For accurate species-level identification of microorganisms, researchers today increasingly use acombination of standard microbiological cultivation and visual observation methods with molecular biological and genetic techniques that help distinguish between species and strains of microorganisms at the levelof DNA or RNA molecules. The aim of this work was to identify microorganisms from the ICG SB RAS Collectionusing an integrated approach that involves a combination of various phenotypic and genotypic characteristics. Key molecular-genetic and phenotypic characteristics were determined for 93 microbial strains from theICG SB RAS Collection. The strains were characterized by means of morphological, physiological, moleculargenetic, and mass-spectrometric parameters. Specific features of the growth of the strains on different mediawere determined, and cell morphology was evaluated. The strains were tested for the ability to utilize varioussubstrates. The strains studied were found to significantly differ in their biochemical characteristics. Physiological characteristics of the strains from the collection were identified too, e.g., the relationship with oxygen,type of nutrition, suitable temperature and pH ranges, and NaCl tolerance. In this work, the microorganismsanalyzed were combined into separate groups based on the similarities of their phenotypic characteristics.This categorization, after further refinement and expansion of the spectrum of taxa and their metabolic maps,may serve as the basis for the creation of an “artificial” classification that can be used as a key for simplified andquicker identification and recognition of microorganisms within both the ICG SB RAS Collection and othercollections Identification of prokaryotes, which are morphologicallyless diverse than eukaryotes, is based on a wide variety ofphenotypic – and in many cases also genotypic – characteristics. During the description and identification of bacteria,researchers study their cultivation properties, morphology,cell organization, physiological and biochemical features,chemical composition of cells, GC content of DNA, nucleotidesequence of the gene coding for 16S ribosomal RNA (rRNA),and other phenotypic and genotypic characteristics. Phenotypic methods of identification are popular mostlybecause of their relatively low cost. Phenotypic reactionsusually include the responses to various chemical compoundsor biochemical markers. Nonetheless, the manifestation ofphenotypic traits of a microorganism – e. g., cell size andshape, sporulation, cell composition, antigenicity, biochemical activity, and sensitivity to antimicrobial agents – oftendepends on the nutrient media and culture conditions beingused. Therefore, in recent years, to improve the classic methods of biochemical identification, investigators developedmodern methods of biochemical identification . Characteristic features of the growth of microorganisms onsolid and liquid nutrient media are categorized as cultivationrelated or macromorphological properties. Morphologicalcharacteristics and bacterial-cell organization include suchtraits as cell shape and size, cell motility, the presence offlagella, flagellation type, and sporogenesis capacity. In bacterial systematics, the top priority is given to Gram stainingand cell wall structure.Research on physiological and biochemical propertiesprimarily includes determination of the nutrition mode of thebacterium being analyzed (photo-/chemo- and auto-/heterotrophy) and the type of energy metabolism . It is important to identify such traits as the relationshipof this bacterium with molecular oxygen, temperature, pH ofthe medium, with salinity, illuminance, and other environmental factors. This group of traits also contains the list ofsubstrates utilized as sources of carbon, nitrogen, and sulfur;requirements for vitamins and other growth factors; formationof characteristic products of metabolism; and the expressionof certain enzymes. For this purpose, special assays are oftenperformed.Many assays employed for the detection of the aforementioned traits  arecrucial for clinical diagnoses and are widely used in medicalmicrobiology. These assays require substantial time, a large number of complicated media and reagents, compliance withstandardized operating procedures, and meticulous execution. To accelerate and facilitate the identification of somemicroorganisms, mostly those that are clinically important,researchers have developed various assay kits, such asMIKROLATEST® ID | Erba Lachema s.r.o. and BioLog. Forinstance, the MIKROLATEST® ID assay is designed for theidentification of enterobacteria and represents a plastic chamber with wells containing colored diagnostic media. Whetherthe result is positive or negative is determined by changes inthe color of a medium or by a reaction after the addition ofspecial reagents .A state-of-the-art phenotypic technology called BioLogyields valuable information about the properties of strains inaddition to species level identification. Molecular techniquessuch as 16S rRNA sequencing and matrix-assisted laserdesorption/ionization time of flight (MALDI-TOF) massspectrometry do not provide information about the properties of a strain. The technology of carbon source utilizationin the BioLog assay allows environmental microorganismsand pathogenic microorganisms to be identified by compilinga characteristic profile or “metabolic fingerprint” after certainassay reactions carried out in a microtiter plate. Suspensioncultures are tested by means of a panel of preselected assays,then are incubated and analyzed on a signal reader, and theresults are queried against databases.Among the modern methods of biochemical identificationis MALDI-TOF mass spectrometry, which is one of the latestmethodologies for microbial identification. Even though thisassay is “phenotypic,” it in a sense eliminates the gap in thereliability of the test results obtained by phenotyping-basedbiochemical assay systems and genotyping-based identification systems. Additionally, the methodology is very rapid andtherefore well exemplifies a “rapid microbiological assay”. Determination of the bacterial-cell chemical compositionalso plays a role in bacterial systematics (chemosystematics). Chemotaxonomic methods may be helpful in particularfor classifying the bacterial taxa whose morphological andphysiological characteristics vary widely and are insufficientfor their satisfactory identification. Additionally, cell wallcomposition determines serological properties of bacteria.This principle underlies the immunochemical techniques fortheir identification.Investigators sometimes also employ the lipid and fatty-acidcomposition of bacterial cells as chemotaxonomic markers.Active research on fatty acids has become possible with advancements in gas chromatography. Differences in the lipidprofile are used for the identification of bacteria at speciesand genus levels. This method, however, has some limitationsbecause fatty-acid content of cells may depend on cultivationconditions and culture age.Analysis of nucleotide sequences of rRNAs has gainedmuch popularity and importance for the identification ofbacteria and for the creation of phylogenetic approaches totheir classificationFounded at a federal publicly funded scientific institution,the Federal Research Center ICG SB RAS, a collection of biotechnologically important microorganisms contains more than1500 strains, cultures of microorganisms, and DNA samplesvaluable for science and biotechnology and is intended foridentification of new microorganisms with promise in termsof biotechnology and bioengineering and for studies on theirgenetics and metabolism. The collection contains representatives of all major superkingdoms  and physiological groups (including anaerobes andextremophiles). Most strains in the collection have been isolated from previously unstudied unique extreme ecosystems:brine lakes, hot springs, soils, offshore areas, and bodies offreshwaterFor accurate species level identification of microorganisms,currently, investigators are increasingly applying a combination of standard microbiological techniques of cultivationand visual observation with molecular biological and geneticmethods, which help distinguish the species and strains ofmicroorganisms at the level of DNA or RNA molecules . For biotechnological purposes,it is important to have information not only about speciesaffiliation of strains but also about their substrate specificity,completeness of metabolic pathways’ implementation, activityof metabolic reactions, and the possibility of their modulation.Consequently, an integrated approach to the identification ofnatural microorganisms will simplify the search for the strainsthat hold promise for bioengineering tasks. The purpose of this study was to identify microorganismsfrom the ICG SB RAS Collection via an integrated approachinvolving a combination of a wide variety of phenotypic andgenotypic characteristics. Phenotypic characterization. The shape and size of liveand stained cells were determined using light and electronmicroscopes Axioskop 2 Plus, Axioskop А1, LIBRA120 (CarlZeiss) at the Multi-Access Center for Microscopic Analysis ofBiological Objects (SB RAS). The samples were prepared bystandard methods . Gram staining wasconducted by means of the Gram Stain Kit according to the manufacturer’s instructions. The optimal temperature and pH for growth, NaCl tolerance, catalase and urease oxidase activities, anaerobic growth,amylolytic and caseinase activities, and other activities as wellas the ability to utilize various substrates were determined according to Netrusov et al. (2005) and Logan & De Vos(2009).Most of the assays were carried out using Lachema and BioLog reagents and assay kits. 4)2SO4, 0.05 % of Tween 20, 0.2 mM each dNTP, 0.3 mMeach primer, and 1 U of recombinant Taq polymerase . DNA sequencing was performed by the Genomics Multi-Access Center, SB RAS.Sequencing of 16S rRNA genes. Taxonomic affiliation(phylogenetic position) of the strains was determined according to the 16S rRNA gene sequence. To this end, bacterialDNA was isolated by the standard phenol method . Amplification of the 16S rRNA gene was conducted with universal bacterial primers 16S-8-f-B (5ʹ-AGRGTTTGATCCTGGCTCA-3ʹ) and 16S-1350-r-B (5ʹ-GACGGGCGGTGTGTACAAG-3ʹ). The reaction mixture consisted of 1.5 mM MgCl2, 65 mM Tris-HCl (pH 8.8), 16 mM(NHhttp://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence alignment wasperformed in the ClustalW software Searches for similar sequences in nucleotide databases wereconducted by means of the software of the Blast series ;after alkaline hydrolysis of lipids, acids were extracted withhexane and were methylated with a methanolic HCl solutionaccording to Schäffer et al. (2002). The mixture of methylesters of fatty acids was analyzed by gas chromatographyon an Agilent Technologies 6890N chromatograph coupledwith an Agilent Technologies 5973N quadrupole mass spectrometer and a quartz DB-1 column. The carrier gas was Heat a constant flow rate of 1 ml/min. Injection temperaturewas 250 °С, and sample (1 µl) injection was performed viaa microsyringe; electron impact ionization was set to 70 eV.Chromatographic-mass-spectrometric analysis of the studiedsolutions was conducted by means of total ion current inSCAN mode in the mass range 10 to 800 Da, with selectiveion monitoring (SIM) of molecular ions of the compoundbeing studied. Identification of methyl esters of fatty acidswas performed through comparison with database NISTMass Spectral Search Program for the NIST/EPA/NIH MassSpectral Library Version 2.0a, build “Jul 2002.”Mass-spectrometric analysis was conducted on an Ultraflex III MALDI TOF/TOF mass spectrometer . The spectra were recorded in linear positive mode ata laser frequency of 100 Hz in the mass range 2000–20000 Da.Accelerating electrode voltage was 25 kV, IS2 voltage23.45 kV, and lens voltage 6 kV, without an extraction delay.For each sample, three spectra were acquired by summingup 500 laser impulses (5×100 impulses at different positionsof the target cell). External calibration was performed withprecise masses of known proteins: Escherichia coli RL36:4365.3 Da, RS22: 5096.8 Da, RL34: 5381.4 Da, RL32:6315.0 Da, RL29: 7274.5 Da, and RS19: 10300.1 Da. Theseries of spectra obtained for each strain were employed togenerate the characteristic spectra in Biotyper 3.0 software,which constituted a list of mass peaks with averaged m/z values and relative peak intensitiesTo identify microorganisms from the ICG SB RAS Collection, the phenotypic and genotypic characteristics determined were analyzed in Statistica 6.0 software. Dendrogramswere constructed by the method of unweighted pairwisearithmetic mean, and two-dimensional graphs by multidimensional scaling. Multivariate analysis was carried out inPast 3, version 3.25 . In accordancewith the requirements of the software, semiquantitative dataon 95 substrates of the BioLog assay system were convertedto numerical values: data on the absence/presence were codedas “0” or “1,” respectively, and undetermined values as “?”In this study on microbial identification, 93 strains fromthe ICG SB RAS Collection were analyzed. Geographiclocations of sampling for the isolation of microbial cultureswere rather diverse: from vineyards of Crimea to geysers ofKamchatka and the Kuril Islands. Ecological types of habitatsvaried widely too: from bodies of freshwater to salty soils;temperature conditions were cold or thermal; environmentalpH levels were neutral, acidic, or alkaline. The samples werecollected both from unaffected natural areas of water and fromanthropogenically polluted rock typesStrains were isolated on various media: e. g., LB, beefextract agar, beef-extract broth, or Pfennig’s medium withsupplements. The cultivation was conducted at 32 to 55 °С.Each strain was subjected to phylogenetic, phenotypic, andmass-spectrometric characterization.Morphology and biochemical propertiesMost strains produced circular white, cream-colored, or yellowcolonies. Colony edges were even or wavy, and the profilewas flat or convex. Colony sizes varied from pinpoint size(less than 1 mm) to >5 mm. Cells of the strains were rodlike.Seventeen strains secreted a pigment into the medium. Thecell wall was gram-positive. Streak growth varied among thestrains: from nondiffuse to highly diffuse and from rosaryliketo solid. Seventy-five strains featured sporogenesis.The temperature ranges for growth tested were within8–70 °С, and the pH ranges tested were within 2–10. Thesuitable temperature range for the growth of thermophilicmicroorganisms was 40–70 °С with an optimum at 60 °С.The suitable range for the growth of mesophilic microorganisms was 25 to 40, 50, and 55 °С, with an optimum mostlyat 35 °С. Strong growth of the strains was noted at a NaClconcentration of 1 g/l. Some strains did not grow or grewpoorly at 5 g/l NaCl in the medium. All the strains studied were tested for the ability to utilizevarious substrates by means of Lachema and BioLog assaysystems. The strains were found to be aerobes and/or facultative anaerobes. In terms of nutrition, the strains turned out to beheterotrophs and chemoorganoheterotrophs. An overwhelming majority of the strains grew well on media with casein,starch, or Tween as a sole carbon source. Seventy-three strainsshowed a well-pronounced caseinase activity, characterizedby the presence of clear zones around colonies after treatment with acetic acid . Furthermore,81 strains had a good amylolytic activity, evidenced by clearzones. In a reaction with iodine, aside from the usual lossof color, in some cases, there was reddening of the mediumaround colonies, indicating the formation of dextrins.2S test). None of the strains except one utilized lysine, and17 strains had a urease activity. None of the strains manifesteda β-glucuronidase activity. The strains either utilized or did notutilize mannitol, trehalose, lactose, cellobiose, arginine, melibiose, sorbitol, salicin, raffinose, inositol, arabitol, adonitol,and dulcite. Twenty strains featured a β-xylosidase activityIt was determined that 40 strains possess a β-galactosidaseactivity. Virtually none of the strains utilized malonate, citrate, ornithine, or sulfur compounds ; branched-chain acids: isomyristic (isoС14:0),isopentadecanoic (isoС15:0), anteiso-pentadecanoic (aC15:0),isopalmitic (isoС16:0), and anteiso-palmitic (aC16:0); andmonounsaturated: palmitoleic acid (С16:1). The profile andratio of fatty acids in the cell wall of bacteria are importanttraits for the identification of these microorganisms. Fig. 1 depicts a phylogenetic tree built from 16S rRNA sequences; it reflects the clustering of bacterial strains by speciesaffiliation. Fig. 2 presents a statistical analysis of 21 strainsfor 96 formalized biochemical parameters determined by theBioLog Omnilog assays. This analysis did not reveal clear-cutclustering, especially judging by the strains of Bacillus subtilis. Ten strains from 21 samples analyzed belong tothe species B. subtilis. Six of them  are components of a relatively loose clusterthat is formed by representatives of the B. subtilis group andB. cereus group. This cluster includes Lysinibacillus macrolides (strain No. 6). No other strains of B. subtilis  clustered with their own species.In prokaryotic systematics, for species identification, researchers utilize such parameters as rRNA sequence, cellmembrane structure, and certain features of metabolism, e. g.,methanogenesis or bacteriorhodopsin-dependent photosynthesis . Our results suggest that thefeatures of metabolism analyzed are not species-determiningbut may play a major role when the usefulness of one or another strain for biotechnology is determined. This is because the possibility of degradation of various substrates is takeninto account.2S. The analysisincluded strains of three Anoxybacillus species, 17 Bacillusspecies, and two Geobacillus species. Most of the strainsformed a relatively tight cluster, regardless of the species ofa microorganism, thus indicating a similarity between the substrates used for growth. The ability to grow on various sugarsand organic acids (which is what most of the substrates testedwere) is typical of the representatives of various taxonomicgroups from the bacterial kingdom, irrespective of their origin.The second cluster  is formed by some strainsof bacteria belonging to the following species: B. simplex,Anoxybacillus spp. ( flavithermus), G. stearothermophilus,B. mycoides, A. pushchinoensis, and B. licheniformis. Thesespecies were found to be represented by multiple strains, butthe other strains of these species ended up in a large clusteraccording to metabolic characteristics. The unification of thesestrains into one cluster by metabolic characteristics points toa similarity between the substrates or possibly to the loss ofthe ability to utilize some of the substrates. In this case, theclustering does not reflect a common evolutionary origin,but rather shows substrate specificity that has developed asa consequence of convergent processes, probably during theadaptation to the substrates. According to the sampling sites,the species that ended up in the small cluster do not havea common origin eitherFig. 3 shows the results of clustering of 61 strains by 29 parameters of metabolism, as determined by MICROLATEST®Lachema assays. We used such parameters as the ability ofbacteria to utilize some sugars , the presence of urease activity, and production of HThere was clustering by morphological characteristics too.For instance, the small cluster highlighted in Fig. 4 is formedby the strains of microorganisms that produced relativelylarge colonies. Evidently, further refinement and expansion of the spectrumof taxa and of their metabolic maps may lay the foundationfor an “artificial” classification that may be helpful as a keyfor simplified and quicker identification and recognition ofmicroorganisms. For 93 strains of microorganisms from the ICG SB RAS Collection, we determined key molecular-genetic and phenotypiccharacteristics. The strains were characterized by morphological, physiological, molecular-genetic, and mass-spectrometricparameters. Specific features of the strains’ growth on variousmedia were determined, and cell morphology was assessed.Next, the strains were tested for the ability to utilize varioussubstrates. It was found that the strains studied substantiallydiffer in their biochemical characteristics. Additionally, weevaluated physiological characteristics of the strains from thiscollection: e. g., the relationship with oxygen, nutrition type,suitable ranges of temperature and pH, and NaCl tolerance.Application of the integrated approach to microbial identification is necessary for solving the problems of targetedsearches for biotechnologically promising strains. In thisstudy, we combined microbes into separate groups/clusterson the basis of similarities of their phenotypic characteristics.This categorization, after further elaboration and expansion ofthe spectrum of taxa and of their metabolic maps, may formthe basis for the creation of an “artificial” classification thatmay be helpful as a key for simplified and faster identificationand recognition of microbes within both the ICG SB RASCollection and other collections.The authors declare no conflict of interest.Church D.L. Biochemical tests for the identification of aerobic bacteria. In: Leber A. (Ed.). Clinical Microbiology Procedures Handbook.Fourth Edn. AMS Press, Washington, 2016;3.17.1.1-3.17.48.3. DOI10.1128/9781555818814.ch3.17.DasSarma P., Capes M.D., DasSarma S. Comparative genomics ofHalobacterium strains from diverse locations. In: Das S., Dash H.R.(Eds.). Microbial Diversity in the Genomic Era. Acad. Press, 2019;285-322. DOI 10.1016/B978-0-12-814849-5.00017-4.Gaudreau A.M., Labrie J., Goetz C., Dufour S., Jacques M. Evaluationof MALDI-TOF mass spectrometry for the identification of bacteriagrowing as biofilms. J. Microbiol. Methods. 2018;145:79-81. DOI10.1016/j.mimet.2018.01.003.Hammer O., Harper D.A., Ryan P.D. PAST: paleontological statisticssoftware package for education and data analysis. Palaeontol. Electronica. 2001;4(1):9.Jenkins R.E., Tanner M.J.A. The structure of the major protein of thehuman erythrocyte membrane. Characterization of the intact protein and major fragments. Biochem. J. 1977;161(1):139-147. DOI10.1042/bj1610139.Kardymon O.L., Kudryavtseva A.V. Molecular genetic methods forintestinal microbiome investigation. Rossiiskiy Zhurnal Gastroenterologii, Gepatologii, Koloproktologii = Russian Journal of Gastroenterology, Hepatology, Coloproctology. 2016;26(4):4-13. DOI10.22416/1382-4376-2016-26-4-4-13. (in Russian)Logan N.A., De Vos P. Genus I. Bacillus. In: Bergey’s Manual of Systematic Bacteriology. Vol. 3. The Firmicutes. Springer, 2009;21-128.Maniatis T., Fritsch E., Sambrook J. Molecular Cloning. Cold SpringHarbor lab., 1982. Netrusov A.I., Egorova M.A., Zakharchuk L.M. Workshop on Microbiology. Moscow: Academy Publ., 2005. (in Russian)Reyes A.T. Morpho-biochemical aided identification of bacterial isolates from Philippine native pig. Adv. Pharmacol. Clin. Trials. 2018;3(5):000148. DOI 10.23880/apct-16000148.Schäffer C., Wugeditsch T., Kählig H., Scheberl A., Zayni S., Messner P. The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a: analysis of its glycosylation. J. 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Data were analyzed using univariate and multivariate approaches. Relative to wakefulness, during anesthesia there was a significant (q < 0.0001) fourfold change in 21 metabolites. During anesthesia 11 of these 21 molecules decreased and 10 increased. The Kyoto Encyclopedia of Genes and Genomes database was used to relate behavioral state-specific changes in the metabolome to metabolic pathways. Relative to wakefulness, most of the amino acids and analogs measured were significantly decreased during isoflurane anesthesia. Nucleosides and analogs were significantly increased during anesthesia. Molecules associated with carbohydrate metabolism, maintenance of lipid membranes, and normal cell functions were significantly decreased during anesthesia. Significant state-specific changes were also discovered among molecules comprising lipids and fatty acids, monosaccharides, and organic acids. Considered together, these molecules regulate point-to-point transmission, volume conduction, and cellular metabolism. The results identify a novel ensemble of candidate molecules in PFC as putative modulators of wakefulness and the loss of wakefulness.Identifying similarities and differences in the brain metabolome during different states of consciousness has broad relevance for neuroscience and state-dependent autonomic function. This study focused on the prefrontal cortex (PFC) as a brain region known to modulate states of consciousness. Anesthesia was used as a tool to eliminate wakefulness. Untargeted metabolomic analyses were performed on microdialysis samples obtained from mouse PFC during wakefulness and during isoflurane anesthesia. Analyses detected 2,153 molecules, 91 of which could be identified. Analytes were grouped as detected during both wakefulness and anesthesia (NEW & NOTEWORTHY The loss of wakefulness caused by a single concentration of isoflurane significantly altered levels of interrelated metabolites in the prefrontal cortex. The results support the interpretation that states of consciousness reflect dynamic interactions among cortical neuronal networks involving a humbling number of molecules that comprise the brain metabolome. Among the most daunting unanswered questions in neuroscience are those that concern the mechanisms by which states of consciousness are generated. The role of the prefrontal cortex (PFC) as a modulator of states of consciousness is undergoing active investigation from an electrophysiology and connectivity perspective . AlthougThe brain metabolome comprises thousands of low-molecular-weight molecules that underlie fundamental cell biology  and haveSleep and anesthesia are different states of consciousness that exhibit similar traits such as altered autonomic regulation, motor hypotonia, and state-specific changes in the electroencephalogram . The braThe present study collected microdialysis samples from mouse PFC during wakefulness and during isoflurane anesthesia and analyzed these samples using UHPLC-HRMS. By holding species, sex, and brain region constant relative to previous studies , it was n = 24) were purchased from the Jackson Laboratory . These mice comprised a different group from those used for previous studies of the PFC metabolome during sleep and wakefulness .Adult male C57BL/6J mice  or during isoflurane anesthesia (n = 12 mice). The stereotaxic aim point was 2.6 mm anterior, 1 mm lateral to the midline, and 1 mm ventral, relative to bregma  at a flow rate of 1 μL/min using a CMA 4004 microdialysis pump. Beginning 30–40 min after probe insertion into the brain, five microdialysis samples (each 25 μL) were collected sequentially on ice from each mouse. For the waking condition, the pump and dialysis probe were connected via a liquid swivel , making it possible for the mouse to move freely during sample collection. The behavior of each mouse was monitored to ensure that samples were collected during wakefulness.This study used a completely randomized design. Microdialysis samples were collected from the PFC during wakefulness  software. Exact mass and retention times of identified metabolites were confirmed using previously analyzed standards purchased from Fisher Scientific, Inc. Orbitrap mass analyzer technology confirmed reliable metabolite identification via measurements of exact mass and retention time  was confirmed by comparing the stained tissue sections with the coordinates of a mouse brain atlas  and the predicted variation of the model (Q2) approached 1.0. Thus, a highly reliable model was supported. To perform fuzzy k-means clustering, a principal component analysis (PCA) was first calculated across the 61 log transformed metabolomic measures of interest using the R “stat” package  was conducted to stabilize heteroscedasticity . The exp package . Three p package . Fuzzy kZ-scores were derived using the following steps. 1) The average and standard deviation within a 25-min microdialysis sampling point were calculated across subjects from both the wakefulness and the isoflurane treatment groups. 2) Raw metabolite intensities were then subtracted from the time point average and subsequently divided by that standard deviation. 3) A time point z-score average was calculated separately for isoflurane and wakefulness subjects, which generated five data points per metabolite and treatment condition. 4) Last, the z-score time point average from the isoflurane treatment condition was subtracted from the wakefulness treatment condition.Heatmaps were constructed using R routines for hierarchical clustering . Heat maP values  and fold change. For each metabolite, the fold change was calculated using the median and dividing the median for isoflurane by the median for wakefulness. The median was used instead of the mean in order to provide a measurement of central tendency robust to the effects of outliers.A volcano plot was generated using the Benjamini Hochberg adjusted 1) amino acids and analogs, 2) nucleosides and analogs, 3) organic acids and derivatives, 4) monosaccharides and derivatives, and 5) lipid and fatty acid derivatives. A final set of analyses quantified how the 21 metabolites with a fold change ≥ 4 and q value P < 0.0001 relate to the biochemical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for mice. Molecules commonly contribute to multiple biochemical pathways. Therefore, organizational criteria were developed to operationally define the relative percent contribution made by a measured molecule to major categories and subcategories of KEGG biofunction. First, the association between the measured molecule and the individual pathways was identified. Second, the frequency with which the molecule occurred in the pathways comprising the major and subcategories of biofunction was summed for each KEGG biofunction category. Third, these sums were expressed as the numerator relative to the total number of times the 21 molecules were identified in a KEGG pathway.Using the Human Metabolome Database (HMDB), molecules were sorted into five chemical groups based on their closest direct/alternate parent compound: D and E).All microdialysis samples were obtained from the PFC . Figure https://figshare.com/s/42669ca81d5079678004). Twenty-three molecules were measured only during wakefulness and seven molecules were identified only during anesthesia .Untargeted metabolomic analyses confirmed detection of 2,153 molecules, 91 of which were identified from a spectral list of more than 300 molecules with previously determined mass and retention time . Sixty-oz-score. The colors in each cell represent average ion intensity as a surrogate for relative molecule concentration. The numbers 1 through 5 on the abscissa of the heat maps correspond to the ordinal sequence of microdialysis samples, with 1 being the first and 5 being the final dialysis sample. The color distribution illustrates no systematic increase or decrease in analytes during the 125 min of microdialysis. Supplemental Table S1 provides the median, maximum, and minimum along with the P and q values for each molecule in For the 61 molecules in Supplemental Table S1, the R2X = 0.466, R2Y = 0.973, Q2 = 0.972). Fuzzy k-means clustering on three PCA scores validated the separation between wakefulness and isoflurane . Of those 21 shared metabolites, 11 metabolites were decreased during isoflurane, and 10 metabolites were increased during isoflurane.The 61 shared metabolites were analyzed using three multivariate techniques: PLS-DA, PCA, and fuzzy k-means cluster analysis on log-transformed data. These analytic approaches all demonstrated a significant separation between the samples collected during wakefulness and during isoflurane anesthesia . PLS-DA oflurane B. Additioflurane . After ioflurane A and fuzoflurane B were seoflurane . A volcaoflurane C indicatleft) and during isoflurane anesthesia (right). left), those measured only during anesthesia (right), and those measured during both states (middle). q < 0.0001) decreased or increased by isoflurane. esthesia Aright. esthesia Bright, h states Bmiddle.q < 0.0001. The most striking finding revealed by N-acetylglutamine was increased during administration of isoflurane. sn-glycerol 3-phosphate (G3P) also was significantly increased during isoflurane administration . In contrast to sleep, during anesthesia there were significant increases in the levels of d-gluconate, glutamate, homovanillic acid, lactate, N-acetyl-β-alanine, N-acetylglutamine, succinate/methylmalonate, and uridine. Supplemental Table S3 reveals no change in levels of orotate and pyruvate. The lack of identity between the PFC metabolome during sleep and anesthesia is consistent with many studies indicating that sleep and anesthesia are distinctly different states , and G3P is a constituent of lipid rafts within the cell membrane. Anesthetics including isoflurane disrupt lipid rafts and, via a phospholipase D2-dependent mechanism, activate two-pore potassium channels , which underlie a membrane-mediated mechanism for volatile anesthetics (sthetics . Autoradsthetics .N-acetylglucosamine (UDP-GlcNAc), fumarate, ascorbate, and xylitol during isoflurane anesthesia  and neurotransmitters  are assoThis work was supported by Departments of Anesthesiology and Psychology, University of Tennessee, Knoxville, TN, and National Heart, Lung, and Blood Institute Grant HL-65272 (R.L.).https://energy.gov/downloads/doe-public-access-plan).This paper has been coauthored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this paper, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (No conflicts of interest, financial or otherwise, are declared by the authors.H.A.B. and R.L. conceived and designed research; A.G.B. and A.K.B. performed experiments; A.G.B., A.K.B, J.M.P., S.R.C., H.A.B., and R.L. analyzed data; A.G.B., A.K.B, J.M.P. S.R.C., D.A.J, H.A.B., and R.L. interpreted results of experiments; R.L., H.A.B., A.G.B., A.K.B., J.M.P., D.A.J., and S.R.C. edited and revised manuscript; A.G.B., A.K.B, J.M.P., S.R.C., D.A.J., H.A.B., and R.L. approved final version of manuscript.


Rhodococcus fascians is an important pathogen that infects various herbaceous perennials and reduces their economic value. In this study, we examined R. fascians isolates carrying a virulence gene from symptomatic lily plants grown in South Korea. Phylogenetic analysis using the nucleotide sequences of 16S rRNA, vicA, and fasD led to the classification of the isolates into four different strains of R. fascians. Inoculation of Nicotiana benthamiana with these isolates slowed root growth and resulted in symptoms of leafy gall. These findings elucidate the diversification of domestic pathogenic R. fascians and may lead to an accurate causal diagnosis to help reduce economic losses in the bulb market. Rhodococcus contains Gram-positive aerobic bacterial species belonging to the phylum Actinobacteria. These bacteria can be isolated from water, soil, and marine habitats, including harsh ecological regions such as the Arctic, deserts, and heavily polluted areas [Rhodococcus species has been increasing because of their biotechnological applications and potential in bioremediation [Rhodococcus species grow epiphytically or endophytically in the roots or leaves of plants, are nonpathogenic, and are beneficial, aiding the host plant in environmental adaptation [The genus ed areas ,2. Interediation ,4. Most aptation ,6,7.Rhodococcus fascians and livestock-pathogenic Rhodococcus equi [R. fascians causes fasciation and leafy gall disease in a wide spectrum of plants, including 87 genera spanning 40 plant families [Agrobacterium infection or under abiotic stress [To date, only two members of the genus are known to be pathogenic: the plant-pathogenic cus equi ,9,10. Pafamilies ,12. The c stress ,14.R. fascians is often identified by its phenotypic characteristics in selection medium [Nicotiana benthamiana [R. fascians contains the linear plasmid pFiD188 with fas, att, and hyp virulence loci, and the cytokine production causes disease symptoms [R. fascians is the vicA gene, which encodes malate synthase, an enzyme in the glyoxylate cycle [R. fascians isolates without virulence genes are often cultured from symptomatic tissues [Pathogenic n medium ,16; by mn medium ,18; or bthamiana . Pathogesymptoms ,21,22. Tte cycle . However tissues .R. fascians has been detected as a result of strict domestic post-entry quarantine measures. A large proportion of these bulbs, the second most traded in South Korea, were thus destroyed, which became a social issue in South Korea [In lily bulbs imported to South Korea, th Korea . Althougth Korea .R. fascians strains carried known virulence genes, such as fasD and vicA, and their infection caused disease symptoms in N. benthamiana. Overall, the accurate identification and characterization of R. fascians strains that induce lily fasciation will be helpful in the control and epidemiological investigation of this pathogen in South Korea.In this study, we aimed to identify the causative agent of fasciation in lily plants grown in Gangwon province, South Korea. The four identified R. fascians infection [R. fascians, the total bacterial genomic DNA isolated from the plant samples was analyzed for the presence of the virulence gene fasD by PCR , including Asian and Oriental lily varieties, were sampled from greenhouses and nurseries . These pnfection . Figure D by PCR C. fasD ians D188  and enco species ,18. SequR. fascians [fasD gene in all symptomatic lily samples and therefore tried to develop chromosome-based pathogenic markers for the accurate screening of causative bacteria. We targeted vicA, the only virulence gene present on the chromosome. However, there are reports that conventional vicA primers (VicA1497F and VicA1990R) [Rhodococcus species [vicA-based diagnostic primers. To this end, we obtained the full sequence of vicA from 50 Rhodococcus species including five pathogenic R. fascians strains from publicly available sources and identified variable regions that distinguish pathogenic species  was used as a positive control. No amplification with these primers was observed for the DNA of the avirulent Rhodococcus species, but amplification was detected for the positive control. Additionally, amplification was detected in symptomatic lily specimens but not in asymptomatic lilies   have pro species ; therefo species , for whi species . The detemplates A. DNA frc lilies B. These R. fascians. As R. fascians has an orange-colored pigment [vicA and fasD. For nucleotide sequence analysis of the fasD gene, a new primer was synthesized and used instead of the conventional fasD diagnostic primer  and fasD (100% identity)  . In termoutgroup , the phy species . All fouromosome ,24. Two Rhodococcus have virulence plasmids; however, two exceptional strains, A21d2 and A25f, have been reported, which are presumed to have virulence genes in their chromosomes [fas operon, including fasD, which is replaced by a novel gene chimera in islands within the chromosome. Therefore, we investigated whether seven orange colonies that produced the pathogenic R. fascians-specific vicA amplicon, but not the fasD amplicon, are related to A21d2. A sequence comparison based on vicA showed that they all belong to the same clade as D188 (data not shown), suggesting that they lost their virulence plasmids during the screening process.Most plant pathogenic omosomes . In partN. benthamiana [vicA-based phylogenetic analysis (N. benthamiana seedlings with YWS3-1 and YWS8-2 significantly stunted root growth (p ≤ 0.05), whereas inoculations with isolates YWS1-1 and YWS4-1 did not alter growth compared with that of mock-treated control seedlings .To verify the pathogenicity of the isolated strains using thamiana , we seleanalysis . Infectieedlings A. Similaeedlings B. On theeedlings B. The baR. fascians D188 are related to the level of expression of the fas operon present in the virulent plasmid [fas transcripts expressed by the four isolates, the symptomatic difference in N. benthamiana may be due to differences in fas expression in these isolates. Another possibility is that the differences in symptom severity might be related to differences in the virulence genes present in their genomes. Indeed, nonpathogenic R. fascians can alter root structure, including the proliferation of root hairs and early lateral roots, without retarding root growth [Differences in the severity of disease symptoms caused by  plasmid ,21,22. At growth . HoweverR. fascians. Publicly available whole genome sequence information of Rhodococcus species allowed us to develop diagnostic primers specific to pathogenic R. fascians by targeting the virulence gene vicA on the chromosome  were collected from greenhouses or nurseries in Gangwon province, South Korea in May 2020  and fasD (encoding isopentenyl transferase) were analyzed by PCR using R. fascians-specific primers. The conventional primer sets for fasD produced relatively short amplicons of ≈200 bp; therefore, a new primer set was designed using Primer Premier 6.00 to obtain a 573 bp PCR product  and cultured at 27 °C for 2–3 days with shaking. Bacterial DNA was isolated using a Wizard genomic DNA purification kit  according to the manufacturer’s instructions for Gram-positive bacteria. For bacterial identification, the 16S rRNA gene was amplified by PCR and analyzed after DNA sequencing. For further identification, the chromosomal virulence locus  product . To deve strains . The spe species . Primer vicA, and fasD obtained from the bacterial isolates were fully sequenced by overlapping reads in forward and reverse directions using PCR primers and internal primers . The resulting sequences were used as queries in BLAST searches (default search parameters) using the NCBI nr database. Based on a similarity of 16S rRNA (≥99%) with the reference sequences, the isolates were considered R. fascians ) and used to construct the phylogenetic tree. The sequences from the Corynebacterium glutamicum strain AS019 (L27123) and nonpathogenic Rhodococcus species strain KB6 [Phylogenetic trees were constructed with the sequences of 16S rRNA, software  and the software . The treNicotiana benthamiana) seedlings was carried out as described previously [R. fascians isolates  of 0.5 in 10 mM MgCl2) and vertically maintained at 26 °C under a 16 h light condition. Plant images were taken 7 days after inoculation. For the leafy gall assay of tobacco, apical meristems of 4-week-old plants were pinched using forceps and inoculated with 10 μL R. fascians suspension (OD600 of 0.5 in 10 mM MgCl2). Plant images were taken 3 weeks after inoculation. For a mock treatment, the plant was inoculated with 10 mM MgCl2. To isolate R. fascians from the infected plants, leafy galls or meristems were excised from plants and ground in half-strength phosphate-buffered saline. Serial dilutions of extracts were plated on nutrient agar plates. After incubation for 3–5 days, orange-colored bacteria were analyzed by PCR using R. fascians-specific primers.The inoculation of tobacco  and an ANOVA . A


Entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line therapies for chronic hepatitis B (CHB) infection. Although both drugs reduce hepatocellular carcinoma (HCC) risk, their comparative effectiveness remains controversial. We aimed to determine whether TDF is superior to ETV in preventing HCC.Q test and I2.PubMed, Embase, and Cochrane Library from inception until June 9, 2020, were searched according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Key terms included entecavir, tenofovir, and hepatocellular carcinoma. The adjusted hazard ratios (HRs) were pooled using a random effects model. Heterogeneity among studies was assessed by the Cochran Thirteen observational studies (4 of which were conference abstracts) were included with 85,008 patients with CHB . TDF was associated with a lower HCC risk . This beneficial effect was present in cirrhotic patients  and retrospective cohort studies using electronic data sets . However, this beneficial effect did not reach statistical significance for noncirrhotic patients  and retrospective/prospective cohort studies using clinical records .TDF was associated with a lower HCC risk compared with ETV among patients with CHB, particularly cirrhotic patients. Further prospective large-scale studies with longer follow-up periods were required to identify specific subgroups that will benefit most from TDF. Chronic hepatitis B (CHB) virus infection is a global public health threat, with 257 million people being affected worldwide in 2016. It can lead to chronic hepatitis, hepatitic flare up, cirrhosis, hepatocellular carcinoma (HCC), and mortality . NucleosTDF is more effective in viral suppression, especially in hepatitis B e-antigen (HBeAg)-positive patients with very high HBV DNA ,8, but iTwo recent meta-analyses did not include most of the recent high-quality studies ,26. In aWe therefore aimed to compare the effect of ETV and TDF in reducing HCC risk by including the more recent high-quality studies, with a detailed subgroup analysis to explain how the conflicting results among various studies could arise.http://links.lww.com/CTG/A384). Potential studies were retrieved after title/abstract screening by the investigator (K.S.C.). All articles were imported to Endnote X9.2 , and duplicates were removed.Three databases including PubMed, Embase, and Cochrane Library were searched following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guideline  from incTwo authors (K.S.C. and L.Y.M.) determined the eligibility of studies independently, and dissonance was resolved by 2 senior authors (M.F.Y. and C.L.L.). The inclusion criteria included the following: (i) study population: patients with CHB; (ii) treatment: ETV vs tenofovir monotherapy; (iii) study design: prospective/retrospective cohort study, case-control study, and RCT; and (iv) outcome: HCC. There was no language restriction.The exclusion criteria were (i) hepatitis C or D virus, or human immunodeficiency virus coinfection; (ii) previous HCC or liver transplantation; (iii) studies that did not report adjusted effect estimate ; and (iv) review articles, meta-analyses, editorials, case reports, and other forms .Studies that did not report adjusted effect estimates were excluded because marked imbalance in baseline characteristics exists between ETV and TDF. As ETV was introduced to the market much earlier with a longer available data of drug efficacy and safety than TDF, it may be preferentially given to patients with more severe liver disease. In addition, patients with underlying renal impairment or at risk of renal impairment/osteoporosis  tend to receive ETV because of TDF-associated renal toxicity. If this indication/selection bias is not taken into consideration by adjusting for liver disease severity, comorbidities, and age, the association could be biased toward favoring TDF in HCC risk reduction.For the eligible articles, we recorded items including first authors, publication year, ethnicity, study design, inclusion/exclusion criteria, exposure of interest, outcome of interest, sample size, age, sex, HBeAg, cirrhosis, alanine aminotransferase (ALT), HBV DNA, previous NA/pegylated-interferon use, follow-up time, and variables taken into consideration in PS matching or multivariable analysis. The PS represents probability of prescribing TDF instead of ETV that is dependent on other covariates  . PS matcWe also recorded data of HR derived by the PS matching or PS regression adjustment/multivariable analysis. Two authors (K.S.C. and L.Y.M.) assessed the quality of observational studies by the Newcastle-Ottawa scale independently . AuthorsQ test to detect heterogeneity among studies, with a P value <0.1 indicating significant heterogeneity. We calculate I2 statistic to measure the proportion of total variation in the study estimates attributed to heterogeneity. I2 values of <25%, 25%–75%, and >75% indicate low, moderate, and high heterogeneities, respectively  statistical software. Continuous variables were expressed as median (interquartile range [IQR]) or mean (±1 SD). Comparisons of outcome were expressed as HR and 95% confidence interval (CI) using the random effects model and were presented as Forest plot. We used the Cochran ectively . Publicaectively .Eight studies reported aHR derived by both PS matching and multivariable analysis, whereas 5 reported aHR by multivariable analysis only.The pooled HR in the current meta-analysis was calculated by pooling individual HR derived by PS matching in various studies. However, if a particular study had not reported HR by PS matching, the aHR by multivariable analysis would be pooled into the analysis. PS matching is preferred to multivariable analysis because PS matching achieves good balance of baseline characteristics between the 2 groups to minimize bias by its quasiexperimental design.A subgroup analysis was performed according to cirrhosis, ethnicity (Asian vs non-Asian), and study design .Sensitivity analyses were performed by (i) leave-one-out analysis  to assess the impact of a single study on the pooled effect estimate. In particular, subgroup analyses were also performed by excluding the study by Choi et al. because it accounted for the largest weight in the pooled analysis; (ii) pooling aHR derived by multivariable analysis instead of PS matching in various studies; (iii) excluding studies that recruited treatment-experienced subjects; and (iv) using fixed effects model.http://links.lww.com/CTG/A384).Figure 10IU/mL . All studies recruited treatment-naive patients, except for the study by Papatheodoridis et al. Although Gordon et al. also recruited both treatment-naive and experienced patients, they provided aHR after excluding treatment-experienced patients . Eight studies provided details on the follow-up time of both the ETV and TDF groups. Only 3 studies showed a minimal difference in follow-up between the 2 groups (ranging from 1 to 3 months), whereas the other studies showed much shorter follow-up time for TDF vs ETV groups (ranging from 10 to 33 months). Five studies did not adjust for HBV DNA. Importantly, Choi et al. also did not adjust for liver function parameters  and platelet count.A total of 85,008 patients with CHB  were included. Male proportion was 76.2%, median age was 49 years , HBeAg was 43.7%, cirrhosis was 22.5%, median ALT was 94 , and median HBV DNA was 5.9 logP = 0.066; I2 = 43.4%). A random effects model yields an aHR of 0.81  . Funnel plot did not suggest publication bias (Egger test: P = 0.624) .Of the 13 studies included, 8 reported the aHR of HCC with TDF compared with ETV by both PS matching and multivariable analysis, and 5 by multivariable analysis only. There was no significant heterogeneity among the studies (P < 0.001) For cirrhosis, 7 studies  were analyzed, with an aHR of 0.73  .For noncirrhosis, 7 studies  were analyzed, with an aHR of 0.83  . There was no significant heterogeneity among the studies .For the Asian population, 10 studies were analyzed, with an aHR of 0.82  . There was no significant heterogeneity among the studies .For the non-Asian population, 4 studies were analyzed, with an aHR of 0.80  . There was no significant heterogeneity among the studies .Three retrospective cohort studies using electronic databases were analyzed, with an aHR of 0.63  . There was no significant heterogeneity among the studies .Eleven retrospective cohort studies using clinical records were analyzed, with an aHR of 0.97  shows the leave-one-out sensitivity analysis using random effects model. Excluding the study by Choi et al.  or Kim et al. has the largest effect on the pooled HR and P value. The pooled HR increased from 0.81 to 0.85, and the P values were >0.10. Contrarily, excluding the study by Kim et al. or Ha et al. caused a change of the pooled HR from 0.81 to 0.77.eTable 2  . There was significant heterogeneity among the studies .Pooling individual aHR derived by multivariable analysis yields an aHR of 0.80  .The subgroup analysis performed by excluding the study by Choi et al. shows that the beneficial effect did not exist for various subgroup, except for limiting analysis to studies using electronic databases . A random effects model yields a pooled aHR of 0.79 .Sensitivity analysis was also performed by excluding the study by Papatheodoridis et al., which recruited both treatment-naive and experienced patients. There was no significant heterogeneity among the studies , but not among non-Asians (log-rank P = 0.87). Gordon et al. also showed a numerical difference in effect estimates between the 2 ethnicities . Although no statistical significance was detected in the stratified analysis, this could be because of underpower (517 Asians only). Whether beneficial effect of TDF over ETV in Asians requires further studies with a large sample size to confirm because the current meta-analysis shows that it was of borderline significance (P = 0.067). We hypothesize that Asians, whether residing in Asia or in other countries, contracted CHB virus early in life from carrier parents, whereas Whites usually get infected during adulthood, which may lead to a lower HCC risk  , in whicHCC risk .Different study designs also likely contributed to the controversy. There was a statistically significant difference for the retrospective cohort studies using electronic data sets but not for prospective/retrospective cohort studies using data from clinical records. The prevalence of nonadvanced cirrhosis may be underestimated in retrospective cohort studies using electronic data sets, identification of which was dependent on the diagnosis code entry, and transient elastography might not be a routine. The management strategy in liver function test, HBV DNA monitoring, and HCC surveillance was not unified, unlike in prospective cohort settings. HBV DNA suppression and development of resistance to ETV were unlikely to be fully adjusted for. For example, Choi et al.  had feweThere are several strengths of our study. Our study addressed the major limitations of 2 recent meta-analyses ,37 that There are some limitations in this study. The most important one is disparity in the follow-up periods between the 2 groups even after PS matching in some of the included studies (the ETV group had longer follow-up than the TDF group—a difference of up to 33 months). This differential follow-up time may spuriously favor a better preventive effect of TDF over ETV because HCC takes years to develop. Second, residual/unmeasured confounding factors are still possible despite PS methodology or multivariable analysis. For instance, drug compliance and HCC surveillance program data could not be captured in electronic data sets. Third, most of the studies had a follow-up time of up to 5 years only. Longer-term studies of >10 years would help to draw more definitive conclusions. Fourth, beneficial effect of TDF over ETV should be interpreted with caution because the statistically significant association was dominated by the study by Choi et al. Sensitivity analysis showed that the association was no longer statistically significant after excluding this study, except for limiting analysis to studies using electronic databases. Fifth, HBV genotype, a known risk factor for HCC with genotype C conferring higher risk than others , was notTDF was associated with a lower HCC risk compared with ETV in patients with CHB, particularly cirrhotic patients. Further prospective studies with larger sample size and longer follow-up period to identify specific subgroups  that benefit most from TDF should be identified.Guarantor of the article: Ching Lung Lai, MD.Specific author contributions: K.S.C.: involved with study concept and design, literature search, analysis and interpretation of data, drafting of the manuscript, and approval of the final version of the manuscript. L.Y.M.: involved with literature search and approval of the final version of the manuscript. S.H.L., W.K.S.: involved with interpretation of data and approval of the final version of the manuscript. M.F.Y., C.L.L.: involved with the study concept and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, study supervision, and approval of the final version of the manuscript.Financial support: None to report.Potential competing interests: C.L.L. has given sponsored lectures for Bristol Myers Squibb and Gilead Sciences. M.F.Y. received speaker honoraria from Bristol Myers Squibb and Gilead Sciences and unrestricted hepatitis B related research grants from Bristol Myers Squibb and Gilead Sciences. There are no competing interests for the other authors.✓ Both entecavir and tenofovir can reduce hepatocellular carcinoma (HCC) risk among chronic hepatitis B (CHB) patients.✓ Tenofovir disoproxil fumarate (TDF) is associated with a lower HCC risk among CHB patients in this meta-analysis.✓ This beneficial effect is most prominent among patients with cirrhosis.


Heliobacteriaceae, genomic analyses of heliobacteria to date have been limited to just one published genome, that of the thermophilic species Heliobacterium (Hbt.) modesticaldum str. Ice1T. Here we present an analysis of the complete genome of a second heliobacterium, Heliorestis (Hrs.) convoluta str. HHT, an alkaliphilic, mesophilic, and morphologically distinct heliobacterium isolated from an Egyptian soda lake. The genome of Hrs. convoluta is a single circular chromosome of 3.22 Mb with a GC content of 43.1% and 3263 protein-encoding genes. In addition to culture-based observations and insights gleaned from the Hbt. modesticaldum genome, an analysis of enzyme-encoding genes from key metabolic pathways supports an obligately photoheterotrophic lifestyle for Hrs. convoluta. A complete set of genes encoding enzymes for propionate and butyrate catabolism and the absence of a gene encoding lactate dehydrogenase distinguishes the carbon metabolism of Hrs. convoluta from its close relatives. Comparative analyses of key proteins in Hrs. convoluta, including cytochrome c553 and the Fo alpha subunit of ATP synthase, with those of related species reveal variations in specific amino acid residues that likely contribute to the success of Hrs. convoluta in its highly alkaline environment.Despite significant interest and past work to elucidate the phylogeny and photochemistry of species of the  Typically, an enzyme encoded by cysD and cysN, sulfate adenyltransferase, catalyzes the assimilation of sulfate as adenosine phosphosulfate (APS) [Hrs. convoluta lacks cysD, but genes encoding the bifunctional enzyme CysN/CysC , which can also perform this function [cysC) and phosphoadenylyl-sulfate reductase  catalyze the subsequent reaction to yield sulfite [Growth studies indicate eduction . Consistte (APS) ,53. Hrs.function , are pre sulfite ,53.Hrs. convoluta may employ an unusual reductase or an alternative mechanism to perform this reaction. Genes encoding all successive enzymes necessary to synthesize cysteine, homocysteine, and methionine from hydrogen sulfide were identified (data not shown). By comparison, the Hbt. modesticaldum genome lacked cysN, cysH, and sulfite reductase, supporting physiological studies indicating that Hbt. modesticaldum requires a reduced sulfur source for biosynthetic purposes [To produce sulfide for amino acid biosynthesis, sulfite must undergo further reduction through sulfite reductase . Howeverpurposes .Hrs. convoluta strain HHT were able to grow well in the presence of high levels of sulfide (10mM), with sulfide oxidation accompanied by the production of elemental sulfur globules during growth [Hrs. convoluta genome appears to lack genes encoding traditional sulfide oxidoreductases, such as the sulfide:quinone oxidoreductase (SQR) from the green sulfur bacterium Chlorobaculum (Chlorobium) tepidum that oxidizes H2S to S0 and reduces quinone [c oxidoreductase from the purple sulfur bacterium Allochromatium vinosum that oxidizes sulfide to sulfur or polysulfides [Hrs. convoluta contains a novel sulfide oxidoreductase for this purpose.Interestingly, cultures of g growth . However quinone , or sulfsulfides . Thus, ig, a pigment absorbing light maximally between 785 and 790 nm, for phototrophic growth [gltX, hemALBCDEN, and bchIDHME) for pigment biosynthesis , which catalyzes the oxidation of protoporphyrinogen to protoporphyrin, was identified in the Hrs. convoluta genome. Moreover, comparisons with hemG from Escherichia coli and hemY from Bacillus subtilis yielded no significant sequence identity to genes in the Hrs. convoluta genome. Due to the anaerobic nature of Hrs. convoluta, an alternative and unidentified enzyme likely acts as a dehydrogenase rather than an oxidase in this step of pigment biosynthesis. Studies with Desulfovibrio gigas, also a strict anaerobe, suggest that electron carriers, such as flavins and pyridine nucleotides, or electron-transport complexes, such as nitrite and fumarate reductases, do not use O2 as the electron acceptor in the conversion of protoporphyrinogen to protoporphyrin [Hbt. modesticaldum [Hrs. convoluta. Heliobacteria synthesize bacteriochlorophyll (BChl) c growth . Accordiynthesis  were annorphyrin ,58. Moreticaldum , and a sHrs. convoluta, genes encoding protochlorophyllide reductase (bchLNB), chlorophyllide reductase (bchXYZ), and bacteriochlorophyll synthase (bchG) are present to facilitate catalysis of subsequent reactions and produce BChl g. Previous work with Hbt. modesticaldum suggested the need for an isomerase in the interconversion between 8-vinyl bacteriochlorophyllide a and bacteriochlorophyllide g [a and circumvent the need for a separate isomerase in the biosynthesis of bacteriochlorophyllide g [Following the synthesis of divinyl protochlorophyllide in yllide g , but moryllide g ,61.a, 81-OH-Chl a, which was observed as a smaller absorption peak at 672 nm in spectrophotometric studies of Hrs. convoluta [g, a bacteriochlorin-type chlorophyll, is reduced at the C-7 and C-8 bond and has an ethylidene functional group at C-8 [1-OH-Chl a, a chlorin, has a double bond connecting C-7 and C-8 with a hydroxyethyl group at C-8 [g and 81-OH-Chl a are putatively synthesized from a common precursor, divinyl chlorophyllide a [Heliobacteria also contain an alternative form of chlorophyll (Chl) onvoluta . Whereasp at C-8 , 81-OH-Cp at C-8 . BChl g yllide a .a is catalyzed by 8-vinyl chlorophyllide hydratase, and bacteriochlorophyll synthase catalyzes the addition of a farnesyl group to produce the mature 81-OH-Chl a [Hrs. convoluta or Hbt. modesticaldum [1-OH-Chl a synthesis includes steps of dehydrogenation and subsequent hydroxygenation of bacteriochlorophyllide g to produce 81-OH-chlorophyllide a [Hrs. convoluta or Hbt. modesticaldum. Yet another possible mechanism for 81-OH-Chl a synthesis would require the irreversible conversion of BChl g into 81-OH-Chl a upon exposure to O2 and light [2, and therefore this mechanism is unlikely as the major pathway for 81-OH-Chl a production [Hydration of the C-8 vinyl group of divinyl chlorophyllide OH-Chl a ,60. Howend light . Howeveroduction ,62.g and 81-OH-Chl a biosynthesis in Hbt. modesticaldum [Hrs. convoluta , a mechanism similar to BChl ticaldum ,7,60 is onvoluta . Many ofrial PGC .c, d, and e of green sulfur bacteria, both BChl g and 81-OH-Chl a of heliobacteria are esterified with farnesol [Hbt. modesticaldum, Hrs. convoluta contained the complete complement of genes for this pathway, beginning with pyruvate and glyceraldehyde-3-phosphate and proceeding to an unidentified but predicted isomerase that could catalyze the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate [g and 81-OH-Chl a or further transformed into the major carotenoids found in Hrs. convoluta [Hbt. modesticaldum [Hrs. convoluta to that of Hbt. modesticaldum suggests a similar activity in the alkaliphile.Like BChls farnesol . A non-mfarnesol . As was hosphate ,64  encoding 4,4′-diapophytoene synthase in both Hbt. modesticaldum [Hrs. convoluta. Presumably, the presence of an enzyme with 4,4′-diapophytoene synthase activity is essential in the proposed biosynthetic pathway for each of the carotenoids found in heliobacteria [crtM was not identified, two nonidentical copies of crtN (FTV88_2648 and FTV88_3059) having a sequence identity of 71% were annotated in the Hrs. convoluta genome, and it is possible that one of their gene products exhibits CrtM-like activity.Experimental work and pigment extraction from carotene ,63. Thescarotene . The synotenoids  is complHrs. convoluta  showed 71% sequence identity to PshA of Hbt. modesticaldum but nearly 96% identity to PshA of Hrs. acidaminivorans . As is the case in Hbt. modesticaldum, pshX (FTV88_2551) is situated outside of the PGC in Hrs. convoluta and encodes a protein consisting of just 31 amino acids. The PshX RC subunit from Hrs. convoluta showed a 74% sequence identity to that of Hbt. modesticaldum.Heliobacteria possess a type I (Fe–S type) photosynthetic reaction center (RC) imbedded in the cytoplasmic membrane ,67,68. AHbt. modesticaldum RC revealed the presence of 54 BChl g molecules, two 81-OH-Chl a molecules, two carotenoids , four BChl g′ molecules  [Hrs. convoluta RC are not yet available. However, with their highly similar PshA and PshX proteins, the geometry and pigment composition of the Hrs. convoluta RC should closely resemble that of the Hbt. modesticaldum RC [The crystal structure of the r, P800) ,71,72, tr, P800) . Experimaldum RC . Neverthaldum RC .Hrs. convoluta exhibited high sequence similarity to those from Hbt. modesticaldum, and thus the overarching mechanism of light-driven energy conservation is likely to be highly conserved across all heliobacterial taxa. Although not experimentally confirmed, it is likely that electrons first enter the chain by the activity of either NADH:quinone oxidoreductase  in Hrs. convoluta (nuo genes show high sequence identity (62–79%) between the two species. As for Hbt. modesticaldum, menaquinone is predicted to shuttle electrons from Complex I to the cytochrome bc complex (PetABCD), and electron transfer through these complexes drives translocation of H+ to the periplasmic space (Proteins of the electron transport chain (ETC) of eductase , a 14-suonvoluta . With thic space , formingidentity 2–79% betbc1 complexes, which are found in a variety of anoxygenic phototrophs and also in eukaryotic mitochondria, consist of a minimum of three protein subunits: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein [b6f complex, which is present in cyanobacteria and chloroplasts, is comprised of cytochrome f (PetA), cytochrome b6 (PetB), the Rieske iron-sulfur protein (PetC), and subunit IV (PetD) [b contains eight transmembrane helices, whereas cytochrome b6 and its associated subunit IV contain four and three transmembrane helices, respectively [b6 shows homology to the N-terminal half of cytochrome b, and subunit IV is homologous to the C-terminal half of cytochrome b [Cytochrome  protein . In contV (PetD) . Having ectively . Cytochrchrome b .bc complex of Hrs. convoluta indicated that it resembles a hybrid of the cytochrome b6f complex and the cytochrome bc1 complex. A comparison of cytochrome b6 and subunit IV proteins from Hrs. convoluta and the model cyanobacterium Synechocystis PCC 6803 showed 48% and 42% amino acid sequence identity, respectively. However, cytochrome b6 and subunit IV from Hrs. convoluta also showed 36% and 30% amino acid identity, respectively, to the N-terminal and C-terminal halves of cytochrome b from the purple bacterium Rhodobacter sphaeroides. Furthermore, whereas subunit IV from Hrs. convoluta is predicted to contain the usual three transmembrane helices, cytochrome b6 from Hrs. convoluta contained a predicted five transmembrane regions instead of the four typically observed in the b6f complex. Notably, cytochrome b6 from Hbt. modesticaldum is predicted to contain the conventional four transmembrane helices. Therefore, considering the above sequence analyses and their total of eight predicted transmembrane helices, the PetB and PetD proteins of Hrs. convoluta may represent a structural and evolutionary intermediate between cytochrome b and cytochrome b6/subunit IV proteins, a distinction perhaps not shared with neutrophilic heliobacteria.An analysis of the cytochrome c  and shows no sequence or structural similarity to cytochrome f [Firmicutes, the diheme cytochrome c has been identified in all heliobacteria studied thus far and is likely a universal feature of these phototrophs. PetA from Hrs. convoluta showed high sequence identity with PetA from Hrs. acidaminivorans (79%), and sequence identities to PetA from neutrophilic heliobacteria  were all near 50%. Based on similarities in its N- and C-terminal domains, the heliobacterial diheme cytochrome c may have been the result of a past gene duplication and subsequent fusion [The PetA protein in heliobacteria is also of interest because it functions as a diheme cytochrome chrome f . Althougt fusion . atpABCDEFGH) was identified in the genome of Hbt. modesticaldum [Hrs. convoluta was identical to that in the Hbt. modesticaldum genome. Kinetic studies with Hba. mobilis and Hbt. modesticaldum and physiological similarity to photosystem I of cyanobacteria suggest that a PMF established by cyclic electron flow drives photophosphorylation in heliobacteria [A single operon containing all eight genes encoding the subunits of ATP synthase  in Hbt. modesticaldum were also identified in the Hrs. convoluta genome. Like Hbt. modesticaldum, Hrs. convoluta lacked the spo0M gene functioning to regulate stage 0 development of endosporulation [spoIIB gene necessary for robust sporulation in B. subtilis [Hrs. convoluta strain HHT [spo0M or spoIIB in B. subtilis results in impairment of endosporulation [cot genes encoding proteins that comprise the protective spore coat for B. subtilis, including cotH required for spore coat assembly [Hbt. modesticaldum [Hrs. convoluta. Likewise, key proteins that coordinate spore coat assembly and composition in Clostridioides (Clostridium) difficile, including CotA and CotB [Hrs. convoluta. Despite these deficiencies, cells of Hrs. convoluta strain HHT were still capable of forming heat-resistant endospores, even if sporadically [Hbt. modesticaldum and Hrs. convoluta that differ in some respects from those that govern endosporulation in species of Bacillus and Clostridium.A likely universal trait of heliobacteria is the ability to form endospores , differerulation  and the subtilis . This marain HHT , as the rulation ,81. Addiassembly , did notticaldum  or Hrs. and CotB , showed adically . These fHrs. convoluta maintains a cytoplasmic pH at or below pH 8, as is true from studies of several alkaliphilic strains of Bacillus [Alkaliphilic bacteria employ several mechanisms to maintain intracellular pH homeostasis in their highly alkaline environments. Experimental work conducted with alkaliphiles revealed that these organisms maintain a lower cytoplasmic pH than their external environment—up to a 2.3 pH unit difference—for optimal enzyme activity and cellular functioning ,85. DespBacillus ,86.Hrs. convoluta is likely supported by the presence of a Na+/H+ antiporter encoded by nhaA (FTV88_0116). To maintain cytoplasmic pH at homeostatic levels, the Na+/H+ antiporter operates in an electrogenic manner, facilitating the import of twice as many H+ as Na+ exported [Hrs. convoluta was found to be 87% identical in amino acid sequence to NhaA from Heliorestis acidaminivorans—also an alkaliphile—but only 50% identical to NhaA from the neutrophile Hbt. modesticaldum. The NhaA enzyme may, therefore, be a good candidate to study which amino acid residues facilitate antiporter activity in alkaline versus neutral environments.Cytoplasmic pH homeostasis in exported ,88. The exported ,88,89. T+/H+ antiporter generates a sodium motive force (SMF) that has been shown to be important for secondary active transport of various substrates [Hrs. convoluta). The use of Na+-coupling for transport is potentially more important in Hrs. convoluta than in its neutrophilic relative, Hbt. modesticaldum, as genes encoding multiple Na+-dependent transporters (FTV88_2418 and FTV88_1400) and a Na+/Ca+ antiporter (FTV88_2739) in Hrs. convoluta showed little to no significant similarity with genes in Hbt. modesticaldum. In addition to its role in cytoplasmic pH maintenance, the Nabstrates ,90,91 , but no corresponding homolog was identified in Hbt. modesticaldum.The more neutral cytoplasm compared to the alkaline extracellular milieu would seemingly create an outward-directed bulk PMF rather than the inward-directed PMF needed to drive ATP synthesis ,90,91. Dsynthase ,92. Alkabacteria , have bebacteria ,95. In asynthase ,97,98. Bc553 (PetJ) of Hrs. convoluta contained 13 more acidic amino acid residues and 11 fewer basic residues than PetJ of Hbt. modesticaldum , with acidic amino acids (aspartate or glutamate) or neutral residues in their place ,100,101.ticaldum A. In lin the PMF . Althougo alpha subunit of ATP synthase in Bacillus pseudofirmus OF4 was determined to favor H+-powered ATP synthesis at an alkaliphilic pH due to its basic properties [182 in the Hrs. convoluta Fo alpha subunit (+ would be retained on the residue side chain at ~pH 7 (below the side chain pKa). This highlights the significance of a glycine residue at the corresponding position in the Fo alpha subunit in neutrophilic bacteria, including Hbt. modesticaldum [In a similar way, several key amino acid residues and motifs in ATP synthase have been found to contribute to optimal functioning of the enzyme at different pH levels ,102,103.operties ,105,106. subunit B can preBacillus spp. concluded that a highly conserved valine residue is present in H+-driven (MotB) flagellar motor protein sequences, whereas a leucine residue takes the place of this valine in Na+-driven (MotS) motor protein sequences [Bacillus spp. contained MotS with the conserved leucine amino acid, allowing these bacteria to use a SMF to power Na+-coupled flagellar motility [Hrs. convoluta and Hbt. modesticaldum, suggesting that a PMF is used to power motility in both alkaliphilic and neutrophilic heliobacteria. Genomic analyses confirmed the presence of a core set of 24 genes  in Hrs. convoluta that are essential for PMF-driven swimming motility in numerous flagellated bacteria [Several alkaliphilic bacteria use a SMF to power flagellar motor proteins, thus reserving the valuable and limited PMF for ATP production ,108. Resequences ,108. Themotility ,108. Intbacteria .Hrs. convoluta has provided further insight into the photoheterotrophic metabolism, nitrogen utilization, sulfur assimilation, and pigment biosynthesis pathways of heliobacteria, as well as molecular adaptations to an alkaliphilic existence. Further biochemical and genetic experimentation with alkaliphilic heliobacteria, including Hrs. convoluta, is necessary to confirm genomics-based predictions regarding the roles of specific genes and the apparent absence of specific enzyme activities.The analysis of the complete genome sequence of


Verticillium dahliae is a soil-borne hemibiotrophic fungus that can lead to plant vascular disease and significant economic loss worldwide. Its hosts include over 400 dicotyledon plant species, such as annual herbs, perennials, and woody plants. The average yield loss of cotton crop caused by Verticillium wilt is approximately 10–35%. As the control of this disease is an urgent task for many countries, further understanding of the interaction between plants and V. dahliae is essential. Fungi can promote or inhibit plant growth, which is important; however, the most important relationship between plants and fungi is the host–pathogen relationship. Plants can become resistant to V. dahliae through diverse mechanisms such as cell wall modifications, extracellular enzymes, pattern recognition receptors, transcription factors, and salicylic acid (SA)/jasmonic acid (JA)/ethylene (ET)-related signal transduction pathways. Over the last decade, several studies on the physiological and molecular mechanisms of plant resistance to V. dahliae have been undertaken. In this review, many resistance-related genes are summarised to provide a theoretical basis for better understanding of the molecular genetic mechanisms of plant resistance to V. dahliae. Moreover, it is intended to serve as a resource for research focused on the development of genetic resistance mechanisms to combat Verticillium wilt. Verticillium dahliae can cause refractory vascular Verticillium wilt in a wide range of crops worldwide due to its highly aggressive pathogenicity and production of melanised dormant structures called microsclerotia, which can survive for several years in the soil [The soil-borne hemibiotrophic phytopathogenic fungus the soil . It prodthe soil , and thuthe soil .V. dahliae can infect a variety of dicotyledonous species including cotton, tobacco, tomatoes, Arabidopsis, and others. It usually invades and colonises the roots of plants, and then spreads throughout the plant [V. dahliae begins to infect the roots of the plant through the soil, and hyphae penetrate the surface of the plant roots to colonise the vascular bundles, leading to plant death [he plant . V. dahlnt death ,6.V. dahliae is an acidic protein–lipopolysaccharide complex [3PO4, and eventually lead to plant death [The main mechanism of its pathogenesis is xylem vessel blockage and toxin production. When the fungus invades the plant body, the mycelium blocks the xylem vessel, affecting the transport of water and nutrients in the plant ,7. Howev complex . It can nt death . Currentnt death ,13.Plants have evolved numerous defence mechanisms to protect themselves from invading pathogens , and plaV. dahliae. Currently, several studies focusing on plant resistance to V. dahliae have been reported. In this review, some key findings and resistance-related genes are summarised to provide a theoretical basis to further understand the molecular genetic mechanisms of plant resistance to V. dahliae.Functional analysis of the key genes involved in growth and pathogenicity is the molecular genetic basis of revealing plant resistance to V. dahliae [CkPGIP1 from Cynanchum komarovii and GhPGIP1 from Gossypium hirsutum in cotton can improve cotton resistance to V. dahliae, which is associated with the upregulated expression of pathogenesis-related proteins (PRs), enhanced disease susceptibility 1 (EDS1), phytoalexin-deficient 4 (PAD4), and isochorismate synthase 1 (ICS1) genes that enhance xylem lignification [In plants, defence-related proteins play a significant role in plant resistance to fungal pathogens. Polygalacturonase, which digests pectin in plant cell walls, contributes to fungal pathogenicity and plays a considerable role in the pathogenicity of  dahliae . A classfication ,25.Nicotiana alata with strong antifungal activity against many filamentous fungi, is associated with significant resistance to V. dahliae after transgenic expression in cotton plants [GbNRX1 gene codes an apoplastic thioredoxin protein from Verticillium wilt-resistant island cotton (G. hirsutum cv Hai 7124), which is associated with an increase in abundance in response to V. dahliae infection. The observed increase in apoplastic reactive oxygen species (ROS) accumulation and reduced V. dahliae resistance in GbNRX1-silenced plants suggest that GbNRX1 can scavenge apoplastic ROS and is pivotal for the apoplastic immune response [Gossypium barbadense contains proline-rich repetitive and Pollen Ole e I domains and negatively regulates the resistance of cotton plants to V. dahliae. GbHyPRP1 silencing was shown to markedly enhance cotton plant resistance to V. dahliae via cell wall thickening and ROS accumulation [NaD1, a plant defensin from n plants . GbNRX1 response . Hydroxymulation .Arabidopsis NPR1 (AtNPR1) gene in cotton significantly increases the resistance of transgenic plants to non-defoliating V. dahliae [StoNPR1, a Solanum torvum NPR1 gene, was previously expressed in V. dahliae-sensitive potato, which increased the resistance of transgenic plants to V. dahliae. Further, ICS1 and PR1a expression was also evidently enhanced in the StoNPR1 overexpression lines and was significantly induced by V. dahliae infection [G. hirsutum, is induced by V. dahliae, jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). GhMLP28 silencing enhances the susceptibility of cotton plants to V. dahliae infection, whereas GhMLP28 ectopic overexpression in tobacco increases disease resistance. A further assay demonstrated that GhMLP28 activates the transcription factor activity of ET response factor 6 (GhERF6), which augmented the expression of some GCC-box (AGCCGCC element) genes, contributing to defence against V. dahliae [Non-expressor of pathogenesis-related protein 1 (NPR1) is a key regulator of systemic acquired resistance (SAR) in plants. When plants lack functional NPR1, their ability to express the PR gene is impaired and they show a near-total lack of an SAR response to pathogen infection . Constit dahliae . StoNPR1nfection . GhMLP28 dahliae .V. dahliae. The expression of genes related to sulphur uptake and assimilation, sulphur-containing defence compounds, and high-affinity sulphate transporter genes are increased in V. dahliae-infected tomatoes during companion cropping [TLP) gene is related to plant biotic and abiotic stress regulation. Transgenic plants with higher expression of the cotton TLP gene (GbTLP1) show enhanced resistance to different stress factors including V. dahliae infection [V. dahliae in vitro [Arabidopsis thaliana increases resistance to V. dahliae [Sulphur plays a considerable role in tomato disease resistance against cropping . The haunfection . A synthin vitro . Further dahliae .StoCYP77A2 is a wild eggplant cytochrome P450 gene and is induced by V. dahliae. Constitutive expression of StoCYP77A2 in tobacco enhances plant resistance to V. dahliae infection. Protein extraction from StoCYP77A2-transgenic tobacco indicated strong antifungal activity, which implies that StoCYP77A2 should participate in the synthesis of some antifungal compounds [GhDIR1 encodes a putative dirigent protein and its overexpression leads to increases in lignin content in transgenic cotton plants, which display enhanced tolerance to V. dahliae infection [GhUMC1, an umecyanin-like gene in cotton, is involved in the resistance of cotton plants to V. dahliae through regulation of the JA signalling pathway and lignin metabolism [ompounds . GhDIR1 nfection . GhUMC1,tabolism .Chi28 silencing significantly impairs cotton plant resistance to V. dahliae. VdSSEP1, a secretory serine protease, was shown to hydrolyse Chi28; however, cotton apoplastic protein CRR1 protects Chi28 from VdSSEP1-induced cleavage [In plants, extracellular enzymes are often the first line of defence against fungal pathogens. Increasing evidence shows that chitinase is a key hydrolytic enzyme, which degrades the fungal cell wall , and itscleavage .GhPMEI3 silencing in cotton leads to enhanced susceptibility to V. dahliae. Moreover, GhPMEI3 and GhPMEs might participate in protein–protein interactions and are important for plant evolution to resist fungal pathogens [Pectins as the main element of the primary plant cell wall play a key role in defence mechanisms against plant pathogens. Pectin methylesterases (PMEs) catalyse dimethyl esterification of the homogalacturonan domains of pectin in the plant cell wall. athogens .GhLAC15, a laccase gene, was demonstrated to be strongly induced by pathogens. Moreover, its overexpression increases Verticillium wilt resistance via increased defence-induced lignification and arabinose and xylose accumulation in the upland cotton cell wall [Lignification in the plant cell wall is a plant innate immune defence response and the lignification of lignin in resistant cotton stems contributes to the resistance of cotton to disease . GhLAC15ell wall .GbSBT1 in G. babardense, which encodes a subtilase that is mainly localised to the cell membrane, is induced by V. dahliae, JA, and ET, as it translocates to the cytoplasm following JA and ET treatment. GbSBT1 gene silencing reduces the tolerance of Pima-90 (resistant genotype) to V. dahliae infection. Moreover, the overexpression of GbSBT1 activates the expression of defence-related genes and increases Arabidopsis resistance to Fusarium oxysporum and V. dahliae [The expression of  dahliae .GhECR-silenced cotton plants are susceptible to V. dahliae infection, indicating that the GhECR gene is related to cotton resistance to different V. dahliae strains [GbANS silencing significantly reduces anthocyanin production and cotton plant resistance to V. dahliae [V. dahliae. As such, silencing GbCAD1, encoding a key enzyme involved in gossypol biosynthesis, compromises cotton plant resistance to V. dahliae [Enoyl-CoA reductase (ECR) plays a crucial role in very-long-chain fatty acid formation.  strains . Cotton  dahliae . A U-box dahliae . The pro dahliae .V. dahliae infection. However, GhLYK1 and GhLYK2 silencing compromises cotton plant resistance to V. dahliae. GhLYK2, but not GhLYK1, can induce ROS bursts in plants. Therefore, GhLYK2 and GhLYK1 might be distinctively dedicated to cotton defence [V. dahliae via the recognition of chitin signals. The three PRR proteins are induced in response to V. dahliae, and their silencing greatly impairs SA, JA, and ROS generation, as well as resistance to V. dahliae [Lysin motif (LysM)-containing proteins are important PRRs in plants, which function in chitin recognition and the activation of defence responses against fungal pathogen attacks ,52,53. G defence . In addiSolanum lycopersicum), Ve encodes receptor-like proteins containing extracellular leucine-rich repeats, and the Ve R-gene locus contributes to Verticillium resistance [Ve1 is involved in the race-specific resistance to Verticillium wilt pathogen infection [Ve locus includes two closely-linked inverted genes, Ve1 and Ve2, encoding the extracellular leucine-rich repeat receptor-like protein (eLRR-RLP) and cell surface receptors [Ve genes encode a class of cell-surface glycoproteins with leucine zipper and receptor-mediated endocytosis-like signals [Ve1 transgene introduction does not alter the endogenous Ve2 expression [In tomato  gene expression, and JA are required for Ve1-mediated resistance to V. dahliae [Ve1-transgenic Arabidopsis is only resistant to race 1, but not to race 2, strains of V. dahliae, V. albo-atrum, and V. longisporum. Importantly, the critical elements for resistance signalling are conserved, and the signalling components ACIF, MEK2, SERK3/BAK1, and SERK1 play a role in Ve1-positive regulation [Ve1-mediated plant defence requires the basal defence signalling elements EDS1, NRC1, and NDR1 [Arabidopsis, cotton, and tobacco plants results in increased resistance to Verticillium wilt [V. dahliae effector protein Ave1 and then triggers a hypersensitive response in tobacco and tomato [Verticillium resistance [Ve1 mediates plant resistance by monitoring the presence of the Ave1 effector in  dahliae . Some re dahliae ,60. Ve1-gulation . The defand NDR1 ,62. The ium wilt . As a cod tomato , but it sistance .Humulus lupulus), potato (S. tuberosum), tobacco (Nicotiana glutinosa), and wild eggplant (S. torvum) have been cloned and characterised [Gbve1, a tomato Ve homolog, was cloned from an island cotton cultivar with resistance to Verticillium wilt and it can be induced by V. dahliae infection via SA, JA, and ET. Gbve1 silencing in resistant cotton decreases the resistance to V. dahliae. Conversely, the overexpression of Gbve1 in Arabidopsis and upland cotton plants enhances resistance to V. dahliae [Phylogenetic analysis also indicates that Ve1 homologues are extensively scattered in land plants, and that Ve1 homologues in hop , PR3, and PDF 1.2; SA-related genes PR1 and PR2; the ET-regulated gene GST2; and ROS and callose accumulation in the early stage after V. dahliae infection [ Hai7124 ,69. Silenfection ,69,70.Ve1 homologous gene, VvVe, was identified in Vitis vinifera, and its overexpression in tobacco significantly increased resistance to V. dahliae and upregulated defence-related gene expression, including the SA-regulated pathogenesis-related protein gene (PR1), ET- and JA-regulated genes (ERF1), and lipoxygenase (LOX), and enhanced the accumulation of ROS, callose, and PAL [Ve1 homolog from S. torvum, and StoVe1-overexpressing potato lines show increased resistance to V. dahliae [StVe, a potential Verticillium wilt disease resistance gene, from S. torvum and SlVe1 from S. lycopersicoides are homologous to Ve1 and Ve2, respectively, and StVe and SlVe1 encode cell surface-like receptor proteins [A  and PAL . StoVe1  dahliae . StVe, aproteins ,74.V. dahliae [Verticillium wilt, and some novel small RNAs have been identified after infection by V. dahliae strains with different virulence, V991 and D07038 [V. dahliae infection [V. dahliae, which is possibly related to the upregulated expression of lipoxygenase 1 and lipoxygenase 2 [GbRLK, a receptor-like kinase gene from the disease-resistant cotton G. barbadense Hai7124, is induced by V. dahliae. Moreover, transgenic cotton and the overexpression of GbRLK in Arabidopsis plants result in resistance against V. dahliae infection [Micro-RNAs (miRNAs) are indispensable regulators of plant responses to biotic and abiotic stresses . RNA sil dahliae . Upland d D07038 . Variantd D07038 . Membersnfection . GhlncNAgenase 2 . Expressnfection .V. dahliae stimulates Ca2+ influx into the cytosol and enhances GhMYB108 expression in cotton root cells. GhMYB108 was demonstrated as interacting with the calmodulin-like protein GhCML11 in a calcium-dependent manner; thus, GhCML11 and GhMYB108 silencing enhances cotton susceptibility to V. dahliae [V. dahliae directly targets CBP60g to inhibit plant immunity, and mutations in Arabidopsis CBP60g and SARD1 compromise resistance to V. dahliae [HDTF1 was found to significantly enhance cotton plant resistance to V. dahliae and Botrytis cinerea, resulting in activation of JA-mediated signalling and JA accumulation [G. barbadense, namely, GbSOBIR1, in vitro and in vivo, and has a positive effect on cotton resistance to V. dahliae [The MYB family of proteins is both large and diverse, with many members functioning as transcription factors. Increasing evidence shows that plant MYB transcription factors partake in defence against pathogen infection. Infection by  dahliae . A stres dahliae . CBP60g  dahliae . A homeomulation . Further dahliae .GaRPL18) gene from G.arboreum mediates plant resistance to V. dahliae via an SA-related signalling pathway. Silencing GaRPL18 impairs cotton plants resistance to V. dahliae, whereas GaRPL18 overexpression enhances Arabidopsis resistance to V. dahliae [GaGSTF9) in Gossypium arboreum was found to be induced by V. dahliae via SA-related signalling pathway. GaGSTF9 silencing in cotton enhances its susceptibility. Conversely, the overexpression of GaGSTF9 increases Arabidopsis resistance to V. dahliae and the accumulation of endogenous SA and GST, indicating that GST might adjust ROS content via catalytic reduction of the tripeptide GSH, which in turn affects SA content [The ribosomal protein L18 . These results suggest that GhPAO contributes to plant resistance to V. dahliae by activating Spm and camalexin signalling pathways [Spermine (Spm) signalling is correlated with plant resistance to abiotic and biotic stresses. Two key rate-limiting enzymes in Spm biosynthesis are Spm synthase (GhSPMS) and S-adenosylmethionine decarboxylase (GhSAMDC), and pathways . The ove dahliae . A polyapathways .GhCDKE) is induced in cotton via V. dahliae infection and MeJA treatment. GhCDKE increases plant resistance to V. dahliae, which is mediated by the JA response pathway. GhCDKE silencing in cotton enhances susceptibility to Verticillium wilt pathogen, whereas GhCDKE overexpression in A. thaliana increases resistance to the pathogen [B. cinerea and V. dahliae. During plant infection by V. dahliae, GbWRKY1 is also a key regulator that mediates the plant defence-to-development transition by activating JAZ1 expression [G. hirsutum negatively regulates cotton resistance to V. dahliae by directly controlling JA biosynthesis. GhCPK33 silencing was shown to constitutively activate JA biosynthesis and JA mediated-defence responses and enhance resistance to V. dahliae infection [to V. dahliae [Cotton cyclin-dependent kinase E  proteins play a key role in plant defence against pathogens. A genome-wide association study indicated that CG02-containing TIR-NBS-LRR domains are the most likely candidate underlying cotton resistance to  dahliae . The islte Vd991 . The ove pathway .G. barbadense GbSTK in Arabidopsis enhances resistance to V. dahliae and elevates PR-4, PR-5, and EREBP expression [Verticillium wilt; GhMKK4, GhMKK6, and GhMKK9 positively regulate, whereas GhMKK10 negatively regulates, cotton resistance [GhMKK2 and GhNDR1 silencing compromises the resistance of cotton plants to V. dahliae infection [Serine/threonine-protein kinase (STK) is involved in responses to pathogen infection and oxidative stress via phosphorylation. The overexpression of pression . Moreovesistance . As suchnfection .V. dahliae resistance have been identified in plants (Verticillium wilt is still an enormous threat to agricultural production. Due to the presence of microsclerotia in V. dahliae, it can survive in the soil for many years, and it rapidly spreads; thus, it is difficult to control once infection occurs. Moreover, there is no fungicide available for plants, further limiting efficient control. Currently, the most effective control measures are genetic resistance. Therefore, it is necessary to further explore the genes related to V. dahliae resistance in plants. Resistance-related genes can be explored from various perspectives such as extracellular enzymes, the cell wall, PRRs, transcription factors, and SA/JA/ET-related signal transduction pathways. The further development of new technology such as host-induced gene silencing can aid in plant protection. At present, many genes remain unidentified, and there are few known genes related to resistance. Therefore, the technology available for genetic research is also extremely limited. In this paper, we summarised the genes related to V. dahliae resistance in plants on the basis of extracellular enzymes, cell walls, PRRs, transcription factors, and SA/JA/ET-related signal transduction pathways. This report provides a good theoretical resource for researchers and could aid in the discovery of additional genes related to resistance by providing a theoretical basis to further understand the molecular genetic mechanisms of plant resistance to V. dahliae. With the development of molecular biology technology and the application of multi-omics integrative analyses to the study plant disease resistance mechanisms, it is possible to study interactions between plants and Verticillium wilt, which will contribute to the discovery of plant disease resistance genes. At the same time, with the in-depth analysis of the molecular mechanisms underlying plant resistance to Verticillium wilt, it will be possible to obtain crops varieties that are resistant to Verticillium wilt through genetic engineering and breeding technology.Currently, numerous genes related to n plants . However