Crataegus pinnatifida Bge.) fruits are rich in polyphenols —active compounds that exert beneficial effects. This review summarizes all information available on polyphenolic content and methods for their quantification in Chinese hawthorn berries and the relationships between individual polyphenolic compounds as well. The influence of species or cultivars, the locality of cultivation, the stage of maturity, and extract preparation conditions on the polyphenolic content were discussed as well. Currently, only fruits of C. pinnatifida and C. pinnatifida var. major are included in the Chinese Pharmacopoeia. Recent trials have demonstrated the efficacy of Chinese hawthorn fruit in lowering blood cholesterol and the risk of cardiovascular diseases. The fruit has also demonstrated anti-inflammatory and anti-tumour activities. This review deals mainly with the biological activity of the fruit related to its antioxidant properties.Chinese hawthorn ( Crataegus spp.—the genus comprises approximately 300 species) have been utilized by many cultures since antiquity for a variety of therapeutic applications [Crataegus monogyna and Crataegus laevigata are the major hawthorn species used in Europe, and Crataegus pinnatifida and Crataegus scabrifolia in China and Asian countries [The medicinal properties of hawthorn  variety are the most important, due to their large and delicious fruits with a characteristic acidic taste [C. pinnatifida and C. pinnatifida var. major are included in the Chinese Pharmacopoeia [Crateagus pinnatifida and Crataegus scabrifolia have been used traditionally as a peptic agent in oriental medicine and recently in a local soft drink product [The Chinese hawthorn is commonly considered to comprise 18 species, of which ic taste ,7. Moreo product , in jams product .Crataegus spp.) have recently attracted increasing attention in the field of food nutraceuticals and medicine because of their widely reported health benefits. Recent trials have demonstrated their efficacy in lowering blood cholesterol and in the reduction of the risk of cardiovascular diseases [Crateagus spp. along with their health promoting activity have been published. This review focuses its attention only in one species—Chinese hawthorn (Crateagus pinnatifida Bge.) and summarises its health benefits.Hawthorn species , the following can be considered as the main constituents: procyanidin B2, epicatechin, chlorogenic acid, procyanidin C1 and rutin [C. pinnatifida var. major can be divided into two groups, one rich in procyanidins and acids and another rich in flavonoids and sugars [In fruits, oligomeric procyanidins and their glycosides represent the major group of phenolic compounds ,20. Inveyanidins ,22,23. P. fruits ,24,25. T. fruits . Among mnd rutin . Accordid sugars .A, O-caffeoylquinic acid (neochlorogenic acid) was reported only in fruits of C. grayana [B, C. pinnatifida. Bge. caffeic acid was only seen in fruit of European hawthorn (C. monogyna) [Chlorogenic acid A,  has been grayana ,25. In a grayana , 4-amino grayana ,27 were onogyna) .Crateagus spp. [O-glycosides like luteolin-7-glycoside, rutin, and the C-glycosides vitexin, vitexin rhamnoside and monoacetylvitexin rhamnoside in Crateagus sp. fruits was also confirmed. Regarding the total contents of flavonol glycosides, the fruits of C. pinnatifida var. major contained less flavonol glycosides (0.4 mg/G DM as average content of 10 cultivars in the species) than those of other species. On the other hand, the fruit of C. pinnatifida var. major contained the highest level (1.1 mg/g DM) among all the tested hawthorn species [To date, more than 50 flavonoids have been isolated from genus gus spp. ,29,30. Dgus spp. . Aside fgus spp. , the pre species .C. pinnatifida var. major was confirmed [C. pinnatifida var. major and were identified as quercetin rhamnosyl hexoside and quercetin (dirhamnosyl hexoside) and isoquercetin [C-glycosyl flavones in hawthorn berries are derivatives of apigein and luteolin [et al. [O-rhamnoside—one of the typical components of Chinese hawthorn leaves.Kaempferol is the dominant flavonol in Chinese hawthorn fruit , howeveronfirmed ,25. Compluteolin . Cui et  [et al.  determinProcyanidins, as the second most abundant group of natural phenolics after lignins, are widespread throughout the plant kingdom where they display multiple biochemical properties, mainly involving interactions with proteins, the chelation of metals and antioxidant activity, which are the basis of their various protective functions for plants ,33,34.A, C. pinnatifida var. major (11.7 mg/g DM) [et al. also identified catechin in fruits of Crataegus spp. in monomer form and as a constituent unit of oligomeric and polymeric procyanidins [Procyanidins are a class of proanthocyanidins consisting primarily of epicatechin as the flavan-3-ol units or catechin. Epicatechin A,  predominmajor 11. mg/g DM mg/g DM) . Svedstryanidins . On the yanidins ,25. For yanidins .et al. identified procyanidins in Chinese hawthorn fruits, based on UV and mass spectra, that were mostly B-type procyanidins (PA) and their glycosides, including aglycons of three dimers, three trimers, eight tetramers, four pentamers, two hexamers and two glycosides of PA monomers, seven glycosides of PA dimers, one glycoside of a PA trimer, two glycosides of PA tetramers, one glycoside of a PA pentamer, and two glycosides of quercetin [The diversity of procyanidins is mainly due to their compositional differences and sequential order of the flavan-3-ol units, as well as variation in the location and stereochemistry (A/B) of the interflavanol bonds. The number of possible isomers increases exponentially with the degree of polymerization. The isomers have the same molecular weight and similar UV and mass spectral characteristics . Liu et uercetin . Accordiuercetin  epicatecuercetin .B and C, D, C. pinnatifida var. major [C. scabrifolia Rehd., and C. brettschneideri Ahneid. have similar procyanidin aglycon profiles, whereas the profile of procyanidin glycosides varied among the mentioned species. Fruits of C. pinnatifida var. major had higher contents of procyanidins, but lower contents of flavonols compared with C. brettschneideri. The fruits of C. scabrifolia contained the highest level of PA dimer hexoside, which was detected in trace amounts in the fruit of C. pinnatifida Bge. The content of this compound in fruit of different cultivars of C. pinnatifida var. major varied from trace amounts to 0.8 g/kg DM [Procyanidins B2 and B5 B and C,  were ider. major ,25. The r. major . Values r. major  which mir. major  and the r. major . C. scabCrateagus sp. fruits were based on the colorimetric Folin-Ciocalteu method. Froehlicher et al. found out that dried and fresh fruit of C. monogyna contained 12 g gallic acid equivalent/kg DM [C. pinnatifida, C. pinnatifida var. major contained 18 and 61 g/kg DM [The first attempts of quantification of total polyphenols content in nt/kg DM . Content g/kg DM . The men g/kg DM .et al. quantified the levels of seven polyphenols  in mature fruits of Chinese hawthorn (C. pinnatifida) by a HPLC-UV method [Other approaches have utilized the HPLC-UV technique. Cui V method . The aveV method .et al. utilized capillary electrophoretic analysis of flavonoids in hawthorn ethanolic extracts of fruits [et al. isolated by this method four flavones from C. pinnatifida with the following recoveries: vitexin-2'-rhamnoside 96.8%, hyperoside 99.9%, rutin 97.1% and vitexin 97.8% [C. pinnatifida fruits or flowers. A method based on capillary electrophoresis with electrochemical detection was also developed for the simultaneous separation and determination of epicatechin, kaempferol, chlorogenic acid and 4-hydroxybenzoic acid [Urbonaviciute f fruits . Liu et in 97.8% . The conoic acid . QuercetRosaceae, Hawthorn fruit (C. pinnatifida Bge.), showed a highest rate of neuroprotective phenols  with the grand total of 437 mg/100g FW. The fruits of C. pinnatifida had almost four times higher neuroprotective phenolics content compared to the plant species Lonicera edulis Turcz. ex. Freyn and Amelenchier canadensis investigated by Gazdik et al. [Nowadays an electrochemical detection method for polyphenolic compounds has been widely utilized in analyses of fruit samples of common and less known fruit species as well ,38,39. Hk et al. . This cak et al. ,43,44,45et al. provided the first systematic study of phenolic compounds in the major Chinese hawthorn variety C. pinnatifida Bge. var. major [O-galactoside), chlorogenic acid (0.2–1.6 mg/g DM) as the main phenolic acid, flavonol glycosides—hyperoside  (0.1–0.8 mg/g dry mass DM), and isoquercitrin (quercetin-3-O-glucoside) (0.1–0.3 mg/g DM), which is in good agreement with the previously published results [C. pinnatifida was higher than had been reported previously in 37 cultivars of C. pinnatifida var. major [C. pinnatifida var. major reported by [et al. [Liu r. major ,25. The  results ,28. The r. major . Procyanorted by , with slet al. developed a simultaneous determination of vitexin-2''-O-glucoside and vitexin-2''-O-rhamnoside (VOR), rutin and hyperoside in the extract of Crateagus pinnatifida Bge. leaves and fruits [Cheng d fruits . Anotherd fruits . In anotd fruits , phenoliet al. developed a HPLC method with high recovery (95.4 ± 1.3%) for the investigation of volatile organic compounds (VOR) by determining malondialdehyde (MDA) in ECV304 cell culture medium [C. pinnatifida Bunge, was described [r) between 0.9997 and 0.9999.Ying e medium . A novelescribed . Under tC. pinnatifida and C. brettschneideri. Geographic locations also had to be considered. Significant inverse correlation between procyanidin contents and the latitude of the geographical origin of the studied cultivars was observed . The time of harvest is also a very important factor [C. pinnatifida Hebei Dajinxing cv. 61 days after blossom, the total polyphenol levels reached their highest point at 1.36 g/100 g FW. Fruit shows changes in levels of procyanidins, flavonoids and chlorogenic acid. In the early bearing period, the chlorogenic acid and flavonoid levels were relatively higher. In the fruit of C. pinnatifida var. major, procyanidins were synthesised rapidly in the early growth stage and reached up to 14 g/kg FW in fruits, thereafter the procyanidin level in fruit decreased gradually, reaching 4 g/kg FW at the optimal maturity stage [C. grayana the content of phenolic compounds were the highest around mid-August and early September (22 g/kg DM) [et al. developed a new extraction procedure that uses ethyl acetate treatment of an ethanol extract to prepare a sugarless extract [et al. [et al. found out that procyanidins eluted faster in chromatograms when a low ratio of acetonitrile to methanol was used as mobile phase, and the phenolic acids gradually eluted faster when the proportion of acetonitrile in the mobile phase was increased [C. pinnatifida Bge. var. major N.E.Br.) fruits were optimized by response surface methodology (RSM). Results showed that 93.4 ± 0.21% of the procyanidins could be recovered. The crude extract was then purified by using a LSA-10 resin column, which showed excellent adsorption and desorption properties for PC purification. A fraction with PC content above 83.2% and mainly consisting of EC, a singly-charged dimer and trimer as identified by HPLC/MS, was obtained by isolation on LSA-10 resin. Liu et al. [C. pinnatifida Bge. The best extraction parameters were obtained using RSM. Under this condition, an extraction yield of 23.5% was obtained and the percentage of phenolic compounds in the extract reached 41.2%. Wu et al. [The phenolic composition in hawthorn fruit varies among species and cultivars ,35 accout factor . The totg/kg DM) . Phenolig/kg DM) . The valg/kg DM) . Cui et  [et al. , procyanncreased . The extu et al.  investigu et al.  utilizedet al. [Temperature was found to markedly influence the stability of polyphenolics in Chinese hawthorn berries. Low temperatures are recommended for storage of fruit. Chang et al.  studied C. pinnatifida Bge. yielded a strong correlation (p < 0.001) between the individual contents of the four procyanidins and total procyanidin contents (r = 0.7413–0.9898), and between flavonoids and the chlorogenic acid (r = 0.5383–0.9212) [et al. [r = 0.49, p < 0.01), and a significant positive correlation was found between the content of epicatechin and the levels of practically all procyanidins, except the PA dimer hexoside. A positive correlation also exists between the contents of PA B2 and PA trimer II. A weak negative corelation between total flavonols and total PAs was also observed (r = −0.16).Correlation analysis of the levels of the seven compounds epicatechin, procyanidin B2, procyanidin B5, procyanidin C1, hyperoside, isoquercitrin and chlorogenic acid in fruits of 37 cultivars of –0.9212) . Moreove [et al.  determinAntioxidant activity of hawthorn berries rich in phenolic compounds have been widely used as both a medicinal and food raw material in China and Europe ,8.C. pinnatifida Bge. have the capacity to quench free radicals and inhibit the oxidation of low density lipoprotein (LDL) in both cells and cell-free systems [2+ mediated LDL oxidation. They were also effective in preventing the peroxy free radical-induced oxidation of α-tocopherol in human LDL. The inhibitory effect of these compounds on oxidation of LDL and α-tocopherol was dose-dependent at concentration 5–40 µM DM. Chu et al. [Crateagus pinnatifida and found its capacity of quenching 1,1-diphenyl-2-picrazyl radicals to be EC = 0.118 mg/mL. Flavonoids (6.9%), procyanidins (2.2%), catechin (0.5%) and epicatechin (0.2%) are responsible for this high antioxidant capacity. A hot water extract also reduced LDL oxidation in a Cu2+-induced cell-free system as well as in sodium nitroprusside (SNP)-treated RAW 264.7 (a mouse leukaemic monocyte macrophage cell line) [C. pinnatifida fruits also protected LDL from Cu2+-induced oxidation in vitro. Studies by Zhang et al. showed that the Chinese hawthorn fruit is an excellent source of constituents with antioxidant properties purified from ethyl acetate extract reduced production of thiobarbituric acid-reactive substances (TBARS) in Cu2+-induced LDL oxidation and inhibited peroxy radical-induced oxidation of α-tocopherol in LDL and in AAPH assay [Antioxidant activity refers to the ability of antioxidants to quench or scavenge free radicals ,53,54,55 systems ,57,58. F systems . The ant systems ,60. All u et al.  studied ll line) . The ethPH assay . In thisC. pinnatifida Bge.) possessed a strong inhibitory effect against DPPH, hydroxyl radicals and lipid peroxidation, as well as strong reducing power [et al. [Biological studies showed that the extract from hawthorn  efficiently inhibit lipid peroxidation during enzymatic hydrolysis of porcine meat at 50 °C for 24 h [C. pinnatifida leaves and fruit exhibited collagenase inhibitory activity (IC50) at concentrations of less than 1 µM [Procyanidins also play a very important role in antioxidant activity . The proin vitro . The antfor 24 h . Three ohan 1 µM .C. pinnatifida Bge. fruits were stronger than the purified one in their antioxidant activity, especially in the solution system. The ethyl acetate extract fraction consisting of procyanidins with DP 1–5 (197 g/kg), chlorogenic acid (12 g/kg DM) and flavonoids (5 g/kg DM) inhibited the activity of prolyl endopeptidase and showed scavenging activities on superoxide and hydroxyl radicals [Crude extracts of radicals . Anotheret al. compared the antioxidant, anti-α-glucosidase and anti-inflammatory activities of Crateagus pinnatifida Bunge var. typica Schneider (CBS) and C. pinnatifida Bunge (CB) [Lonicera kamtschatica Sevast. Pojark), Saskatoon berry . and Chinese hawthorn (Crateagus pinnatifida Bge.), Hawhorn fruit showed the highest antioxidant capacity by both the TAC and DPPH assay methods [Li nge (CB) . The CB  methods .Crataegus sp.) are used both in traditional and folk medicine to improve digestion, avoid food retention, promote blood circulation and resolve blood stasis [C. pinnatifida Bge.) berries displaying a high level of flavonoids and procyanidins [Crataegus spp. have mainly been attributed to their individual polyphenolic compounds such as oligomeric procyanidins (OPCs), abundant in hawthorn [Generally, fruits of hawthorn  and triacylglycerols (TAG) level in animal models ,71,72,73p < 0.05) [p < 0.05). Polyphenolic extract of hawthorn fruit lowered serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) and inhibited the accumulation of hepatic TC and triglyceride (TG), improved antioxidant status in cholesterol—enriched diet (CED) for 12 wkd in male ICR mice. Also hepatic histopathological examinations showed markedly decreased fatty deposits in the liver of mice treated with extract [After 12 weeks, serum total cholesterol (TC) and triglyceride (TG) were 23.4 and 22.2% lower, respectively, in the Chinese hawthorn fruit group of New Zealand white rabbits on a high cholesterol diet compared with the control group ( < 0.05) . In a si extract .et al. [Panax ginseng C.A. Meyer) and Crateagii fructus  are also very interesting. Treatment of RG and CIF significantly reduced blood triglyceride (TG) and total cholesterol (TC) levels in Triton WR-1339-induced hyperlipidemic mice and serum TG levels, decreased blood HDL cholesterol in corn oil-induced hyperlipidemic mice.Results of comparative studies by Min et al.  of the aet al. showed that the hypolipidemic effects could be associated with flavonoid and triterpene saponins in fruits as well [et al. isolated 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors from hawthorn fruit (C. pinnatifida) and evaluated them for their antihyperlipidemic effect in mice subjected to a high-fat diet [In vivo results also revealed that the mixture had a more significant lipid-lowering efficiency than monomers. Structure-activity relationships revealed the inhibitory activity and lowering-lipid ability of quercetin, hyperoside and rutin decreased with increasing glycoside numbers.Rajendran  as well . In addifat diet . They diet al. [C. laevigata per 100 g body weight per day for 6 weeks was associated with a significant increase of hepatic LDL-receptor activity, resulting in greater influx of plasma cholesterol into the liver. Extract also prevented the accumulation of cholesterol in the liver by enhancing cholesterol degradation to bile acids and by simultaneously supressing cholesterol biosynthesis. This probable mechanism was also supported by a study by Ho et al. [et al. determined whether hawthorn fruit reduced the absorption of cholesterol and investigated the ACAT activity in the intestine of rabbits [C. pinnatifida) could suppress intestinal acyl-Coa:cholesterol acyltransferase (ACAT) activity in rabbits. The hypolipidaemic effects of the hawthorn phenolic extract were associated with the activation of peroxisome proliferator-activated receptor α (PPAR α) in the adipose tissue of hamsters and were reversed by treatment with MK 886, a PPAR α antagonist [Recent studies have been aimed at clarifying the bioactive substances in Chinese hawthorn fruit responsible for this action and the underlying mechanism. In the study reported by Rajendran et al. , supplemo et al. , who inv rabbits . Studiestagonist . The hyptagonist . The oratagonist .C. monogyna or C. laevigata), are used as dietary supplements and herbal medicines for treating heart failure and mild forms of arrhythmia [Crataegus sp. Genus, including C. pinnatifida, are recommended for treatment of myocardial weakness, paroxysmal tachycardia, hypertension and arteriosclerosis [et al. recommended Crateagus spp. as an effective and safe therapeutic alternative for this indication [et al. found out that hawthorn treatment modifies left ventricular remodelling and counteracts myocardial dysfunction in early pressure overload-induced cardiac hypertrophy [Aqueous alcohol extract of hawthorn fruits, especially standardised extracts of the European hawthorn , hyperoside (66%) and rutin (66%), as well as an increase of the relaxation velocity by luteolin-7-glucoside (104%), hyperoside (62%) and rutin (73%) were markedly effected of flavonoids from Crateagus species observed at a maximum concentration of 0.5 mmol/L [C-glycosides of vitexin, vitexin rhamnoside and monoacetylvitex rhamnoside. The possible mechanism of cardiac action of flavonoids extracted from hawthorn berries suggest the inhibition of 3'-5'-cyclic adenosine monophosphate phosphodiesterase [Many studies have demonstrated the beneficial effects of extracts of hawthorn berries on the blood circulation system . Hawthor5 mmol/L . Similaresterase , while aesterase .et al. investigated potential interference of hawthorn in serum digoxin measurements using immunoassays as well as pharmacodynamic interaction between hawthorn and digoxin [Current research to date suggests that hawthorn may potentially represent a safe, effective, nontoxic agent in the treatment of cardiovascular disease (CVD) and ischemic heart disease (IHD) . Because digoxin . They foPanex notoginseng, Ginko biloba and C. pinnatifida Bge is widely used. Iwaoka et al. showed the preventive effects of the medicine in hypertensive rats, whereby systolic blood pressure in MR-treated rats was significantly decreased, cerebral blood flow in the MR treated group was significantly higher than in control group [Shan Zha) is known in Traditional Chinese Medicine (TCM) for its effects on reducing food stagnancy and blood stasis, improving blood circulation and is used to treat hypertension [Shan-Zha fruit extract showed merit in improving obesity and hyperlipidemia. Studies with fatty rats and hamsters show that after administration of Shan-Zha significant decreases of body weight, plasma TG, TC  and FFA (free fatty acids), liver TG, TC and FFA, plasma alanine transferase (ALT) and aspartic aminotransferase (AST) compared with control group occurred [Shan Zan inhibited the fat droplet accumulation in adipocytes. The clinical efficacy of the Chinese therapeutic food  on dyslipidemia was evaluated in a placebo-controlled, double blind, paired clinical trial conducted in Melbourne, Australia. The results indicated that a four-week intake of the compound increased the serum HDL-c levels by 5% (p = 0.026) and decreased the ratio of TC/HDL-c and LDL-c/HDL-c . The intake of hawthorn fruit and Chinese kiwifruit extract compoundd may increase the serum levels of HDL-c and decrease the ratios of TC/HDL-c and LDL-c/HDL-c, therefore, may reduce the risk of cardiovascular disease [Currently, hawthorn leaves, flowers and both green (unripe) and red (ripe) berries are used to make herbal preparations to treat patients with cardiovascular disease . In Chinol group . Among trtension . As a heoccurred ,93. Shan disease .et al. demonstrated that the flavonoid contents of dried fruits of C. pinnatifida present in vitro and in vivo anti-inflammatory potential, and may play a role in hepatoprotection [Crateagus flavonoids (CF-Fs) (0.25–0.75 mg/mL) decreased the release of PGE2 and nitric oxide (NO) as induced by lipoplysaccharide (LIPS and endotoxin) in macrophage RAW 264.7 cells. In addition, oral administration of Crataegus spp. fruit extract caused a dose-dependent antiinflammatory effect in a model of carrageenan-induced rat paw edema.Kao otection . The preCrataegus sp. extract revealed its moderate bactericidal activity, especially against G-positive bacteria Micrococcus flavus, Bacilus subtilis and Lysteria monocytogenes with no effect on Candida albicans [Antimicrobial testing of the albicans .Crataegus fruit extract was investigated using an ethanol—induced acute stress ulcer in rats model, with rantidine as reference drug. Hawthorn extract produced dose-dependent gastroprotective activity . Consumption of hawthorn (C. pinnatifida) altered the digestive enzymes of the stomach and cholesterol metabolism of the liver [Gastroprotective activity of he liver ,68.O-rhamnoside (VOR) is the polyphenolic compound presented in the leaves of C. pinnatifida Bge.var. major and in minor proportion in fruit. VOR plays an important role in preventing human pathologies related to oxidative stress [C. pinnatifida exhibited collagenase inhibitory activity (IC50) at a concentration of less than 1 mµ/M [et al. [C. pinnatifida fruit. They use JB6 mouse epidermal cell model to investigate the molecular events specific to tumour promotion. The achieved results of experiment demonstrated that polyphenolic fraction of dried fruit of C. pinnatifida inhibits TPA (12-0-tetradecanoylphorbol-13-acetate) induced tumour transformation by blocking the AP-1 and NF- B signals, protein expression of COX-2/iNOS, generation of H2O2, activation of MPO and tumour promotion by decreasing inflammation and oxidative stress [Procyanidins show cytotoxic potential on SK-OV-3 cancer cell lines. Vitexin-2''-e stress . Oligomen 1 mµ/M . Kao et  [et al.  proved ce stress .C. pinnatifida fruit showed notable growth inhibitory activity against chloroquine-sensitive strains of P. falciparum with IC50 values of 2.7 μM . This is the first report on the antiplasmodial activity of these oligomeric procyanidins from Chinese hawthorn fruits [The oligomeric procyanidins fraction of n fruits .Crataegus pinnatifida Bge.) fruit, responsible for its hypolipidemic effects and reduction of the risk of cardiovascular diseases  and cancer. Polyphenols responsible for free radical scavenging activity are epicatechin, hyperoside and chlorogenic acid, and these compounds are considered to be the best antilipoperoxidants. Procyanidins, especially procyanidin B2, procyanidin B5 and procyanidin C1 also play a very important role in its antioxidant activity, as the procyanidin fraction from fruits of C. pinnatifida var. major scavenges superoxide and hydroxyl radicals and inhibits lipid peroxidation in vitro. The polyphenolic content of Chinese hawthorn fruit is dependent on the cultivars, the locality of cultivation, the stage of maturity, the conditions of extract preparation and the method of chemical determination of polyphenols.There has been an increasing interest in investigating polyphenols from lesser known fruit species because of their potential health benefits in the prevention of chronic diseases. Polyphenols represent the predominant group of biologically active substances in the Chinese hawthorn (


Porphyromonas gingivalis is proposed as a keystone pathogen in chronic periodontitis causing both dysbiosis and discordant immune response. The present review focuses on the growing recognition of a relationship between P. gingivalis and orodigestive cancers. Porphyromonas gingivalis has been recovered in abundance from oral squamous cell carcinoma (OSCC). Recently established tumorigenesis models have indicated a direct relationship between P. gingivalis and carcinogenesis. The bacterium upregulates specific receptors on OSCC cells and keratinocytes, induces epithelial-to-mesenchymal (EMT) transition of normal oral epithelial cells and activates metalloproteinase-9 and interleukin-8 in cultures of the carcinoma cells. In addition, P. gingivalis accelerates cell cycling and suppresses apoptosis in cultures of primary oral epithelial cells. In oral cancer cells, the cell cycle is arrested and there is no effect on apoptosis, but macro autophagy is increased. Porphyromonas gingivalis promotes distant metastasis and chemoresistance to anti-cancer agents and accelerates proliferation of oral tumor cells by affecting gene expression of defensins, by peptidyl-arginine deiminase and noncanonical activation of β-catenin. The pathogen also converts ethanol to the carcinogenic intermediate acetaldehyde. In addition, P. gingivalis can be implicated in precancerous gastric and colon lesions, esophageal squamous cell carcinoma, head and neck  carcinoma, and pancreatic cancer. The fact that distant organs can be involved clearly emphasizes that P. gingivalis has systemic tumorigenic effects in addition to the local effects in its native territory, the oral cavity. Although coinfection with other bacteria, viruses, and fungi occurs in periodontitis, P. gingivalis relates to cancer even in absence of periodontitis. Thus, there may be a direct relationship between P. gingivalis and orodigestive cancers.There is increasing evidence for an association between periodontitis/tooth loss and oral, gastrointestinal, and pancreatic cancers. Periodontal disease, which is characterized by chronic inflammation and microbial dysbiosis, is a significant risk factor for orodigestive carcinogenesis.  Porphyromonas gingivalis is regarded as a keystone pathogen in adult periodontitis [P. gingivalis, the most important virulence factors are lipopolysaccharide (LPS), fimbriae, gingipains and outer membrane vesicles. Major pathogenic mechanisms include ability to create a dysbiotic microbiota and a dysregulated immune defense [P. gingivalis can invade oral epithelial and endothelial cells [Over the years, a number of bacteria have been associated with periodontitis . Among tdontitis –9. This dontitis ,11. In P defense –18. It ial cells –21 and ial cells .P. gingivalis in the etiology of oral, gastrointestinal, and pancreatic cancers [P. gingivalis IgG, independent of periodontal disease. This indicates that P. gingivalis can have an important role in the development of orodigestive carcinogenesis irrespective of periodontitis. A review of the emerging role of bacteria in oral carcinogenesis was recently published by Perera et al. [P. gingivalis and orodigestive cancers  constitute more than 90% of oral cancers, listing among the top 10 most common types of cancer worldwide –29. An eSeveral epidemiological and clinical studies have found a positive relationship between periodontal disease or tooth loss and the progression of cancers such as oral cancer, gastric cancer, pancreatic cancer, and even gastric precancerous lesions –39. In aPorphyromonas gingivalis occurred in significantly higher levels in sampled gingival SCCs than in normal gingival tissue (p < 0.05) [Streptococcus gordonii, which is a non-invasive oral bacterium [P. gingivalis was before or after cancerogenic transformation of cells. < 0.05) . It was acterium . No infoP. gingivalis and Fusobacterium nucleatum, together with administration of the oral carcinogen 4-nitroquinoline-1-oxide (4NQO), promoted transformation of oral squamous carcinogenic cells and increased signaling along the interleukin-6 (IL-6)-signal transducer and activator of transcription 3 (STAT3) axis [P. gingivalis activating the Janus kinase 2 (JAK2) and Glycogen synthase kinase 3 beta (GSK3β) pathways [P. gingivalis and F. nucleatum stimulate tumorigenesis by direct interaction with oral epithelial cells through activation of Toll-like-receptors (TLRs) causing IL-6 production from the epithelial cells. In addition, oral pathogens stimulated proliferation of OSCC and expression of key molecules in tumorigenesis such as cyclin D1, Matrix Metalloproteinase-9 (MMP-9) and heparanase [In a newly established murine model for periodontitis-associated oral tumorigenesis, it was demonstrated that chronic infection induced by T3) axis . This inT3) axis ,43. The pathways ,44. Resupathways  indicateparanase .in vitro model exposing human immortalized oral epithelial cells to P. gingivalis at a low multiplicity of infection for 5–23 weeks. Persistent exposure to P. gingivalis induced cell morphological changes, increased the proliferation ability with a higher S phase fraction in the cell cycle, and promoted cell migration and invasion. It was suggested that tumor-related genes such as NNMT, FLI1, GAS6, IncRNA CCAT1, PDCD1LG2, and CD274 are key regulators in tumor-like transformation under long-time exposure to P. gingivalis. Thus, chronic infection with P. gingivalis may be regarded as a potential risk factor for oral cancer.Geng et al.  establisP. gingivalis strains (W83 and ATCC 33277T), both B7-H1 and B7-DC receptors were upregulated on oral squamous carcinoma cells, SCC-25, and BHY, and on human primary gingival epithelial cells [S. salivarius K12 did not cause upregulation. However, the implementation of S. salivarius K12, a nonpathogenic oral commensal probiotic bacterium, as a sole control microorganism rather stands incomplete. Additional inclusion of pathogenic and/or opportunistic oral microorganisms that are also Gram-negative is warranted to challenge and substantiate the physiological importance and specificity of these findings.Expression of B7 homolog 1 (B7-H1) receptors occurs in most human cancers causing anergy and apoptosis of activated T cells. The B7-H1 receptors are upregulated on cancer/transformed cells, and the B7-H1 receptors then interfere with PD1 receptors on tumor-infiltrating lymphocytes (TILS), blocking the PD1 receptors’ cytotoxic activity against the cancerous epithelial cells. This may enable tumor cells to overcome the host response. After infection with two separate al cells . After sP. gingivalis caused upregulation of the immune-regulatory receptor PD-L1 (B7-H1) in oral squamous carcinoma cells and gingival epithelial cells [P. gingivalis infection.In culture experiments, the membrane fraction of al cells . The totP. gingivalis in the mouth, OSCC cells were exposed to P. gingivalis twice a week for 5 weeks [In order to mimic chronic irritation by  5 weeks . The expp < 0.01) in human primary epithelial cells during the course of P. gingivalis infection [p < 0.01) in protein and mRNA expression. The protein expression mesenchymal intermediate filament, vimentin, was significantly enhanced over the 120-h period of infection with P. gingivalis. When the adhesion molecule E-cadherin was analyzed, a significant decrease (p < 0.05) in expression and loss of membrane localization together with ß-catenin were seen in the oral epithelial cells. In contrast, MMP-2, −7 and −9 were markedly increased during long-term infection. Porphyromonas gingivalis infection also promoted migration of P. gingivalis-infected cells. In contrast, there was only a slight increase following coinfection with F. nucleatum [P. gingivalis [P. gingivalis survival in the primary epithelial cells strongly correlated with the aforementioned phenotypic changes, it would be interesting to see whether bacterial secreted effectors such as nucleoside-diphosphate-kinase (NDK) or structural virulence factors such as fimbriae and LPS could contribute to EMT independently [An important regulator of EMT, phospho-GSK3ß, was found to increase significantly -Ets1, p38/Heath-shock-protein 27 (HSP27) and Protease activated receptor 2 (PAR2)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) pathways to produce proMMP-9 expression. The gingipain proteases (Arg- and Lys-gingipains) of P. gingivalis subsequently activated the proenzyme, which promoted cellular invasion and metastatic abilities of cell lines derived from OSCC. ProMMP-9 was produced by highly invasive/metastatic cells but not by cells with low invasiveness. In contrast, F. nucleatum did not perform such activities. Inhibitors of SAS cells reduced proMMP-9 over-expression and cellular tumorigenic invasion. In addition, P. gingivalis mutants that lacked gingipain proteases, failed to activate MMP-9. Accordingly, these findings, based on disruption of normal cell signaling, indicated perhaps a new mechanism involved in the progression and metastasis of OSCC associated with periodontitis. Further, this investigation highlighted the increase of HSP27 in oral cancer cells infected with P. gingivalis and HSP27’s role in the activation of MMP9 in infected cells [P. gingivalis-NDK – which confers an anti-apoptotic phenotype to human primary oral epithelial cells [P. gingivalis in the oral mucosa. Further, NDK-HSP27 interaction could point to targeted therapeutic strategies against cancers recently associated with P. gingivalis.While proMMP-9 was secreted continuously from carcinoma SAS cells, he cells . Porphyred cells . HSP27 ied cells ,57. Henced cells ,58,59. Aal cells . HSP27 mP. gingivalis increased the invasive ability of cells derived from OSCC such as OSC-20 and SAS by upregulating interleukin 8 (IL-8) and matrix metalloproteinases, particularly MMP-1 and MMP-2 [P. gingivalis. However, when IL-8 was directly applied upon SCC-25 cells, these cells’ invasive ability and MMP level increased significantly. In contrast, downregulation of IL-8 in P. gingivalis-infected OSC-20 and SAS cells reduced their invasive potentials and levels of MMP. Thus, P. gingivalis increased the invasiveness of OSCC cells by IL-8-dependent upregulation of matrix metalloproteinases. Interestingly, MMP-9 activation and spreading of OSCC could be prevented by inhibitors such as apple polyphenol (AP), hop bracht polyphenol (HBP) and high-molecular fractions of HBP (HMW-HBP). These inhibitors could be candidate cytostatic agents to prevent such cancerous invasion [Exposure to nd MMP-2 . This wainvasion .Porphyromonas gingivalis induced changes in the level and phosphorylation status of proteins that display multilevel control on the eukaryotic cell cycle involving cyclins, p53 and Phosphatidylinositol-4,5-bisphosphate 3-kinase (P13K) [P. gingivalis affects the cell cycle of human gingival epithelial cells by promoting the G1/S transition, which implies upregulation of cyclin D and cyclin E. Accordingly, contribution from cell proliferation-enhancing bacteria to carcinogenesis should not be overlooked, although the latter is a protracted multistage and multifactorial process.Bacteria regulate the cell cycle of their host cells to be able to survive and express their virulence factors within the host. e (P13K) . The ince (P13K)  demonstrP. gingivalis inhibited apoptosis by upregulating the anti-apoptotic molecule Bcl-2 and downregulating the pro-apoptotic Bad protein [P. gingivalis blocks apoptosis and increases survival of primary gingival epithelial cells through activation of the pPI3K/Akt survival pathway. Later, Mao et al. [P. gingivalis can block apoptosis in gingival epithelial cells is through manipulation of the P13K/Akt and JAK/Stat pathway that controls intrinsic mitochondrial cell death pathways. In addition, sequestration of pro-apoptotic Bad, a member of the Bcl-2 family, through Akt, has been demonstrated [P. gingivalis. The latter can also inhibit apoptosis in primary gingival epithelial cells induced by a danger molecule, i.e. ATP by ligation of P2X7 receptors [P. gingivalis suppresses SOC6 that has an important role in modulating mitochondrial dynamics and subsequent apoptotic events [P. gingivalis in periodontal tissues and a key feature of its pathogenicity. In addition, spread of this bacterium to other cells could be achieved by protection from the immune system through reduced inflammation in epithelial tissues [In primary cultures of short-term gingival epithelial cells,  protein . Yilmaz  protein  reportedo et al.  suggestenstrated . Akt is eceptors . Agents eceptors . In addic events ,71. Reguc events . Accordi tissues ,73.P. gingivalis on oral epithelial cells, little is known about its effect on cancer cells. When oral cancer cells were invaded by P. gingivalis FDC 381, proliferation of the cells were inhibited through arrest of the G1 cell cycle. There was no effect on apoptosis, but macro autophagy was increased [P. gingivalis within the tumor, serving as a survival mechanism that limits bacterial toxicity. The expression of cyclin D1 and cyclin-dependent kinase 4 (cdk4) was reduced in the oral cancer cells, while the Cdk inhibitor – p21- was upregulated, compared to non-infected controls. The increased autophagy response probably contributed to the suppressed proliferation. This response was activated by formation of reactive oxygen species (ROS). Researchers have also found that P. gingivalis increases cell proliferation by accelerating cell cycling or by pro-survival signaling via modulation of apoptosis [P. gingivalis infection of human primary oral epithelial cells has been shown to modulate and inhibit both mitochondrion- and membrane (NADPH-oxidase)-derived ROS to secure successful intracellular growth and survival [P. gingivalis employs a selective form of autophagy  that is rich in ER-networks in order to replicate and persist in the human primary epithelial cells [Although several published studies have dealt with the effect of ncreased . It was poptosis . The repsurvival ,76. Moreal cells .P. gingivalis promoted distant metastasis of oral cancer to the lungs together with development of resistance to anti-cancer agents [P. gingivalis-infected cells from OSCC exhibited a higher resistance to the chemotherapeutic agent Taxol through Notch intracellular domain (NICD) 1 activation. It suggested that P. gingivalis might have a role in the development of chemoresistance towards OSCC. Similarly, when OSCC cells were exposed repeatedly with P. gingivalis for 5 weeks, resistance to the chemotherapeutic agent Taxol was developed [Sustained infection with r agents . This waeveloped . Intereseveloped ,79.P. gingivalis and Aggregatibacter actinomycetemcomitans increased proliferation properties of oral tumor cells by affecting gene expression of human defensins. While incubation of oral tumor cells with P. gingivalis and human α-defensins led to increased cell proliferation, A. actinomycetemcomitans caused increased cell death. Accordingly, these periodontal pathogens had opposite primary effects on the proliferation of oral tumor cells, but both caused similar secondary effects on the proliferation rate by modifying expression levels of oncogenic relevant α-defensin genes. It was suggested that anti-microbial peptides could function as a molecular link between tumorigenesis and infection since human defensins affect cell proliferation via Epidermal Growth Factor Receptor (EGFR)-dependent signaling.Hoppe et al.  showed tP. gingivalis to contribute to tumorigenesis. It could be so, because noncanonical activation of β-catenin and disassociation of the β-catenin destruction complex by gingipain might induce a proliferative phenotype. This could be a novel mechanism for P. gingivalis to contribute to disruption of oral tissue homeostasis by proteolytic processing.Zhou et al.  reportedin vitro salivary ACH production from ethanol compared to individuals with good oral hygiene [Porphyromonas gingivalis converts ethanol to acetaldehyde at levels capable of inducing DNA damage, mutagenesis, and hyperproliferation of the epithelium [Acetaldehyde (ACH), which is the first metabolite of alcohol, is a group 1 carcinogen to humans when associated with intake of alcoholic beverages . Subject hygiene ,84. Porpithelium . This maithelium .P. gingivalis LPS markedly regulates genes involved in epigenetic mechanisms [Chronic periodontitis is one of the most common inflammatory diseases in humans. Stimulation and permanency of inflammation are regulated by complex mechanisms among which epigenetic pathways have received special attention. Epigenetic modification includes chemical changes in DNA and associated proteins. This can lead to remodeling of the chromatin and activation/inactivation of gene transcription . Such chchanisms .Porphyromonas gingivalis is currently known as the only pathogen that produces peptidyl-arginine deiminase (PAD) [se (PAD) . This ense (PAD) ,90–92. Pse (PAD) ,93. The se (PAD) –97. Thusse (PAD)  reportedse (PAD) ,98,99, aP. gingivalis in ESCC, i.e. 61% in ESCC and 12% in adjacent tissues, while it was undetected in the normal esophageal mucosa. A similar distribution of lysine-specific gingipain and 16S rDNA of P. gingivalis was detected. Median serum levels of IgA and IgG against P. gingivalis were significantly higher in ESCC than in esophagitis and healthy controls [P. gingivalis were correlated with a worse prognosis in ESCC patients, particularly in those with stage 0–II or negative lymph node metastasis. Patients with both high IgA and IgG had the worst prognosis. Therefore, combining IgA and IgG may improve diagnosis and lead to better prognosis. The findings of this study implied that P. gingivalis might be involved in the pathogenesis of ESCC and that combination of several biomarkers is better than any single biomarker for its diagnosis. Therefore, P. gingivalis serum biomarkers could be important for detection of ESCC at an early stage and may improve diagnostic and prognostic performance.Esophageal cancer is the eighth most frequent cancer and the sixth leading cause of cancer death worldwide . There hcontrols . Conventcontrols . IgA forcontrols . FurtherP. gingivalis abundance tended towards a higher risk for ESCC, Tannerella forsythia was associated with a higher risk of esophageal adenocarcinoma (EAC). Other bacteria such as Neisseria and S. pneumoniae were associated with lower EAC risk. Porphyromonas gingivalis was also associated with ESCC lymph node metastasis and reduced survival time [Peters et al.  found inval time .P. gingivalis was detected preferentially and frequently in esophageal cancer and dysplasia of the esophagus, but rarely in matching non-cancerous portions. This bacterium was quite low or absent in cancers from the cardia or remaining stomach. The variance was ascribed to less tolerance of P. gingivalis to acidic pH values.In a study by Yuan et al. , P. gingP. gingivalis LPS-activated macrophage-conditioned medium promoted invasion of primary and metastatic cell lines that had been derived from stage II, III and IV HNSCO. Proliferation of HN4 cells (cell line from HNSCC) was inhibited. The tumorigenic invasion may have been mediated by induced macrophage Nitric oxide (NO) generation through the EGFR signaling pathway, but needs further study.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer world-wide with a 50–60% survival rate ,103. UsiAmong patients who undergo gastroscopy with biopsy for clinical indications, approximately one in 19 with dysplasia will develop gastric cancer within 20 years . In indiP. gingivalis may be important to the development of pancreatic cancer [ABO genetic variant [P. gingivalis and A. actinomycetemcomitans together with decreased relative abundance of the phylum Fusobacteria and its genus Leptotrichia were related to an increased risk of pancreatic cancer [P. gingivalis had a 59% higher risk of developing cancer than those without, and patients with A. actinomycetemcomitans had a 50% risk. Noticeable was that the oral microbial dysbiosis  preceded cancer development [P. gingivalis ATCC 53978, which is strongly associated with destruction of the periodontium, were associated with a > 2-fold increased risk to pancreatic cancer after adjusting for known risk factors, compared to those with lower antibody levels (≤200 ng/mL) [P. gingivalis ATCC 53978 were the best antibody marker for a high bacterial load and aggressive periodontitis. In addition, Ahn et al. [P. gingivalis can reach the pancreas and contribute in pancreatic carcinogenesis.Globally, about 338,000 people had pancreatic cancer in 2012, which made it the 11th most common cancer with the highest incidence and mortality rates found in developed countries . Positivc cancer ,111. Per variant . A prospn et al.  found inP. gingivalis in conjunction with established risk factors e.g. alcohol consumption, may play a role in increased cancer development. It has been suggested that in the presence of risk factors such as alcohol and tobacco abuse, the commensal oral microbiota may act synergistically in the oral cancer pathogenesis [P. gingivalis, a keystone pathogen in periodontitis, are discussed in the current review. Porphyromonas gingivalis is probably not the only carcinogenic agent in the oral microbiota where coinfection with other bacteria, e.g. F. nucleatum and Prevotella intermedia occurs. Not every person either carries P. gingivalis that may induce cancer, irrespective of periodontitis and may be associated with worse prognosis. Indeed, serum antibodies to P. gingivalis may be used to improve cancer diagnosis and prognosis. The fact that pancreatic and gastric cancers can be associated with P. gingivalis suggests that oral microorganisms, bacterially-secreted effectors, inflammatory cells and mediators travel with saliva and blood to distant sites and induce systemic carcinogenic effects. This is also supported by the finding that a greater serum level of P. gingivalis IgG can be associated with orodigestive cancer. It is compelling to propose that there may be a direct relationship between P. gingivalis and orodigestive cancers, where the contribution to carcinogenesis may be due to secondary intrusion of the oral microorganism outside of its primary location , yet still within anatomically continuous regions. More molecular epidemiological and mechanistic studies with larger cohorts and better controls will be important to answer these emerging questions.There has been an increasing appreciation of a direct relationship between oral infection/inflammation and cancer. The most convincing evidence for such a relationship applies to periodontitis and orodigestive cancers. This relationship exists even after confounding factors, e.g. smoking, body mass index, and socioeconomic status have been accounted for. The underlying molecular mechanisms have not yet been fully clarified, but murine models of periodontitis-associated carcinogenesis have recently shown that long-term chronic bacterial infection promotes OSCC through direct interaction with cancerous and pre-cancerous oral epithelial cells via toll-like receptors. Members of the oral microbiota may directly stimulate OSCC proliferation and induce expression of key molecules  that are implicated in tumorigenesis. It seems very possible that ogenesis . Althoug


Hymenolepis microstoma and combine this with spatial gene expression analyses of a selection of genes.Tapeworms are agents of neglected tropical diseases responsible for significant health problems and economic loss. They also exhibit adaptations to a parasitic lifestyle that confound comparisons of their development with other animals. Identifying the genetic factors regulating their complex ontogeny is essential to understanding unique aspects of their biology and for advancing novel therapeutics. Here we use RNA sequencing to identify up-regulated signalling components, transcription factors and post-transcriptional/translational regulators  in the transcriptomes of Larvae and different regions of segmented worms in the tapeworm H. microstoma v.2 genome assembly, demonstrating that the transcriptome profiles captured a high percentage of predicted genes. Contrasts made between the transcriptomes of Larvae and whole, adult worms, and between the Scolex-Neck, mature strobila and gravid strobila, resulted in 4.5–30% of the genes determined to be differentially expressed. Among these, we identified 190 unique GOI up-regulated in one or more contrasts, including a large range of zinc finger, homeobox and other transcription factors, components of Wnt, Notch, Hedgehog and TGF-β/BMP signalling, and post-transcriptional regulators . Heatmap clusterings based on overall expression and on select groups of genes representing ‘signals’ and ‘switches’ showed that expression in the Scolex-Neck region is more similar to that of Larvae than to the mature or gravid regions of the adult worm, which was further reflected in large overlap of up-regulated GOI.RNA-seq reads collectively mapped to 90% of the > 12,000 gene models in the Spatial expression analyses in Larvae and adult worms corroborated inferences made from quantitative RNA-seq data and in most cases indicated consistency with canonical roles of the genes in other animals, including free-living flatworms. Recapitulation of developmental factors up-regulated during larval metamorphosis suggests that strobilar growth involves many of the same underlying gene regulatory networks despite the significant disparity in developmental outcomes. The majority of genes identified were investigated in tapeworms for the first time, setting the stage for advancing our understanding of developmental genetics in an important group of flatworm parasites.The online version of this article (10.1186/s13227-018-0110-5) contains supplementary material, which is available to authorized users. Hymenolepis species and other tapeworms contribute to further morbidity, particular of children, and frequently co-occur with other helminth infections  summarised Gene Ontology biological process terms associated with differentially expressed gene models. Table S3.1. Larvae cf. Whole Adult. Table S3.2. Scolex-Neck cf. Mid. Table S3.3. Scolex-Neck cf. End. Table S3.4. Mid cf. Scolex-Neck. Table S3.5. Mid cf. End. Table S3.6. End cf. Scolex-Neck. Table S3.7. End cf. Mid.Additional file 4: Table S4. Zinc finger transcription factor genes of interest.Additional file 5: Figure S1. Vitellarium-associated and additional expression foci of the putative zinc finger transcription factor Hmic-zf621400. A Enlarged view of vitellarium expression in mature segments (boxed region of inset). B–C Punctate expression is also seen in some specimens, especially in immature segments . D Expression by vitelline cells distributed with ova becomes visible in the uterus of mature segments. Abbreviations: esv, external seminal vesicle; gp, genital pore; isv, internal seminal vesicle; sr, seminal receptacle; t, testis; u, uterus; vt, vitellarium. scale bars 100 μm , 200 μm .Additional file 6: Table S5. Gene-specific primers and predicted protein sequences of transcripts examined by WMISH.


Pterolophia sp. ZJY-2019. The origins of these intergenic spacers could be explained by the slipped-strand mispairing and duplication/random loss models. A conserved motif was found between trnS2 and nad1 gene, which was proposed to be a binding site of a transcription termination peptide. Also, tandem repeat units were identified in the A + T-rich region of all five mitogenomes. The monophyly of Lamiinae and Prioninae was strongly supported by both MrBayes and RAxML analyses based on nucleotide datasets, whereas the Cerambycinae and Lepturinae were recovered as non-monophyletic.Cerambycidae is one of the most diversified groups within Coleoptera and includes nearly 35,000 known species. The relationships at the subfamily level within Cerambycidae have not been convincingly demonstrated and the gene rearrangement of mitochondrial genomes in Cerambycidae remains unclear due to the low numbers of sequenced mitogenomes. In the present study, we determined five complete mitogenomes of Cerambycidae and investigated the phylogenetic relationship among the subfamilies of Cerambycidae based on mitogenomes. The mitogenomic arrangement of all five species was identical to the ancestral Cerambycidae type without gene rearrangement. Remarkably, however, two large intergenic spacers were detected in the mitogenome of  Cerambyydalinae  whereas speridae . Even ifThe mitochondrial genome is widely considered to be an informative molecular marker for species identification, molecular evolution, and comparative genomic research  due to iMordella atrata (Coleoptera: Mordellidae) and Naupactus xanthographus (Coleoptera: Curculionidae) (Phrixtothrix hirtus (Coleoptera: Phengodidae) and Teslasena femoralis (Coleoptera: Elateridae) (Pyrocoelia rufa (Coleoptera: Lampyridae), a 494-bp region in Hycleus chodschenticus (Coleoptera: Meloidae) and two large intergenic spacers of more than 30 bp in Hycleus species  . In additeridae) . The mitteridae) . Neverth species . Previou species .The phylogenetic relationships within Cerambycidae have yet to be fully resolved due to a lack of adequately convincing taxon sampling, and the monophyly of subfamilies within Cerambycidae need further discussion . With thOberea yaoshana, Thermistis croccocincta, Blepephaeus succinctor, Nortia carinicollis, Pterolophia sp. ZJY-2019) were captured from Jinxiu, Guangxi Zhuang Autonomous Region, China and were stored at −40 °C in the lab of JY Zhang . The specimens were identified by Dr. JY Zhang based on morphology. Total genomic DNA was extracted from the thorax muscle using Ezup Column Animal Genomic DNA Purification Kit .Five longicorn beetle specimens  (http://tandem.bu.edu/trf/trf.html)  . Two rRNndex.py)  based onndex.py) . The nucndex.py) . We usedndex.py)  to find ndex.py) . Composindex.py) . Tandem rf.html)  was usedPaleosepharia posticata, Diabrotica barberi and Diabrotica virgifera served as the outgroups (nad2 gene) from 38 complete or nearly complete mitogenomes  of the 13 protein-coding genes of 26 complete mitogenomes was used  accordinutgroups . To veriogenomes . These iogenomes . Each ofogenomes . Conservogenomes . Proteinogenomes  based onogenomes  and 3. BP26 of thogenomes  with theogenomes .O. yaoshana, T. croccocincta, B. succinctor, N. carinicollis, Pterolophia sp. ZJY-2019) were determined. Structures of the five newly sequenced entire mitogenomes are shown in T. croccocincta to 16,063 bp in Pterolophia sp. ZJY-2019. Every mitogenome of the five species possessed similar compositional profiles and featured the typical gene arrangement and orientation that have been hypothesized for most coleopteran insects (trnW-trnC-trnY triplet (N. carinicollis) to 79.1% (O. yaoshana) of the base pairs. A comparison of AT-skew and GC-skew showed that the AT skew of all mitogenomes was positive and the GC-skew was negative , but the nad4 and nad5 genes of O. yaoshana, T. croccocincta, B. succinctor used a single T residue as the terminal codon. The cox1 and cox2 genes of O. yaoshana, T. croccocincta and Pterolophia sp. ZJY-2019 also used a single T residue as the terminal codon. Functional terminal codons can be produced by partial terminal codons in polycistronic transcription cleavage and polyadenylation processes , AUU (Ile), UUU (Phe), and AUA (Met) as the most frequently used codons. All codons contained A or T nucleotides, indicating that the strong AT mutation bias obviously influenced the codon usage . FurthertrnL and trnV whereas the 12S rRNA gene was between trnV and the A + T-rich region. Due to the impossibility of faultless determination by DNA sequence alone, the terminus of the rRNA genes in coleopteran mitogenomes has been presumed to stretch to the border of the flanking genes  in the Pterolophia sp. ZJY-2019 mitogenome and the lowest in the N. carinicollis mitogenome (75.7%). The AT-skew of 16S rRNA and 12S rRNA showed great positivity, whereas the GC-skew was somewhat negative (The two expected rRNAs (16S rRNA and 12S rRNA) were found in the mitochondrial genomes of all five longicorn beetles. The 16S rRNA gene was situated between ng genes . Therefo beetles . The siznegative , which inegative .trnS1 displayed the classic clover-leaf secondary structure, whereas trnS1 lacked the dihydrouridine (DHU) arm and formed a simple loop  can also be observed  possessed 21 similar copies of tandem repeats consisting of a 58-bp fragment. The A + T-rich region of Priasilpha obscura (Coleoptera: Phloeostichidae) is known to possess 6 tandem repeats of a 132-bp fragment and Psacothea hilaris (Coleoptera: Cerambycidae) possesses 7 identical copies of a 57 bp tandem repeat  near the 12S rRNA gene and the other stretch was 17-bp in length . Previous studies have confirmed that the two poly-T stretches were structural signals for the recognition of proteins that performed a role in replication initiation  and some Hycleus species (Coleoptera: Meloidae). Coincidentally, a 5 bp consensus motif (TACTA) exists in the intergenic regions situated between trnS2 and nad1 of all five species studied here. This pentanucleotide motif is conserved across coleopteran lineages (Evania appendigaster (Hymenoptera: Evaniidae) possessed a 6 bp motif ‘THACWW’ and Chilo suppressalis  possessed a 7 bp motif ‘ATACTAA’, respectively  in both ends of the intergenic region. The formation of an intergenic region may be explained by slipped-strand mispairing ) was supported. However, T. gebleri as the basal position of Lamiinae with a high value, and B. lineolata and Apriona swainsoni were reliably recovered as a sister group. Our results concurred with the suggestion that B. lineolata was closely related to A. swainsoni, rather than T. gebleri. The results also placed Pterolophia sp. ZJY-2019 as a sister group of all remaining Lamiinae. Moreover, our results suggested that O. yaoshana clustered with Trachypachus holmbergi, as a sister group of T. gebleri. For the relationship within Cerambycinae, M. raddei, A. oenochrous and Obrium sp. NS-2015 were gathered into one clade and most closely related to the subfamily Prioninae rather than the remaining Cerambycinae, consistent with the morphological and molecular analyses in previous reports (The phylogenetic relationships were reconstructed based on the nucleotide data (13P26) with BI and ML methods . BI and  reports .The results from the BI trees of the nucleotide dataset showed that Lepturinae cluster with the clade (Cerambycinae + Prioninae) with a high support value . HoweverThe most controversial point in our results was in Cerambycinae , which ws. l. In addition, BI and ML analyses recovered the monophyly of Prioninae including Callipogon relictus, Dorysthenes paradoxus and Aegosoma sinicum, as proposed by Spiniphilus spinicornis (Vesperidae). It has been well accepted that Necydalinae and Lepturinae have a close relationship. The monophyly of Lepturinae was recovered in both BI and ML analyses of the 13P26 dataset. However, BI and ML trees from the 12P38 dataset returned a paraphyletic Lepturinae, due to a sister relationship between Necydalis ulmi  and Brachyta interrogationis  with BI and ML methods . Prioningationis .S. spinicornis as a species of Vesperinae in Cerambycidae  and the genes outside the map are coded on the majority strand (J-strand) whereas the genes inside the map are coded on the minority strand (N-strand). The second circle shows the GC content and the third shows the GC skew. Both GC content and GC skew are plotted as the deviation from the average value of the total sequence.Click here for additional data file.10.7717/peerj.7633/supp-3Figure S2The first circle shows the gene map  and the genes outside the map are coded on the majority strand (J-strand) whereas the genes inside the map are coded on the minority strand (N-strand). The second circle shows the GC content and the third shows the GC skew. Both GC content and GC skew are plotted as the deviation from the average value of the total sequence.Click here for additional data file.10.7717/peerj.7633/supp-4Figure S3The first circle shows the gene map  and the genes outside the map are coded on the majority strand (J-strand) whereas the genes inside the map are coded on the minority strand (N-strand). The second circle shows the GC content and the third shows the GC skew. Both GC content and GC skew are plotted as the deviation from the average value of the total sequence.Click here for additional data file.10.7717/peerj.7633/supp-5Figure S4The first circle shows the gene map  and the genes outside the map are coded on the majority strand (J-strand) whereas the genes inside the map are coded on the minority strand (N-strand). The second circle shows the GC content and the third shows the GC skew. Both GC content and GC skew are plotted as the deviation from the average value of the total sequence.Click here for additional data file.10.7717/peerj.7633/supp-6Figure S5The first circle shows the gene map  and the genes outside the map are coded on the majority strand (J-strand) whereas the genes inside the map are coded on the minority strand (N-strand). The second circle shows the GC content and the third shows the GC skew. Both GC content and GC skew are plotted as the deviation from the average value of the total sequence.Click here for additional data file.10.7717/peerj.7633/supp-7Figure S6O. yaoshana (2): T. croccocincta (3): B. succinctor (4): N. carinicollis (5): Pterolophiasp. ZJY-2019 A: trnI ; B: trnQ; C: trnM; D: trnW; E: trnC; F: trnY; G: trnL (UUA); H: trnK; I: trnD; J: trnG; K: trnA; L: trnR; M: trnN; N: trnS (AGN); O: trnE; P: trnF; Q: trnH; R: trnT; S: trnP; T: trnS (UCN); U: trn L (CUA); V: trnV.(1): Click here for additional data file.10.7717/peerj.7633/supp-8Supplemental Information 2Click here for additional data file.10.7717/peerj.7633/supp-9Supplemental Information 3Click here for additional data file.10.7717/peerj.7633/supp-10Supplemental Information 4Click here for additional data file.10.7717/peerj.7633/supp-11Supplemental Information 5Click here for additional data file.10.7717/peerj.7633/supp-12Supplemental Information 6Click here for additional data file.10.7717/peerj.7633/supp-13Supplemental Information 7Click here for additional data file.


Anopheles funestus is one of the 3 most consequential and widespread vectors of human malaria in tropical Africa. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis in this important mosquito.A. funestus reference genome (AfunF3) assembled using 240× coverage of long-read single-molecule sequencing for contigging, combined with 100× coverage of short-read Hi-C data for chromosome scaffolding. The assembled contigs total 446 Mbp of sequence and contain substantial duplication due to alternative alleles present in the sequenced pool of mosquitos from the FUMOZ colony. Using alignment and depth-of-coverage information, these contigs were deduplicated to a 211 Mbp primary assembly, which is closer to the expected haploid genome size of 250 Mbp. This primary assembly consists of 1,053 contigs organized into 3 chromosome-scale scaffolds with an N50 contig size of 632 kbp and an N50 scaffold size of 93.811 Mbp, representing a 100-fold improvement in continuity versus the current reference assembly, AfunF1.Here we present a new high-quality A. funestus reference genome assembly will serve as an improved basis for future studies of genomic variation and organization in this important disease vector.This highly contiguous and complete  Many insect genomes remain a challenge to assemble, and mosquito genomes have proven particularly difficult owing to their repeat content and structurally dynamic genomes. These issues are compounded by the fact that long-read sequencing technologies typically require >10 μg of DNA for library construction. As a result, it is often impossible to construct a sequencing library from a single individual. Instead, it has been necessary to sequence a pool of individuals from an inbred population . HoweverAnopheles species using short-read Illumina sequencing [Anopheles assemblies are highly fragmented collections of relatively short scaffolds, causing gene annotation problems such as missing genes, missing exons, and genes split between scaffolds or sequencing gaps. Thus, one of the consequences of fragmented assemblies is that it is difficult to estimate gene copy number, which may be linked to important phenotypic traits  [Motivated by the goal of genome-enabled malaria control, a large international consortium previously sequenced and assembled the genomes of 16 quencing , 4. Althistance) , 6. GenePlasmodium infection rates [A second major limitation of fragmented insect assemblies is that they are rarely scaffolded into chromosomes, owing to difficulty and lack of funding for physical or linkage mapping. Among other consequences, the unknown placement of scaffolds along chromosome arms means that their position within or outside of chromosomal inversions is difficult or impossible to determine. Many anopheline species are highly polymorphic for chromosomal inversions, which tend to occur disproportionately on particular chromosome arms . In a heon rates , 26. ThuAnopheles funestus (NCBI:txid62324) is one of the 3 most important and widespread vectors of human malaria in tropical Africa [Anopheles gambiae with which it broadly co-occurs, it is a relatively neglected species. It is considered even more highly anthropophilic and endophilic than A. gambiae and amenable to conventional indoor-based vector control such as bed nets and indoor spraying of houses with residual insecticides. Indeed, historical house spraying campaigns in eastern and southern Africa not only locally eliminated this species, but the effect was maintained for several years following the cessation of spraying, due to the apparent inability of A. funestus to recolonize some areas. Likewise, A. funestus was eliminated from a humid forest and degraded forest areas in West Africa where malaria is meso- or hypoendemic [l Africa , and unloendemic . Howeveroendemic , 32–34.A. funestus, but it remains understudied; information on its behavior and genetics lags far behind that of A. gambiae. At least part of the reason for its neglect may be the historical lack of laboratory colonies, a problem solved with the establishment of the FUMOZ colony and its registration with the Anopheles program of BEI Resources [. A. funestus shares with A. gambiae not only a broad sub-Saharan distribution and major vector status but also abundant chromosomal inversion polymorphism and shallow range-wide population structure [A. funestus, named Kiribina and Folonzo, seem to differ in larval ecology, and—importantly—they also differ in adult behaviors affecting vectorial capacity, most notably indoor resting behavior. Mechanistic understanding of the genomic determinants of these and other epidemiologically important phenotypic and behavioral traits ultimately depends on upgrading the A. funestus reference to a chromosome-based assembly in which the unanchored scaffolds are united, ordered, and oriented on chromosome arms.Mastery over malaria will require tackling esources . A. funetructure . Howevertructure . These cA. funestus genome (AfunF3), we first assembled contigs from long, single-molecule reads and then scaffolded these contigs into chromosome-scale scaffolds using Hi-C proximity ligation data. A similar strategy was recently used to improve the genome of Aedes aegypti [To achieve a complete and highly contiguous assembly of the  aegypti . An init aegypti . These aA. funestus chromosomes: 2, 3, and X  was then scaffolded using Hi-C Illumina reads to first bin the contigs into 3 chromosomes, followed by ordering and orientation of the contigs using the Proximo method . The final scaffolded assembly (AfunF3 primary) contains 210.82 Mbp of sequence and a scaffold N50 of 93.81 Mbp. The resulting scaffolds represent the entirety of the 3 Because single-molecule PacBio data are prone to insertion and deletion errors, all AfunF3 contigs were polished twice with Arrow  using thA. gambiae reference genome (AgamP4) confirmed a known reciprocal whole-arm translocation between 2L and 3R, as well as substantial intra-chromosomal shuffling  expression data to the assemblies and computed the number of concordant paired-end reads. A better assembly is expected to have both a higher fraction of mapped reads (completeness) as well as a higher fraction of correctly spaced and oriented pairs . Both primary and complete AfunF3 assemblies have better agreement of mapped read pairs as well as a higher overall mapping rate versus the AfunF1 assembly  cells of PacBio RSII sequencing using the P6-C4 chemistry were run by the core facility at the Icahn School of Medicine at Mount Sinai , resulting in 173× coverage (assuming a 250-Mbp genome size). A previous study generated 70× coverage of the same colony using the older PacBio P5-C3 chemistry sequencing [RRID:SCR_015880) [PacBio contig assembly was performed with Canu v1.3   using th_015880)  using deRRID:SCR_005476) [RRID:SCR_012091) [RRID:SCR_011798) [A. gambiae is known to be on the X chromosome [A. funestus assemblies.To order and orient the primary contigs along the chromosomes, Hi-C reads were aligned using Bowtie2   and scaf_012091) . This as_012091)  to the A_011798)  and mumm_011798) , and comromosome , this isBecause diploid and population variation introduces indels in the Arrow polishing process , the finA. funestus or Culicidae. The remaining sequences were primarily unannotated or annotated at a higher taxonomic level (3.76%), from possible bacterial/human sources , and had slightly lower guanine-cytosine (GC) content  [RRID:SCR_013048) [RRID:SCR_008992) [Paired-end RNA-Seq for the _015530)  and asse_013048)  with def_008992) . TranscrRCWQ00000000. The version described in this paper is version RCWQ01000000. Supporting data and materials are available in the GigaScience GigaDB database [Raw genomic sequence reads are available in the NCBI Sequence Read Archive under project accession PRJNA494870. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession database .bp: base pairs; BUSCO: Benchmarking Universal Single-Copy Orthologs; FISH: fluorescence in situ hybridization; GC: guanine-cytosine; GWAS: genome-wide association studies; kbp: kilobase pairs; Mbp: megabase pairs; NUCmer: NUCleotide MUMmer; PacBio: Pacific Biosciences; rDNA: ribosomal DNA; RNA-Seq: RNA-sequencing; NCBI: National Center for Biotechnology Information; QV: quality value; SMRT: single-molecule real-time; SRA: Sequence Read Archive.The authors declare that they have no competing interests.https://hpc.nih.gov).Physical mapping and data production were supported by the US National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) grant R21 AI112734 to N.J.B. S.T.S. and N.J.B. received support from NIAID grant R21 AI123491 and Target Malaria, which receives core funding from the Bill & Melinda Gates Foundation and from the Open Philanthropy Project Fund, an advised fund of Silicon Valley Community Foundation. J.G., S.K., and A.M.P. were supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health. This work utilized the computational resources of the NIH HPC Biowulf cluster (A.M.P. and N.J.B. conceived and coordinated the project. J.G., S.K., S.T.S., and A.M.P. performed the genome assembly, validation, and comparative analyses. S.R. provided the 10X Genomics data and analysis. P.H. provided FUMOZ samples for sequencing. J.G., A.M.P., and N.J.B. drafted the manuscript. All the authors have read and approved the manuscript.giz063_GIGA-D-18-00496_Original_SubmissionClick here for additional data file.giz063_GIGA-D-18-00496_Revision_1Click here for additional data file.giz063_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giz063_Reviewer_1_Report_Original_Submission -- Monika Gulia-Nuss1/30/2019 ReviewedClick here for additional data file.giz063_Reviewer_2_Report_Original_Submission -- Brad Main2/8/2019 ReviewedClick here for additional data file.


To compare hemodialysis (HD) internal jugular vein (IJV) versus subclavian vein (SCV) catheters in terms of procedural complications, patients’ comfort, tolerance and cost effectiveness.Sixty six consecutive eligible adult patients planned for hemodialysis @ 3 sessions/ week for maximum 42 days in a private hospital at Sialkot, Pakistan were documented between March 2017 and April 2018. A group, IJV or SCV catheter was allotted to alternate subjects. The catheters were inserted as per practice guidelines. Record of catheter-related complications (CRCs) was computerized. Similarly, patients’ uncomfortability and expenditures on management of CRCs were recorded.M = 47, range 24-75 years) or catheter stay time (M = 40 days). The rate of vein damage or artery puncture was found higher in IJV than SCV group  during catheterization. The difference also existed in late CRCs such as bacterial infection (32.3 vs. 16.1%), or device dysfunctioning (9.7 vs. 3.2%) with an exception of mechanical kinking. All the patients of IJV or SCV group with missed (19.4 vs. 6.5%) or shortened HD sessions (22.6 vs. 12.9%) reported CRCs-based discomfort as a cause of the regularity. Moreover, the participants of IJV group consumed 69% of the total expenditures on CRCs management.Of 66 cases, 62  successfully completed the study. Baseline information showed male predominance , age (SCV is a better site for HD catheterization as it has comparatively lesser likelihood of complications, patients’ feel comfortable and it is also cost-effective.than IJV. A remarkable reduction in the excretory function of the human kidney can be observed on chronic kidney disease2st step towards efficient extracorporeal blood flow for hemofiltration. Similarly, the device acts as a bridge while switching from one type of permanent vascular access e.g. arteriovenous (AV) fistula to other type ‘AV graftHemodialysis (HD) is recommended as renal replacement therapy to sustain the life of the sufferer with low grade morbidity. Insertion of readily operational percutaneous double-lumen tunneled cuffed central venous catheter (CVC) at suitable vessel e.g. internal jugularviz. insertion, stay in period, and removal (rarely). Arterial puncture or catheter-site bacterial infection usually emerges just after catheter’s placement; hence termed as early complications. However, central venous stenosis,The experienced practitioner inserts the device carefully using National guidelines and modern technologies. However, likelihood of short or long term CVC-related complications still exists at any of the three stages viz. CVC-related complications , noncompliance with therapy due to patients’ feeling uncomfortable, or cost-effectiveness. To fill the gap, present work was planned. The aim of the study was to compare the outcomes of the HD catheterization at IJV and SCV in terms of the 3 areas. The findings will be useful for the professionals in general practice.Open-accessed literatureThis experimental cross sectional study was conducted between March 2017 and April 2018 in a setting ‘The Kidney Centre’, Sialkot, Pakistan.Consecutive patients (aged>18 years) of either sex who were recommended for urgent hemodialysis (HD), shifted from peritoneal dialysis to HD, or needed change in position of the catheter on dysfunctioning of the previous vascular access were included. However, patients with missing previous medical record, reporting prophylactic administration of antibiotics, renal carcinoma, hemodialysis frequency (<3> per week), or severe psychological/mental issues were excluded. Group IJV or SCV catheter was assigned to alternate subjects (n = 66 i.e. 33 cases/group) from computer-generated list.+ level was managed by inj. Calcium-Sandoz diluted I/V, inj. Sodabicarb 100 ml and 25% D/W 10 ml (2 ampoules) plus 6 units plain insulin I/V. Blind (i.e. without ultrasound guide) aseptic catheterization was commenced at locally anaesthetized site in vein of interest following Practice GuidelinesPre-assessment and management of the subjects was conducted by physical examination along with laboratory work for urea/creatinine, complete blood count, serum electrolytes; ultrasonography and ECG. The raised KInternational Classification of Diseases, Ninth Revision, Clinical Modification). The non-adherents were asked to give response (yes/no) against a question “Do CRCs cause non-adherence?” to assess the device-related uncomfortability. In each study group, the financial expenditures on management of the complications were pooled before %age estimation for cost-effectiveness.Baseline information of the subjects e.g. duration of dialysis were recorded. The infections were identified before classification vide codes of ICD-9-CMThe study was conducted after getting approval from the hospital ethics committee. Moreover, participation consent was mandatory for participants.Data of age (continuous variable) was processed for mean+/-SD (range) values using statistical tool in SPSS ver. 16.0 . Non-adjusted odd ratio (OR) were calculated for different complications to visualize the comparative efficiency of the catheters.SD = 14, range 24-75) years. More than 50% population was on urgent HD. However, others switched from peritoneal dialysis to HD, or on replacement of dysfunctional central venous access i.e. AV fistula/graft. On the average, the patients held the temporary subcutaneous double-lumen tunneled cuffed device for 40 days. was found, as: 1, 34], 4, 44], 4, and 0 .Unpleasant experience of HDM = 47, range 24-75) years and an Indian studyDominance of male population (47 out of 62) is in good agreement to demographic pattern of previously published dataSurvival of the patient lies in successful HD for patients awaiting/not awaiting kidney transplant.Catheter dysfunctioning is referred to failure in extracorporeal blood flow (Qb) of 300 mL/minutes. The reasons behind the problem include mechanical issues and/or thrombosis.Procedural complications and physical inconvenience in handling the catheter leads to missed and shortened dialysis sessions i.e. noncompliance/non-adherence to the scheduled RTT. The irregularities exert pressure on the clinicians to reduce the pathetic morbidityThe catheter-related complications (CRCs) e.g. malfunctioning of the deviceSubclavian vein is a better site for HD catheterization as it involves comparatively lesser likelihood of device-related complications, patients’ feeling uncomfortable and cost-effectiveness than internal jugular vein. The patients’ compliance with renal transplant therapy is comfort dependent. However, patient’s consent should also be respected while deciding the placement site. Randomized trials on mega level are needed before rational decision-making approach about suitable place for the device insertion.MNS: Conceived, designed, collected data, financing, and manuscript’s final editing.HA and MM: Collected data, and supported in manuscript writing.MH: Applied statistical analyses, edited (language) and reviewed the manuscript.


Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus are phenotypically and genotypically closely related, and together comprise the L. casei group. Although the strains of this group are commercially valuable as probiotics, the taxonomic status and nomenclature of the L. casei group have long been contentious because of the difficulties in identifying these three species by using the most frequently used genotypic methodology of 16S rRNA gene sequencing. Long used as the gold standard for species classification, DNA–DNA hybridization is laborious, requires expert skills, and is difficult to use routinely in laboratories. Currently, genome-based comparisons, including average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH), are commonly applied to bacterial taxonomy as alternatives to the gold standard method for the demarcating phylogenetic relationships. To establish quick and accurate methods for identifying strains in the L. casei group at the species and subspecies levels, we developed species- and subspecies-specific identification methods based on housekeeping gene sequences and whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral pattern analysis. By phylogenetic analysis based on concatenated housekeeping gene sequences , 53 strains were separated into four clusters corresponding to the four species: L. casei, L. paracasei and L. rhamnosus, and Lactobacillus chiayiensis sp. nov. A multiplex minisequencing assay using single nucleotide polymorphism (SNP)-specific primers based on the dnaK gene sequences and species-specific primers based on the mutL gene sequences provided high resolution that enabled the strains at the species level to be identified as L. casei, L. paracasei, and L. rhamnosus. By MALDI-TOF MS analysis coupled with an internal database and ClinProTools software, species- and subspecies-level L. casei group strains were identified based on reliable scores and species- and subspecies-specific MS peaks. The L. paracasei strains were distinguished clearly at the subspecies level based on subspecies-specific MS peaks. This article describes the rapid and accurate methods used for identification and classification of strains in the L. casei group based on housekeeping gene sequences and MALDI-TOF MS analysis as well as the novel speciation of this group including L. chiayiensis sp. nov. and ‘Lactobacillus zeae’ by genome-based methods. Lactobacillus is the largest genus of the family Lactobacillaceae. As of May 2018, it consists of 196 validly published species1, and these species are commonly isolated not only from environments associated with fermented food, such as fruits, meat, sourdough, vegetables, and wine, but also from the gastrointestinal and vaginal tracts of humans and animals  (L. casei strain Shirota and L. rhamnosus GG) are used worldwide in fermented dairy products or food supplements and as probiotics to enhance host health  . The widt health . AlthougL. casei group has been altered numerous times. In the Approved Lists of Bacterial Names ; (ii) L. paracasei comprising two subspecies, L. paracasei subsp. paracasei  and L. paracasei subsp. tolerans (including the previous subspecies L. casei subsp. tolerans); and (iii) L. rhamnosus (including the previously classified subspecies L. casei subsp. rhamnosus).The taxonomy of the al Names , L. casebspecies : (i) L. Lactobacillus has mostly depended on morphological, physiological, and biochemical traits, and scientists have principally depended on commercial identification kits, such as, the API 50 CHL system  stated that the current taxonomy of the L. casei group is comprised of three closely related species: L. casei (type strain: ATCC 393), L. paracasei subsp. paracasei (type strain: ATCC 25302) and L. paracasei subsp. tolerans (type strain: ATCC 25599), and L. rhamnosus (type strain: ATCC 7469) (L. casei group based on whole-genome sequencing (WGS), multilocus sequence analysis (MLSA), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), phenotypic characterization and species-specific PCR.On the basis of analyses of cellular soluble protein patterns, randomly amplified polymorphic DNA (RAPD) fingerprinting, and DNA–DNA hybridization (DDH), CC 7469) . Huang eL. casei group strains. DDH has long been used as a method for delineation of bacterial species because of its status as a conventional gold standard genotypic assay , L. casei group , Lactobacillus plantarum group  and Lactobacillus sakei group  are indistinguishable using 16S rRNA gene sequencing because of the high degree of similarity (as high as 99%) of the 16S rRNA gene sequences among the species  and RNA polymerase alpha subunit (rpoA) has been used to distinguish closely related LAB species of the genera Lactobacillus, Enterococcus, Leuconostoc, Pediococcus, and Weissella  is a reliable method of examining deep-level phylogenies and complex species groups , with the neighbor-joining, maximum-likelihood, and minimum-evolution methods used for phylogenetic analysis. The results showed that a novel species, L. chiayiensis sp. nov. (strains BCRC 81062T and BCRC 18859), comprised an independent cluster notably distinct from L. casei and ‘L. zeae.’ Furthermore, a split network tree based on the concatenated sequences of five housekeeping genes  was performed, and the 53 strains of the L. casei group were divided into three principal clusters: Cluster A ; Cluster B (comprising 27 strains of L. paracasei); Cluster C (comprising 21 strains of L. rhamnosus). Split network analysis revealed that Cluster A comprised three subclusters: subclusters A-1 (L. casei), A-2 (‘L. zeae’), and A-3 (L. chiayiensis sp. nov.) . Thus, multilocus phylogenetic analysis of the sequence data of ‘L. zeae’ indicated that this microorganism could be reclassified as an independent species or a subspecies of L. casei.L. casei group member  on the basis of variation in the V1 region of the 16S rRNA gene. These primer pairs were validated using 63 Lactobacillus isolates from cheeses. However, ambiguous results were obtained when these primers were used to analyze 76 wild isolates from dairy samples (milk and differently ripened cheeses) . These nine regions exhibit substantial sequence diversity among bacteria. The hypervariable regions contain species-specific sequences that are suitable for diagnostic assays . Ward ancheeses) . A specihamnosus . Protein methods . Studies members .L. paracasei subsp. tolerans, L. rhamnosus, and ‘L. zeae’ within the L. casei group  markers through RAPD-PCR analysis. SCARs are detected by PCR using specific primers designed based on the sequence data of RAPD fragment. Multiplex PCR assay of SCAR makers is useful method for reducing the costs and time for routine identification. A combination of RAPD-PCR and the SCAR technique has been applied to develop the species- and subspecies-specific primers for ei group .L. casei group by using rpoA gene sequence and performed a SNaPshot minisequencing assay to identify species belonging to the L. casei group. A SNaPshot minisequencing assay using dnaK as a target gene was also developed, and five species-specific SNP primers were subsequently designed by analyzing the conserved regions of the dnaK gene sequences. The specificity of this assay was evaluated using 63 L. casei group strains. SNaPshot minisequencing assay combined with group-specific PCR was confirmed to quickly, accurately, and inexpensively identify the L. casei group at the species level. By using species-specific PCR based on dnaJ gene sequences with SNaPshot minisequencing, strains of L. rhamnosus were identified with high accuracy at the species level; furthermore, the method determined the SNP haplotypes (SNaPshot is a primer extension-based multiplex system for detecting single nucleotide polymorphisms (SNPs) by using fluorescently labeled dideoxynucleotides (ddNTPs). Each fluorescent ddNTP emits a unique wavelength, which is subsequently represented as a particular color in a base-dependent manner. The extension products with fluorescent labeling can be visualized using capillary electrophoresis with automated sequencing. plotypes .Multiplex minisequencing is a method for determining the exact nucleotide located at a particular site, particularly when numerous SNPs are screened simultaneously within a single reaction tube. Only 40 min is required for minisequencing products to undergo automated fluorescent capillary electrophoresis, making this method quicker than direct sequencing. A total of 2.5 h is required for sequencing products to undergo capillary electrophoresis.5–107 cells) are required for protein extraction and analysis.MALDI-TOF MS is a phenotype-based method that can be used for the identification, classification, and dereplication of large numbers of microorganisms on the basis of the specific proteomic profiles of these microorganisms . AppliedBacillus pumilus and Bacillus safensis, Lactobacillus johnsonii and Lactobacillus gasseri, and Lactobacillus plantarum subspecies plantarum, and Lactobacillus plantarum subspecies argentoratensis)  . Thus, ratensis) . A ribosei group .L. casei group strains at the species and subspecies-level by using ethanol/formic acid/acetonitrile for protein extraction in a routine protocol  for compiling an in-house database given the optimized culture conditions . The reference strains of L. casei group were used to validate the in-house database. Forty-eight (100%) strains had high scores (mean: 2.45 ± 0.1) and were accurately identified at the species level, which was consistent with the results obtained using genotypic methods (housekeeping gene sequencing and species-specific PCR).protocol  with MS T was isolated from cow manure collected in Chiayi, Taiwan. Strain BCRC 18859 (=NRIC 1947) was isolated from coconut juice from the Philippines, which was distributed by the NODAI.During a study whose objective was to isolate lactobacilli from different environmental samples, strain BCRC 81062L. paracasei subsp. tolerans. These two strains could not be clearly identified as any recognized species of the genus Lactobacillus by 16S rRNA gene sequencing analysis.Research Institute Culture Collection  as L. casei group were divided into five distinct clusters: Cluster A comprised two novel L. chiayiensis strains (BCRC 81062T and BCRC 18859) and the ‘L. zeae’ strain; Cluster B comprised two strains of L. casei; Cluster C comprised 10 L. rhamnosus strains; Cluster D comprised 12 L. paracasei subsp. paracasei strains; Cluster E comprised two L. paracasei subsp. tolerans strains. Cluster A was divided into two subclusters: ‘L. zeae’ (BCRC 17942T) was the single strain located in independent subcluster A-1. Two strains of L. casei were located in subcluster A-2 . This result was in good concordance, with the exception of the subspeciation of L. paracasei in the MLSA analysis based on housekeeping gene sequences. In addition, species- and subspecies-specific MS peaks were observed in the MS profiles, and these peaks served as the specific markers for differentiating L. casei group strains at both species and subspecies levels (Table 1).Detecting and describing taxonomic novelty and reclassifying microbes has also been accomplished using MALDI-TOF MS .in silico methods based on genome-to-genome comparison can be used instead of the usual wet-lab DDH and conventional method for determining the DNA G+C content and sequencing of 16S rRNA and housekeeping genes of type strains  in the L. casei group based on a core genome phylogenetic tree, DNA G+C content analysis, and pairwise genome distances (ANIb and TETRA). These researchers also suggested that the heme-dependent catalase gene and the SOD-encoding gene play a role for molecular markers for the accurate identification of L. casei and L. paracasei.T and BCRC 18859). The ANI values were estimated using orthologous average nucleotide identity (OrthoANI) . A split network tree based on the fragmented all-against-all comparison of the whole genome sequences using Gegenees software to separate the novel strains and four type strains in the L. casei group could be separated into four clusters: Cluster A consisting of L. casei ATCC 393T (Accession No. GCA_000829055.1) and ‘L. zeae’ DSM 20178T (GCA_001433745.1); Cluster B consisting of two novel L. chiayiensis strains ; Cluster C consisting of L. rhamnosus JCM 1136T (GCA_000615245.1); Cluster D consisting of L. paracasei subsp. paracasei JCM 8130T (GCA_000829035.1) and L. paracasei subsp. tolerans DSM 20258T (NZ_AYYJ00000000) . The ANI and dDDH values between L. chiayiensis sp. nov. BCRC 81062T and type strains in the L. casei group ranged from 77.4 to 88.5, and 22.3 to 36.1%, respectively, which were markedly lower than the commonly used cut-off threshold  for delineation of prokaryotic species. Phenotypic and chemotaxonomic characterization as well as MLSA based on three housekeeping genes , species-specific PCR and whole-cell MALDI-TOF MS spectral pattern analyses revealed that the novel strains (BCRC 81062T and BCRC 18859) represented a single, novel species within the L. casei group, for which the name L. chiayiensis sp. nov. is proposed.rthoANI) , and dDDrthoANI) . The disrthoANI)  was plotL. casei group showed that 148 (4.9%) and 109 (3.6%) of the ORFs in ‘L. zeae’ DSM 20178T were orthologous to those of L. casei ATCC 393T and L. chiayiensis sp. nov. BCRC 81062T, respectively. This result indicated that ‘L. zeae’ DSM 20178T is more closely related to these two strains than other strains . However, the results of polyphasic characterization implied that the current L. casei group has at least four distinct species; L. casei, L. chiayiensis sp. nov., L. paracasei, and L. rhamnosus. L. paracasei comprises two subspecies: L. paracasei subsp. paracasei and L. paracasei subsp. tolerans . The species ‘L. zeae’ was described by L. casei ATCC 393T and ‘L. zeae’ DSM 20178T, were 94.6 and 57.3%, respectively, which were lower than the species delineation threshold. This has a possibility that ‘L. zeae’ DSM 20178T could be reclassified as the independent species or subspecies.Venn diagram analysis based on genome sequences of six type strains in the L. casei group species) involves two steps. First, 16S rRNA gene sequencing is used to ascertain the species-group level of an unknown strain. This is subsequently the foundation used to define the housekeeping genes  used to ascertain the species level of the strain by MLSA. Group- and species-specific primers in combination with conventional PCR and SNaPshot minisequencing analyses are used; these techniques are useful for direct identification and differentiation of strains in the L. casei group, in particular, the multiplex primer set based on housekeeping gene is the simplest and quickest method. Nevertheless, proteomic identification of species within the L. casei group, based on the in-house database and applying ClinProTools to MALDI-TOF MS data, exhibits superior discriminatory power (at the subspecies level) and requires shorter analysis duration than DNA sequencing and PCR-based methods. This method also facilitates efficient quality control of probiotic products. Due to technological advancement, DNA sequencing can be performed inexpensively, and high-quality, useful bioinformatic tools are available for classifying and identifying prokaryotes. Whole-genome-based comparisons, such as ANI and dDDH, have become the gold standards for demarcation of phylogenetic relationships as well as for delineating new species; these methods can replace traditional DDH, conventional determination of DNA G+C content, and 16S rRNA and housekeeping genes sequencing.Identifying closely related taxonomic groups (e.g., the C-HH analyzed the data and wrote the manuscript. S-WL analyzed the genome data. LH reviewed and revised the manuscript. KW designed this review, wrote the manuscript, and is a corresponding author. All authors read and approved 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.