0.01, 0.001, 0.001, 0.001, 0.001, 0.001, and 0.01, respectively. One-step growth curve analyses revealed that all of the phages had a latency period of about 5 min . The burJs98virus, Tevenvirinae, Myoviridae; IME339 and IME340 were most closely related to T4virus, Tevenvirinae, Myoviridae; IME338 was most similar to Felix01virus, Ounavirinae, Myoviridae; IME18 and IME347 were most closely related to T1virus, Tunavirinae, Siphoviridae, and IME253 was most closely related to Rtpvirus, Tunavirinae, Siphoviridae (The genomes of phages IME18 (GenBank Accession No. MH051911), IME253 (GenBank Accession No. KX130960), IME281 (GenBank Accession No. MH051913), IME338 (GenBank Accession No. MH051914), IME339 (GenBank Accession No. MH051915), IME340 (GenBank Accession No. MH051916), IME341 (GenBank Accession No. MH051917), and IME347 (GenBank Accession No. MH051918) were 50,354, 46,717, 170,531, 85,675, 164,366, 165,549, 172,379, and 50,048 bp in size, respectively, with GC contents of 45.6, 44.2, 39.4, 38.7, 35.6, 35.5, 39.5, and 49.7%, respectively. Phylogenetic analysis based on the amino acid sequence of the large subunit of the terminase from each phage showed that IME281 and IME341 were most closely related to oviridae .E. coli mutants that were insensitive to each of the isolated phages were randomly selected for whole-genome sequencing. The mutations putatively responsible for phage resistance were then identified by whole genome comparison with the sequence of phage-sensitive strain BL21 (DE3). We searched for phage receptor-associated variations in the genomes of the BIMs, with all results listed in fhuA, fepA, ompF, waaG, tsx, ompA, fadL, and yncD were present in the strains showing resistance to phages IME18, IME253, IME281, IME338, IME339, IME340, IME341, and IME347, respectively. fhuA, fepA, ompF, tsx, ompA, fadL, and yncD all encode different bacterial outer membranes proteins, while waaG participates in the synthesis of LPS. These findings indicate that the identified genes play an important role in the infection of BL21 (DE3) by the corresponding bacteriophages.Three fhuA, fepA, ompF, waaG, tsx, ompA, and fadL were phage receptor-related genes with a role in the observed phage resistance, we constructed deletion mutants of each of these genes using the Scarless Cas9 Assisted Recombineering system. FhuA is the receptor for bacteriophage T1, and the infection process requires the function of TonB . The receptor for phage IME339 is Tsx, which serves as a substrate-specific channel for nucleosides and deoxynucleosides. Structures of Tsx with bound nucleosides show that there are at least three distinct binding sites in the channel . Mutations Phe27Leu, Gly28Arg (Glu), Ser217Arg, Gly239Asp, and Gly240Asp result in a Tsx protein defective in nucleoside transport  receptor region, and substitutions Asn249Lys and Asn254Lys (Tyr) strongly impaired the phage T6 receptor function of Tsx  found to affect the ability of OmpA to function as a phage receptor  binding, with loops 2 and 4 being the most critical . The receptor for phage IME281 is the osmotically regulated cation-selective OmpF protein, which consists of three monomeric channels  and ΦR1-RT  is dependent on temperature-regulated expression of OmpF  by phages IME18 and IME253 was TonB-dependent, with FhuA and FepA used as receptors, respectively. The FhuA amino acid mutation sites of the phage IME18-resistant strain and PR8 were Ser675_Trp704del, Thr629fs, Phe519fs, and Met416_Arg417del. Residues Ser675_Trp704 and Met416_Arg417 are related to receptor function of phage IME18. In the phage IME253-resistant strain and PR8, fepA was deleted. FhuA and FepA belong to the family of TonB-dependent receptors. FhuA is mainly involved in the binding and absorption of ferrichrome and colicin M, and is a receptor for Siphoviridae bacteriophages T1, T5, phi80, and UC-1  type LPS, while LPS lacking the O-antigen is referred to as rough (R) type. Lipid A is located on the innermost side of LPS and is usually conserved, while the polysaccharide polymer composed of O-antigen, the structural composition of which is highly variable, can extend to the outside of the cell membrane. Generally, the host range of phages capable of cleaving S-type strains is broader than that of phages targeting R-type cells . waaG is the LPS . Deletioreceptor .T4virus phages (IME339 and IME340), two Js98virus phages (IME281 and IME341), one Felix01virus phage (IME338), two T1virus phages (IME18 and IME347), and one Rtpvirus phage (IME253), all of which used different receptors for infection of E. coli BL21 (DE3). We confirmed that the receptors for phages IME18, IME253, IME281, IME338, IME339, IME340, IME341, and IME347 are FhuA, FepA, OmpF, LPS, Tsx, OmpA, FadL, and YncD, respectively, and that none of the receptors are shared. We then identified a polyvalent phage-resistant BL21 (DE3) mutant strain, designated PR8, using a screening assay based on a phage cocktail consisting of the eight identified phages; PR8 is resistant to 23 tested phages. Strain PR8 not only resists infection by multiple phages but also has the ability to express high levels of recombinant protein, indicating that it is likely to be a valuable strain for production of recombinant protein. However, the mechanisms of interactions between phages and their hosts are not fully understood, and further research is needed to provide a theoretical basis for phage contamination control. Replacement of all UAG termination codons in E. coli with UAA enhances host resistance to T7 phage (In this study, we identified two T7 phage . TherefoT7 phage . In the YT, JW, and PL conceived and designed the experiments. PL carried out the experiments and wrote the manuscript. All authors analyzed the data, read, and approved the final manuscript.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.


DDX3Y, KDM5D, ZFY, EIF2S3Y, EIF1AY, LOC110255320, LOC110257894, LOC396706, LOC100625207, and LOC110255257. Conceptuses that presented reads CPM sum for these genes (ΣCPMchrY) greater than 400 were classified as males and those with ΣCPMchrY below 2 were classified as females. It was demonstrated that the sex identification can be performed at early stages of pig development from RNA-sequencing analysis of genes mapped on Y chromosome. Additionally, these results reinforce that sex determination is a mechanism conserved across mammals, highlighting the importance of using pigs as an animal model to study sex determination during human prenatal development.Sexual dimorphism is a relevant factor in animal science, since it can affect the gene expression of economically important traits. Eventually, the interest in the prenatal phase in a transcriptome study may not comprise the period of development in which male and female conceptuses are phenotypically divergent. Therefore, it would be interesting if sex differentiation could be performed using transcriptome data, with no need for extra techniques. In this study, the sex of pig conceptuses (embryos at 25 days-old and fetuses at 35 days-old) was determined by reads counts per million (CPM) of Y chromosome-linked genes that were discrepant among samples. Thus, ten genes were used:  Sus scrofa is a prolific specie with a relatively short developmental period, besides being a cheaper and easier mammalian model organism to maintain . The experimental protocols used in this study have followed ethical principles in animal research  and were approved by the Ethical Committee on Animal Use of the All experimental protocols were performed according to the experimental design described in Costa et al. . Brieflyn = 5) and five females of ARG (n = 5) were rendered unconscious using head-only electrical stunning  and immediately exsanguinated. The same procedure was followed for four females of CONT (n = 4) and six females of ARG (n = 6) at 35 days of gestation. After slaughter, four conceptuses were collected per female at each gestational age, totaling in average 20 conceptuses per treatment. At 25 days of gestation, five females of CONT (n = 3) per treatment, since at least three biological replicates are recommended for describing results in RNA-seq experiments [n = 3 gilts) and three embryos from each ARG gilt (n = 3 gilts) at 25 days of gestation  and three fetuses from each CONT gilt (n = 3 gilts) and three fetuses from each ARG gilt (n = 3 gilts) at 35 days of gestation , totaling 36 samples, were randomly chosen for RNA-seq analysis. Afterwards, these samples were transported in liquid nitrogen to the Animal Genetics Laboratory at the Embrapa Swine and Poultry National Research Center, Concordia, SC, Brazil, for further RNA extraction and library preparation.The conceptuses were quickly washed with PBS  solution, individually identified, stored in liquid nitrogen and transported to the Animal Biotechnology Laboratory (LABTEC) at the Department of Animal Science, UFV. At LABTEC, each conceptus (embryos from gilts slaughtered at 25 days and fetuses from gilts slaughtered at 35 days) was entirely and separately macerated in liquid nitrogen. Although an average of five gilts was initially used per treatment in the original trial described in Costa et al. , subsequeriments ,23. Moreg. The eluate was discarded and 700 μL RW1 buffer were added, followed by centrifugation for 15 seconds at 8000× g. Two washes with 500 μL RPE buffer were done and, finally, RNAs were eluted in 50 μL of RNase free water.Total RNA extraction of the 25- and 35-day-old conceptuses from the ARG and CONT females was performed with TRIzol . The macerated conceptuses (100 mg) and TRIzol (1 mL) were mixed with vortex and then incubated for 5 minutes at room temperature . Then, 200 µL of chloroform were added, shaking vigorously for 15 seconds and incubated at RT for 5 minutes. Centrifugation was performed at 11,000× g at 4 °C for 15 minutes. Approximately 600 μL of the clear upper aqueous phase containing only RNA were carefully removed and transferred to a new tube, and 600 μL of 70% ethanol were added and homogenized by inversion. This volume was added to the silica column RNeasy mini kit  and centrifuged for 15 seconds at 8000× After RNA extraction, the quantification was performed in a QUBIT fluorimeter  and the integrity was determined in 1.0% agarose gel. In addition, the Agilent 2100 BioAnalyzer  was used for integrity measurement, in which samples with RNA integrity number (RIN) higher than eight were used for library preparation. In this sense, one fetus sample was excluded from subsequent analysis due to low RIN score (RIN < 8). Therefore, the 35 remaining conceptuses samples (nine ARG and nine CONT at 25 days and eight ARG and nine CONT at 35 days) were submitted to RNA-seq library preparation using the TruSeq Stranded mRNA Library Prep Kit , followed by purification of the poly-A tail using 2 μg of total RNA, according to the manufacturer´s recommendations.Universidade de São Paulo, Piracicaba, SP, Brazil. The FASTQ files were deposited in the SRA database, with Bioproject number PRJNA576701 and Biosample numbers SAMN13003023, SAMN13003024, SAMN13003025, SAMN13003026, SAMN13003027, SAMN13003028, SAMN13003029, SAMN13003030, SAMN13003031, SAMN13003032, SAMN13003033, SAMN13003034, SAMN13003035, SAMN13003036, SAMN13003037, SAMN13003038, SAMN13003039, SAMN13003040, SAMN13003041, SAMN13003042, SAMN13003043, SAMN13003044, SAMN13003045, SAMN13003046, SAMN13003047, SAMN13003048, SAMN13003049, SAMN13003050, SAMN13003051, SAMN13003052, SAMN13003053, SAMN13003054, SAMN13003055, SAMN13003056 and SAMN13003057.The libraries were sequenced in Illumina HiSeq2500 , following the 2 × 100 bp paired-end protocol, at the Functional Genomics Center, ESALQ, Sus scrofa, v. 11.1) and the reads counting for each feature was based on Ensembl annotation release 95.The BAQCOM pipeline   was usedn = 18 embryos and n = 17 fetuses) was performed using discrepant reads counts per million (CPM) of Y chromosome-linked genes from the conceptuses RNA-seq dataset. After the samples classification as males or females, the HTseq files containing all samples transcripts were renamed and used to build a multi-dimensional scale (MDS) plot using plotMDS function of EdgeR package [The conceptuses sex determination , with 83.8% and 79.8% of the reads counted into genes for embryos and fetuses, respectively. An average of 0.05% of the reads was uniquely mapped on Y chromosome. At 25 and 35 days of age, male and female conceptuses could not be phenotypically differentiated, since gonadal phenotype could not be observed , KDM5D (Lysine demethylase 5D), ZFY , EIF2S3Y (eukaryotic translation initiation factor 2 subunit 3), EIF1AY , LOC110255320 (lysine-specific demethylase 6A-like), LOC110257894 (gamma-taxilin-like), LOC396706 , LOC100625207  and LOC110255257 . The CPM sum for these genes (ΣCPMchrY) was calculated for each conceptus in order to determine its sex. In this way, conceptuses that presented ΣCPMchrY greater than 400 were classified as males and those with ΣCPMchrY below 2 were classified as females. Male conceptuses showed mean (standard deviation), minimum and maximum ΣCPMchrY of 499.08 (44.74), 427.29, and 573.09, respectively (chrY of 0.38 (0.42), 0.00 and 1.75, respectively , which is expressed during a critical period of embryonic development, and other genes that are important for spermatogenic function, as the ZFY, EIF1AY and KDM5D. The importance of these Y-linked genes during male development has been elucidated for mammals [SRY, EIF1AY, KDM5D, ZFY, EIF2S3Y, and DDX3Y [Several studies have also used Y chromosome-linked genes to determine the sex of pigs at early development stages ,18,19, h mammals ,32,33,34 mammals . The MSYnd DDX3Y ,35,36. SRY was not used to differentiate male and female conceptuses (data not shown). This gene seems to follow a strict expression pattern during mammalian embryonic development. In mouse, its expression starts at 10.5 days post coitum (dpc), reaches a peak at 11.5 dpc and then wanes by 12.5 dpc [SRY expression is detectable in the embryos genital ridge around 21 and 23 days, with faint expression at 26 days and absence of expression at 31 days [SRY expression phase, reinforcing the critical time window during embryonic development in which SRY is expressed. In this study, among all abovementioned genes, 12.5 dpc ,37. In p 31 days . ConsequSRY gene, other male-specific genes are important to maintain testicular development and spermatogenesis [KDM5D, ZFY, and EIF1AY. The KDM5D gene encodes a histone demethylase enzyme that acts removing tri- and di-methylations of lysine 4 of histone H3 at the start site of transcription in actively transcribed genes [ZFY, the first coding gene identified in the human Y chromosome, regulates the transcription of a number of Y-linked genes [EIF1AY is requested for a high rate of protein biosynthesis and the absence of this gene may contribute to failures in the spermatogenic process [Subsequently to the sex determination by the ogenesis . Accordiogenesis , during ed genes . The traed genes  and medied genes . The tra process . EIF2S3Y gene, located in the short arm of the Y chromosome. This gene has been identified as a key mouse-specific regulator of spermatogonia proliferation and differentiation [DDX3Y gene, along with KDM5D and EIF1AY, are azoospermia factors (AZFa) [DDX3Y is the major AZFa gene and belongs to a highly conserved subfamily of the DEAD-box RNA helicase family [KDM5D, ZFY, EIF1AY, EIF2S3Y, and DDX3Y genes showed discrepant reads CPM among samples and have important spermatogenic and gonadal functions, being crucial to differentiate male and female pig conceptuses in the present study. Similar pattern has already been observed in humans blastocyst [Another important translation initiation factor during spermatogenesis is the ntiation . Finallys (AZFa) . Azoospes (AZFa) . The DDXe family . This gee family . Therefoastocyst , which hThe reads CPM observed for the Y-linked genes in female conceptuses may be considered technical artifacts from RNA-seq, since mammalian X and Y chromosomes share regions of high sequence similarity, causing mismapping of short reads to a reference genome . In thisLOC110255257, LOC100625207, LOC110255320, LOC110257894, and LOC396706 genes are located in the Y chromosome, however, they have not yet been fully characterized in Sus scrofa genome. According to Skinner et al. [The r et al. , there aWe have demonstrated that sex identification can be performed from read counts per million of genes mapped in the Y chromosome at early stages of pig development using already available conceptuses RNA-seq data, with no need for extra techniques. Additionally, the results reinforce that molecular sex determination is a mechanism conserved across mammalian species, since the role of these genes is also important during male development in humans, highlighting the applicability of pigs as an animal model to study sex determination during human prenatal development.


E. coli isolates recovered from 179 outbreaks of enteric colibacillosis from pig production farms in Spain during a period of 10 years. Most samples were of diarrheagenic cases occurred during the post-wean period (PWD) which showed to be significantly associated with ETEC (67%) followed by aEPEC (21.7%). On the contrary, aEPEC was more prevalent (60.3%) among diarrheas of suckling piglets, followed by ETEC (38.8%). STEC/ETEC or STEC were recovered in 11.3 and 0.9% of PWD and neonatal diarrhea, respectively. Detection of the F4 colonization factor was not significantly different between isolates recovered from neonatal pigs and those recovered post wean (40.5 versus 27.7%) while F18 was only present among PWD isolates . We also found a high prevalence of resistance to colistin related to the presence of the mcr-1 gene (25.6% of the diarreagenic isolates). The characterization of 65 representative mcr-1 isolates showed that all were phenotypically resistant to colistin (>2 μg/ml), and most (61 of 65) multidrug-resistant (MDR). Six ETEC and one STEC mcr-1 isolates were also carriers of ESBL genes. In addition, other seven mcr-1 isolates harbored mcr-4 (three ETEC) and mcr-5 (two ETEC and two aEPEC) genes. In the phylogenetic analysis of the 65 mcr-1 diarrheagenic isolates we found that more than 50% (38 out of 65) belonged to A-ST10 Cplx and from those, 29 isolates showed the clonotype CH11-24. In this study, we also recovered 18 ST131 isolates including seven mcr-1 carriers. To the best of our knowledge, this would be the first report of ST131 mcr-1 isolation in pigs. Worryingly, the swine mcr-1 ST131 carriers also showed MDR, including to trimethoprim-sulfamethoxazole, tobramycin, gentamicin and ciprofloxacin. In the PFGE-macrorestriction comparison of clinical swine and human ST131, we found high similarities (≥85%) between two pig and two human ST131 isolates of virotype D5. Acquisition of mcr-1 by this specific clone means an increased risk due to its special feature of congregating virulence and resistance traits, together with its spread capability. Here we show a potential zoonotic swine source of ST131.This is a wide epidemiological study of 499  Neonatal and post-weaning diarrheal (PWD) disease affecting pigs during the first weeks after birth result in significant economic losses for the pig industry due to mortality, decreased weight gain, costs derived of treatment and handling, and vaccinations .Escherichia coli pathotypes commonly implicated in enteric diseases in piglets, namely, Shiga toxin-producing E. coli (STEC) and enterotoxigenic E. coli (ETEC). STEC isolates carrying the shiga toxin type 2e (Stx2e) are the causative agents of edema disease in weaning piglets, while ETEC isolates encoding the heat stable  and/or heat labile (LT) enterotoxins cause secretory diarrhea in newborn and weaned piglets (E. coli (EPEC) is another pathotype found in pigs with enteric colibacillosis  or other beta-lactamase genes (such as carbapenemase) are being increasingly identified in isolates from different origins  10, ST69, ST131, ST405, ST410, or ST648 (Colistin (polymyxin E) is one of the few cationic antimicrobial peptides commercialized in both human and veterinary medicine. In humans, colistin is used as a last resort against infections of multidrug-resistant (MDR) Gram-negative bacteria . Howeverate 2015 , severalescribed . Livestoescribed . Besides origins . In addior ST648 . Of partor ST648 , and whoor ST648 .E. coli isolates obtained in Spain during the period 2006–2016 from pigs suffering enteric colibacillosis with three main aims: (i) to define clonal groups of clinical importance in swine enteric colibacillosis; (ii) to analyze the rates of antibiotic resistance in pig farming in Spain, including mobile resistance to colistin (mcr gene); and (iii) to gain knowledge about the presence and zoonotic potential of ST131 isolates of livestock origin.In the present study, we characterized a collection of Escherichia coli (LREC), in Lugo, Spain. The samples were collected in farms from different Spanish regions between 2006 and 2016, mainly of diarrheas after weaning (73%) and the remaining (27%) from suckling piglets.A total of 464 rectal samples from pigs suffering diarrhea were tested for routine diagnosis of enteric colibacillosis at the Reference Laboratory of rfbO25 using specific primers described by E. coli-like colonies from LMAC and/or CTSMAC plates were plated on tryptone soy agar (Oxoid) and individually analyzed by PCR. Those colonies showing different genetic characteristics for the selected targets were stored at room temperature in nutrient broth (DifcoTM) with 0.75% nutrient agar (DifcoTM) for further characterization. In total, 499 E. coli isolates were examined in this study representing 179 diarrheagenic outbreaks.Swabs were plated on lactose MacConkey agar (LMAC) and sorbitol MacConkey agar (Oxoid) supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CTSMAC), and incubated at 37°C for 18–24 h. Afterward, the confluent growth of all plates were tested to detect the presence of ETEC, STEC, and EPEC by PCR based on specific genes encoding toxins , fimbriae , intimin (Eae), and bundle-forming pilus (BFP) . ConflueDetermination of O:H antigens was carried out following the method described by E. coli isolates were investigated for the presence of mcr-1 gene by PCR as detailed elsewhere  according to the manufacturer’s instructions. The antibiotics tested included ticarcillin, aztreonam, ceftazidime, cefepime, ampicillin-sulbactam, piperacillin-tazobactam, imipenem, meropenem, amikacin, gentamicin, tobramycin, levofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, fosfomycin, colistin, minocycline, and tigecycline. Additionally, resistance to ampicillin, cefotaxime, chloramphenicol, and nalidixic acid was determined by disk  diffusion assays. All results were interpreted according to the CLSI  using the MicroScan WalkAwaythe CLSI . Geneticthe CLSI .E. coli phylogenetic groups  was performed using the quadruplex phylogroup assignment method described by arpA, chuA, yjaA, and TspE4.C2  were amplified and sequenced using published criteria and primers  and 489-nt sequence of the fumC and fimH genes, respectively  and fimH allele designations was used as the CH “type.” Finally, a neighbor-joining tree was constructed by MEGA6  status if positive for two or more of five markers, including papAH and/or papC, sfa/focDE, afa/draBC, kpsM II, and iutA .Isolates confirmed as ST131 by MLST were additionally investigated for extraintestinal virulence markers. Based on the results, the isolates were considered to conform the extraintestinal pathogenic and iutA , and theand yfcV . The virE. coli clonal group ST131 was established comparing the XbaI-PFGE profiles of the isolates which were obtained following the PulseNet protocol,3 and imported into BioNumerics  to perform a dendrogram with the UPGMA algorithm based on the Dice similarity coefficient and applying 1% of tolerance in the band position.The similarity within the P values <0.05 were considered statistically significant.Differences were compared by a two-tailed Fisher’s exact test. E. coli isolates analyzed in this work had been obtained from 464 fecal samples of 179 diarrheagenic outbreaks occurred in different geographic areas of Spain. By PCR, 481 of those 499 isolates shown diarrheagenic pathotypes, while 18 isolates recovered by means of the rfbO25 screening and negative for the enteric virulence genes were later investigated concerning the ST131 clonal group.The 499 eltA, and/or estA, and/or estB) and aEPEC  positive for eae but negative for bfpA . The remaining isolates were assigned as STEC/ETEC  positive for both Stx2e and enterotoxin-encoding genes (stx2e and estB and/or estA) and STEC  positive for stx2e. According to the age of the affected animals, aEPEC was the most prevalent pathotype in suckling piglets (60.3%) followed by ETEC (38.8%). By contrast, ETEC isolates were the most prevalent in PWD (67.0%) followed by aEPEC (21.7%) (P < 0.001 for both comparisons). Furthermore, STEC/ETEC were recovered in 9.0% of PWD versus 0.9% of neonatal diarrheas (P < 0.005); and STEC only in piglets after weaning (2.3%). The colonization factors identified among ETEC isolated from neonatal diarrhea were F4, F5 + F41, and F6 ; while for PWD no isolates were positive for F5, F6, or F41; however F4 was detected at a level not significantly different to neonatal diarrhea isolates . Finally, F18 was only detected in PWD (51.5%) including 45.5% of ETEC, 82.1% of STEC/ETEC, and 80.0% of STEC isolates.Specifically, the major pathotypes found among the 481 diarrheagenic isolates were ETEC  positive for genes encoding enterotoxins  isolates that did not react with any of the O1 to O185 antisera were classified as non-typeable (ONT). In spite of this variability, we found a significant association between pathotypes and certain serogroups. Thus, O108, O138, O141, O149, and O157 accounted for 75.1% of ETEC; O26, O49, O80, and O111 for 39.1% of aEPEC; O138 and O141 for 57.6% of STEC/ETEC; and O139 accounted for 46.7% of STEC  ; nevertheless, there was a significant association with the age of the affected animals  (P < 0.001). The 123 isolates showed 23 serogroups, although 75.6% belonged to only 8   out of 481 diarreagenic isolates harbored the mcr-1 E. coli involved in swine colibacillosis, a representative group of 65 isolates was selected for further characterization. The selection included approximately 50% of each pathotype , as well as the serogroups representing 75.6% of the mcr-1 isolates  together with other less prevalent .From the 123 mcr-1 isolates showed 11 different O:H combinations being O157:HNM, O141:H4, and O138:H14 the most prevalent ; the five STEC/ETEC isolates were O141:H4; and the three STEC showed three different serotypes   . High rates of resistance were also found against ampicillin (75.4%), ticarcillin (73.8%), trimethoprim-sulfamethoxazole (72.3%), ampicillin-sulbactam (64.6%), nalidixic acid (60.0%), and chloramphenicol (58.5%) (Table mcr-1 isolates (61 of 65) met the definition of MDR  Table . Furthern of MDR  being reoxazole 7.3%, ampimcr-1 isolates which were typed by sequencing as CTX-M-14 and SHV-12 . The PCR screening of other mcr variants among the 65 mcr-1 positive isolates determined that three ETEC isolates carried both mcr-1/mcr-4 genes and other four isolates (two ETEC and two aEPEC) were simultaneous carriers of mcr-1/mcr-5 (Tables Extended-spectrum beta-lactamase genes were detected in six ETEC and one STEC of the 65 5 Tables .mcr-1 diarrheagenic isolates belonged to phylogroups A (46 isolates), B1 (11 isolates), and E (eight isolates), with 18 different STs including a new one , being CH11-24 the most prevalent  which was here associated to STs 10 and 5,796 of the ST10 Cplx, and to different pathotypes . Besides, three of the 65 mcr-1 isolates were fimH negative , and the remaining showed virotypes D2 (two isolates) and D-non typeable . Importantly, all exhibited the ExPEC and UPEC status. Clonotyping showed eight different clonotypes: CH40-22 (nine isolates), CH40-161 (two), CH40-326 (one), CH40-330 (one), CH40-332 (one), CH40-336 (one), CH40-338 (one), and CH40-374 (two); being five of them  single locus variants of fimH22 and two of them (fimH336 and fimH374) two loci variants of fimH22  presently implicated in swine diarrheas, and how the use of antibiotics (including colistin) could have affected the selection and emergence of resistant strains. Most samples (73%) were of diarrheagenic cases occurred during the post-wean period which showed to be significantly associated with ETEC (67%) followed by aEPEC (21.7%). By contrast, aEPEC was more prevalent (60.3%) among diarrheas of suckling piglets, followed by ETEC (38.8%). STEC/ETEC or STEC were recovered in 11.3 and 0.9% of PW and neonatal diarrhea, respectively. It is noteworthy that the F4 colonization factor was identified among neonatal ETEC without significant differences compared to PWD ETEC (40.5 versus 27.7%) while, as expected, F18 was only present among PWD isolates.Pig diarrhea caused by  weaning . Currentthotypes . In the P < 0.001). The reported association of F18 E. coli isolates with PWD in other countries varied widely, from 1.7% in Australia (2estx gene (60.1% of 648 isolates)  were positive for F18.Complete data on the prevalence, serotypes, and pathotypes are not frequently available, which makes comparisons difficult. However, previous analysis had already suggested differences between countries . A recenustralia , 35% in ustralia , 39.3% iustralia , 53% in ustralia , to 62% ustralia . High F1solates)  which deThe majority of ETEC associated with diarrhea in pigs appear to belong to a limited number of serogroups, being O8, O138, O139, O141, O147, O149, and O157 the most commonly reported worldwide. Similarly, STEC causing disease primarily belong to O138, O139, and O141 . The majeae-positive isolates in relation to the pathogenic potential, such as sero/intimin type O123:H11/eae-β1 or O45/eae-β1 (both determined in our collection) with AE activity on ileal villi and frequently occurring in diarrheagenic pigs  as presumptive agents of the clinical condition, and significantly associated with neonatal samples. For atypical EPEC, both animals and humans can be reservoirs and are known as pathogenic for children and young animals . aEPEC hnic pigs . In prevnic pigs .mcr-1 gene in late 2015, its detection has been reported globally, but its detection rate has been variable depending on the geographic region, source, and method of identification. Overuse of colistin in food animals is believed to have trigged the emergence and spread of mcr-1, consistently with the higher figures found in poultry and pig industry  met the MDR definition of mcr-1 positive isolates recovered from PWD in Italy also showed genetically diverse pathotypes and all but one were resistant to ≥3 different antimicrobial families ; besides, resistance profiles of mcr-1 positive E. coli were more extensive than those of mcr-1 negative isolates. In our study, it is also of concern the finding of 11 out of the 65 mcr-1 isolates with MIC >32 mg/L for fosfomycin. Fosfomycin resistance is uncommon and primarily associated with specific chromosomal mutations, however plasmid-mediated resistance in livestock has been detected in Asian countries  and mcr-5 (two ETEC and two aEPEC) genes. We have not investigated if the double carriage of our isolates was in the same or different plasmids. Previous studies have reported the co-existence on the same plasmid of other resistance genes, such as CTX-Mbla, CMYbla, TEMbla, fosA, qnrS, floR, and oqxAB, in various combinations, alongside mcr-1  belonged to A-ST10 Cplx and from those, 29 isolates of different pathotypes showed CH11-24. The ST10 Cplx of E. coli, widely disseminated among animal and human intestinal samples both as a commensal or as a pathogen, is commonly encountered as antimicrobial susceptible but also linked with MDR and ESBL, and recognized as an emerging food-borne ExPEC lineage  might function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10 . Additionally, two aEPEC belonged to the clonal groups O103:H2-B1-ST20 (eae-β1) and O145:H28-E-ST1034 of the ST32 Cplx (eae-γ1), respectively. Enterohemorrhagic E. coli (EHEC) is the causative agent of bloody diarrhea, the hemolytic-uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP). Besides the intimin (Eae) which confers the ability to cause AE lesions, EHEC harbors bacteriophage-encoded stx genes. Currently, the vast majority of EHEC infections are caused by isolates belonging to five O serogroups, namely O157, O26, O103, O111, and O145 . Ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin, and tobramycin resistances were significantly associated with fimH30 isolates in comparison with fimH22 isolates. In fact, they only detected one and eight isolates resistant to ciprofloxacin and trimethoprim-sulfamethoxazole, respectively, among 20 ST131-H22. fimH alleles with the virulence profile, being all 20 fimH22 isolates of virotype D, subtypes D1, D2, D4, and D5. In the present work, we found high genetic variability within the eight fimH alleles of 18 ST131 isolates; however, fimH22 was the most prevalent (nine isolates) and the other seven alleles were fimH22 variants whose nucleotide and aminoacidic differences were compared with those reported by mcr-1 also showed a higher number of resistances, including to trimethoprim-sulfamethoxazole (seven isolates), tobramycin (six isolates), gentamicin (six isolates), and ciprofloxacin (one isolate), more associated to the fimH30 subclone according to A highly important finding of the present study was the recovery of 18 ST131 isolates among the 464 fecal samples of diarrheanic pigs. As far as we know, there are only two reports of pig origin for this clonal group: the first obtained from pork meat in Denmark in 2003 , and thesistance , 2014. Sken meat , then inken meat  and in aken meat . To the escribed . Here, wte Table . In a nate Table . The majE. coli isolates recovered from enteric swine colibacillosis in Spain provides valuable epidemiological information about the current pathotypes involved in this economically important pathology, as well as alerts about the worrisome presence of MDR clones such as ST131. Acquisition of mcr-1 by this specific clone means an increased risk due to its special feature of congregating virulence and resistance traits, together with its spread capability. Here, we give evidences of the potential human pathogenicity of ST131 D5 isolates of swine origin due to their genetic identity.In conclusion, the comprehensive characterization of this wide collection of AM and JB conceived and designed the experiments. IG-M, VG, DD-J, SF-S, and MA performed the experiments. AM, JB, VG, IG-M, JB, and MB analyzed and interpreted the data. AM, VG, JB, and IG-M drafted the manuscript. AM, JB, VG, IG-M, JB, MB, MA, DD-J, and SF-S provided critical input and approved the final manuscript.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.


NFKB1, TNFAIP3, and TTC37), whereas in six patients, we identified seven variants of possible pathogenic potential classified as variants of unknown significance . In the remaining 11 patients, we did not identify possible genetic causes. Genetic findings were correlated to clinical disease presentation, clinical immunological phenotype, and disease complications. We suggest that the variants identified in the present work should lay the ground for future studies to functionally validate their disease-causing potential and to investigate at the mechanistic and molecular level their precise role in CVID pathogenesis. Overall, we believe that the present work contributes important new insights into the genetic basis of CVID and particular in the subset of CVID patients with a complex phenotype involving not only infection, but also autoimmunity, autoinflammation, and malignancy.Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by recurrent bacterial infections and defined by reduced levels of IgG, IgA, and/or IgM, insufficient response to polysaccharide vaccination, and an abnormal B-cell immunophenotype with a significantly reduced fraction of isotype-switched memory B cells. In addition to this infectious phenotype, at least one third of the patients experience autoimmune, autoinflammatory, granulomatous, and/or malignant complications. The very heterogeneous presentation strongly suggests a collection of different disease entities with somewhat different pathogeneses and most likely diverse genetic etiologies. Major progress has been made during recent years with the advent and introduction of next-generation sequencing, initially for research purposes, but more recently in clinical practice. In the present study, we performed whole exome sequencing on 20 CVID patients with autoimmunity, autoinflammation, and/or malignancy from the Danish CVID cohort with the aim to identify gene variants with a certain, possible, or potential disease-causing role in CVID. Through bioinformatics analyses, we identified variants with possible/probable disease-causing potential in nine of the patients. Of these, three patients had four variants in three different genes classified as likely pathogenic ( Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by recurrent bacterial infections and defined by reduced levels of IgG, IgA, and/or IgM, insufficient response to polysaccharide vaccination, and an abnormal B-cell immunophenotype with significantly reduced fraction of isotype-switched memory B cells. CVID affects an estimated 1 in 25,000 individuals, with an equal distribution of males and females and a broad age range of diagnosis –7. Thus,The term “common variable immunodeficiency” was first used in 1971 by a World Health Organization committee to distinguish a heterogeneous group of antibody deficiency syndromes from others with a more specific clinical description and well-defined pattern of inheritance . The firPreviously, we described the demographic, infectious, and non-infectious phenotype of the Danish CVID cohort . We idenPatients with a diagnosis of CVID in the Department of Infectious Diseases, Aarhus University Hospital, were included under the following criteria:A clinical phenotype of sinopulmonary infections and an autoimmune, inflammatory or malignant phenotype. More specifically, patients had decreased IgG and IgM and/or IgA, insufficient vaccine responses to polysaccharide vaccination (when assessed) or reduced isohemagglutinins, and varying degrees of abnormal B-cell phenotype, particularly reduced isotype-switched memory B cells, on flow cytometric analysis. Out of a population of all CVID patients in our clinic—in total 62, we focused on those with a complicated CVID phenotype, that is, those not within the “infectious-only” CVID phenotype with clinical signs and symptoms of autoimmunity, inflammation, granulomatous disease, and/or malignancy. We identified and included a total of 20 patients fulfilling these criteria in the present study.The study was approved by the Danish Ethics Committee (#1-10-72-354-13). All patients provided informed written consent to participate in the study.TruSeq DNA sample preparation was performed according to the manufacturer's recommendations (Illumina), targeting of exomes with EZ Human Medexome Plus Library v3.0 (Roche). Libraries were quantified employing KAPA v.3.13 quantification (Kapabiosystems), and sequencing was performed on NextSeq2, paired end 2X150 bp indexed. Adapters were identified and removed; reads were mapped to hg19 employing BWA mem. PCR and optical duplicates were identified and marked. The alignment file (bam) was re-aligned, employing GATK to refine the alignment. The alignment was recalibrated using GATK. Single nucleotide polymorphisms were called employing HaplotypeCaller from the GATK package. Variant call files (VCF) were uploaded to Ingenuity Variant Analysis and filtered.Total variants are filtered for confidence  for deleteriousness , filtered for presence in a comprehensive list of genes known to be involved in PID or in relevant pathways (list of genes in + cells in a lysis-no-wash approach in BD Truecount tubes (FACSCanto) or by direct volumetric flow cytometry (NovoCyte). Concentrations of the additional lymphocyte subsets were calculated from their frequency relative to the CD3+ population.For each analysis, 100 μl heparinized peripheral blood was washed twice in phosphate-buffered saline by centrifugation (5 min at 500 g) before cell labeling with florescent-coupled monoclonal murine anti-human antibodies. Antibodies used for B-cell phenotyping: Anti-CD21[PE-Cy7] , -IgD[PE] , -CD19[Super Bright 600] , -CD27[VioBright FITC] , and -CD38[BV421] . Antibodies used for T-cell phenotyping: Anti-CD3[APC-Cy7] , -CD8a[BV785] , -CD27[BV421] , -CD4[BV570] , -CD197(CCR7)[PE] , and -CD45RA[APC] . After 30 min incubation, leukocytes were fixed, and red blood cells were lysed by addition of BD FACS Lysing Solution (BD Biosciences). Remaining cells were washed once in PBS, resuspended in PBS, and then analyzed by multicolor flow cytometry . Matrices for compensation were generated monthly (using BD CompBeads together with the relevant fluorescent-coupled antibodies) or whenever instrument configurations were altered. Data were analyzed using FlowJo software  or NovoExpress Software (ACEA Biosciences). Lymphocyte phenotyping was based on recommendations , six had been diagnosed with malignancy, three of which were diffuse large B cell lymphoma , one was splenic marginal zone lymphoma (P17), one was cervical adenocarcinoma (P5), and one was esophagus squamous cell carcinoma (P4) . SplenomNFKB1, STAT3, TNFAIP3, and TTC37 and rare variants in IL-17F, IRAK4, DDX41, NLRC3, TNFRSF1A, and PLCG2 were identified , and the distribution of females:males was 11:9. A total of 635,900 variants were identified in patient exomes. Variants were filtered based on call quality of 20.0, allele frequency above 25%, and deleteriousness to identify significant variants within a comprehensive list of 564 immune deficiency and immune pathway-related genes , resultientified . The rementified , Resultsentified , Additioentified , Immunopentified , and T-centified .NFKB)1 gene were identified  domain, and are predicted to have an impact on the function of the protein. The mutation was verified by Sanger sequencing, and it was de novo—the healthy parents did not carry the variant neither did a healthy daughter. Other variants in NFKB1 in CVID have been described as the most common cause of CVID with non-infectious complications, including lymphadenopathy, splenomegaly, and autoimmune disease, and all reported patients exhibit deficient B-lymphocyte differentiation with increased CD21low B-cell numbers 3 variant (p.R107W)  , 3 prediescribed . A recenescribed .TNFAIP)3 gene (c.997dupG) resulting in a frameshift (p.A333fs*2) was found in P10  characterized by hemolytic anemia, thrombocytopenia, autoinflammation, and autoimmune lymphoproliferative syndrome, the latter presenting with lymphadenopathy and hepatosplenomegaly  to avoid neutropenia. A variant in the tumor necrosis factor, alpha-induced protein 17F gene, was identified (IL17F gene (1.05) however indicates that selection against missense mutations is not strong. Nevertheless, variants in the IL17F cytokine gene have been reported in autosomal dominant candidiasis with a hypomorphic S65L allele 4 gene encoding Interleukin-1 receptor-associated kinase 4  however indicates that selection against missense mutations is not strong. However, a potential role of a heterozygous variant working by haploinsufficiency or dominant negative effect in this patient cannot be fully excluded.P12 was a man with marked complications, including pneumonias, sinusitis, enteropathy, and more recently a diffuse large B cell lymphoma in the gut. A rare potentially damaging missense variant was identified in the interleukin-1 receptor-associated kinase kinase 4 , 3. The children . The o/e*14 variant in tetratricopeptide repeat domain (TTC)37 was found in P15 (TTC37 gene (0.63) does indicate some selection against LoF. The variant was also detected in the mother, a brother and a sister, all with similar symptoms as P15, whereas two healthy brothers and the healthy father did not carry the variant.P15 was a 34-year-old patient diagnosed with “atypical CVID” at the age of 26 and with a history of hypogammaglobulinemia, multiple sclerosis, recurrent miscarriages, and inflammatory bowel disease (Mb. Crohn) with an episode of autoimmune pancreatitis. A novel likely deleterious frameshift S1330fsd in P15 , 3. Her d in P15 . HoweverDDX)41 (p.R353W), not present in 60,000 control samples [gnomAD v2.1.1(controls)] was present (DDX41 encodes an ATP-dependent RNA helicase and variants predispose to myeloproliferative/lymphoproliferative neoplasms (OMIM #616871) (DDX41 gene (0.68).P16, a 48-year-old woman, suffered from cervical dysplasia and lymphocytosis. In P16, a possibly deleterious variant in DEAD-Box Helicase   and thusNLRC)3 c.1189G > A; p.V397M was identified  stimulation in mice    , 31. The = 0.64) , 29. AltNFKB1 . MP and JJ performed flow cytometry to determine immune cell phenotyping. MC and TM drafted the first version of the manuscript. All authors read, commented, and approved the final version of the manuscript.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.


A previously healthy 38-year-old man presented with a severe headache that had lasted for 3 days. The headache had a continuous pressing quality and bilateral involvement. One month earlier, he had eaten raw pila snail and freshwater shrimp. He was afebrile, and no nuchal rigidity was detected. The findings of other neurological examination were unremarkable. A blood test revealed a white blood cell (WBC) count of 11,150 cells/mm3 with 21% eosinophils. Computed tomography of the brain without contrast administration suggested generalized brain swelling. A lumbar puncture revealed an opening pressure of 250 mm H2O. Cerebrospinal fluid (CSF) analysis showed a WBC count of 368 cells/mm3 with 60% eosinophils, a protein level of 156 mg/dL, and a glucose level of 44 mg/dL (concurrently a blood glucose level of 125 mg/dL). The results of Gram staining, India ink preparation, and culture for bacteria were negative. Blood and CSF serological study for angiostrongyliasis using immunoblotting were positive for the 29-kDa antigenic band (2 He was treated with a 2-week course of prednisolone and supportive treatment,3 and the headache improved. However, at a follow-up 2 weeks later, he complained of numbness of the left side of his face and the left upper limb. Magnetic resonance imaging of the brain showed a focal lesion at the body of corpus callosum, which exhibited low signal intensity on T1-weighted images, high signal intensity on T2-weighted images and fluid-attenuated inversion recovery images, and nodular enhancement after administration of gadolinium (The etiologies of diseases involving the corpus callosum include congenital factors, demyelinating, infection, trauma, neoplasm, and ischemia.nic band .2 He wasdolinium . Other sdolinium .