The biologically active phytochemicals are sourced from edible and medicinally important plants and are important molecules being used for the formulation of thousands of drugs. These phytochemicals have great benefits against many ailments particularly the inflammatory diseases or oxidative stress-mediated chronic diseases. Eugenol (EUG) is a versatile naturally occurring molecule as phenolic monoterpenoid and frequently found in essential oils in a wide range of plant species. EUG bears huge industrial applications particularly in pharmaceutics, dentistry, flavoring of foods, agriculture, and cosmeceutics. It is being focused recently due to its great potential in preventing several chronic conditions. The World Health Organization (WHO) has declared EUG as a nonmutant and generally recognized as safe (GRAS) molecule. The available literature about pharmacological activities of EUG shows remarkable anti-inflammatory, antioxidant, analgesic, and antimicrobial properties and has a significant effect on human health. The current manuscript summarizes the pharmacological characteristics of EUG and its potential health benefits. Eugenia caryophyllata (clove), Zingiber officinale (ginger), bark and leaves of Cinnamomum verum (cinnamon), Curcuma longa (turmeric), and peppers (Solanaceae) [Cinnamomum verum (true cinnamon), Ocimum basilicum (basil), Myristica fragrans Houtt. (nutmeg), and Cinnamomum loureirii Nees. (Saigon cinnamon). The major natural sources of EUG are Eugenia caryophyllata (syn Syzygium aromaticum) which comprises 45-90% [Eugenol (EUG) or 4-allyl-2-methoxyphenol is a phenylpropanoid having an allyl chain-substituted guaiacol . EUG, a anaceae) , as wells 45-90% .Escherichia coli, Corynebacterium sp., and Bacillus cereus [EUG can be prepared synthetically by guaiacol allylation with allyl chloride or produced through biotransformation process that involves microorganisms like s cereus . The phas cereus . The ants cereus , 6. In as cereus , 8.Medicinally important plant species are a good source of active and potent drugs bearing extensive pharmacological properties. There is a huge list of pharmacological activities and bioactivities of EUG along with industrial utilization. In spite of its strong antioxidant properties, EUG protects neuron cells and recues the chances of the oxidative stress-oriented diseases as well as inflammatory diseases. Moreover, EUG is also used in local anesthesia to check the multiple pathological pains other than its wide utilization in dental clinics. Keeping in view the broad-spectrum utilization of EUG, the current review is aimed at highlighting and summarizing the recent advancements using EUG and exploring its pharmacological properties in a broader way. Moreover, this review will also discuss the roles of EUG in inflammatory and chronic diseases, in its antioxidant potential, and in neuroprotection.The mechanism behind the therapeutic potential of EUG has been explained in huge literature. EUG is effective against a number of diseases such as reproductive disorders, nervous system disorders, blood glucose and cholesterol irregularities, microbial infections, tumorigenesis, hypertension, inflammations, and digestive complications . Herein,Cancer usually forms tumors by accumulation of cells and involves uncontrolled cell division. It is the second major cause of death worldwide, with a 6 million fatality rate annually , 11. CelDifferent studies on EUG demonstrated its strong potential in combating colon cancer, prostate cancer, skin tumors, and gastric cancers , 18–20. in vitro study, use of little quantity of EUG in combination with gemcitabine potentiates the effects of the drug with no side effects on healthy cells [Drug combination therapies are mostly used in combating cancer . EUG shohy cells , 11, 25.Breast cancer is ranked second among most common cancers in women, and it is classified as the fourth common cause of cancer-linked deaths worldwide . MammaryMelanoma or malignant melanoma develops from melanocytes and is a sort of skin cancer . Among aCervical cancer arises from the cervix. In 2002, cervical cancer resulted in 274,000 deaths . More thAntioxidants protect body from the harms of free radicals by eliminating ROS or scavenging free radicals –39. UnneL-ascorbic acid [EUG shows dual properties, i.e., antioxidant as well as prooxidant actions, and later is the cause of cytotoxicity . EUG behbic acid . Kaur anHyperlipidemia is the most common social issue in common people and causes cardiovascular diseases (CVDs) and lipid-related diseases . CVDs anCVD death and disease rate can be eradicated by less intake of lipids in the diet . ReducedDyslipidemia is another example which is linked with oxidative stress and causes disregulation in working of cellular antioxidant stress response system . Rice brHowever, it is preferable to use natural and safer drugs in as minimum quantities as possible. EUG has a great antihypercholesterolemic and antiatherogenic potential. Venkadeswaran et al.  reportedEUG undergoes dose- and endothelium-dependent reversible vasodilator responses . In hypeHypercholesterolemia also causes an imbalance in the serum concentration of malondialdehyde  and superoxide dismutase . This effect was studied by Munisa et al. , by the α-glucosidases after which it intercepts the development of advanced glycation end (AGE) products [α-glycosidase compounds like EUG, dietetic complex carbohydrates do not undergo liberation of absorbable monosaccharides. The inhibitors prevent any drastic rise in meal-induced glucose level in blood by delaying the absorption of glucose into the bloodstream [Hyperglycemia is a condition after which there is an outbreak of a degenerative disease called diabetes mellitus caused by abnormalities in glucose metabolism. Studies have shown that EUG is capable of curing several metabolic illnesses owing to its various pharmacological activities. EUG is a strong antidiabetic bioactive substance . Accordiproducts . In the odstream .EUG enhances the activities of carbohydrate metabolism enzymes like glucose-6-phosphate dehydrogenase (31.05%) and instance hexokinase (62.25%). Studies show that a diminishing effect has also been observed by the application of EUG on creatine kinase (38.57%), glucose-6-phosphatase (24.45%), blood urea nitrogen (34.01%), and fructose-1,6-bisphosphatase .aromaticum and Cuminum cyminum (C. cyminum) following the α-amylase enzyme assay method. Starch is converted to simple sugars by α-amylase which is a human enzyme and is involved in causing diabetes. The rate at which glucose absorbs into the blood stream can be reduced by the application of inhibition of α-amylase enzyme. The enzyme retards the digestion of carbohydrates ultimately causing a decrease in blood glucose level. The antidiabetic activity is dose dependent. S. aromaticum depicted more antidiabetic potential than C. cyminum, as S. aromaticum is chiefly composed of EUG (18.7%) while the main constituent of C. cyminum is composed of á-pinene (18.8%) [S. aromaticum was found to be 95.30% which is greater than 83.09% of C. cyminum. These results proved that EUG has a great antidiabetic potential compared with many related essential oils [Tahir et al.  demonstr (18.8%) ; the antial oils .Srinivasan et al. experimented male diabetic rats by inducing streptozotocin drug (40 mg/kg per B.W.) in them. The activity of major enzymes taking part in the glucose metabolism was studied to examine efficacy of EUG. In contrast, only a 10 mg/kg per B.W. dosage of EUG greatly enhanced hepatic glycogen and insulin in plasma (46.15%) samples with a reduction in glycosylated haemoglobin 25.70%) and blood glucose (70%) [% and bloTrypanosoma cruzi, Giardia lamblia, and Leishmania donovani [S. haematobium, S. mansoni, S. japonicum, and S. mansoni) and also familiar as bilharzia or snail fever which is somewhat a neglected tropical disease [in vivo study using mouse models, the drug-resistant parasite Schistosoma mansoni showed a great reduction (19.2%) following a synergistic effect of EUG in combination with conventional drugs [Leishmania are the major cause of leishmaniasis in humans, and the pathogens are being transferred through the bite of infected phlebotomine female sandflies [The majority of human parasites are a key cause of increased mortalities compared with rest of the issues excluding tuberculosis (TB) and AIDS . Certaindonovani . Schisto disease –75. Schi disease . EUG canal drugs . Moreoveal drugs . Protozoandflies . Leishmaandflies . Most ofandflies . Followiandflies  primarilandflies , 79.Pseudomonas aeruginosa, Salmonella choleraesuis, Yersinia enterocolitica, Helicobacter pylori, and E. coli) and gram-positive  bacterial strains. EUG has proven to be active against several bacterial enzymes like histidine, amylase, ATPase, and proteases [E. coli, Yersinia enterocolitica, S. aureus, and Bacillus cereus and proven that gram-negative bacteria are more resistant than its counterpart gram positive [EUG have been well known for its antimicrobial potential and uses. Majority of food-borne diseases are attributed to the microorganisms and are an escalating health issue of global public. About one-third of the population in industrial countries are facing food-borne diseases issues. In the US, 31 notable pathogens give rise to approximately 9.4 million cases of food-borne diseases per annum . EUG canroteases , 83. Theroteases . EUG extroteases , 86 follroteases , 83, androteases . Catheriroteases  tested tpositive . The compositive , 89. Thepositive , 90. Thepositive , 91.Eugenia caryophyllus extracts rich in EUG which were applied in combination with acyclovir (ACV) showed an anti-HSV-1 potential in the brain and skins of mice [Herpes simplex virus (HSV) infects persons 50 years and is a frequent sexually transmitted infection . The med of mice , 100. In of mice . In HSV- of mice  which sh of mice . EUG disFusarium graminearum, F. moniliforme, Tricophyton rubrum, Penicillium citrinum, Candida tropicalis, C. krusei, and Aspergillus ochraceus . EUG either causes cell cycle arrest or affects fungal cell membrane integrity by disruption [C. albicans and found extraordinary effectiveness [B. cinerea which showed much irregular structural features such as broken or shriveled hyphae, large vesicles, and leaked intracellular contents. In addition to morphological alterations, EUG induces the accumulation of ROS especially hydrogen peroxide (H2O2) in the fungal cells to generate oxidative stress which ultimately bursts the fungal hyphae [EUG has shown lethal effects on the growth of different strains of fungi such as sruption . Owing tsruption , 104. Ansruption . Combinativeness . Furtherl hyphae .α (TNF-α), and antioxidant enzymes [Inflammation is called as the body's adaptive immunity response, triggered by noxious stimuli, tissue infection, and injury . It can  enzymes . Generalα and also maintained the proinflammatory mediators. Magalhães et al. [in vivo. A decrease of proinflammatory cytokines like TNF-α, NF-κB-mediated signalizing pathway, and macrophage infiltration were reported, eventually leading to an improvement in the function and structure of the lungs due to a decrease in inflammation [α and cyclooxygenase-2 (COX-2) expression [κB-stimulating macrophages mainly due to TNF-α and COX-2 actions. COX-2 effect is enhanced by lipopolysaccharides (LPS) [Biswas et al.  studied s et al.  investigammation . EUG inhpression . Its antes (LPS) . The expβ. It shows a significant antifever action than acetaminophen when induced intragastrically, centrally, and intravenously. When given intragastrically, EUG in addition to treating fever, induces slight hypothermia unlike acetaminophen. Acetaminophen reduced fever by 68% at a dose of 1.3 mM/kg while EUG reduced 68% fever at a much less dosage. When given peripherally, it shows marked results in inhibiting fever. When given intravenously, respiration of rabbits and vasodilation in ears were increased. Increased respiration depicts that EUG have a positive action on the central nervous system (CNS). If prostaglandins and sodium archidonate mediate fever in the CNS, acetaminophen and EUG can inhibit fever by retardation of prostaglandin R and sodium archidonate synthesis. Both these drugs show the same antipyretic mechanism but EUG shows more potent behaviour as compared to acetaminophen [The antipyretic efficacy of EUG was examined by Feng and coworkers , where tminophen .+, K+, and Ca2+ voltage-dependent channels [2+ channel (HVACC) inhibition by EUG plays an important role in pain-relieving effect. It inhibited HVACC currents in both capsaicin-insensitive and capsaicin-sensitive dental primary afferent neurons [+ and Ca2+ channels [EUG is significantly efficient in relieving pain by reducing the responses that are linked with pain. It suppresses several responses to histamine, norepinephrine, and stimulation of periarterial sympathetic nerves  and inhichannels , 120. Th neurons . TRPV-1 channels .2+-dependent release of neurotransmitters and also retards interleukin (IL-)1β and PGE2 synthesis [EUG is an antagonist towards NMDA (N-methyl-D-aspartate) and gamma-aminobutyric acid (GABA); both are involved in pain transmission , 124. Itynthesis . Park etynthesis  studied Ferland et al.  performein vivo [β peptide (A-β) induced cell death through the abnormal blockage of Ca2+ (resulted from A-β) [EUG in addition to acting on the periphery also performs actions in the central nervous system . The hydin vivo . EUG guain vivo . EUG shorom A-β) . Its abirom A-β) . The inhrom A-β) .β peptide. In experimental diabetes, EUG improves vascular and neuronal complications and suppresses the transmission of action potential in sciatic nerves [Depression, a neurodegenerative disorder, causes common psychological disorders, and 10-20% of the general population suffer through it. EUG showed a marked effect as an antidepressant in force swimming test (FST) and tail suspension test (TST) comparable to an antistress drug imipramine . The mecc nerves . It alsoc nerves . EUG is c nerves . EUG hasc nerves .Regulation of release of neurotransmitters by EUG eventually moderates different brain functions. Rats with 4 h restraint stress model were studied by Garabadu et al. , and ant2+ levels are also diminished in the brain. It is further demonstrated that EUG and isoeugenol potentially restrict acrylamide-mediated neuropathic condition in rats and due to this factor, they can be directly used through diet to soothe different neuropathic conditions in humans [The neuroprotective efficacy of EUG and isoeugenol in response to acrylamide-mediated neuropathy in male albino rats was studied by Prasad , where in humans .Rhizoma acori graminei extracts enriched with EUG [Neurological disorders include a number of conditions whose inception results from subsequent degradation of normal function and morphology of neurons, mainly in the CNS . Globallwith EUG .β-pleated sheets), calling this group of illness called as brain amyloidosis [The neurodegenerative disorder AD is described characterized by the loss of neurotransmitters and neurons and is accompanied by depression and PD . Neuronaloidosis .The stratum corneum of the skin is composed in a way to control the passive movement of substances across the skin. A common way for enhancing drug permeation through the skin is the transdermal drug delivery. Use of small electric charge for the transport of a substance (drug) across the skin is called iontophoresis and is used to compensate for impermeability of the skin, in addition to chemical permeation enhancers like essential oils , 143. Anin vivo studies. However, little or no information is available in humans [EUG toxic behaviour has been studied in multiple n humans . EUG toxn humans . The pron humans , and Medn humans  reportedn humans  and maken humans .EUG, due to its antioxidant, anti-inflammatory, anesthetic, and analgesic potentials, is used extensively in dentistry at low dosage. In dental emergencies, it is used as an anodyne . It is uIn vivo examination finds that diarrhea caused by castor oil was reduced by EUG. Also, the intestinal aggregation of fluid induced by PGE2, the rate of intestinal transit, and the tone of isolated gut muscle and myometrium were also suppressed [At high concentrations, EUG has also been reported to provide anesthetic effects during a pharmacokinetic study using male Sprague-Dawley rats . Tajuddippressed . Karmakappressed  reportedThe current review summarized the potential health benefits and effectiveness of EUG as a therapeutic agent which can be used in medicines and food for the treatment of inflammatory and oxidative stress-oriented disorders. The antioxidant, anti-inflammatory, antipyretic, analgesic, antiparasite, and antimicrobial properties of EUG are well described. It has a great role in neuroprotection, enhances skin permeability, relieves pains, and has a role in temporary dental filler formation (ZnO+EUG). EUG has no known toxicity in smaller quantities, but at higher concentrations, it behaves as prooxidant; hence, a strong anticancer activity is shown by this molecule. Furthermore, diverse applications of EUG such as its pharmacological importance in regulating blood cholesterol and lipid levels are also discussed. Future studies involving a specified dose range of EUG to cure different ailments are recommended to highlight this molecule for the development of drugs.


Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid  biosynthesis and proteins involved in cell envelope re-arrangement  and a redistribution of carbon flux through fermentative pathways . Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs , RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels.  The consequences of massive exploitation of fossil fuels on global warming and climate changes have prompted research toward alternative energy sources with lower environmental impact. Biofuels produced by microbial fermentation of plant biomass have attracted substantial interest based on their potential to benefit current environmental, economic, and societal issues . It has Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium (n-butanol (hereinafter referred to simply as butanol). Butanol (four carbon chain) has a longer carbon backbone than other established biofuels such as ethanol (two carbons) or methanol (one carbon), which gives it fuel properties more similar to that of gasoline, such as high combustion energy, low volatility and corrosivity , ethanol, H2 and CO2  typically can attain 15–20 g/L butanol titer   which liol titer . However butanol . Recentlvolution . Althougvolution . The devvolution . Enhancevolution , whole gvolution  and adapvolution  and;   . Furtherptation)  and Pseus putida , or genehia coli  and Synestis sp.  with resstis sp. , responsstis sp. .C. cellulovorans responses to butanol challenge and identifying genes possibly involved in tolerance to this chemical. A comparative proteomic analysis was performed on C. cellulovorans cultures grown in butanol-supplemented medium and in control medium  leading to identification of 307 differentially expressed proteins. This protein dataset will help future rational metabolic engineering strategies for obtaining butanol-hypertolerant strains to be exploited in large scale production of this biofuel.However, microbial responses to solvent stress are complex and species-related. The present investigation aimed at digging into Clostridium cellulovorans was grown anaerobically as described previously (D-glucose as the main carbon source (5 g/L). Additionally, butanol was supplemented at a concentration ranging from 1 g/L to 8 g/L, when required. Inocula were grown until exponential growth phase and then transferred into 500 mL butyl-stoppered bottles containing 400 mL of medium supplemented with the selected butanol concentration. Cultures were incubated at 37°C in continuous agitation (200 rpm). For each growth condition, three independent cultures were performed. Hourly, microbial growth was assessed through estimation of the optical density at 600 nm (OD600 nm).eviously  with D-gg for 10 min. Cell pellets were re-suspended in 100 μL of 0.2 M NaOH and incubated at 100°C for 10 min. Protein quantification was performed by using the Bradford reagent  following the manufacturer’s instructions. Bovine serum albumin was used as the standard. In addition, in order to calculate the biomass amount of each culture, a conversion factor, previously determined in our laboratory, was used: the ratio between proteins (g) and biomass (g) is 0.261.For determination of total protein cell content, 1 mL culture samples were centrifuged at 16,000 Glucose content in cell free supernatants was measured through glucose oxidase/peroxidase reaction as previously described .2SO4 at a flux of 0.5 mL/min and a temperature of 50°C.Acetate, lactate, formate, butyrate, ethanol, and butanol in culture supernatants were quantified by high-pressure liquid chromatography , equipped with an Aminex HPX-87H column . The detection was performed with a UV–Vis detector set at 210 nm for organic acids and a refractive index detector for alcohols. The mobile phase was 5 mM H® One Solution  as previously described . Cell pellets were washed twice with 0.9% NaCl. The soluble proteins were extracted as previously described . The supernatant was centrifuged again . Proteins were precipitated from supernatants by methanol chloroform method, then resuspended in 25 mM NH4HCO3 with 0.1% SDS (w/v). Protein concentration was measured by the 2-D Quant kit , following the manufacturer’s instructions. Protein samples were subjected to in-solution digestion with trypsin  as previously described  and then analyzed as previously described  were collected during the exponential growth phase (4 h after culture inoculation)  . The LC–C. cellulovorans proteins  was adopted. The label-free quantification was performed as previously described  and Mascot v. 2.4 , as previously described . The Uniescribed . Brieflyt-test using MarkerView 1.2 software  and proteins were considered differentially expressed for p-value < 0.05 and fold change > 1.5 or <0.67   stored in the EggNOG   database(v2.0.1) . The funThe percentage of up-/and down-regulated proteins was computed for each COG-extracted functional category. Fold enrichment of each category in up- and down-regulated proteins was computed as the ratio between the number of up- or down-regulated proteins belonging to a certain category and the number of proteins in the proteome belonging to the category.C. cellulovorans genes encoding differentially regulated proteins or genes which were found to be their best hits by performing standard protein BLAST analysis2. All main speculations were reliant on direct or indirect evidences retrieved in literature.Differentially expressed proteins assigned to each COG category were further investigated by querying the Uniprot database  for funcC. cellulovorans  were harvested  4 h after culture inoculation , washed twice with 0.9% NaCl, and extracted combining RNAprotect® Bacterial Reagent  and RNeasy Mini Kit® . Briefly, an equal amount of cell pellet (5 × 108 cells) was transferred in a sterile plastic tube in which 1 mL of RNAprotect® Bacterial Reagent was added. The mixture was incubated for 5 min at room temperature (RT) then centrifuged for 10 min at 5000 g. Following centrifugation, the supernatant was removed, and 200 μL of lysis buffer  was added. The samples were mixed by vortexing, incubated for 5 min at RT and vortexed every 10 s for 2 min. Then, total RNA was isolated using the RNeasy Mini Kit®, following manufacturer’s instructions. Total RNA quantity was measured using an UV/visible spectrophotometer Ultrospec 3000  while the quality was checked by 1% (w/v) agarose gel electrophoresis.For gene expression analysis, cell pellets of 2O and 5 μL of SYBR-Green I  were used and the thermal conditions were as reported in One μg of total RNA was reverse-transcribed using Maxima H Minus First Strand cDNA Synthesis Kit , following the manufacturer’s instructions. The resulting cDNA was employed as a template for quantitative real-time PCR using the QuantStudio 3 Real-Time PCR system . For each qPCR reaction primers 0.3 μM, 4.1 μL of nuclease-free HPrimers for both target and reference genes, listed in C. cellulovorans growth and metabolism was determined. Growth was only slightly affected by 1 g/L butanol (The effect of supplementing different butanol concentrations (1–8 g/L) on  butanol , while h butanol . These rC. cellulovorans. For this purpose, a butanol concentration that significantly inhibits growth efficiency but allows enough biomass production for proteomic analyses is ideal. Cultures with 4–7 g/L butanol showed comparable growth rate and maximum biomass and were considered suitable for proteomic analyses. Among these, cultures with 6 g/L butanol were finally chosen.Eventually, these analyses were aimed at finding the most suitable butanol concentration for studying butanol-stress response in C. cellulovorans grown in control condition as compared to 6 g/L butanol-enriched cultures (0.46 ± 0.03 g/L) after 4 h  oxidation by pyruvate ferredoxin oxidoreductase (PFOR), leading to production of acetyl-CoA and reduced ferredoxin (possibly used for hydrogen production); (ii) conversion to formate and acetyl-CoA by pyruvate formate lyase (PFL) and; (iii) reduction to lactate by lactate dehydrogenase (LDH) with concomitant consumption of NAD(P)H. According to fermentation end-product determination, LDH reaction accounts for very minor carbon flux, since no detectable lactate amounts were produced, and most pyruvate flux is expectedly taken in charge by PFL and PFOR reactions. The decrease of formate yield in butanol-challenged cultures is indicative of an increase in the carbon flux through the PFOR reaction in this condition. Higher amount of reducing equivalents generated by the PFOR reaction may also have promoted acetyl-CoA-to-butyrate pathway [which requires 3 NAD(P)H per butyrate molecule]. In a number of studies on cellulolytic clostridia, it has been reported that acetate is preferentially accumulated in conditions promoting slower growth (Almost a double amount (0.84 ± 0.18 g/L) of glucose was consumed by fter 4 h . This sefter 4 h  and withfter 4 h . Howeversumption , 2. Butasumption  as well sumption . The oppsumption . No signr growth . This obC. cellulovorans responses to butanol stress is depicted in p-value ≤ 0.05) in butanol-treated cultures versus control ones were considered as differentially expressed. The analysis identified 203 up-regulated and 104 down-regulated proteins  for proteomic analyses were harvested 4 h after culture inoculation, that is during exponential growth phase . A schemproteins  in butanproteins . It is wfunction , namely function . Apart fF, and G .C. acetobutylicum and opposite patterns between proteomic and transcriptomic results were ascribed to post-transcriptional regulation of gene expression. The present results indicate an overall good quality of our proteomic data but suggest that post-transcriptional regulation mechanisms may also occur in C. cellulovorans. Additional comments to specific genes/proteins will be given in the following sections.Expression profile of thirteen key genes was validated by means of qRT-PCR analysis . SelecteDifferentially expressed proteins found in the present investigation will be thoroughly discussed in the next sections.C. acetobutylicum detected a high number of point mutations in genes encoding rRNAs which likely affect the structure and function of ribosomes ppGpp that mediates the so called stringent response  which mediates the formation of functionally inactive 100S ribosomes in bacteria (namely ribosome dimers formed through interactions between their 30S subunits), thus contributing to decrease translation rate (B. subtilis [its transcription is activated by (p)ppGpp]   among the proteins overexpressed by butanol-challenged ynthesis . These rutylicum , C. beijerinckii , Staphyl warneri , and Synstis sp.  which restis sp. . Down-restis sp. . Althougt stress . The latresponse . (p)ppGpresponse . Increasresponse . Clocel_ mucosae . A very  GTPases . In addiion rate . Overexpp)ppGpp] . Altogets (HSPs) . The reahfq-deleted Acinetobacter baumannii was dramatically compromised when exposed to stresses including ethanol and temperature . Hfq is a global modulator of the activity of small regulatory RNAs (sRNAs) . sRNAs aperature . In addi mobilis . Mutatiosolvents . Butanolutylicum .C. cellulovorans cultures encompass 11 members of the COG category O (molecular chaperones and related functions). HSPs comprise two major classes, namely chaperones binding to denatured or misfolded proteins and promoting refolding to their native structure, and ATP-dependent proteases which catalyze hydrolysis of irreversibly damaged proteins    HSPs, namely ClpP (Clocel_1566) and ClpC . ClpC and ClpP form the ClpCP ATP-dependent protease , while McsB phosphorylates CstR making it inactive and promotes its proteolysis by ClpCP (thus enabling transcription of genes under CstR regulation)  and HtrA2 peptidase  from C. cellulovorans show some sequence identity with C. acetobutylicum HtrA . HtrA belongs to class V  HSPs and is thought to be a membrane-anchored protease acting on non-native proteins within or on the outer face of the cytoplasmic membrane (C. acetobutylicum (Proteins overexpressed in butanol-supplemented proteins . In Grampression . Some of operons . The strerinckii . Consistel_0510) . Transcrel_0510) . A searcutylicum  and indiubtilis) . Curiousubtilis) . C. cellprotease  (Schumanprotease . In addis tested . In partulation) , 2016. Omembrane . These rutylicum . Furtherutylicum .C. cellulovorans. COG category E was the second most represented class among overexpressed proteins in butanol-challenged C. cellulovorans. A similar observation was reported on butanol-exposed S. warneri  the differentially expressed proteins involved in amino acid metabolism and transport identified in this study were up-regulated in butanol-stressed  warneri . Up-regu warneri . In addiB. subtilis  imreported  and;  exposed to butanol and other alcohol stress , and CodY regulation mechanisms (either negative or positive) may be highly variable. This makes computational approaches for predicting putative CodY-regulated genes hardly conclusive , UDP-N-acetylglucosamine pyrophosphorylase (Clocel_3808), two D-alanine/D-alanine ligases (Clocel_3085 and Clocel_0693), mur ligase , peptidoglycan transferase (Clocel_2098), cell wall hydrolase/autolysin (Clocel_2663), and a zinc metalloprotease (Clocel_1781)  . PreviouPCC 6803 . EnzymesPCC 6803 . D-alanicus oeni . Studiesthinning .C. cellulovorans genome was down-regulated. Globally, these data indicate that fatty acid biosynthesis is improved in butanol-challenged cells, which is likely related to a change in fatty acid composition of the cell membrane. Extensive up-regulation of enzymes involved in fatty acid biosynthesis has been reported in a number of bacteria upon butanol stress, such as S. warneri .Because of its chaotropic effects, butanol increases biological membrane fluidity . It has  warneri . Up-reguC. cellulovorans overexpresses a couple of proteins belonging to the MreB/Mrl family (Clocel_3042 and Clocel_2768) which are involved in cell shape regulation  with some ATP-dependent permeases found in other Clostridia . Increased levels of membrane transporters have previously been observed in other bacteria under butanol stress or as involved in butanol tolerance  and MgtE (Clocel_1328). NatB mediates Na+ extrusion from cells and was overexpressed by ethanol in B. subtilis (Clostridium phytofermentans (2+ transporters in bacteria and involved in Mg2+ uptake (2+ transporter has been described among the genetic traits of an ethanol-hypertolerant C. phytofermentans (2+ supplementation has been shown to reduce dissipation of membrane ΔpH caused by butanol in C. beijerinckii (+ and Mg2+ are employed by bacteria to maintain membrane ΔpH by means of Na+/H+ antiporters and Mg2+-dependent H+-translocating ATPases (C. acetobutylicum tolerance to butanol (btrR-btrT-btrM-btrK-like gene cluster was found in the C. cellulovorans genome (Clocel_4202-5) consistently with other clostridia  were up-regulated in butanol-challenged olerance . As mentolerance . The chaolerance , which polerance . It thersubtilis  and possrmentans . MgtE is+ uptake . A mutatrmentans , while Merinckii . It has  ATPases . A recen butanol . This ra butanol , althougostridia  althoughFinally, proteins up-regulated by butanol exposure include two components of an ABC transporter putatively involved in dipeptide/oligopeptide transport (Clocel_1355-6). This finding seems consistent with increased requirement of amino acids as suggested by the activation of pathways for amino acid biosynthesis described above.1 subunits K, Clocel_1656; C, Clocel_1658; F, Clocel_1659; A, Clocel_1660; and B, Clocel_1661) and three (out of eight) subunits of a F-type ATPase  were overexpressed by C. cellulovorans cells grown in butanol-supplemented medium. Expression profile of Clocel_1656 and Clocel_1660 was confirmed by qRT-PCR also . Both enzymes are reversible ATPases/synthetases since they function as molecular motors that hydrolyze or synthesize ATP depending on the physiological conditions (+ or Na+) gradient across the cell membrane, while consumption of electrochemical gradient is used to synthesize ATP (1/V1) catalyzing ATP synthesis/hydrolysis and a membrane moiety responsible for ion translocation across the membrane (Fo/Vo) (Five (out of nine) subunits of a V-type ATPase  .As mentioned above, butanol is known to inhibit membrane-bound ATPases, diminish the membrane ΔpH  and Δψ , and lowC. cellulovorans . It is worth noting that only enzymes involved in de novo biosynthesis of purine and pyrimidine were down-regulated in butanol-challenged C. cellulovorans cultures, while, except for the product of Clocel_3712, enzymes catalyzing nucleotide salvage pathways  were not identified  and pyrimidine (7 proteins) biosynthesis was significantly lowered by butanol challenge . This colovorans . As regalovorans . This pihallenge , althoughallenge . Down-reonounced . Differeonounced . Howeversubtilis . XMP is subtilis , and repentified . De novopathways . Hence, i-dependent phosphorylation of Ser46 of HPr, a protein of the PEP-dependent sugar phosphotransferase system (PTS), and also its dephosphorylation , triose phosphate isomerase (Clocel_0721) and pyruvate phosphate dikinase (Clocel_1454) were down-regulated in butanol-challenged C. cellulovorans. Interestingly, up-regulation of some/most key glycolytic enzymes was reported in other bacteria  in response to butanol stress , a sensor enzyme involved in the regulation of sugar uptake and carbon metabolism in several bacteria . HPrK/P rylation . Conditirylation . P-Ser46rylation . On the activity . In addil stress .C. cellulovorans fermentative pathways  was significantly altered by butanol exposure.The amount of some key enzymes involved in C. acetobutylicum  was two-fold overexpressed in butanol-supplemented cultures, which is consistent with increased acetate yield observed in this condition . It is tutylicum .C. cellulovorans  out of ten involved in acetyl-CoA conversion to butyrate were down-regulated  identified two motif instances upstream (–23 and –88 from Clocel_2976 transcription start site) of the Clocel_2972-2976 gene cluster further supporting the hypothesis that Rex regulates its expression. Additional putative Rex binding motif instances were found upstream of acetyl-CoA acetyltransferase encoding genes (Clocel_0192 and Clocel_3058) but not upstream of genes encoding butyrate kinase and phosphate butyryltransferase . These data suggest that Rex may be involved in the regulation of gene expression in butanol-challenged C. cellulovorans. Interestingly, it was recently reported that the Rex regulon was not affected by butanol stress in C. acetobutylicum  and thiolase after butanol shock was reported, which corresponds to an opposite trend with respect to what observed in the present study on C. cellulovorans. It is worth remembering that Rex is a gene transcription repressor whose activity depends on low intracellular NADH/NAD+ ratio . Currently, an explanation for the up-regulation of these proteins in butanol-challenged C. cellulovorans is therefore elusive. A more detailed characterization of C. cellulovorans ADHs, with particular attention on those encoded by Clocel_4197 and Clocel_1949, aimed at determining their substrate(s), cofactor(s) and metabolic role(s) seems important for improving understanding of C. cellulovorans physiology and strategies to tolerate butanol.In addition, two alcohol dehydrogenases  were two-fold overexpressed in butanol-enriched cultures. The nditions . A numbenditions  or theirnditions . Recentlnditions  althoughC. cellulovorans include a desulfoferrodoxin (Clocel_4154) and a nitroreductase (Clocel_4148). Desulfoferredoxin catalyzes NAD(P)H-dependent reduction of superoxide anion to hydrogen peroxide  stress has been observed in different microorganisms . Proteinperoxide  and was utylicum . Nitroreutylicum .C. cellulovorans. Up-regulation of isocitrate dehydrogenase was reported as part of the butanol response of other gram positive bacteria   cycle, namely citrate synthase (Clocel_3688) and isocitrate dehydrogenase (Clocel_2469) were up-regulated in butanol-challenged bacteria . The funes NADH) .C. cellulovorans. However, determination of cellular ATP content showed that ATP levels were generally similar in control- and butanol-supplemented cultures of C. cellulovorans   increasing membrane permeability to ATP; (ii) inhibiting membrane-bound ATPases; (iii) increasing membrane permeability to protons and other ions thus leading to dissipation of the proton motive force; (iv) inducing energy-consuming adaptation mechanisms  . In addilovorans . Interes upshift . It is wchanisms . Althougchanisms , direct 4% (v/v) . However-decanol . It has C. cellulovorans thus confirming previous observations made in other microorganisms. These responses encompass adaptation mechanisms at the cell wall, cell membrane and cytoplasmic levels:The results obtained by the present investigation indicate that butanol exposure elicits complex responses in •C. cellulovorans dedicated a massive effort in the biosynthesis of proteins involved in protein translation, folding and degradation (28% of the up-regulated proteins). However, lower total cell protein levels measured in butanol-supplemented C. cellulovorans cultures  and membrane composition occurs in butanol-stressed rganisms .•C. cellulovorans. These proteins include Na+  and Mg2+  transporters and several subunits of a V-type ATPase  and a F-type ATPase .Many membrane transport proteins, and in particular ion transporters possibly involved in ATP synthesis and/or generation of transmembrane electrochemical gradient were up-regulated in butanol-challenged •C. cellulovorans. This observation possibly correlates with the up-regulation of a putative glucose PTS transporter (Clocel_2778). In addition, pyruvate fate was significantly perturbed by butanol-stress in C. cellulovorans. Formate production was inhibited , and a higher proportion of the acetyl-CoA was driven to acetate instead of butyrate (Multiple metabolic pathways were significantly affected by butanol exposure including: (i) up-regulation of enzymes involved in amino acid biosynthesis; (ii) down-regulation of several enzymes involved in pyrimidine and purine biosynthesis; (iii) modulation of the expression of multiple enzymes involved in glycolysis and fermentative pathways. As regards amino acid biosynthesis, the enhancement of enzymes catalyzing biosynthesis of branched-chain amino acids  could possibly refer to additional mechanisms to modulate cell membrane fluidity . In factbutyrate . The latbutyrate . AlthougC. cellulovorans through rational metabolic engineering. The complexity of microbial responses to butanol stress makes identification of priority gene modifications aimed at these strategies not trivial. Furthermore, a significant number of previous studies reported involvement of genes with previously unknown/poorly characterized function in butanol tolerance , htpG , dnaJ  and clpC . Recently, an increasing attention has been devoted to the role of sRNAs in response to a variety of stresses including butanol, and studies involving overexpression or suppression of some of them were effective in improving butanol tolerance (C. cellulovorans since the RNA chaperone Hfq (Clocel_2035) was among the most up-regulated protein under butanol-stress (FC = 3.09). Therefore, overexpression of Hfq is another interesting strategy that would be worth testing. Recently, overexpression of several sRNA regulators was used to improve butanol-tolerance of E. coli  is currently unknown. Our study thus points at interesting gene candidates for engineering butanol-hypertolerant C. cellulovorans and stimulates further studies aimed at characterizing still poorly characterized proteins.It is worth noting that the function of four among the ten most up-regulated proteins in butanol-stressed The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/RM conceived the study. PC and GU performed the bacterial cultures and prepared extracts for proteomic analysis. MM performed the proteomic analysis. PC, GU, and AR analyzed the proteomics results. RM, GM, and CB performed the qRT-PCR analysis. PC, GU, AR, MM, GM, CB, and RM prepared the figures and tables. PC, GU, AR, CB, EP, and RM interpreted the results. All the authors contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PDZ-binding kinase (PBK) encodes a serine/threonine protein kinase related to the dual specific mitogen-activated protein kinase kinase (MAPKK) family. There is evidence that overexpression of this gene is associated with tumorigenesis. However, the role of PBK in hepatocellular carcinoma (HCC) remains unclear. Therefore, we evaluated the prognostic role of PBK and its correlation with immune infiltrates in hepatocellular carcinoma.The expression of PBK in pan-cancers was studied by Onconmine and TIMER. The expression of PBK in HCC patients and its relationship with clinicopathological characteristics were analyzed using The Gene Expression Profiling Interactive Analysis (GEPIA), The human protein atlas database (HPA), The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) databases. Receiver operating characteristic (ROC) curve was used to determine the diagnostic value of PBK in HCC patients. The relationship between PBK and prognosis of HCC was performed by GEPIA and Kaplan Meier plotter web tool. The correlations between the clinical characteristics and overall survival were analyzed by Univariate Cox regression and Multivariate Cox hazards regression to identify possible prognostic factors for HCC patients. LinkedOmics was applied to investigate co-expression associated with PBK and to analyze Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The network map of PBK and related genes is constructed by GeneMANIA. Finally, TIMER and TISIDB were used to analyze the correlations between PBK and tumor-infiltrating immune cells.P=0.0089), age (P=0.0131), and race (P=0.0024) of HCC patients. The receiver operating characteristic (ROC) curve analysis showed that PBK had high diagnostic potential to HCC in GSE76427 (AUC=0.8799), GSE121248 (AUC=0.9224), GSE62232 (AUC=0.9975), and GSE84402 (AUC=0.9541). Multivariate Cox hazards regression showed that high expression of PBK may be an independent risk factor for overall survival in HCC patients . The Protein–protein interaction network showed that PBK significantly interacted with LRRC47, ARAF, LGALS9B, TTK, DLG1, and other essential genes. Furthermore, enrichment analysis showed that PBK and co-expressed genes participated in many biological processes, cell composition, molecular functions, and pathways in HCC. Finally, the immune infiltration analysis by TIMER and TISIDB indicated that a significant tightly correlation between PBK and macrophages, neutrophils, as well as chemokines and receptors.Multiple database analysis shows that PBK was highly expressed in many types of tumors, including hepatocellular carcinoma, and was significantly related to tumor stage (High expression of PBK is significantly correlated with poor survival and immune infiltrates in hepatocellular carcinoma. Our study suggests that PBK can be used as a biomarker of poor prognosis and potential immune therapy target in hepatocellular carcinoma.The online version contains supplementary material available at 10.1186/s12957-021-02479-w. Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related death in the world . HepatocWith the development of DNA sequencing technology, precision medicine has made great progress, and many new therapeutic targets have been recognized. PDZ-binding kinase (PBK) is a member of the mitogen-activated protein kinase (MAPK) family, which acts on oncogenes as upstream kinases in some important signaling pathways . PBK is In this study, we revealed the expression of PBK in pan-cancers and HCC. The results showed that PBK expression was extremely higher in most common tumor tissues, including liver cancer, from Oncomine and TIMER databases Fig.S A&B. TheThe RNA-seq data of HCC from The Cancer Genome Atlas (TCGA) was downloaded from the cBioPortal platform , 15. Clihttps://www.oncomine.org/) [P value:0.05, the fold change: 2, gene rank: top10%, data type: DNA+mRNA. TIMER (http://timer.comp-genomics.org/) [p value < 0.05; **: p value <0.01; ***: p value <0.001).To investigate the expression of PBK gene in pan-cancers, two online databases were used respectively—Oncomine and TIMER. There are 715 datasets and 86733 specimens collected by Oncomine web applications (ne.org/) . These dcs.org/) , 18 is ahttp://lifeome.net/database/hccdb/) [http://gepia.cancer-pku.cn/) [https://www.proteinatlas.org/ )[, the correlation analysis of PBK and clinicopathological features according to the TCGA-LIHC dataset were examined.HCCDB (/hccdb/)  is a datpku.cn/)  is a newas.org/ ) provideshttp://kmplot.com/analysis) [n = 155) group and PBK-low (n = 156) group. The Kaplan-Meier analysis was performed to investigate the prognosis of patients in the two groups. Univariate Cox regression and Multivariate Cox hazards regression analysis were performed to identify possible prognostic factors for HCC patients.GEPIA can provide an association between PBK gene expression level and prognosis in patients with LIHC. The analysis indicators of GEPIA included Overall Survival and Disease-Free Survival, and the group cut-off is median. Kaplan Meier plotter   is a frihttp://genemania.org/ )[GeneMANIA (ia.org/ ) is an onhttp://www.linkedomics.org/ )[LinkedOmics (cs.org/ ) is an opcs.org/ ). We usedThe version of TIMER2.0 provides immune infiltrates' abundances estimated by multiple immune deconvolution methods, and allows users to generate high-quality figures dynamically to explore tumor immunological, clinical and genomic features comprehensively . Online http://cis.hku.hk/TISIDB/) [TISIDB (TISIDB/)  is an onT tests were used to analyze the differential expression levels of PBK mRNA between the HCC tissues and the normal tissues from the GEO databases. ROC curve was performed to detect the diagnostic value of PBK in HCC. Kaplan-Meier and log-rank tests were conducted to assess the effect of PBK on survival. Univariate and multivariate analyses were performed using Cox proportional hazards regression model. Statistically significant differences were considered when P < 0.05.All statistical analyses and graphs were performed in SPSS21.0, GraphPad Prism 8 software, and web resources. The measurement data are presented as mean ± SD. Unpaired This study was conducted according to the flow chart shown in Fig. We used 4 independent GEO databases to verify PBK mRNA expression in HCC and matched normal tissues. Compared to normal tissues, PBK mRNA levels were upregulated in HCC tissues, based on GSE76427 , tumor stage (P=0.0089), and age (P=0.0131), except for gender (P=0.2998) in HCC patients.The association between PBK mRNA expression and the clinicopathological characteristics of HCC patients including gender, race, tumor stage, and age was investigated using TCGA data. Based on the median values of PBK mRNA level, 155 patients were classified as high PBK expression group, and 156 patients were classified as low PBK expression group. As shown in Fig. P=0.0061) and DFS (Logrank P=0.006)   Fig. A, B. Accn = 155) group and PBK-low (n = 156) group. As shown in Fig. P = 0.007).The Kaplan-Meier analysis was performed to investigate the relationship between PBK expression and overall survival in 311 HCC patients in the TCGA cohort. Median expression of PBK of all samples was chosen as a cut-off to divide samples into the PBK-high (P < 0.001) and PBK expression  were identified to be related to the overall survival of 311 HCC patients in the TCGA training set. In the Multivariate Cox regression model, tumor stage  and PBK expression  were likewise confirmed to be independent prognostic factors of overall survival in HCC patients  network to analyze the interaction between PBK and other genes. PPI network showed that PBK significantly interacted with LRRC47, ARAF, LGALS9B, TTK, DLG1, and other essential genes. Biological function of these genes may include cell cycle G2/M phase transition, cell cycle checkpoint, regulation of nuclear division, mitotic cell cycle checkpoint, negative regulation of mitotic cell cycle, mitotic nuclear division and G2/M transition of mitotic cell cycle Fig. . Fig. 5PIn addition, the co-expression genes in TCGA-LIHC that are closely related to PBK were explored on the Linkedomics website. As shown in Fig. GO and KEGG analysis of PBK and co-expression genes were further analyzed using the GSEA in Linkedomics Fig. . ConsequP=0.0175) and CCR10 , and was negatively correlated with CCR7  and CXCR1. In addition, the result showed that 12 types of chemokines were negatively correlated with the expression of PBK, and 6 types of chemokines were positive , CD8+T cells , B cells , macrophages , neutrophils , and dendritic cells  , which also confirmed our previous results. Furthermore, the expression of macrophages and neutrophils was positively correlated with poor prognosis , meanwhile, there was no correlation between the remaining immune cells  and prognosis (P>0.05)  and neutrophils  were also significant independent risk factors among the six types of immune cells  in HCC. The results show that a significant positive correlation between PBK and CD4+T cells , also known as PDZ-binding kinase (PBK), is a novel mitotic serine/threonine protein kinase, which was confirmed to be associated with the development, progression, and metastasis of malignancies and to be a potential therapeutic target in cancer therapy . StudiesMany studies about the possible role of PBK in tumor immune cell infiltration have emerged in recent years. In Esophageal squamous cell carcinoma, immune infiltration analysis showed that infiltration of dendritic cells was significantly negatively correlated with PBK expression levels . Zhou etIn conclusion, multiple studies have identified PBK as a crucial gene for HCC. The difference between our study and previous studies was that we reported the relationship between PBK expression and clinicopathological characteristics. We also identified the diagnostic value of PBK and as an independent prognostic factor in HCC. In addition, another advantage in our study was the first time to evaluate the correlation between PBK expression in pan-caners and tumor-infiltrating lymphocytes (TILs), chemokines, and receptors. However, there were few limitations in the present study. Experimental verification is also needed to elucidate the molecular mechanisms in vivo and in vitro. Thus, results in our research may provide useful information for prospective research on HCC immunotherapy and targeted therapy.Additional file 1: Fig. S1 The expression of PBK in pan-cancers and HCC. A: PBK mRNA levels in 20 types of human cancers analyzed by Oncomine. Red means increased expression and blue means decreased expression. The numbers indicated the amounts of dataset satisfying the threshold in the colored cell. B: PBK mRNA levels analyzed by TIMER. . C: The expression of PBK in HCC tissues from HCCDB. D: PBK expression profile in HCC based on GEPIA database. (*P<0.05) E-F: Protein expression and distribution of PBK from HPA.Additional file 2: Fig. S2A: Relations between receptors and expression of PBK in pan-cancers (red is positive correlated and blue is negative correlated). B: PBK expression was positively closely related with CCR6 and CCR10, and was negatively correlated with CCR7 and CXCR1.Additional file 3: Fig. S3 A: Relations between chemokines and expression of PBK in pan-cancers (red is positive correlated and blue is negative correlated). B: PBK expression was positively or negatively closely related with chemokines .Additional file 4: Table S1. Multivariate Cox analysis of immune cells infiltration.


B and T lymphocyte attenuator (BTLA) is one of the most important cosignaling molecules. It belongs to the CD28 superfamily and is similar to programmed cell death-1 (PD-1) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) in terms of its structure and function. BTLA can be detected in most lymphocytes and induces immunosuppression by inhibiting B and T cell activation and proliferation. The BTLA ligand, herpesvirus entry mediator (HVEM), does not belong to the classic B7 family. Instead, it is a member of the tumor necrosis factor receptor (TNFR) superfamily. The association of BTLA with HVEM directly bridges the CD28 and TNFR families and mediates broad and powerful immune effects. Recently, a large number of studies have found that BTLA participates in numerous physiopathological processes, such as tumor, inflammatory diseases, autoimmune diseases, infectious diseases, and transplantation rejection. Therefore, the present work aimed to review the existing knowledge about BTLA in immunity and summarize the diverse functions of BTLA in various immune disorders. On cells coexpressing BTLA and HVEM, BTLA can interact with HVEM in cis, whereas trans interaction occurs when BTLA and HVEM are expressed on different cells  is a member of the CD28 superfamily. Its gene is localized in the q13.2 region of chromosome 3 and consists of 5 exons with a total length of 870 bp. The protein structure of BTLA is similar to programmed cell death-1 (PD-1) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4), which includes extracellular domain, transmembrane domain and cytoplasmic domain . In addition, these cells exhibit a poorly differentiated phenotype, reduced cytolysis, and increased proliferation potential among patients with diffuse large B-cell lymphoma (DLBCL). Further, increased BTLA levels are related to an advanced disease stage in DLBCL patients  and is often associated with impaired anti-tumor immune response. Upregulated BTLA expression in gallbladder cancer (GBC) plays a role in inhibiting anticancer immunity, and an increased proportion of BTLApatients . In hepa T cells , 46. Bes T cells . BTLA+ Tpatients . BTLA expatients , 49. Fur T cells . BTLA ex T cells . In addi T cells . However T cells . Notably T cells . Althougma cases . This tema cases , 56. Besma cases , chronicma cases  and esopma cases . Five BTma cases . Howeverma cases .-/- mice experience a prolonged duration of allergic airway inflammation and enhanced recruitment of inflammatory cells relative to those in wild-type mice  cases, while the remission of fingolimod-induced relapsed-remitted MS is related to a significantly increased number of CD19+/BTLA+/IL-10+ B lymphocytes  , 78. BTLphocytes . In patiecreased . Sawaf eactivity . BTLA deactivity . Howeveractivity . Anti-BTactivity . Transfeseverity . In rheu T cells . Lin et  T cells . Besides T cells .+ and CD8+ T cells from COVID-19 patients , BTLA-/- mice show markedly improved spleen and liver injuries and reduced mortality  infection, as manifested by increased cell proliferation and cytokine secretion, suggesting that BTLA expression in αβT cells is involved in protective immune memory against Mtb infection among patients with active pulmonary tuberculosis  resulting from allogeneic bone marrow transplantation , 105. Ind kidney , 107. Agoduction . Furtheroduction . Partialoduction . Agonistoduction , 111. Mooduction . Howeveroduction , 114. Rooduction . Besidesoduction .BTLA has been found to affect the function of a variety of immune cells and plays an important role in many immune-related diseases Table 1.+ T cells , Doctoral Startup Fund of Jining Medical University (NO. 2017JYQD24), Research Fund for Academician Lin He New Medicine (NO. JYHL2018MS11), and Chinese and foreign cooperation cultivation project of Jining Medical University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bifidobacterium dentium, could regulate important neurotransmitters, we examined Bifidobacteria genomes and explored neurotransmitter pathways in secreted cell-free supernatant using LC-MS/MS. To determine if B. dentium could impact neurotransmitters in vivo, we mono-associated germ-free mice with B. dentium ATCC 27678 and examined fecal and brain neurotransmitter concentrations. Results: We found that B. dentium possessed the enzymatic machinery to generate γ-aminobutyric acid (GABA) from glutamate, glutamine, and succinate. Consistent with the genome analysis, we found that B. dentium secreted GABA in a fully defined microbial media and elevated fecal GABA in B. dentium mono-associated mice compared to germ-free controls. We also examined the tyrosine/dopamine pathway and found that B. dentium could synthesize tyrosine, but could not generate L-dopa, dopamine, norepinephrine, or epinephrine. In vivo, we found that B. dentium mono-associated mice had elevated levels of tyrosine in the feces and brain. Conclusions: These data indicate that B. dentium can contribute to in vivo neurotransmitter regulation.Background: Accumulating evidence indicates that the gut microbiota can synthesize neurotransmitters as well as impact host-derived neurotransmitter levels. In the past, it has been challenging to decipher which microbes influence neurotransmitters due to the complexity of the gut microbiota. Methods: To address whether a single microbe,  The communication between the intestine and the brain, known as the gut–brain axis, has recently attracted attention for its crucial role in human health. Intestinal microbes are known to modulate both the enteric nervous system (ENS) and central nervous system (CNS) and have been linked to intestinal motility ,7,8,9,10Bifidobacterium (phylum Actinobacteria) [Bifidobacteria species dominant in human infants include B. longum, B. longum subspecies infantis, B. breve, B. bifidum, B. catenulatum, B. adolescentis, B. angulatum, B. animalis, and B. dentium [Bifidobacteria have been shown by several groups to beneficially modulate the gut–brain axis. For example, B. longum strains NCC3001, 1714, and R0175 can modulate anxiety in animal models [B. longum NCC3001 was found to improve depression scores in patients with IBS [B. infantis improved depressive-like behaviors in a rat model of maternal separation [B. longum 1714 was shown to enhance object recognition and cognition in a mouse model [Bifidobacterium species  affects the CNS synaptic plasticity, microglia activation, and rodent behavior, including anxiety and hyperactivity [B. dentium administration to adult mice modulates the serotonergic system and adult behavior [Bifidobacteria modulate the gut–brain axis remains to be discovered.The communication between the gut and brain is speculated to be particularly important during early life. During this early developmental period, the gut microbiota is dominated by the genus acteria) ,36,37,38 dentium ,41,42,43l models ,45,46, awith IBS . Likewisparation , while Bse model . Our preactivity ,51. We hbehavior . AlthougBifidobacteria species in vitro and in vivo. We selected B. dentium because it is found in infants and adults [B. dentium. To effectively address the contribution of B. dentium on neurotransmitter concentrations in vivo, we used a gnotobiotic model (defined microbiota applied to germ-free mice); a well-established model for studying the effects of specific microorganisms on the gut–brain axis [B. dentium colonization is associated with a unique gut and neuronal neurotransmitter profile.To address this knowledge gap, we sought to characterize the neuroactive compounds produced by a single human d adults , has thed adults , possessd adults ,56, and d adults . The impain axis ,21,57. OBifidobacterium dentium ATCC 27678  was cultured in Man, Rogosa, and Sharpe  media in an anaerobic workstation (Anaerobe Systems AS-580) at 37 °C with a gas mixture of 5% CO2, 5% H2, and 90% N2. After culturing in MRS media, B. dentium was sub-cultured into a chemically defined media, termed ZMB1 [600nm) of 0.1 and cultured anaerobically at 37 °C for 18 h. Bacterial cultures were centrifuged at 5000× g for 5 min and the cell-free supernatant was sterile-filtered  for LC-MS/MS analysis.Diet.com) and water. Adult male germ-free Swiss Webster mice (6–9 months of age) were gavaged with sterile MRS  or 3.2 × 108 CFU (in 0.2 mL) B. dentium ATCC 27678 in MRS  and maintained in isolators for 17 days as previously described [Gnotobiotic experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Baylor College of Medicine, Houston, TX, USA. The germ-free mice had ad libitum access to irradiated food , incubated with crystal violet, gram iodine, decolorized, and counter-stained with gram safranin. Sections were then incubated with picric acid-acetone. After dehydration through a series of alcohols  and xylene, sections were cover-slipped and imaged on an upright Nikon Eclipse 90i microscope at 20× using a Plan Apo (NA 0.75) differential interference contrast (DIC) objective.Bifidobacteria members to generate neuro-active metabolites was estimated using the Integrated Microbial Genomes (IMG) database (http://img.jgi.doe.gov (accessed on 17 January 2021)). The IMG annotates publicly available sequence data and the total number of Bifidobacteria genomes queried are shown in The ability of ® (Ver. 1.6.2), and quantitative analysis was performed using MultiQuant™ (Ver. 3.0.1).The liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was comprised of a Shimadzu Nexera X2 MP Ultrahigh-Performance Liquid Chromatography (UHPLC) system  coupled to a Sciex 6500 QTrap hybrid triple-quadrupole/linear ion trap MS system from Danaher . Operational control of the LC-MS/MS was performed with AnalystFecal and tissue homogenization and extraction procedures, and targeted LC-MS/MS-based metabolomics methods used for the quantitative analysis of the glutamate cycle and tyrosine pathway metabolites are described in the Calibration standards  for the glutamate cycle and tyrosine pathway methods were prepared at concentrations of 0.98, 3.9, 15.6, 62.5, and 1000 ng/mL for all metabolites measured in each method. In each instance, a consistent concentration of method-specific deuterated IS compounds were added to each calibrator and unknown sample—the assay-specific concentration levels for each deuterated IS compound is specified in the p < 0.05.GraphPad Software  was used to generate graphs and analyze data for statistical significance. Comparisons between the groups were made with one-way or two-way Analysis of Variance (ANOVA), using the Holm-Sidak post-hoc test comparisons, * Bifidobacteria are known to colonize the mammalian gastrointestinal tract, but the characterization of Bifidobacteria-specific neuro-active compounds remains incomplete. We first focused on the neurotransmitter GABA, which serves as the major inhibitory neurotransmitter of the ENS and CNS [B. dentium harbored the genes for the ECs associated with GABA production, we queried the B. dentium Bd1 genome in the KEGG database (https://www.genome.jp/kegg (accessed on 17 January 2021)) and found that this genome contained the complete enzymatic pathway to generate GABA from glutamate/glutamine and succinate  database (http://img.jgi.doe.gov (accessed on 17 January 2021)), contained all five genes for GABA production from glutamine/glutamate and succinate  needed to convert succinate to GABA, although these species could still produce GABA from glutamate. and CNS . Select  and CNS . In thisuccinate A. Four ouccinate B. B. denBifidobacteria members, we examined the genomes of 83 Bifidobacteria species   for converting succinate to GABA. Eighteen different Bifidobacteria species contained the gene for 4-aminobutyrate-2-oxoglutarate transaminase (EC 2.6.1.19), but not for succinate-semialdehyde dehydrogenase. Seventy-nine Bifidobacteria species (94.1%) possessed the genes to generate GABA from glutamate or glutamine , suggesting that the majority of Bifidobacteria species can only generate GABA from glutamate and not succinate , B. aemilianum (1 genome), B. catulorum (1 genome), and B. xylocopae (1 genome) did not have the ability to generate GABA from glutamate (EC 4.1.1.15) or succinate (EC 2.6.1.19), suggesting that GABA production may not be essential for these strains. In this genome comparison, we found that B. dentium (6 genomes) and 10 other Bifidobacteria species contained all genes required to convert glutamine, glutamate, and succinate into GABA. These findings indicate that B. dentium is well equipped to produce GABA.To see how common this pathway was among other genomes) . Thirty uccinate C. Four BB. dentium could generate GABA in vitro, we grew B. dentium ATCC 27678 in a fully defined bacterial media, termed ZMB1, for 18 h and examined GABA, glutamine, and glutamate levels by LC-MS/MS . However, we did observe a significant increase in colonic GABA. We also examined GABA, glutamate, and glutamine in the whole brain homogenate  Bifidobacteria species did not have tyrosine aminotransferase (EC 2.6.1.1), which converts 4-hydroxy-phenylpyruvate into tyrosine, indicating that tyrosine production is species dependent. We did not observe any genes related to L-dopa, dopamine, norepinephrine, or epinephrine production in our genome analysis. Next, we examined tyrosine concentrations in our fully defined ZMB1 media , these levels were unchanged in response to B. dentium colonization. These data suggest that B. dentium colonization in the gut does not influence neuronal GABA concentrations.Gnotobiotic models serve as a simple exploratory technique to determine how individual microbes influence the host. Using this approach, we previously found that in cells . HoweverB. dentium elevated tyrosine concentrations in the gut and brain. Tyrosine is a non-essential amino acid that can be transported from the intestine into the circulation, cross the blood–brain barrier, and enter neurons, where it is metabolized into catecholamine neurotransmitters. We found that B. dentium harbored the genes to generate tyrosine. We did not observe elevated levels of tyrosine in our ZMB1, which had incredibly high tyrosine levels at baseline. We speculate that the high levels of tyrosine in the ZMB1 limited the production of tyrosine by B. dentium in vitro. Future work optimizing the concentration of tyrosine for growth and secretion would provide an interesting baseline for confirming bacterial tyrosine production. Although we did not observe tyrosine production in vitro, we observed elevated concentrations of tyrosine in the feces of B. dentium-associated mice when compared to the germ-free controls. Since epithelial cells do not appear to have a mechanism to apically secrete tyrosine, we believe that the elevated tyrosine levels in B. dentium-associated mouse stool samples reflect B. dentium synthesis of this amino acid. Although select facultative anaerobes such as Bacillus, Escherichia, and Proteus have been found to produce dopamine and norepinephrine [B. dentium-conditioned ZMB1 or in our B. dentium mono-associated mouse feces. These findings are consistent with our KEGG pathway analysis and indicate that B. dentium is unable to synthesize these neurotransmitters.Another interesting finding was that nephrine , we did B. dentium mono-associated mouse brains, we surprisingly did not see any changes in dopamine, epinephrine, or norepinephrine. This observation could be due to the fact that we examined whole brain neurotransmitter levels. Another possibility is that dopamine concentrations could be elevated in specific brain compartments, but these distinct levels are averaged in a whole brain homogenate. It is also possible that the neuronal cells which generate dopamine require additional metabolite signals generated by other gut microbes to convert tyrosine to dopamine. Finally, since our mice were colonized as adults, it may be that early-life colonization is necessary for the proper wiring of these circuits. Additional studies are necessary to fully tease out this information.In vivo, tyrosine is an important regulator of the neuronal catecholamine neurotransmitters, including dopamine, epinephrine, and norepinephrine. Although we saw elevated tyrosine, we did not observe increased concentrations of these catecholamine neurotransmitters in the stool. We speculate that this is because dopamine-producing cells are not commonly found in the intestinal epithelium. Consistent with this notion, we have previously examined a panel of neurotransmitters in human jejunum organoids, also known as enteroids, transduced to increase enteroendocrine cells and we did not detect dopamine, epinephrine, or norepinephrine neurotransmitters . PreviouBifidobacteria–host modulation and should be explored in the future.In addition to neurotransmitter production, there are several methods by which the gut microbiota can influence the gut–brain axis, including vagal nerve stimulation, activation of neuropods/direct stimulation of afferent nerves, altering the activity of the stress-associated hypothalamic–pituitary–adrenal (HPA) axis, regulation of the immune cells, and modulating the permeability of the blood–brain barrier ,72,73,74In conclusion, this work has provided a unique glimpse into neurotransmitter profiles using germ-free and mono-associated gnotobiotic animals. Our data is among the first to demonstrate that a single microbe is sufficient to modulate select gut and neuronal neurotransmitters in vivo. Previous work with gut microbes has revealed that many microbial effects are strain-specific. As a result, it is important to delineate which microbes are associated with certain effects. We speculate that mono-associated animals and LC-MS/MS analyses may allow researchers to dissect the essential pathways by which commensal microbes influence the gut–brain axis. A better understanding of these pathways may provide novel adjuvant strategies for both gastroenterological and neurologic disorders.


Talipariti tiliaceum is an evergreen mangrove associate species distributed throughout the world. In this study, the complete chloroplast genome sequence of T. tiliaceum was assembled and characterized using high-throughput sequencing data. The chloroplast genome was found to be 161 766 bp in length, consisting of large single-copy (LSC) and small single-copy (SSC) regions of 89 273 and 19 551 bp, respectively, which were separated by a pair of 26 471 bp inverted repeat (IR) regions. The genome contained 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. A phylogenetic tree including 66 chloroplast genomes from various species revealed that T. tiliaceum was most related to T. hamabo of the same genus. Talipariti tiliaceum (syn. Hibiscus tiliaceus), commonly known as the coast cotton tree or yellow mallow tree , Yunxiao County, Fujian Province, China. Specimens were stored at the Herbarium of Taizhou University  under the voucher number TZU-20200818HT01. The total genomic DNA was extracted from the leaves using a modified CTAB protocol as described by Doyle and Doyle (T. hamabo genome (GenBank accession no. NC_030195) as a reference , containing two IR regions of 26,471 bp, separated by LSC and SSC regions of 89,273 and 19,551 bp, respectively. The overall GC content of the genome was 36.9% and it was predicted to contain 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Six protein-coding genes , seven tRNA genes , and four rRNA genes  were duplicated in the IR regions. Nine genes contained two exons and four genes contained three exons.The complete chloroplast genome of T. tiliaceum within the genus Talipariti and other related groups. All the chloroplast genome sequences were aligned using MAFFT v7.307 (Katoh and Standley Arabidopsis thaliana and Brassica napus were used as out-groups. The phylogenetic analysis showed that T. tiliaceum was most closely related to T. hamabo (T. tiliaceum may contribute to a better understanding of the evolution of Talipariti species within Malvaceae family.A total of 65 complete chloroplast genomes were downloaded from GenBank to investigate the phylogenetic relationships of . hamabo . The seq


Ornithobacterium rhinotracheale is one of the most important bacterial agents of respiratory diseases in poultry. For correct identification and characterization of this fastidious bacterium, reliable diagnostic tools are essential. Still, phenotypic tests are used to identify O. rhinotracheale and serotyping is the most common method for characterization, despite known drawbacks and disadvantages such as divergent results, cross-reactivity between strains, or the non-typeability of strains. The intention of the present study was to evaluate MALDI-TOF MS and whole genome sequencing for the identification and characterization of O. rhinotracheale. For this purpose, a selection of 59 well-defined reference strains and 47 field strains derived from outbreaks on Austrian turkey farms were investigated by MALDI-TOF MS. The field strains originated from different geographical areas in Austria with some of the isolates derived from multiple outbreaks on farms within a year, or recurrent outbreaks over several years. MALDI-TOF MS proved a suitable method for identification of O. rhinotracheale to genus or species level except for 3 strains representing serotypes M, K and F. Phylogenetic analysis showed that most strains grouped within one cluster even though they were comprised of different serotypes, while serotypes F, K, and M clearly formed a different cluster. All field isolates from turkey farms clustered together, independent of the origin of the isolates, e.g., geographical area, multiple outbreaks within a year or recurrent outbreaks over several years. Whole genome sequencing of serotype M, K and F strains confirmed the extraordinary status and deviation from known fully-sequenced strains due to a lack of sequence similarity. This was further confirmed by alignments of single genes (16S-RNA and rpoB) and multilocus sequence typing although the demarcation was less obvious. Altogether, the results indicate that these three serotypes belong to a different species than O. rhinotracheale, and might even be members of multiple new species. Ornithobacterium rhinotracheale is a gram-negative rod-shaped bacterium firstly described in 1994 [Ornithobacterium hominis, isolated from humans. O. rhinotracheale is an economically important bacterial pathogen of turkeys and chickens worldwide [O. rhinotracheale are also reported from many other bird species [ in 1994  proposedorldwide , and it  species ,11,12,13 species .The clinical picture is, besides respiratory symptoms, mainly associated with growth retardation, reduction of egg production, increased mortality and higher condemnation rates . Gross pO. rhinotracheale are primarily diagnosed by bacterial isolation followed by identification and/or detection of antibodies [O. rhinotracheale. Differentiation of strains based on phenotypic methods often resulted in heterogeneous outcomes or non-typeable strains [Infections with tibodies . Since i strains ,15. Sero strains ,17. Sero strains ,18. To o strains ,13,19,20 strains ,10.In the last decade, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has become popular in routine diagnostic laboratories due to its fast and reliable direct identification of microorganisms. Thus, progressively replacing all the biochemical  and phenotypic tests ,22 for sO. rhinotracheale strains. The study is based on a selection of well-defined reference strains to see if proteomics has the power to identify and distinguish O. rhinotracheale serotypes. Furthermore, selected field strains from O. rhinotracheale outbreaks in turkey flocks originating from (a) different geographical areas in Austria, (b) multiple outbreaks within a year on the same farm, (c) recurrent outbreaks over several years noticed on the same farm were included in the study to investigate the relationship of strains. Additionally, whole genome sequencing (WGS) was used to characterize serotype F, K and M strains in order to gain more insights into the genetic diversity of O. rhinotracheale.In the present investigation, we report for the first time the application of MALDI-TOF MS to analyze and differentiate numerous O. rhinotracheale reference strains were obtained from LMG . Information regarding the serotype was provided for 19 strains comprising 15 (A–O) different serotypes . Before investigation, the strains were thawed and cultivated on blood agar , at 37 °C for 24 h under microaerophilic conditions .m/z values) of the spectrum. For species identification, the MALDI Biotyper output is a log (score) in the range of 0 to 3.0, computed by comparison of the peak list for an unknown isolate with the reference Main Spectra (MSP) in the reference database. A MALDI score between 1.7 and 2.0 represents genus identification, while a MALDI score ≥2.0 represents identification at species level. Anything less than 1.7 was rated as non-identifiable by the software. For database construction or MSP creation, from each bacterial strain acid-soluble proteins were spotted on the MALDI target plate eight times and each spot was measured three times ending with 24 spectra for each strain. FlexAnalysis (v. 4) software  was used for visual inspection of the mass spectra and atypical spectra were excluded from further analysis . For creating a new database entry or an MSP, 20 to 24 mass spectra were processed with the software functionality and standard settings. The spectral peak lists for a particular strain were transferred into MSP containing information on average peak masses, average peak intensities and peak frequencies. Similar MSP result in a high matching score value. Each MSP is compared with all MSP of the analyzed set. The list of score values is used to calculate normalized distance values between the analyzed species, resulting in a matrix of matching scores. The visualization of the respective relationship between the MSP is displayed in a dendrogram using the following settings of the MALDI Biotyper 4.1 software: distance measure was set to correlation, linkage to average and score threshold value for a single organism at 700. Based on Sauer et al. [Sample preparation for MALDI-TOF MS was performed as previously described in detail . Bacterir et al. , clusterGenomic DNA of strains was isolated using QIAGEN DNeasy kits. Libraries were sequenced in a multiplexed 300 bp paired-end run on an Illumina MiSeq . For assembly, adapter sequences and low-quality nucleotides were first removed from the 3′ ends using BBduk  as a reference for the rRNA operons. Shortly, Riboseed uses Barrnap [As the library fragment sizes turned out to be too small for assembling the ribosomal RNA operons, we used riboSeed (v. 0.4.65)  with the Barrnap  to scan  Barrnap  in an itbacteroidetes_odb9 dataset. For comparability, we also screened the three already published O. rhinotracheale genomes  ,28 with  genomes .The final contigs were then submitted to GenBank under the BioProject PRJNA501809 with sample IDs SAMN10346233, SAMN10346254, and SAMN10346265, and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (v. 4.6) . The assThe genomes were scanned for antibiotic resistance genes using the online and the command-line version of the RGI tool (v. 5.0) for the CARD database (v. 3.2) ,31 Addi.O. rhinotracheale and Cand. O. hominis (O. sp. OH-22767 & OH-2280), first the average nucleotide identity (ANI) between pairs of sequences was calculated using the OrthoANI tool v. 0.93.1 [To examine the relatedness of the newly sequenced bacteria to characterized strains of . 0.93.1 .O. rhinotracheale strains and Cand. O. hominis using the genome of Riemerella anatipestifer  as an out-group. UBCG consists of a predefined set of 92 core genes, which have been found to occur in single copy in an extensive collection of fully sequenced bacterial genomes. The UBCG tool matches and extracts sequences homologous to these 92 core genes, aligns them individually and constructs both single gene and concatenated phylogenetic trees. For construction of the various trees RAxML v. 8.2.12 [tig (Trigger factor) was not identified in any genome, so in the end, only 91 core genes were used.The UBCG  tool v. 3.0  was used. 8.2.12  with a grpoB) from the fully sequenced O. rhinotracheale genomes were aligned to 16S rRNA sequences of various O. rhinotracheale strains using MAFFT (v. 7.429) [O. hominis (O. sp. OH-22767 & OH-2280) as an out-group.For a more defined grained look, the sequences of the 16S rRNA and the beta subunit of RNA polymerase (. 7.429)  with ite. 7.429) . The max. 7.429)  with a gO. rhinotracheale MLST regions were downloaded from pubmlst [To compare the newly sequenced genomes to already known multilocus sequence types (MLST) , we trie pubmlst ,39 and t pubmlst  and extr pubmlst  with a m pubmlst  with a gGraphics were created using R , pheatmaps . PhylogeAvibacterium and Gallibacterium [O. rhinotracheale strains investigated. Signal patterns obtained were compared with data in the Bruker Biotyper reference database version 4 consisting of more than 7000 microorganisms. This resulted in an identification of all strains at the species level, except for the reference strain representing serotype M (LMG 19032), which was identified only to the genus level (MALDI score 1.90), and reference strains of serotypes K and F , which were not identifiable   .O. rhinotracheale population is predominantly clonal [All 47 field isolates were identified to species level by MALDI-TOF MS . The orgy clonal ,14,20,44O. rhinotracheale strains and, while clustering into one branch, also quite different from each other.Both figures show that serotypes F, K and M are different from all other It has been shown that MALDI-TOF MS database used for identification should include field strains to improve identification results ,45. ThusO. rhinotracheale strains, DSM 15997 (GenBank: GCA_000265465.1), H06 030,791 (GenBank: GCA_000754515.1) and UMN 88 (GenBank: GCA_000756505.1) were used for comparison. While the resulting genomes were substantially more fragmented than the previously published O. rhinotracheale genomes, the gene content was similar, with all strains containing around 2300–2500 genes  sequencing on an Illumina MiSeq in 300bp paired-end mode was performed. Assembly of the genomes was done by using SPAdes and riboSeed ,25. The 00 genes . We also genomes ,28. As aO. rhinotracheale outbreaks [Antibiotic treatment is an important intervention strategy in the case of utbreaks . In addiutbreaks ,48,49,50utbreaks ,24 . The ANI of common sequences is closely correlated with the level of DNA-DNA hybridization (DDH), and a 95 to 96% ANI value is commonly used as a genomic measure of prokaryotic species delineation [O. hominis strains show high sequence similarity (ANI: 98.7%) to each other but, are distant from the other strains (ANI ~69) , they show only low similarity to the F, K and M serotype strains (ANI 89.3–90.2%), well below the usual species demarcation value of 95–96%. The F, K and M serotype strains are more similar to one another, but still slightly below 95% (ANI: 94.7–94.8%). Together, this indicates that these three newly sequenced strains belong to a different Ornithobacter species than O. rhinotracheale, they might even constitute multiple new species. In agreement with this assumption are reports from vaccination studies that showed cross-protection for the most frequently isolated serotypes but not for strains belonging to serotypes F, K and M. Until now it was concluded that these serotypes may be less virulent and play an inferior role in outbreaks as they are only occasionally isolated [We then calculated the overall average nucleotide identity (ANI) between all fully sequenced ineation ,51. As eANI ~69) . Unexpecisolated ,52.Ornithobacter strains and species, we used the UBCG  tool [O. rhinotracheale and Cand. O. hominis strains, as well as Riemerella anatipestifer  as an out-group, was constructed. UBCG constructs a tree from a concatenation of all genes, as well as single-gene trees, and gives the number of gene trees supporting each branch in the concatenated tree as a gene support index (gsi), as a measure of robustness. As one gene-tig-was not identified in any of the genomes, the maximal gsi for our study was 91. The concatenated UBCG tree shows a clear separation between the O. rhinotracheale and Cand. O. hominis strains (gsi: 91), as well as a distinct separation into two clades between the previously sequenced and the actual LMG strains (gsi: 87)  tool . With thgsi: 87) .O. rhinotracheale strains. For this, we compared the sequences of the 16S rRNA and the beta subunit of the RNA polymerase (rpoB) extracted from the genomic sequences with published sequences from various reference and field strains [O. hominis strains were used as an outgroup  data from PubMLST [As the 16S rRNAs show little divergence, and only a subset of known strains have  PubMLST ,39. Whil PubMLST , which oO. rhinotracheale. By including a high number of field strains in the in-house MALDI-TOF MS reference database the identification level could be substantially improved. MALDI-TOF MS confirmed previous findings regarding the regional clonality of O. rhinotracheale strains which seem to stay stable over years. Although the distinction of serotypes was not possible with MALDI-TOF MS we were able to show that strains belonging to serotypes F, M and K do not belong to the species O. rhinotracheale. This finding was confirmed by whole genome sequencing data indicating that these three serotypes are clearly different to the other O. rhinotracheale strains, and probably belong to different Ornithobacter species.In the present study, we demonstrate that MALDI-TOF MS can be used for the identification and characterization of


To compare the effect of burrhole craniostomy with and without a postoperative course of dexamethasone on recurrence rate of chronic subdural hematoma (CSDH).The study was conducted at the Department of Neurosurgery, Pakistan Institute of Medical Sciences, Islamabad, from September 2017 till May 2018. Adult patients diagnosed with CSDH and advised burrhole craniostomy were enrolled in this prospective randomized controlled trial. Participants were randomized into Group-1 (receiving two weeks dexamethasone), and Group-2 (no dexamethasone). Clinical assessment and Brain CT were done preoperatively, second postoperative day, sixth and twelfth postoperative week, with outcome assessed at twelfth postoperative week. Complications of treatment and recurrence rate were recorded.P=0.646) and radiological outcome  was similar in both groups. Complication rate was higher in Group-1 but not significantly different . Most frequent complication was pneumocephalus, with mortality rate equal (n=one). Recurrence was observed in 2.2% (n=1/46) patients in Group-1 and 4.3% (n=2/46) in Group-2 (P=0.557), which was not statistically significant.Ninety-two  patients were enrolled. Improvement in neurological (95.7% vs 93.5%; Neurological and radiological outcome, and mortality rates were similar in both groups. The recurrence rate was lower and complications slightly higher in Group-1 but these were not statistically significant. Chronic subdural hematoma (CSDH) is a self-perpetuating inflammatory process of dura matter, wherein fluid collects within the dural border cell layer of the meninges. Brain atrophy causes stretching of subarachnoid and associated bridging veins making them susceptible to minor trauma, resulting in blood or cerebrospinal fluid leaking into the dural border cell layer causing local dissection.CSDH usually presents in the elderly, many with pre-existing history of mild head injuryth September 2017. Study was conducted from September 2017 till May 2018. Total of ninety-two (n=92) patients were included .This study was conducted at Department of Neurosurgery, Pakistan Institute of Medical Sciences, Islamabad, Pakistan after approval from ethical and scientific board. Ethical Review Board approval reference number is F. 1-1/2015/ERB/SZABMU/, approved on 26Neurological status was assessed by GCS score, degree of hemiparesis, or presence of aphasia. It was considered improved if these parameters were better at last follow-up compared to at presentation, or deteriorated if worsening occurred at any time. Midline shift of brain was measured on Brain CT. Hematoma volume was calculated by measurements on Brain CT with formula [½x(a)x(b)x(c)], ‘a’ being maximum width, ‘b’ maximum length and ‘c’ number of consecutive 5 mm slices of Brain CT with hematoma. Radiological outcome was considered improved if decreased volume of hematoma was observed at last follow-up compared to the second postoperative day, and worsened if any increase occurred.P-value of ≤ 0.05 was considered significant.Both groups received postoperative antibiotics and analgesics for one week. Minimum postoperative hospital stay was three days, or longer depending on recovery. Follow-up visits with Brain CT at sixth and twelfth postoperative week were scheduled to ascertain neurological and radiological status. Outcomes were measured at twelfth postoperative week. Complications of treatment and recurrence were recorded. Data was analyzed using SPSS version 23. Paired sample t-test was used to compare changes in continuous variables from baseline at different time intervals. Chi-square test was used to compare categorical variables between both groups. P=0.646 and 0.646 respectively, P=0.535, P=0.557).Age and gender distribution were similar in both groups . Mean GCPatients that developed complications were administered treatment accordingly. Recurrence required repeat surgery. Superficial surgical site infections were treated with culture-sensitive antibiotics and pneumocephalus with supplemental oxygen. Decreased diabetic control was managed with intensified insulin regimens, and referred for medical consultation after discharge. Patients with subdural empyema were administered intravenous antibiotics and operated for empyema evacuation.Two deaths occurred during this study period, one in each group. One was a 65 years old lady with diabetes and hypertension presenting with hemiparesis and randomized into Group-2. Her hemiparesis improved postoperatively and was discharged in satisfactory condition on third postoperative day. She presented again three weeks later with fever, decreased conscious level, and worsening hemiparesis. Brain CT revealed subdural collection. She was re-operated via previous burrholes and confirmed to have subdural empyema. Subdural drain was placed and samples sent for culture-sensitivity, and appropriate antibiotics were initiated. No improvement occurred postoperatively. She developed a severe lower respiratory infection and died due to respiratory failure and sepsis in the sixth postoperative week. The second death was a 70 years old gentleman presenting with decreased conscious level and hemiparesis with no comorbidities and randomized into Group-1. He did not improve postoperatively, and second day Brain CT showed pneumocephalus which was treated with supplemental oxygen. Repeat Brain CT after one week revealed a subdural collection. He was operated on via small craniotomy, wherein diagnosis of subdural empyema was confirmed, and was evacuated. Subdural drain was placed and samples sent for culture-sensitivity, and appropriate antibiotics were administered. No improvement occurred postoperatively. He developed severe lower respiratory infection, and died in third postoperative week due to respiratory failure and sepsis.Several international studies on corticosteroids in CSDH have been published. Steroid treatment in CSDH has been proposed in patients with comorbidities.Various studies suggest corticosteroids help in the treatment of CSDH and lower recurrence rates.22In our study, patients receiving dexamethasone had lower recurrence rates and more adverse effects , both ofTo our knowledge, this is the first study conducted in Pakistan concerning adjunctive dexamethasone treatment in CSDH. Dexamethasone is an easily available inexpensive drug, and safe when used in the short-term.Our sample size was relatively small, yet sufficient to draw an inference. The follow-up period was short, being only twelve weeks, therefore could not assess long-term outcomes. This study was single-blinded and investigators aware of patient allocation. Different surgeons were involved in treatment, but all had similar clinical experience. A larger double-blinded study with longer follow-up duration might provide further information on recurrence rates and patient outcomes in both groups, and better able to highlight differences in statistically significant terms.We recommend a further prospective randomized controlled trial with a larger sample size and longer duration of follow-up to better determine the effectiveness of dexamethasone on recurrence rates and patient outcomes.Neurological and radiological outcome, and mortality rates were similar in both groups. The recurrence rate was lower and the complication rate higher in the dexamethasone group but both were not statistically significant.JT conceived, designed study, collected data, statistical analysis, manuscript writing, editing, responsible/accountable for accuracy/integrity of study.SNB supervision, critical review, editing and manuscript approval, responsible/accountable for accuracy/integrity of study.