Aedes larvae compared to chemical insecticides. Substantial reliance on chemical insecticides caused insecticide resistance in mosquito populations. Thus, research for alternate chemical compounds from natural products is necessary to control Aedes larvae. This study explores the analysis of chemical compositions from Areca catechu nut as a potential larvicide for Aedes (Diptera: Culicidae).There is a growing need to use green alternative larvicidal control for Areca catechu nut collected from Ipoh, Perak, Malaysia was grounded into powder and used for Soxhlet extraction. The chemical analysis of the extracts and their structures were identified using the GCMS-QP2010 Ultra (Shimadzu) system. National Institute of Standards and Technology (NIST) Chemistry WebBook, Standard Reference Database 69 (https://webbook.nist.gov/chemistry/) and PubChem (https://pubchem.ncbi.nlm.nih.gov/), the two databases used to retrieve the synonyms, molecular formula, molecular weight, and 2-dimensional (2D) structure of chemical compounds. Next, following WHO procedures for larval bioassays, the extracts were used to asses larvicidal activity against early 4th instar larvae of Aedes aegypti and Aedes albopictus.The th stage larvae with different concentrations in the range from 200 mg/L to 1600 mg/L. The LC50 and LC95 of Aedes aegypti were 621 mg/L and 2264 mg/L respectively; whereas the LC50 and LC95 of Aedes albopictus were 636 mg/L and 2268 mg/L respectively. Mortality was not observed in the non-target organism test. The analysis using gas chromatography and mass spectrometer recovered several chemical compounds such as Arecaidine, Dodecanoic acid, Methyl tetradecanoate, Tetradecanoic acid <n->, and n-Hexadecanoic acid bioactive components. These chemical constituents were used as additive formulations in pesticides, pest control, insect repellent, and insecticidal agents.The larvicidal activities were observed against early 4Areca catechu nut and it deserves further investigation in relation to chemical components and larvicidal actions between different species of Aedes mosquitoes. Even though all these findings are fundamental, it may have some interesting potentials to be developed as natural bio-larvicidal products.Our study showed significant outcomes from the extract of  Aedes mosquitoes and it has been an unresolved problem in the tropical regions. During the Covid-19 pandemic period, based on dengue updates by World Health Organization [Mosquitoes became major vector-borne diseases such as dengue, malaria, yellow fever, Zika, chikungunya, and lymphatic filariasis. Dengue is a major disease transmitted by Aedes aegypti and Aedes albopictus are the major vectors of the dengue virus(DENV) [The us(DENV) . Controlus(DENV) . Biologius(DENV) . Mosquitus(DENV) . There aus(DENV) , 5.Areca catechu is a potential plant to be used as a bioinsecticide due to its wide availability in tropical and subtropical regions. Areca catechu is also known as betel nut or areca palm. The nut of A. catechu can be applied as an additive material and also a stimulant [A. catechu nuts [Aedes, Anopheles, and Culex mosquito species [A. catechu caused significant mortality towards Aedes aegypti larvae [A. catechu nut extract on the larvicidal activity of Ae. aegypti are limited. There are also no related studies on the larvicidal effects of A. catechu nut extract on Ae. albopictus. Thus, further studies on A. catechu nut are essential as it may become a potential bio-insecticide. Following this concept, the objective of this study is to elucidate the analysis of chemical compounds from A. catechu nut extracts and their larvicidal action against the early 4th instar larvae of Aedes aegypti and Aedes albopictus.After several studies on mosquito resistance issues, researchers have decided to work with bioinsecticides as an alternative approach , 5. Bioitimulant . Polyphechu nuts . Previou species . It has i larvae . Until nAll the experimental protocols in this paper were based on World Health Organization (WHO) larval susceptibility test method guidelines . All expAreca catechu  at University Science Malaysia (USM) Penang, Malaysia [Aedes eggs were placed in dechlorinated water for 24 hours until they hatched. Emerged larvae were reared at separate plastic trays with not more than 200 larvae per tray [th instar stages of mosquito larvae were used for bioassay test [The eggs of Malaysia , 14, 15.per tray . The larper tray , 17, 18.say test , 18.Aedes larvae were separated into disposable plastic cups with 200 mL dechlorinated water for each concentration. Five replicates were used for each concentration with methanol (CH3OH) set as control [The larvicidal bioassays test were conducted following World Health Organization guidelines,. A total control , 19. Fur control . The met control , 17. Tot control , 18. Fol control , the morth instar larvae of Aedes aegypti and Aedes albopictus [An optical microscope (Leica USM) with 40-400x magnification was used to view the condition of early 4bopictus .Poecilia reticulata) were used in three replicates. Groups were tested with the larvicidal lethal concentrations of LC95 of 3000 mg/L, 2700 mg/ L, and 2500 mg/L for 24 hours with frequent observation on abnormalities or mortality of the fishes [Ten Guppy fishes  and PubChem (https://pubchem.ncbi.nlm.nih.gov/) were the two databases used to retrieve synonyms, molecular formula and molecular weight of the chemical compounds. PubChem (https://pubchem.ncbi.nlm.nih.gov/) and ChemSpider (http://www.chemspider.com/) were also used to retrieve some chemical and physical properties of chemical compounds such as colour, form, odour, boiling point, melting point, and solubility. Google Patent (https://patents.google.com/) was used to retrieve the patented lists on A. catechu nut for pesticide formulations.National Institute of Standards and Technology (NIST) Chemistry WebBook, Standard Reference Database 69 , chi-square, and degree of freedom (df) value were calculated and tabulated as in There was a significant increase in the mortality percentage with the increase in concentrations of ectively . The bioectively . The graA. catechu nut extracts inside the midgut regions.Poecilia reticulata, showed no mortality with A. catechu extracts ranging from 2500 mg/L to 2700 mg/L but with the concentration of 3000 mg/L, it was 1% mortality rate after 24 hours observations. The outcome from 2700mg/L clearly showed no mortalities and was safe for the guppy fishes which comply with the concentration limit range for larvicidal activity in this study. However, if the concentration value for A. catechu nut extracts exceeds the point of 2700ppm till 3000ppm, there could be 1% mortality. Additionally, no mortalities were observed at all controls. Fish liver histology analysis from all concentrations, including the control test in this study showed no signs of damages in terms of necrosis and vacuolization  on the midgut of Ae. aegpti and Ae. albopictus; as shown in Azolla pinnata and Clitoria ternatea showed larvicidal efficacies for Ae. aegpti and Ae. albopictus between LC50 and LC95 values of 1000ppm till 2500ppm but in this study, a lower LC50 and LC95 values of 620ppm till 2300ppm were reported [A. catechu nut may be the best candidate for natural bio-larvicidal applications in Malaysia. Besides that, according to a review by Silverio et. [Ae. aegpti mosquito larvae. The vast differences in LC50 and LC95 values are due to plant locality, plant resources, extraction techniques and stability of plant based chemical components. Furthermore, A. catechu nut extract did not cause toxic effects to non-targeted organisms such as guppy fish. Based on the test performed the highest concentration used against larvae which were 2700 mg/L, did not bring toxic effect to the non-targeted organisms but by increasing the concentration to 3000 mg/L it had a mild effect on the fishes and caused a mortality rate of 1% within 24 hours. Similarly, to recent studies on non-target organisms test conducted by Ravindran [A previous study with g et al.  showed tavindran  and Raviavindran , A. catereported , 18, 36.erio et. , there aA. catechu nut were mostly from fatty acids, fatty acid methyl esters and flavonoids. All these chemical compounds had been included in various patents for pesticide formulations and applications. Both, Anderson et al. [A. catechu nut extracts can be tested on various life stages of Aedes mosquitoes.Larger components of chemical compounds from n et al.  and Reidn et al.  had inven et al. , adjuvann et al.  and Reidn et al.  also belNigella sativa. These fatty acids were utilized in their invention to produce the synergistic pesticides constituents [Aedes aegypti mosquitoes. Based on the information on these patented inventions, all these fatty acids, fatty acid methyl esters, and flavanol are having the potential for the development of larvicidal products against Aedes mosquitoes using A. catechu nut extracts.Another interesting finding, fatty acids such as oleic acid, palmitic acid, and tetradecanoic acid were also found within the oil of black seed tituents . Besidestituents  inventedtituents  and Ali tituents , some ofA. catechu nut. Some previous studies reported that the toxic properties of oleic acid (unsaturated fatty acids) towards A. albopictus larvae were higher compared to palmitic acid and tetradecanoic acid (saturated fatty acids) [A. aegypti, A. albopictus and C. pipiens pallens mosquitoes showed favourable results for mortality. However, the unsaturated fatty acids (oleic acid) were more responsible for toxicity against mosquito larvae as compared to saturated fatty acids  [A. aegypti and C. quinquefasciatus [Some of the chemical components such as oleic acid, palmitic acid, and tetradecanoic acid were the fatty acids found within y acids) . The laric acid) . Tetradeasciatus .A. catechu nut extract against the fourth instar larvae of Ae. aegypti and Ae. albopictus mosquitoes with lower concentrations below 2268 mg/L. There are also no significant effects on A. catechu nut extracts for the non-target organism test. However, continuity of this research work is required to isolate the specific chemical compounds which may be responsible for its bio-insecticidal interactions. Additionally, the chemical components analysis from A. catechu nut extract had proven a baseline data for its future research on the field-based applications and their long-term effects on other aspects of human health.Current study had shown the effectiveness of S1 File(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


In the last few decades, hyperuricemia has drawn increasing attention owing to its global prevalence. Observational surveys have manifested that there is a relation between hyperuricemia and increased risks of hypertension, chronic kidney disease, cardiovascular events, metabolic disorders, end stage renal disease, and mortality. As alternatives, Traditional Chinese medicinal herbs have demonstrated concrete effects in mitigating hyperuricemia in different experiments. Researchers have made efforts to investigate the role of herbal medicine in attenuating hyperuricemia. This review focuses on traditional Chinese herbal medicines that have been reported to ameliorate hyperuricemia in experimental studies. Hyperuricemia, by definition, is an elevated concentration of serum urate which is over the solubility threshold (approximately 6.8 mg/dl) . There iUrate, the end-product, in human, of the purine metabolism in liver hepatocytes, excretes via the liver or go through the blood and excretes via kidney or the intestines. In general, exogenous factors for hyperuricemia can roughly be attributed to high intake of dietary purine, while endogenous causes are generally ascribed to enhanced uric acid production and a decreased uric acid excretion. Damaged excretion of uric acid is due to abnormality in kidney urate transporter, while increased uric acid production is because of an excess of active XOD .Urate-lowering therapy is currently the major medicinal therapy for hyperuricemia. Urate-lowering agents can be classified based on different mechanism of effects: xanthine oxidase inhibitors such as allopurinol and febuxostat via lowering production of urate; uricosuric drugs such as benzbromarone and probenecid, increasing kidney urate excretion through inhibiting its reabsorption; and recombinant uricase inhibitors which enable degrade of uric acid . CurrentTo our knowledge, there are some review articles concerning hyperuricemia-associated disease, such as a systematic review and meta-analysis of clinical studies about TCM treatment of hyperuricemia , a systeHerbs that show hepatic effects (mainly effects of XOD inhibitors) are as follows.XOD, mainly found in the liver, spleen, serum, lung, kidney and intestinal epithelium, and milk of mammals , is important in the production of uric acid. XOD catalyzes oxidation process of hypoxanthine to xanthine and finally to uric acid.Cinnamomum cassia/Cinnamomum cassia (L.) J. Presl, a major herb in many prescriptions, has long been utilized to treat hyperuricemia and gout. Experiments have shown [Cinnamomum cassia (L.) J. Presl possesses various biological activities in mitigating uric acid related disorders. Kong et al. [ve shown  that Cing et al.  concludeg et al. , it was g et al. .Cinnamomum osmophloeum Kaneh. leaf essential oil demonstrates plenty of biological activities, such as anti-inflammatory activities. A study in Taiwan [C. osmophloeum leaves' essential oil exhibited the most potent inhibitory effects on XOD compared to ethanolic and hot water extracts. No marked XOD inhibitory effects were observed in these two extracts. In addition, researchers found that cinnamaldehyde exhibited potent inhibitory effects against XOD compared to the other main compounds of C. osmophloeum leaves essential oil. Subsequently, cinnamaldehyde was further investigated and found to markedly decrease uric acid levels in potassium oxonate-induced mice. Impressively, the constituents of Cinnamomum osmophloeum Kaneh. leaf essential oil are similar to those of Cinnamomum cassia. Indeed, the inhibition of XOD can reduce the production of uric acid, so this ability to lower uric acid is, at least partly, achieved by XOD inhibition activity.n Taiwan  found thLonicera hypoglauca Miq. were reported to have the ability to suppress XOD, which was examined in a recent study [Lonicera hypoglauca Miq. leaves (LH-crude) and its derived EtOAc-soluble subfractions (LH-EA) exerted significant inhibitory effects on XOD activity. In addition, in potassium oxonate induced hyperuricemic mouse model, LH-EA decreased serum urate (sUA) levels. Finally, researchers isolated a novel bisflavonoid, loniceraflavone, which significantly inhibited XOD. Thus, loniceraflavone might be the main active component.The extracts of nt study . Both thCurcuma longa L., is a natural hydrophobic polyphenol. In the treatment of hyperuricemia, it has been utilized as an anti-inflammatory and antioxidant drug and seems to be efficacious [Curcumin, derived from the rhizomes of icacious . Recent icacious .Smilax china L. showed hypouricemic and renoprotective effects in hyperuricemic animals. In hyperuricemic mice, the ethyl acetate fraction (EAF) of Smilax china L. is stronger in attenuating urate levels than the other four fractions  [β1 (TGF-β1) and connective tissue growth factor (CTGF) and showed a renoprotective action through suppressing formation of monosodium urate and production of prostaglandin E2 (PGE2) and interleukin-1 (IL-1). The renoprotective effects may make Smilax china L. more applicable in clinics with the knowledge of many side effects caused by allopurinol or febuxostat; however, this needs to be further verified.raction) . The EAFraction) . This straction) . Furtherraction) . From thraction) . They shraction) . Of noteraction) , researcAurantii fructus immaturus, named Zhi Shi in Chinese, is derived from citrus plants and has long been widely utilized in China. Carbon dots are derived from charcoal used in TCM. Carbon dots derived from Aurantii fructus immaturus carbonisata, a nanomaterial, has been shown to decrease sUA levels via suppression on XOD activity in vitro and in hyperuricemic rats .Cordyceps militaris (L. ex Fr.) Link is a kind of Ascomycetes (a fungus). In potassium oxonate-induced hyperuricemic mice model, the ability of exopolysaccharides produced by Cordyceps militaris (L. ex Fr.) Link (EPCM) to ameliorate hyperuricemia was tested. In hyperuricemic mice, compared to allopurinol, EPCM (400 mg/kg) exerted equal effects on serum uric acid levels, BUN levels, and liver XOD activity. The antihyperuricemic effect of EPCM is due to a reduction in urate production and XOD inhibitory activities. The renal protection of EPCM, compared to allopurinol, was also demonstrated in this study [is study .Phellodendron chinense Schneid. (Rutaceae)/phellodendri cortex (15 g), and rhizome atractylodis/Atractylodes lancea (Thunb.) DC (Asteraceae)/(15 g), is widely utilized in China for hyperuricemia and gout treatment. Ermiao wan has shown antihyperuricemic action in normal mice and hyperuricemic mice as well in vivo [Ermiao wan, which is composed of  in vivo . This st in vivo . Ermiao  in vivo .Uric acid excretion is handled primarily by urate transporters of the kidney or intestine. For the kidney, the handling of uric acid can be generally separated into four steps: glomerular filtration, tubular reabsorption, tubular secretion, and reabsorption after tubular secretion. The whole process is regulated via urate transporters (reabsorptive and secretory) in the proximal tubule in the kidney, including reabsorptive urate exchanger (URAT1), glucose transporter (GLUT9), organic anion transporter (OAT1), and secretory exchange transporter . In the intestine, there is also a secretory exchange transporter (ABCG2), which can also assist the excretion of uric acid.Some herbs can exert renal actions .Cordyceps militaris (L. ex Fr.) Link. in traditional Chinese books is able to influence serum uric acid levels. As EPCM was discussed in “liver XOD inhibition” [Cordyceps militaris exhibits antihyperuricemic actions, and the possible mechanism is through the inhibition activity on URAT1 protein levels. The water extract of Cordyceps militaris exhibits excellent hypouricemic activity, and it has no adverse impact on liver, spleen, and renal functions [μmol/L, respectively (P < 0.01). The pivotal functional residues might be LYS145, ARG477, ARG325, and ASP168 according to molecular docking examinations. Four compounds were identified by virtual screening of C. militaris as a potential active ingredient [C. militaris may ameliorate hyperuricemia in multiple ways, which need to be further investigated.ibition” , researcunctions . In hypegredient . These iHerbaceous peony/Paeonia lactiflora Pall. flowers were utilized in diet therapy in many nations including China. Previous reports have suggested that herbaceous peony flowers have antioxidant activity. Total glucosides are derived from herbaceous peony root and are widely utilized clinically to treat gouty conditions. It was reported to show effectiveness in lowering uric acid levels [α, MCP-1, UA, Cr, BUN and, XOD and improved weight loss in rats with urate nephropathy [d levels . To testhropathy . Moreovehropathy . It dimihropathy . In concDioscorea nipponica Makino, is a commonly utilized traditional Chinese medicinal herb in treatment of arthrodynia, arthritis, and arthroncus. Total saponins of Rhizoma Dioscoreae Nipponicae reversed changes induced by potassium oxonate effectively in mouse renal OAT1, OAT3, URAT1, and GLUT9 mRNA and protein levels, leading to an increase in kidney urate excretion in potassium oxonate-induced mice [Rhizoma Dioscoreae Nipponicae, which is derived from ced mice . In concced mice .Fraxinus chinensis subsp. rhynchophylla (Hance) A.E.Murray, also known as Qinpi in TCM, has been utilized to treat hyperuricemia and has antioxidant, anti-inflammatory, and diuretic effects. Clinically, Qinpi from different regions exert significantly different therapeutic effects. Zhou et al. [Fraxini Cortex/u et al.  demonstru et al. . In ChinPhellodendron amurense Rupr. (120 g), atractylodis rhizome/Atractylodes lancea (Thunb.) DC (60 g), Achyranthes root/Achyranthes bidentata Blume. (60 g), and coix seed/Coix lacryma-jobi L. var mayuen (Roman.) Stapf (120 g), is a very frequently used prescription in TCM and can be utilized to treat hyperuricemia and gout. In hyperuricemic mice, Simiao pill markedly increased FEUA levels and decreased serum uric acid levels dose-dependently [The Simiao pill, which consists of phellodendri cortex/endently . Moreoveendently .Polyporus umbellatus (Pers.) Fries (180 g), Wolfiporia cocos (Schw.) Ryv. & Gibn (180 g), Alisma plantago-aquatica subsp. orientale (Sam.) Sam. (300 g), Cinnamomi Cortex/Neolitsea cassia (L.). Kosterm. (180 g), and Atractylodes macrocephala Koidz. (180 g), is a famous prescription in TCM. It promotes kidney function and diuresis. In a study [κB signaling and activation of mitogen-activated protein kinases. Furthermore, Wuling San, in fructose-fed hyperuricemic mice, suppressed the activation of NLRP3 inflammasome and thus reduced renal secretion of IL-1β. In high fructose-fed hyperuricemic mice model, Wuling San modulating kidney organic ion transporters and thus reducing serum uric acid levels might explain the inhibition of inflammatory reaction. The molecular mechanism might be related to the suppression of TLR4/MyD88 signaling and activation of NLRP3 inflammasome to decrease IL-1β excretion [Wuling San, which is composed of  a study  in 2013, a study  in 2015,xcretion .Viola yedoensis Makino/Viola philippica Cav., Lobelia chinensis Lour, Taraxacum mongolicum Hand.-Mazz., and Isatis indigotica Fort. In 2018, the aqueous extracts of EF which contain 24 g raw materials/kg  were investigated in a study [Viola yedoensis Makino was the only herb that exhibits hypouricemic effect, while all four herbs of the EF had analgesic and anti-inflammatory effects. The urate-lowering effect might be achieved by suppressing mRNA expression of URAT1 and enhancing that of OAT3. This study provides pharmacological views into the part of the formula and single herbs in uric acid excretion. Erding granule (EDG) is the granular form of EF. To compare the efficacy of each EDG extracts on concentration of serum uric acid in mice with hyperuricemia, a study [The Erding formula (EF) is recorded in the Chinese Pharmacopeia, which consists of  a study . This st a study  in 2019 Smilax glabra Roxb., 18 g of Rhizoma Dioscoreae Hypoglaucae/Dioscorea tokoro Makino ex Miyabe, 15 g of Pseudobulbus Cremastrae seu Pleiones/Cremastra appendiculata (D. Don) Makino, 10 g of Semen Vaccariae/Gypsophila vaccaria (L.) Sm, and 10 g of Radix Achyranthis Bidentatae/Achyranthes bidentata Blume. [Compound Tufuling granules consist of 35g of rhizoma smilacis glabrae/a Blume. . It is ua Blume. . It was a Blume. . This efa Blume.  that comSmilax glabra Roxb., rhizome  (35 g), Heterosmilax japonica Kunth, rhizome  (18 g), Cremastra appendiculata (D. Don) Makino, rhizome  (15 g), Achyranthes bidentata Blume., root  (10 g), and Vaccaria hispanica (Mill.) Rauschert, seed/Gypsophila vaccaria (L.) Sm  (10 g). In basic research, previous study [Xie-Zhuo-Chu-Bi-Fang, which is utilized clinically in a Guangzhou Hospital, is composed of us study  showed trhizoma)  g, HeterDioscorea septemloba Thunb. (30 g), Zea mays L./Maydis stigma (15 g), Coix lacryma-jobi var. ma-yuen (Rom. Caill.) Stapf/coix seed (30 g), Alisma plantago-aquatica Linn./Alismatis rhizome (15 g), Humulus scandens (Lour.) Merr./Humulus scandens (15 g), Taxillus sutchuenensis (Lecomte) Danser/Parasitic loranthus (15 g), Siegesbeckia orientalis L./Herba Siegesbeckiae (18 g), Curcuma longa L./turmeric (12 g), Corydalis yanhusuo (Y.H.Chou & Chun C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu/Corydalis Rhizoma (18 g), and Citrus medica Linn./Citrus medica (12 g). QZTBD is a formula prescribed to hyperuricemia, without serious adverse effects reported. Chen et al. [The Quzhuotongbi decoction (QZTBD) is composed of Smilax glabra Roxb./Glabrous Greenbrier Rhizome) (60 g), n et al.  mentionen et al. . They meDioscorea septemloba (Diocoreacea)/Dioscorea tokoro Makino ex Miyabe, is utilized for rheumatism, joint pain, and diuresis. The action of decreasing uric acid as well as increasing urate excretion was demonstrated in hyperuricemic mice. In vivo, the dioscin metabolite, tigogenin, was considered as an active constituent with antihyperuricemic effect. The hypouricemic effects are at least partially achieved by inhibiting URAT1 and promoting ABCG2 [Dioscin, the rhizoma of ng ABCG2 .Cichorium intybus L., recognized in common as chicory, comes from the Asteraceae family. A study found that there was a close relationship between the hypouricemic activities of chicory and the main effective compounds, aesculin, chicoric acid, chlorogenic acid, 14(15)-diene-3α-ol, 13,14-seco-stigma5(6), and isochlorogenic acid A/B/C, [d A/B/C, . In a prd A/B/C,  in 2017,d A/B/C,  in 2018,Smilax glabra Roxb. (SG), “Tufuling,” in TCM, is used to treat conditions akin to hyperuricemia and gout. To verify the hypouricemic effect of S. glabra and determine the active compounds, total flavonoids of S. glabra, involving neoastilbin, astilbin, neoisoastilbin, and isoastilbin, were obtained in a previous study [Smilax glabra Roxb. have potent hypouricemic effects via suppressing actions of XOD and upregulating the expression of OAT1, organic cation/carnitine transporter 2(OCTN2), and the mRNA (mOAT1 and mOCTN2) in kidney tissue. S. glabra Roxb. also exhibits antioxidative ability, which will be explicated in the “antioxidative” part of this study.us study . The stuGnaphalium pensylvanicum willd. is used in China to treat rheumatoid arthritis, cough, and inflammation. G. pensylvanicum extract reduced the uric acid effect by downregulating GLUT9 and URAT1 expression, upregulating OAT1 expression, and inhibiting XOD activity in hyperuricemic mice induced by potassium oxonate. Additionally, G. pensylvanicum extracts proved to be renoprotective. Caffeoylquinic acid derivatives, the main active components of G. pensylvanicum extracts, may partially explain the uricosuric or XOD inhibition effects [ effects .Smilax riparia/Smilax riparia A. DC is usually used in symptomatic treatment of gout and is reported to be effective. One study demonstrated that S. riparia could enhance uric acid excretion due to downregulation of renal mURAT1 by Smilax riparia total saponins [Smilax riparia A. DC roots, named smilaxchinoside A and smilaxchinoside C. They found that these two constituents exhibit great uricosuric effects. Meanwhile, the inhibition of XOD and the reduction in renal mURAT1 were also observed, which led to an enhancement in uric acid excretion as well as a decrease in kidney dysfunction induced by hyperuricemia [S. riparia exhibited synergistic effects with allopurinol. A later study proved that S. riparia can improve allopurinol's actions in vivo by reducing serum uric acid levels [saponins . The totsaponins . Subsequsaponins , the resuricemia . In a thuricemia , they fouricemia . In addid levels .Morus alba L. and was recorded to treat gout in TCM literature. It has been proven to possess a strong suppressing effect on renal reabsorption of urate in rats. Urate uptake was observed in the brush border membrane of the renal proximal tubule, where there is a urate-anion transporter (URAT1) that is mainly responsible for the uricosuric effect in rats. Therefore, URAT1 has a lot to do with the effects in this study, but the regulation of URAT1 needs to be further investigated. In addition, morin demonstrated inhibitory effects on XOD. The dual actions of morin, uricosuric effect, and XOD inhibitory activity could at least partly explain why morin could lower uric acid levels [Morin is derived from the twigs of d levels .Paederia scandens (Lour.) Merrill (Rubiaceae), which has long been used in TCM, consists of flavonoids, iridoids, triterpenes, sterols, and essential oils. P. scandens is a medicinal herb applied in conditions akin to gout in TCM. In an investigation [P. scandens was shown to be a potent inhibitor of XOD and has inhibitory effects on renal urate reabsorption. The dual effects of this ingredient account for its hypouricemic effect in vivo. Notably, the serum uric level was lowered not in normal mice, but only in mice with hyperuricemia. The antihyperuricemic effect of allopurinol was seen in both groups. Indeed, elevated serum uric acid levels beyond the threshold could possibly induce many pathological conditions, including gout. However, from a different perspective, uric acid is an antioxidant and is not harmful in any case. The uricase gene missing in humans can be for evolutionary benefits, such as a longer lifespan. This additional advantage of this compound may be more suitable in this regard as a hypouricemic agent [tigation , the extic agent .Camellia sinensis (L.) Kuntze (GTPs) are the major active fractions in green tea with various pharmacological effects. A previous study demonstrated that GTPs can decrease serum uric acid levels in mice with PO-induced hyperuricemia via two pathways. First, GTP markedly decreased the expression of XOD in the liver and its activity in the serum as well as liver, thus inhibiting UA production. Second, GTP substantially decreased expression of URAT1 and promoted expression of OAT1 and OAT3 and accordingly increased secretion of urate and suppressed reabsorption of urate [Green tea polyphenols/of urate .Alisma plantago-aquatica L. and rhizoma smilacis glabrae/Smilax glabra Roxb. (RSG) has been used for its urate-lowering effect. A study confirmed in hyperuricemic rats that Alismatis Rhizoma/Alisma plantago-aquatica L. and RSG alone or in combination are capable of lowering serum uric acid levels, with a more marked effect in the combination groups. This mechanism might have something to do with its suppression on XOD activity, improvement of kidney damage, and downregulation of URAT1 expression in the kidney [The combination of Alismatis Rhizoma/e kidney .Achyranthes bidentata Blume., consists of the achyranthes root, phellodendri cortex, and atractylodes rhizome. Sanmiao wan exhibits dual actions in lowering uric acid by suppression on hepatic XOD action to inhibit uric acid production and by downregulating kidney mURAT1 to promote excretion of urate and reduce reabsorption of urate in mice with hyperuricemia [Sanmiao wan, a common formulation in TCM, is widely utilized to treat hyperuricemia and gout. Sanmiao wan, actually Ermiao wan added to achyranthes root/uricemia .D. nipponica/Dioscorea nipponica Makino, 15 g of P. cuspidatum/Reynoutria japonica Houtt., 30 g of honeysuckle rattan/Lonicera japonica Thunb., 15 g of Radix cyathulae/Cyathula officinalis K.C. Kuan, 15 g of glabrous greenbrier/Smilax glabra Roxb., 15 g of Radix saposhnikoviae/Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., 15 g of Radix clematidis/Clematis chinensis Osbeck, 15 g of Rhizoma chuanxiong/Conioselinum anthriscoides “Chuanxiong,” 15 g of coix seed/Coix lacryma-jobi var. ma-yuen (Rom.Caill.) Stapf, 6 g of Glycyrrhizae radix et rhizome/Glycyrrhiza uralensis Fisch. ex DC., in addition, 2 g of sodium alginate was added. In a later study, for convenience and patient compliance, the researchers developed a granule formulation and slightly modified formulation by adding cassia twig/Neolitsea cassia (L.) Kosterm. (10 g). Borneol (0.3 g) was added to the original one [The Chuanhu anti-gout mixture was previously demonstrated to be noninferior to colchicine in treating acute gouty arthritis . It is cinal one . The oriinal one . In addiPlantago asiatica L. (60 g), white mustard seeds/Sinapis alba L. (30 g), vaccaria seeds/Gypsophila vaccaria (L.) Sm 30 g, and abutilon seeds/Malva verticillata L. (30 g), is a compound that our research group usually applied to the treatment of hyperuricemia and hyperuricemia-related diseases, such as urate nephropathy. The clinical efficacy and safety of lowering uric acid levels were confirmed in our previous clinical study [The ShiZhiFang (SZF), a TCM formula which consists of plantago seeds/al study . In our al study .Dendrobium officinalis/Dendrobium officinale Kimura & Migo (the specific dose was not seen in this article), is in accordance with the theory of TCM. In a recent study [Dendrobium officinalis six nostrum (DOS), the transformation of the TCM nostrum SiMiao Wan by adding nt study , DOS wasUrate-lowering therapy is the main, but not the only, drug therapy for hyperuricemia. Long-term hyperuricemia conditions intrinsically include pathological changes, such as oxidation and inflammation. Through activating complex cascades of proinflammation that impair cells and tissues, elevated uric acid levels beyond the threshold enhance the expression of inflammatory proteins . OxidatiSmilax glabra Roxb. (RSG), the rhizome of the Liliaceae plant, is called Tufuling in TCM. RSG water extract showed potent capacity to alleviate hyperuricemia and gout in murine model induced by monosodium urate and potassium oxonate [Rhizoma smilacis glabrae/ oxonate . Hong et oxonate  that uri oxonate , they foAlpinia officinarum Hance, a traditional Chinese medicinal plant. Galangin has been used as an antioxidant and anti-inflammatory agent. Lu et al. found that galangin can effectively improve nephric inflammation through inhibiting inflammatory reactions, which may have a lot to do with the suppression NLRP3 inflammasome, nuclear factor-kappa B, and phosphatidylinositol 3 kinase/AKT (protein kinase B) signaling pathway activation [Galangin belongs to the flavonol class of flavonoids and is obtained from tivation . Of notetivation .In summary, this paper surveys experimental research that TCM, in simples or formulas, could mitigate hyperuricemia, via playing a different role in the liver, kidney, and intestine. TCM, like most botanical medicines, contains a variety of active constituents, which act as anti-inflammatory, antioxidation, regulating uric acid transporter, and so on. Many of these herbs work through multiple modes of activity (MOAs) and multiple constituents, more than just a single active constituent hidden in a phytochemical matrix. Conventional western approaches utilizing anti-inflammatory agents with xanthine dehydrogenase and XOD inhibitors could be supplemented with natural drugs in therapeutic cocktails. These observations urge the wider application of TCM in treatment protocols for hyperuricemia. In fact, to able to be more scientific and persuasive, there are things to be done. Firstly, establishment for hyperuricemia model should be standardized. A proper hyperuricemia mouse model asks for a stable, consistent, and high serum urate concentration, while sometimes it is not easy to determine the hyperuricemia for mice. Secondly, qualification control of TCM might as well be uniformed and the adoption of TCM, such as origins and storage, means of administration, and so on. Thirdly, evaluation of safety of mouse research should be involved, say, acute toxicity tests and long-term toxicological tests of herbs, for example, the injuries of kidney. All in all, there is still a long way to go.


Clostridium perfringens is an important opportunistic pathogen that may result in toxin-mediated diseases involving food poisoning/tissue gangrene in humans and various enterotoxaemia in animal species. It is a main etiological agent for necrotic enteritis (NE), the most financially devastating bacterial disease in broiler chickens, especially if raised under antibiotic-free conditions. Importantly, NE is responsible for losses of six billion US dollars annually in the global poultry industry. To investigate the molecular mechanisms of C. perfringens-induced pathogenesis in the gut and its microbiome mRNA levels in C. perfringens-infected and non-infected hosts, we used RNA sequencing technology to perform transcriptional analysis of both host intestine and microbiome using our NE model. The growth rate was significantly impaired in chickens infected by C. perfringens. In total, 13,473 annotated chicken genes were differentially expressed between these two groups, with ninety-six genes showing statistical significance . Genes involved in energy production, MHC Class I antigen, translation, ribosomal structures, and amino acid, nucleotide and carbohydrate metabolism from infected gut tissues were significantly down-regulated. The upregulated genes were mainly engaged in innate and adaptive immunity, cellular processes, genetic information processing, and organismal systems. Additionally, the transcriptional levels of four crucial foodborne pathogens were significantly elevated in a synergic relationship with pathogenic C. perfringens infection. This study presents the profiling data that would likely be a relevant reference for NE pathogenesis and may provide new insights into the mechanism of host-pathogen interaction in C. perfringens-induced NE infection in broiler chickens. Clostridium perfringens is a Gram-positive, spore-forming anaerobe bacterium inducing opportunistic infection in humans and livestock worldwide [C. perfringens induced-necrotic enteritis (NE) results in histotoxic illnesses with various clinical symptoms ranging in severity from diarrhea to enteritis or enterotoxaemia [C. perfringens strains in the hosts are mainly attributable to their survival patterns including oxygen sensitivity, sporulation, germination, and rapid proliferation capacity [C. perfringens accounts for its virulence and pathogenesis in the host intestine or other organs [C. perfringens isolates are categorized into seven types (A-G) [C. perfringens is one of the primary causes of non-foodborne diarrheal and foodborne diseases, with annual infected cases of approximately one million. The most common vehicle for C. perfringens infections is poultry and undercooked meat [C. perfringens is responsible for 4% of hospitalizations, 10% of illnesses, and 5% of outbreaks for food poisoning diseases [orldwide . C. perftoxaemia . The succapacity . An arser organs . On the es (A-G) . In the ked meat ,7. Epidediseases .C. perfringens infections in domestic animals, including equine, swine, bovine, and poultry, has caused substantial global economic losses and has been regarded as a notable constraint on the livestock industry [C. perfringens-induced NE has been unequivocally recognized as one of the most leading intestinal diseases in turkeys and chickens, characterized by decreased growth rate and elevated feed conversion subclinically, and diagnosed with gaseous lesions and mucosa necrosis of the small intestine at post-mortem [C. perfringens proliferation and NE infection [C. perfringens multiplication that damages the intestinal epithelial layer [Regardless of the outbreaks in human hosts, the widespread occurrence of industry . In poult-mortem ,10. Thert-mortem . In mostnfection ,13. In aal layer ,15.C. perfringens isolates from the field poses a significant threat to public health, given their capability to generate a variety of lethal toxins [C. perfringens infection was associated with a cascade of effector mechanisms. The interactions of C. perfringens pathogen-associated molecular patterns with host Toll-like receptors may trigger the activation of immune cells like T helper (Th) cells and dendritic cells, thereby resulting in the secretion of cytokines like interleukin (IL)-10, IL-17, and interferon-γ [C. perfringens infection on the host small intestine at the molecular level and explore the impact of C. perfringens infection on the major foodborne microbiota members in the gut. With next-generation RNA-Seq sequencing technology, we identified a plethora of differentially expressed genes (DEGs) in the small intestinal mucosa in response to C. perfringens infection. Many fundamental molecular processes associated with NE pathogenesis were also deciphered. In addition, we revealed the specific changes of other foodborne pathogens from the gut microbiota profiling during C. perfringens infection. The profiling data presented here would be a relevant reference for the studies of C. perfringens-induced NE infection in broiler chickens and may enhance our understanding of host-pathogen interaction in NE pathogenesis.The emergence of antimicrobial resistance in l toxins . For susl toxins . A numbel toxins . Howeverl toxins ,18. Therl toxins . Prelimirferon-γ ,20,21,22rferon-γ . Althougrferon-γ . Hence, ad libitum. The Institutional Animal Care and Use Committee of Beltsville Agricultural Research Center approved the animal studies .Ross 708 broiler chicks at the age of one day were obtained from Longenecker’s Hatchery . They were housed in Petersime starter brooder units and later in finisher units, and were supplied with feed and water C. perfringens strain netB+/tpeL+ LLY_TpeL17 was used as the challenge inoculum to infect the broiler chickens as described elsewhere [netB+/tpeL+ LLY_TpeL17 strains were propagated in CMG medium  and then in BYC medium  at 37 °C for 20 h. Twenty-four healthy broiler chicks (One-day-old) were randomly assigned to the uninfected sham control (NV) and C. perfringens infection (CP) groups, with twelve birds per group. An antibiotic-free starter diet with 20% protein  was provided to the birds from days 1 to 14 post-hatch. From the 15th-day, a high-protein diet consisting of 10% protein diet and 50% fish meal was used to feed the birds. On day 20, the chickens were orally infected with 2.0 mL of C. perfringens culture (2.0~4.0 × 109 colony-forming units per chicken), twice daily for five consecutive days, while uninfected sham control birds were orally given an equivalent volume of BYC culture medium. All the birds were weighed and sacrificed on day 5 post-first infection, and the sections (around 2.5 cm) of mid jejunum  were collected and instantly soaked in RNALater solutions . The homogenized lysates of jejunum tissues with a Polytron homogenizer were incubated with 100 µL of Phenol:Chloroform:Isoamyl Alcohol  for 15 min on ice after vigorous vortexing. A RNeasy PowerMicrobiome Kit  was then used to extract RNA according to the manufacturer’s instructions. Freshly isolated RNAs from three randomly selected pooled samples in each group were sent to Genomic Resource Center (GRC), University of Maryland  for RNA sequencing. A 6-point scoring system was used to define gross pathological lesions in the intestines [The lsewhere . Brieflytestines .® Ultra™ II RNA Library Prep Kit for Illumina®  was used to generate strand-specific libraries. The AMPure SPRI-select beads  were applied for the size selection of the library. Glycosylase digestion of the adapter and 2nd strand synthesis was carried out. Multiplexed libraries were sequenced on an Illumina NovaSeq 6000 S1 flowcell using a 101bp paired-end run. Data analysis was performed by Novogene Inc . The software GOseq, topGO, and hmmscan release 2.12 were used to process whole-transcriptome sequencing data and implement differential gene expression (DGE) analysis, Gene Ontology (GO), and GO enrichment analysis as similarly described elsewhere [All RNA-sequencing services, including RNA quality determination, ribosomal RNA (rRNA) removal, library construction, and sequencing in HiSeq 2500 machine, were performed by GRC in a standard protocol . Brieflylsewhere . GO annoThe growth rate was calculated in the relative body weight gain percentage (RBWG%). GraphPad Premier 8.0 software  was used to perform the statistical analysis of the RBWG%, lesion scores, and transcriptional read counts of 5 major foodborne pathogens to total gut microbiota reads in small intestines by unpaired Student’s T-test. The identification of DEGs between the two different conditions was carried out using DEGseq software (v.1.10.1). The statistical significance for DEGs was set at the 0.05 significance level.C. perfringens-induced NE results in the reduction in feed intake and lower growth rate of infected broiler chickens, the pattern of reduced body weight gain of inoculated chickens was expected in this study. When chickens were infected with pathogenic C. perfringens, a significantly lower growth rate in RBWG% was observed compared to the uninfected sham controls   (Given that  < 0.05) A. In add 0.0001) B.p-value estimation by negative binomial distribution model, false discovery rate (FDR) value estimation based on multiple hypothesis testing, and evaluation of gene expression levels with fragments per kilobase of transcript per million base pairs sequenced. The top 500 most variable genes across samples were graphically illustrated in the heat map , among which 48 upregulated genes and 48 down-regulated genes were shown between these two groups  at day 5 post C. perfringens infection in chicken jejunum  increase in the jejunum epithelial layer are summarized in chickens , indicatp-value FDR ≤ 0.05) at 5th day post C. perfringens inoculation in bird jejunum layer , Escherichia coli O157 (p = 0.008), C. perfringens (p = 0.02), and Listeria monocytogenes (p = 0.041), as shown by the relative ratios of pathogenes’ transcriptional read counts to total read counts (Staphylococcus aureus between these two groups (p = 0.14)  . It is iC. perfringens during infection could initiate a series of events leading to the transcriptomic alteration at the pathogen-host interface. Parreira et al. [netB-positive CP1 strain on ligated intestinal loops by washing away the commensal microbiota. They found that C. perfringens gene expression profiles in vivo resembled those of C. perfringens grown in nutritionally deprived conditions, indicating that pathogenic C. perfringens responds well under distinct environmental conditions [C. perfringens infection significantly impacted the gene expressions of the transcriptome in the small intestinal mucosa. Forty-eight genes in the host were upregulated with significant differences following C. perfringens infection, including top enriched CCTα, Chromobox 3, and Filamin B. Inflammation and lipid signaling are intermingled modulators of homeostasis and immunity. Therefore, inflammatory signaling is able to significantly affect lipid metabolism in the multiple tissues in infection [C. perfringens-infected chicken samples. As suggested, upregulation of CCTα could enhance the production of memory B cells and plasma cells in the development of humoral immunity in reaction to C. perfringens invasion, since B cell-specific deletion of CCTα resulted in lowered IgG production and increased IgM secretion to initiate humoral immunes [Understanding the host-bacteria interaction during disease pathogenesis is vital for the development of early intervention measures. The interaction between the chicken intestine and a et al.  performenditions . The resnfection . It is anfection . CCT is nfection . CCTα, anfection , is reponfection . In this immunes .C. perfringens infection could be related to the increased activities of a cascade of cellular processes, including DNA repairing. The detailed mechanism may warrant further investigation. Filamin B (FLNB) is a cytoplasmic protein that controls the cytoskeletal network [C. perfringens infection, FLNB and SPEG were significantly upregulated, suggesting the association with increased cytoskeletal network activities and myocyte repairing/development.CBX3 belongs to the heterochromatin protein 1 (HP1) family encoded by the chromobox genes. The HP1 family members are evolutionarily conserved and may function in many cellular events, including telomere function, gene regulation, and DNA repair. CBX3 protein has been shown to promote optimal co-transcriptional RNA processing through the initiation of the splicing machinery . Upregul network , and mut network ,36. Stri network . In our C. perfringens infection. The internalized epsilon-toxin of C. perfringens was once reported to be moved from early endosomes to late endosomes and lysosomes maximized at 60 min in MDCK cells with confocal microscopy [C. perfringens infection suggests that the host may increase processing activities to maintain the integrity of cell adhesion and reduce the negative impact caused by C. perfringens challenge. In addition, the upregulated DAMM2 gene may be related to the Wnt signaling pathway and planar cell polarity pathway.In this study, a late endosome and lysosome associated marker was also upregulated, possibly related to the late stage of some antigens/toxin internalization in vacuoles at Day 5 post croscopy . WD-40 rcroscopy . The up-C. perfringens infection, some genes in the host intestinal mucosa were differentially down-regulated, for example, Coronin-1C, Receptor Protein-Tyrosine Phosphatases (RPTPs), and Prosaposin. Coronin-1C (Coronin-3) is a homotrimeric F-actin binding protein. Coronin-1C protein is implicated to participate in various cellular events, such as cell migration, gene regulation, apoptosis, signal transduction, cell cycle progression, tumor cell metastasis, and the regulation of leukocyte-specific signaling events in innate immunity [Following immunity ,41,42. Rimmunity ,44. Prosimmunity . Meanwhiimmunity , affectiimmunity , regulatimmunity , programE coli O157, L. monocytogenes, and C. jejuni, were also found to be increased following C. perfringens infection in the small intestine tissues. In a normal healthy situation, microorganisms in gut microbiota have co-developed with the host in a symbiotic relationship to maintain colonization resistance by metabolic benefits, promotion of immune homeostasis, and immune protection against pathogen colonization [C. perfringens. Introduction of pathogenic C. perfringens may result in a lowered overall microbial diversity with a reduction of beneficial symbionts and an increase of pathobionts [C. perfringens and C. jejuni in cecal contents in the chickens [C. perfringens might likely be required for C. jejuni ‘s Ni/Fe hydrogenase for growth, enhancing C. jejuni colonization [C. perfringens and other foodborne pathogens in the host mucosal immune system, so as to timely control the occurrence of pathogenic microorganism overgrowth.In this study, the transcriptomic read counts for several critical foodborne pathogens, such as nization . Therefohobionts . Besideshobionts . For exachickens . It was nization ,50. It mC. perfringens mainly infected the small intestine, our current study was focused on the field of the mid-section of the jejunum. Nevertheless, it demonstrated a comprehensive regulation of the complex responses to C. perfringens infection through our methodology. It would be interesting to further investigate the detailed host-pathogen interaction based on the findings from our current data.Since C. perfringens pathogen in the chicken NE model. Through comparative analysis of DEGs between uninfected and C. perfringens-infected jejunum samples, we identified many gene transcriptional alterations of the host in inflammation, adaptive immunity, muscle skeletal development, cellular process, metabolism, and energy production. In addition, it appeared that the transcriptional levels for three important foodborne pathogens were also significantly elevated in a synergic relationship with C. perfringens infection. In general, the results may enhance our understanding of the molecular mechanisms of host-C. perfringens interactions in chicken NE.A comprehensive overview of the transcriptomic profiling was obtained to study the interaction between host and


Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified. Burkholderia gladioli genome sequences determined as part of this study are deposited under BioProjects PRJEB35318 and PRJEB9765. Complete genomes are available for 4 strains  and 206 draft genomes were determined with the accession numbers provided in Table S1 (available in the online version of this article).Burkholderia gladioli is a fascinating bacterium with detrimental traits of plant pathogenicity, food-poisoning toxicity and the ability to cause lung infections in people with cystic fibrosis (CF). In contrast, because B. gladioli can produce multiple antimicrobial specialized metabolites, it can protect insects and plants, and has been recently exploited as an antibiotic producer. To understand the genomic and metabolic basis of this diversity, we genome-sequenced a collection of 206 B. gladioli isolates, and examined the specialized metabolites they encode and produce. While all agriculturally defined B. gladioli disease pathovars (pv.) were found to represent a single species, B. gladioli pv. allicola and B. gladioli pv. cocovenenans were evolutionarily distinct. In addition, the latter food-poisoning strains were unified as a group because they all encoded the potent toxin bongkrekic acid. Worryingly, 13 % of B. gladioli recovered from CF lung infection also encoded the toxin and could produce bongkrekic acid when grown in conditions mimicking CF sputum. Although identification of toxin-positive strains represents a potential new risk factor for CF, we showed that the clinically used antibiotic trimethoprim was able to suppress toxin bongkrekic acid production. Our genomic and metabolic analysis of B. gladioli has broad impacts for agriculture, biotechnology, chemistry and medicine.Burkholderia contains important plant, animal and human pathogenic bacteria  with a mean size for B. gladioli of 8.28 Mb. The mean GC content was 68 % and the genomes contained a mean of 6872 protein-encoding genes  [P. marginata (ATCC10248) [B. gladioli is supported by the genomic taxonomy (Table S1) [Since the assignment of isolates within the genus olderia  generallifically , has und species . This coG 18113)  and P. mable S1) .B. gladioli (n=27) comprised three closely related sub-groups: 1A, containing the reference B. gladioli pv. cocovenenans strains, 1B and 1C; each subgroup was distinct in terms of their ANI relatedness  contained both the B. gladioli pv. agaricicola and B. gladioli pv. gladioli reference isolates . The significance of core gene differences between the lineage remains to be determined, but a defining conserved feature of the group 1 genomes was the presence of the bongkrekic acid BGC.To investigate the evolutionary linkages behind the ANI groupings , we consd by ANI  were alsd by ANI . The thrclade 1A . These g strains , which c strains . All thrB. gladioli pv. allicola, and grouping of pv. gladioli and pv. agaricicola strains in a separate clade , this disease source was also the major origin for each of the clades, demonstrating that opportunistic human pathogenicity was also a shared species phenotype . The group 1 B. gladioli strains , but was absent from the other strains examined , gladiolin (83 strains), or no specialized metabolite (80 strains) BGCs. Upstream and downstream of this polyketide BGC insertion point were blocks of conserved and syntenic genomic DNA. These surrounding regions of the B. gladioli second genomic replicon did not contain mobile DNA markers, indicating the BGC insertion point as a gene capture hotspot.The enacyloxin BGC was randomly distributed across ladioli . Its preladioli . Genomicladioli . This coB. gladioli-related food poisoning [in vitro, but to varying extents . Four of these B. gladioli strains  were subsequently grown in artificial CF sputum medium [B. gladioli isolates containing the bongkrekic acid BGC, as a potential clinical risk marker for CF. Application of this PCR  to 122 B. gladioli CF isolates prior to their genome sequencing identified 13 positives. Subsequent genome sequencing demonstrated that all contained a complete bongkrekic acid BGC , we are now in a strong position to understand whether the toxin plays a role in poor clinical outcome for infected CF patients. Also, since we have shown that trimethoprim acts to suppress toxin production . The ecological significance of herbivorous Lagriinae and other beetles in distributing the such bacterial symbionts across continental ranges will be fascinating to understand.By combining genomics with analytical chemistry, we have also been able to map the repertoire of bioactive specialized metabolite BGCs across the abolites . The wid species . The conl attack . The beeolin BGC  and has olin BGC , also knolin BGC . Since tsymbiont  must be B. gladioli by indirectly sampling the opportunistic infections the bacterium causes in people with CF. Although our B. gladioli collection was mainly derived from people with CF living in the USA , the 206 genomes examined were genetically diverse and comprised 133 unique MLST sequence types (Table S1). In the absence of patient-to-patient or common source transmission, the natural environment is the main source of Burkholderia lung infections in CF patients [B. gladioli clades appear widely distributed across the North American continental range , with the majority being represented by genetically unique strains (Table S1). Soil, the rhizosphere and terrestrial freshwater environments are common sources of Burkholderia [B. gladioli. Recent findings of close associations with insects [B. gladioli in the natural environment. By defining a systematic framework of population biology and metabolite production, a basis from which to understand the diverse ecology of B. gladioli is now in place.We were able to gain an insight into the ecological distribution of olderia  or food- insects  point toClick here for additional data file.


Staphylococcus aureus and Streptococci. Spontaneous gram-negative SEA and spondylodiscitis especially Klebsiella pneumoniae are very rare. We report a 71-year-old Thai male with diabetes, presenting fever, enlarged neck mass, and progressive painful swallowing a week before admission. MRI of the whole spine demonstrated epidural abscess along the anterior thecal sac from C2 to C7 levels with spinal meningitis; multiple rim-enhancing lesions at the left sternocleidomastoid/levator scapulae, splenius capitis, semispinalis capitis, and bilateral scalene muscles; and rhombencephalitis with brain abscess. Klebsiella pneumoniae was isolated from blood culture. CT of the whole abdomen showed unremarkable intra-abdominal lesion. Intravenous ceftriaxone was administered, but the patient was unable to undergo surgical drainage due to unstable condition and died after two weeks of admission. Spontaneous SEA and spondylodiscitis caused by K. pneumoniae are very rare but sometimes fatal. In the case of SEA and spondylodiscitis, even when K. pneumoniae is uncommon, it should be also considered as a pathogen, especially when the patient had important risk factors.Spinal epidural abscess (SEA) is a rare but sometimes life-threatening condition. The principal organisms in SEA and spondylodiscitis are gram-positive bacteria, e.g.,  Staphylococcus aureus and Streptococci, whereas identifiable gram-negative bacteria were far rarer in SEA [Klebsiella pneumoniae are the family members of Enterobacterales, which are capable of causing various types of community-acquired and healthcare-associated infection (HAI), e.g., pneumonia, internal organ abscess, and bacteremia [K. pneumoniae SEA [K. pneumoniae spondylodiscitis and SEA involving C1-C7 complicated with rhombencephalitis in a patient with diabetes.Spinal epidural abscess (SEA) is a rare but severe condition and sometimes life-threatening. The incidence of SEA was 5.1 cases per 10,000 admissions, and bacteremia was the most common finding . SEA devr in SEA . Klebsieniae SEA -14. HereA 71-year-old Thai male presented to the emergency department with three days of progressively painful swallowing. Two weeks before admission, he reported soreness at both trapezii, shoulders, and scapular region. One week before admission, he developed a high-grade fever and then visited a private hospital, where he received cefdinir and clindamycin without any improvement. Three days before admission, he noticed an enlarged neck mass and experienced progressively painful swallowing.His history was significant for stage III hypopharyngeal cancer with concurrent chemoradiation therapy and was in complete remission for two years. He presented coronary artery disease, well-controlled diabetes mellitus, and lumbosacral stenosis. He denied using herbal medication, smoking, and consuming alcohol, including illicit drug abuse.On physical examination, his blood pressure was 130/58 mmHg, pulse rate 96/minute, respiratory rate 26/minute, and temperature 34.6°C. He presented a swollen left anterior neck erythema extending to the angle of the mandible and marked tenderness. Neurological examination was within the normal limit.Klebsiella pneumoniae. On the second day of admission, he developed progressive choking and hypoxemic cardiac arrest with successful resuscitation. He was in a stuporous condition, and his left-sided motor level was grade I. The initial blood test revealed a white blood cell count of 21,000/µL, comprising 96% neutrophils, a hemoglobin level of 9.1 g/dL, and a platelet level of 208,000/µL. AST was 61 IU/L, ALT was 64 IU/L, and ALP was 551 IU/L. MRI of the whole spine demonstrated epidural abscess along the anterior thecal sac from C2 to C7 levels with spinal meningitis; multiple rim-enhancing lesions at the left sternocleidomastoid/levator scapulae, splenius capitis, semispinalis capitis, and bilateral scalene muscles , ceftriaxone was started due to positive blood culture from the private hospital report for  Figures . CT of tIn the first week of ICU admission, the patient still presented progressive quadriparesis . The patient was unable to undergo surgical drainage of SEA due to unstable condition and a very high risk of surgery. The abscess at the left supraclavicular region was drained, and culture was sent for investigation. The hemoculture sent on the admission date was negative, and no organism was cultivated from the left scalenus muscle.After two weeks of conservative therapy with ceftriaxone, he remained in a progressively debilitating condition. His motor power in all extremities comprised grade I, and his level of consciousness was comatose. Finally, he died after two weeks of admission.S. aureus and Streptococci. The concurrent adjacent spondylodiscitis commonly occurs owing to the contiguous spreading of microorganisms from the adjacent bone and disc, the most common route of infection [K. pneumoniae, which often presents as a secondary infection from another organ and postneurosurgical conditions, remain very rare [Spinal epidural abscess is a rare but life-threatening condition. The most common causative pathogens are nfection ,10. Alconfection . Spontanery rare .K. pneumoniae SEA and four cases described as spontaneous SEA . hvKP infection often exhibited metastatic spread resulting in multifocal organ involvement and increased ability to involve the central nervous system and ocular regions [K. pneumoniae. Diabetes mellitus is recognized as a significant risk factor in developing both SEA and hvKP infection [K. pneumoniae SEA is mostly reported in Asia and possibly correlated with the hvKP strain (Table The community-acquired  regions . hvKP innfection . hvKP inin Table . UnfortuK. pneumoniae SEA from related reports encountered favorable outcomes after combined surgical and medical therapy [Among gram-negative SEA and spondylodiscitis, promptly combined therapy of both surgical drainage and antimicrobial therapy remains crucial . Eight o therapy -10,13,14 therapy ,11. NeveK. pneumoniae are very rare but sometimes fatal. Early diagnosis and prompt combined medical and surgical therapy constitute a considerable strategy. However, in the case of SEA and spondylodiscitis, uncommon organisms such as K. pneumoniae should also be considered, especially when important risk factors are established.In conclusion, spontaneous SEA and spondylodiscitis caused by


Lactobacillus plantarum lipoteichoic acid (Lp.LTA) on a representative dental cariogenic pathogen, Streptococcus mutans. Since LTA structure varies in a bacterial strain-specific manner, LTAs from various L. plantarum strains may have differential anti-biofilm activity due to their distinct molecular structures. In the present study, we isolated Lp.LTAs from four different strains of L. plantarum  and compared their anti-biofilm effects on the dental pathogens, including S. mutans, Enterococcus faecalis, and Streptococcus gordonii. All Lp.LTAs similarly inhibited E. faecalis biofilm formation in a dose-dependent manner. However, their effects on S. gordonii and S. mutans biofilm formation were different: LRCC 5310 Lp.LTA most effectively suppressed the biofilm formation of all strains of dental pathogens, while Lp.LTAs from LRCC 5193 and 5194 hardly inhibited or even enhanced the biofilm formation. Furthermore, LRCC 5310 Lp.LTA dramatically reduced the biofilm formation of the dental pathogens on the human dentin slice infection model. Collectively, these results suggest that Lp.LTAs have strain-specific regulatory effects on biofilm formation of dental pathogens and LRCC 5310 Lp.LTA can be used as an effective anti-biofilm agent for the prevention of dental infectious diseases.Bacterial biofilm residing in the oral cavity is closely related to the initiation and persistence of various dental diseases. Previously, we reported the anti-biofilm activity of  Bacterial biofilms are defined as the aggregation of microorganisms attached to a surface of various biotic and abiotic materials . BiofilmStreptococcus mutans, Enterococcus faecalis, and Streptococcus gordonii are considered as major Gram-positive, facultative anaerobic dental pathogens predominantly found in the oral cavity of patients with apical periodontitis or dental caries  is known as a major cell wall component found in Gram-positive bacteria involved in bacterial growth, adhesion, biofilm formation, and stimulation of host immunity (L. plantarum possesses poly-glycerolphosphate (Gro-P) backbone, which is composed of phosphate and glycerol containing D-alanine, glucose, or galactose residues, linked with triacylated glycolipid moiety, while LTA from Staphylococcus aureus has Gro-P backbone with N-acetylglucosamine or D-alanine residues linked with diacylated glycolipid anchor  showed the most potent anti-biofilm activity against dental pathogens, such as E. faecalis and S. mutans, suggesting that LTA structure may determine its anti-biofilm capacity  for 48h at 37°C in a capped bottle without agitation. A total of 12 strains of dental pathogenic bacteria, including four strains each from L. plantarum  were purified as previously described (L. plantarum harvested by centrifugation was resuspended in 0.1M sodium citrate buffer (pH 4.7) and disrupted by ultrasonication . The bacterial lysates were mixed with an equal volume of n-butanol, and the lower phase was collected by centrifugation. The collected lower phase was then dialyzed in a semi-permeable dialysis membrane  against endotoxin-free distilled water . The dialyzed extracts were equilibrated with 15% n-propanol in 0.1M sodium acetate buffer and subjected to hydrophobic interaction chromatography using a column filled with octyl-Sepharose . After washing out unbound materials with 15% n-propanol in 0.1M sodium acetate buffer, the bound materials were eluted with 35% n-propanol in 0.1M sodium acetate buffer in a series of 7ml aliquots using a fraction collector . According to the result of inorganic phosphate assay, the column fractions containing phosphate, that were considered as LTA-containing elutes, were pooled and dialyzed again under the aforementioned condition. The dialyzed fractions were equilibrated with 30% n-propanol in 0.1M sodium acetate buffer and subjected to ion-exchange chromatography using a column filled with DEAE-Sepharose  equilibrated in 30% n-propanol in 0.1M sodium acetate buffer. A series of 7ml fractions were further eluted with a linear salt gradient (0 to 1M NaCl in the equilibration buffer). Based on the results from inorganic phosphate assay, the column fractions containing phosphate were pooled, dialyzed, and lyophilized. The quantity of LTA was then determined by measuring the dry weight of purified LTA. Possible impurities, such as endotoxins, nucleic acids, and proteins, were examined in the Lp.LTA preparations using Limulus amoebocyte lysate assay kit , spectrophotometer , and BCA protein assay kit (Thermo Fisher Scientific), respectively.Highly pure and structurally intact LTAs from four strains of escribed . The bacThe LTA-containing fractions were identified by inorganic phosphate assay as previously described . BrieflyWestern blot analysis for the prepared Lp.LTAs was conducted as previously described . BrieflyE. faecalis, S. gordonii, and S. mutans on 96-well plates (1×107CFU/ml) were grown in THY medium supplemented with 0.06–0.3% glucose  or 0.01–0.2% sucrose (for S. mutans) in the presence or absence of each Lp.LTA at 37°C for 24h. The biofilms were gently washed with phosphate-buffered saline (PBS) and stained with 0.1% crystal violet solution for 20min at room temperature. The stained biofilm was then dissolved in dissociation buffer (95% ethanol and 0.1% acetic acid in distilled water), and optical density at 600nm was measured using a microplate reader (Molecular Devices).Biofilm formation of dental pathogenic bacteria was determined by crystal violet staining as previously described . All strE. faecalis, S. gordonii, and S. mutans were formed on 24-well plates (1×107CFU/ml) in the presence or absence of various concentrations of LRCC 5310 Lp.LTA at 37°C for 24h. After washing the biofilm with PBS, it was scraped off and resuspended in 1ml PBS. To titrate bacterial CFU within the biofilm, serially diluted bacterial resuspensions (from 10−1 to 10−5) were seeded on the THY agar plate and incubated at 37°C for 48h. Colony formations were then examined, and CFUs were calculated based on the dilution fold.Colony-forming unit (CFU) formation assay was conducted as previously described  with minE. faecalis (KCOM 1083), S. gordonii (KCOM 1967), and S. mutans (KCOM 1223) on confocal glass-bottom dishes (1×107CFU/ml) were grown in THY medium supplemented with 0.3 and 0.03% glucose  or 0.03% sucrose (for S. mutans KCOM 1223) in the presence or absence of 30μg/ml of LRCC 5310 Lp.LTA at 37°C for 24h. The biofilms were gently rinsed with PBS to remove planktonic bacteria and stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher Scientific) containing SYTO9 and propidium iodide according to the manufacturer’s instructions. After washing with PBS, the stained biofilms were visualized by a confocal laser scanning microscope . Simultaneous dual-channel imaging was used to display green (for SYTO9 at 480/500nm) and red fluorescence (for propidium iodide at 490/635nm). The biomass and thickness distribution of biofilms were quantified using COMSTAT2 software . Human dentin slices were prepared as previously described . BrieflyE. faecalis (KCOM 1083), S. gordonii (KCOM 1967), and S. mutans (KCOM 1223) were grown on dentin slices in THY medium supplemented with 0.3 and 0.03% glucose  or 0.03% sucrose (for S. mutans KCOM 1223) in the presence or absence of 30μg/ml of LRCC5310 Lp.LTA at 37°C for 24h. After the incubation, dentin slices were gently rinsed with PBS to remove planktonic bacteria and prefixed with 2.5% glutaraldehyde and 2% paraformaldehyde in PBS at 4°C overnight. The dentin slices were further fixed with 1% osmium tetroxide for 90min and subsequently dehydrated with gradually higher concentrations of ethanol . After drying gold-coated sputter with hexamethyldisilazane, the biofilm formation on dentin slices was examined by a scanning electron microscope .Biofilm formation of dental pathogenic bacteria on human dentin slices in the presence or absence of Lp.LTA was visualized by scanning electron microscopic analysis as previously described . Brieflyt test. Asterisks (*) indicate experimental groups that are significantly different (p<0.05) from the designated control.Values are expressed as mean values±standard deviations from triplicate of each treatment group. Statistical significance between indicated Lp.LTA treatment and non-treatment groups was analyzed with Student’s E. faecalis, S. gordonii, and S. mutans, we initially purified LTAs from four different strains of L. plantarum  under the same condition. Inorganic phosphate assay was then performed for each column fractions from the hydrophobic interaction and ion-exchange chromatography because phosphate contents of column fractions are linearly correlated with their LTA contents  and three dental clinical isolates  by crystal violet staining. As shown in E. faecalis strains in a dose-dependent manner and LRCC 5310 Lp.LTA most effectively inhibited biofilm formation of all strains of E. faecalis compared with other Lp.LTAs. However, LRCC 5194 Lp.LTA showed the anti-biofilm activity in a clinically isolated E. faecalis strain, KCOM 1161, but not in other strains. To determine whether the anti-biofilm activity of Lp.LTAs resulted from changes in bacterial number within the biofilm, we further examined the CFU formation of the biofilms cultured under the aforementioned condition. When four strains of E. faecalis biofilms were formed in the presence of various concentrations of LRCC 5310 Lp.LTA, CFU of E. faecalis biofilms were dose-dependently decreased by LRCC 5310 Lp.LTA, suggesting that the anti-biofilm activity of Lp.LTAs is mediated through the reduction of bacterial number  and three dental clinical isolates , LRCC 5193, 5195, and 5310 Lp.LTA dose-dependently reduced the biofilm formation of all S. gordonii strains  and 5194 . On the other hand, we also examined the CFU formation of S. gordonii and S. mutans biofilms cultured in the presence or absence of various concentrations of LRCC 5310 Lp.LTA. Similar to the results from CFU of E. faecalis biofilm, CFUs from both S. gordonii and S. mutans biofilm were dose-dependently attenuated by LRCC 5310 Lp.LTA , S. gordonii (KCOM 1967), and S. mutans (KCOM 1223). As shown in ex vivo model, E. faecalis (KCOM 1083), S. gordonii (KCOM 1967), and S. mutans (KCOM 1223) were grown on human dentin slices in the presence or absence of 30μg/ml of LRCC 5310 Lp.LTA for 24h, and biofilm formations were then visualized by scanning electron microscope. As shown in E. faecalis, S. gordonii, and S. mutans, suggesting that LRCC 5310 Lp.LTA can be used as an alternative anti-biofilm agent for controlling major Gram-positive dental pathogenic bacteria, that are closely related to pathogenicity of periodontitis, gingivitis, and dental caries.The anti-biofilm activity of LRCC 5310 Lp.LTA, which showed the most effective anti-biofilm activity, was then confirmed by confocal laser scanning microscopic analysis against clinical isolates of L. plantarum inhibited the biofilm formation of clinically isolated dental pathogens, including E. faecalis, S. gordonii, and S. mutans at in vitro level. Among the tested LRCC Lp.LTAs, LRCC 5310 Lp.LTA showed the most effective anti-biofilm activity against all strains of dental pathogens. Furthermore, the result from ex vivo experiments indicated that LRCC 5310 Lp.LTA effectively suppresses biofilm formation of dental pathogens on human dentin slices.In this study, we demonstrated that LRCC Lp.LTAs purified from four different strains of L. plantarum JCM 1149 LTA has 110 repeating units of Gro-P backbone with 42% D-alanine content, while L. plantarum L137 has 96 repeating units of Gro-P with 50% D-alanine content, suggesting that Lp.LTA has relatively lower D-alanine contents as length of Gro-P repeating units increased , which induce EPS production by creating α-1,3- and α-1,6-linked glucan chains using sucrose and glucose  gene needed for production of polysaccharide intercellular adhesion  against the biofilm formation of E. faecalis ATCC 29212 ; LRCC 5194 (OK493618); LRCC 5195 (OK493619); LRCC 5310 (OK493620).The studies involving human participants were reviewed and approved by Institutional Review Board of Seoul National University Dental Hospital, Seoul, Republic of Korea (CRI 17010). The patients/participants provided their written informed consent to participate in this study.DL and SH designed the research. DL, JI, DP, MP, and SY carried out the experiments. DL, JI, DP, SJ, JP, and SH analyzed and interpreted the data. DL, JI, SJ, and SH prepared and reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Lotte Confectionary Co. Ltd. and the National Research Foundation (NRF) funded by the Korean government . The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.MP, SY, and JP are employed by Lotte R&D Center.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coffea arabica L. is an economically important agricultural crop and the most popular beverage worldwide. As a perennial crop with recalcitrant seed, conservation of the genetic resources of coffee can be achieved through the complementary approach of in-situ and ex-situ field genebank. In Ethiopia, a large collection of C. arabica L. germplasm is preserved in field gene banks. Here, we report the whole-genome resequencing of 90 accessions from Choche germplasm bank representing garden and forest-based coffee production systems using Illumina sequencing technology.C. arabica L. reference genome. A total of 11.08 million variants were identified, among which 9.74 million (87.9%) were SNPs (Single nucleotide polymorphisms) and 1.34 million (12.1%) were InDels. In all accessions, genomic variants were unevenly distributed across the coffee genome. The phylogenetic analysis using the SNP markers displayed distinct groups.The genome sequencing generated 6.41 billion paired-end reads, with a mean of 71.19 million reads per sample. More than 93% of the clean reads were mapped onto the Resequencing of the coffee accessions has allowed identification of genetic markers, such as SNPs and InDels. The SNPs discovered in this study might contribute to the variation in important pathways of genes for important agronomic traits such as caffeine content, yield, disease, and pest in coffee. Moreover, the genome resequencing data and the genetic markers identified from 90 accessions provide insight into the genetic variation of the coffee germplasm and facilitate a broad range of genetic studies.The online version contains supplementary material available at 10.1186/s12870-022-03449-4. Coffea [Coffea arabica L. and C. canephora Pierre [C. canephora and C. eugenioides [C. arabica L. is valued for its superior beverage quality [Coffee (Rubiaceae) is an important agricultural crop and is mainly grown as a cash crop in several tropical countries , 2. PresCoffea , the pria Pierre . Arabicaenioides . The culenioides . Besides quality –11 and aCoffea arabica L. is the only coffee species cultivated and exported from Ethiopia [C. arabica L. gene pool, the germplasm found in the country is valuable [C. arabica L. is threatened by various factors that could affect the genetic base for the future breeding program [Ethiopia . It is tEthiopia  and servEthiopia –14. The Ethiopia –18. PresEthiopia . Hence, valuable , and couvaluable –22. Notavaluable , 23. Spevaluable . Despite program . The los program , climate program , 26, and program .Presently, global ex-situ coffee germplasm conservation programs have been implemented in various countries including Ethiopia . Coffee Currently, NGS technology has enabled the identification of genetic variation in germplasm collections . ParticuC. arabica L., studies with SNP markers are scarce and a small number of SNP markers are available for this species [C. arabica L. by whole-genome resequencing. The genomic data and SNP generated in this study could be of great relevance for undertaking various genetic studies.SNP represent single nucleotide change in DNA sequence and are considered the most abundant form of genetic variation , 40. Pre species . This stC. arabica L. accessions that have been maintained at the Choche germplasm bank of the Ethiopian Biodiversity Institute  were used in this study. Genomic DNA was isolated from Silica gel dried leaf material of the 90 accessions originally collected from the garden and forest-based coffee production systems using MagicMag Genomic DNA Micro Kit . The quantity and quality of isolated DNA were checked and analyzed with the NanoDrop2000 spectrophotometer  and 1.0% agarose gel electrophoresis, respectively. Detailed information about each accession included in this study is shown in Additional file The Paired-end libraries with approximately 350 bp insert sizes were constructed from 1 μg of genomic DNA from each accession using Illumina TruSeq or Nextera  kits according to the Illumina manufacturer’s specifications. Whole-genome resequencing was performed for 90 coffee accessions at the Beijing Genomics Institute  using the Illumina Hiseq 2500 Platform .C. arabica L. reference genome using a burrows wheeler aligner (BWA) with default parameters [http://broadinstitute.github.io/picard/) to remove duplicate reads. The Illumina sequencing reads of each accession is deposited under accession number from SRR17316330 to SRR17316419.The filtering of raw reads was accomplished using the FASTQC (version 0.11.3) program. The clean reads were aligned onto the rameters . SAMtoolrameters . Then, tThe mapped reads were used to detect variants (SNP and InDels) using the Genome Analysis Toolkit (version 3.6) software . The annC. arabica L. was performed with the Illumina sequencing platform. The genome sequencing generated 6.41billion paired-end reads, with a mean of 71.19 million reads per sample. After removing low-quality reads, high-quality reads of each accession were mapped onto the C. arabica L. reference genome using a BWA aligner. The percentage of reads mapped onto the reference genome varied from 88.93 to 98.11%. The detailed resequencing information was provided in Additional file Whole-genome resequencing of 90 accessions of The high-quality sequences were used for the identification of SNP, and a total of 9.74 million SNPs were identified in all the 90 coffee accessions. We used the SnpEff  program Based on the nucleotide substitutions, the SNPs identified in the coffee genome were classified into two classes, namely, transitions (A/G and C/T) and transversions . The total transitions and transversions detected were 204,665,968 and 103,693,961, with a transitions/transversions (Ts/Tv) ratio of 1.97. The transition frequency of C/T was more than G/A. The transversion frequency of C/A was higher, similarly within transitions; the C/T transition was higher in number , contained six accessions, of which two were collected from the northwest, while four were collected from the southwest. Cluster II (Purple) consisted of accessions collected from the southwest and the north part of the country. Cluster III (Blue) mainly consisted of accessions sampled from the southwest parts of the country i.e., GUG5, YCG2 and GNG4 sampled from the country’s north, south and southeastern parts, clustered together. Almost all of the south and southeast accession were clustered in group IV (Green). The garden coffee accessions collected from similar geographical areas consistently clustered together in the same group . Only, GNG, GUG, YCG, SAG, GMG and WEG garden coffee samples showed exceptional grouping patterns (Fig. We inferred the phylogenetic relationships of the 90 accessions of C. arabica L. nuclear, mitochondrial and chloroplast genomes yielded a total of 9.74 million SNPs and 1.34 million InDels across 90 accessions. These variants were heterogeneously distributed across the eleven chromosomes of C. arabica L. Such uneven dispersal of variants has also been reported in other plant species such as in Solanum melongena and C. canephora [Presently, whole-genome resequencing is the most convenient approach for genome-wide SNP identification. It provides information of great relevance for crop genetics and breeding . Besidesanephora , 48.C. arabica L. Among these, the Kyoto Encyclopedia of Genes and Genome pathways analysis discovered 11 SNPs that have direct associations with genes encoding enzymes involved in caffeine biosynthesis pathways. These studies demonstrated the importance of SNP in identifying the genetic basis of traits of interest. The resequencing efforts of [Capsicum annuum. Furthermore, [The variant identified by the resequencing study could be used for various studies including marker-assisted selection, genome-wide association studies, phylogenetic and diversity analyses . SNPs alforts of , identifhermore,  highlighhermore, . Thus, tPhenotypic variations in crop plants are the result of variation at the DNA level . SNP is The variant effects that might affect the protein-coding sequences include: synonymous/non-synonymous amino acid replacement, start codon gains or losses, stop codon gains or losses . In thisCoffea spp. into five distinct groups. Further, Silvestrini et al. [C. arabica L. [SNP markers have been employed for analyzing the phylogenetic relationships and differences between genotypes . In thisi et al.  reportedabica L. , 17, 63.Most of the garden coffee accessions collected from the north parts of the country are grouped with southwest forest-based accessions. Whereas, the majority of garden coffee accessions sampled from southern parts of the country are grouped with southeast forest-based accession. Delsuc et al.  reportedC. arabica L. varieties also found the grouping of varieties into different clusters regardless of their geographic origin. The clustering pattern found in this study could indicate the presence of a high level of genetic diversity within coffee accessions sampled from the same geographic origin. Furthermore, the garden coffee accessions sampled from the neighboring regions were consistently grouped with few exceptions (Fig. C. arabica L. germplasm found in Ethiopia has a broad genetic base, and is valuable in developing varieties that could sustain global coffee production.A recent study by Benti et al.  on the Eons Fig. . This miThe availability of reference genomes and the continuous improvements of genetic data analysis methods are fostering resequencing studies. In this study, we performed the resequencing of coffee accessions, which has allowed the identification of genetic markers, such as SNPs and InDels. The SNPs discovered in this study might contribute to the variation in important pathways of genes for important agronomic traits such as caffeine content, yield, disease, and pest in coffee. Moreover, the genome resequencing data and the genetic markers identified from 90 accessions provide insight into the genetic variation of the coffee germplasm and facilitate a broad range of genetic studies.Additional file 1: Table S1. The details of the analyzed 90 accession of C. arabica L.Additional file 2: Table S2. Summary of the C. arabica L. whole-genome sequencing data.Additional file 3: Table S3. Amino acid changes identified by SnpEff software. Rows are reference amino acids and columns are changed amino acids. E.g., Row 'A' column 'E' indicates how many 'A' amino acids have been replaced by 'E' amino acids.Additional file 4: Table S4. Summary count of SNPs with effects on the genome.