Streptococcosis is an economical important bacterial disease that can seriously cause huge losses in the global aquaculture sector. In recent years studies have focused on to use extracts or essences of medicinal herbs and plants to control or treat the disease outbreaks and, in most cases the results were promising. The essential oils of the herbs or plants are more effective than the extracts and, the extracts examined have moderate efficacy in term of increasing fish survival against fish streptococcosis that could be due to the enhancement of fish immunity by the herb bio-compounds. The lack of dosage optimization, toxicity and bioavailability assays of a specific herb/plant or its bioactive compound in fish organs make it difficult to judge the validation of clinical efficacy of a particular herb/plant against fish streptococcosis, and thus, required further investigations. S. iniae and S. agalactiae, is a major re-emerging bacterial disease seriously affecting the global sustainability of aquaculture development. Despite a wide spread of the disease in aquaculture, few studies have been directed at assessing the in vitro antagonistic activity and in vivo efficacy of medicinal herbs and other plants against streptococcal agents. Most in vitro studies of plant extractives against S. iniae and S. agalactiae have found antibacterial activity, but essential oils, especially those containing eugenol, carvacrol or thymol, are more effective. Although essential oils have shown better anti-streptococcal activity in in vitro assays, in vivo bioassays require more attention. The extracts examined under in vivo conditions show moderate efficacy, increasing the survival rate of infected fish, probably through the enhancement of immunity before challenge bioassays. The available data, however, lack dosage optimization, toxicity and bioavailability assays of a specific plant or its bioactive compound in fish organs; hence, it is difficult to judge the validation of clinical efficacy for the prevention or treatment of fish streptococcosis. Despite the known bioactive compounds of many tested plants, few data are available on their mode of action towards streptococcal agents. This review addresses the efficacy of medicinal plants to fish streptococcosis and discusses the current gaps.Streptococcosis, particularly that caused by  Streptococcus species, Streptococcus iniae, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus parauberis, and Streptococcus phocae, are serious re-emerging bacterial diseases in humans and a wide range of terrestrial animals, fish, and marine mammals. Fish streptococcosis is one of the major infectious diseases in freshwater and marine aquaculture, affecting the sustainability of aquaculture development worldwide , factors for immune evasion , superoxide dismutase, D-alanylated lipoteichoic acid, adhesins, hyaluronate lyase, and methionine transport regulator [S. agalactiae in humans [Several virulence factors have been identified in pathogenic egulator ,83,84. Mn humans , no dataS. dysgalactiae, the M-like protein is the most extensively studied virulence factor. This protein can opsonize both adherence to and entrance into host cells [gfba, fnB, fbBA, fnBB, lmb and gapC) are known to mediate binding to fibronectin [gfba gene can also assist bacterial entry into host endothelial cells and intracellular persistence [S. dysgalactiae isolated from affected human and other warm-blooded animals. In addition, protein G, a known virulent factor in S. dysgalactiae strains, can bind with circulating immunoglobulins and, hence, interfere with the host humoral immune response [speG [S. dysgalactiae. In st cells ,87 and aronectin ,89,90,91sistence ,93. Mostresponse . Furtherresponse ,96 and sse [speG , the strS. uberis and S. parauberis in warm-blooded animals [S. parauberis carrying capsuled polysaccharide genes have recovered from diseased fish [Several potential virulence factors, including hyaluronic acid capsule, hyaluronidase, uberis factor, antiphagocytic factors , plasminogen activator/streptokinase factor, surface dehydrogenase protein, CAMP factor, lactoferrin binding protein and surface adhesion molecule, have been identified in both  animals ,99,100, sed fish , but theS. iniae, S. dysgalactiae, S. uberis and S. parauberis. Biofilms can facilitate the survival and proliferation of bacteria in hostile environments, such as aquaculture recirculation bio-filtration systems [The capacity to form biofilms has been reported for  systems ,102,103,S. phocae are mostly studied in marine mammals and rarely in fish species. Strains of S. phocae with antiphagocytic capsule ability are identified in experimental infections of Atlantic salmon  [S. phocae isolates responsible for outbreaks in Atlantic salmon. More detailed studies of these properties are, however, required, as no mortalities or histopathological findings were seen in the fish injected with extracellular products. Other virulence factors, including fibronectin-binding proteins, the toxin SLS, genes encoding for a capsule [S. phocae pathogenesis in aquatic animals, but these studies have mostly been in marine mammalians [Virulence factors of o salar) ,104,105.o salar) , cell-su capsule  and the Streptococcosis were 150 and 250 million USD in 2000 and 2008, respectively [S. iniae, and rainbow trout, yellow tail (Seriola quinqueradiata), Asian seabass  and Nile tilapia (Oreochromis niloticus) are highly susceptible species ,110StrepStreptococcus agalactiae is a globally emerging fish pathogen causing huge economic losses in many freshwater and saltwater species [Micropogonius undulatus), killfish (Menhaden spp.) and silver pomfret (Pampus argenteus) [ species . The bacgenteus) ,114,115.S. dysgalactiae subsp. dysgalactiae was reported in vaccinated and non-vaccinated farmed amberjack/yellowtail in Japanese fish farms [Seriola lalandi), grey mullet , basket mullet , cobia (Rachycentron canadum), hybrid red tilapia (Oreochromis sp.), pompano (Trachinotus blochii), white spotted snapper (Lutjanus stellatus), Amur sturgeon (Acipenser schrenckii), golden pomfret (Trachinotus ovatus) and Nile tilapia from Brazil, Japan, China, Malaysia, Indonesia, Taiwan and Iran [The first outbreak by the α-hemolytic Lancefield group C sh farms . Later, and Iran ,118,119.S. parauberis (formerly known as S. uberis type II) [Scophthalmus maximus), olive flounder, sea bass (Sebastes ventricosus), striped bass (Morone saxatilis) and ram cichlid (Mikrogeophagus ramirezi) [S. parauberis classified as serotype III has been reported as the cause of streptococcosis in different turbot farms in China, and the isolates are different from those that infect flounder  in Japan and South Korea but similar to strains in Spain and the USA [The first study reporting streptococcosis infection by type II)  in fish type II) . Subsequamirezi) ,124,125. the USA . S. uberis is an important causative agent of bovine mastitis worldwide. Although [S. uberis in mandarin fish (Siniperca chuatsi) in China, the first isolation and characterization of this streptococcal species from fish was reported by [S. uberis was isolated from the gills of healthy fish [Although  documentorted by  in Iranithy fish , and thuthy fish  is in doS. phocae subsp. salmonis was first isolated from clinical specimens from harbor seal (Phocae vitulina) by Skaar et al. [S. phocae has been recognized as an important pathogen of marine mammals, gray seal , ringed seal (Phoca hispida), Cape fur seal , southern sea otters (Enhydra lutris nereis), harbor porpoise (Phocoena phocoena) and other cetaceans, causing pneumonia or respiratory infection [Eumatopias jubatus) and skin abscesses in southern sea otters (Enhydra lutris nereis) [S. phocae is a serious disease in marine mammals.r et al.  before br et al. ,64. In anfection ,130,131. nereis) ,133, and nereis)  suggesteS. iniae causing bacteremia, cellulitis, meningitis and osteomyelitis [S. agalactiae [S. dysgalactiae subsp. equisimilus and S. dysgalactiae subsp. dysgalactiae [S. uberis in humans, although the accuracy of this identification is arguable [Some streptococci species are serious zoonotic pathogens, with myelitis , neonataalactiae ,136 and alactiae ,141,142.arguable . A problarguable ,144. In 3 and NO2. Overfeeding, overstocking and overhandling can also cause outbreaks of streptococcosis with high cumulative mortality [The immune system of aquatic animals can inevitably be suppressed by various stressors, which increases the animal’s susceptibility to pathogenic agents ,145. Strortality ,149,150.ortality . These portality , antibioortality . TreatmeS. iniae strains, with the ethyl acetate extraction being most efficacious [S. agalactiae strains [S. iniae isolated from diseased rainbow trout was inhibited by the herb's essential oil [S. iniae. At concentrations below those which affected bacterial growth , the essential oil decreased the hemolytic activity of S. iniae supernatant and significantly suppressed the transcription of the sagA gene of bacterial isolates [S. iniae infection via the inhibition of the production of streptolysin S. Rosemary contains bioactive compounds, including camphor, a-bisabolol, 1,8-cineole, terpineol-4-ol, a-terpineol and limonene, which possess antibacterial activity ,154. Sixicacious . The eth strains . The grotial oil ,158, alttial oil , the antOreochromis sp.) with powdered rosemary leaves  and ethyl acetate extract  significantly increased fish survival following S. iniae challenge  [w/w). Yilmaz et al. [O. mossambicus) for 45 days with 1% rosemary leaves, obtaining a survival rate of 83.37% following challenge with S. iniae, whereas Gültepe et al. [S. iniae challenge when the fish were fed herb leaves at 8% and 16%, but survival rates in both treatment dosages were comparable with oxytetracycline-treated fish  and were significantly higher than control fish  [S. agalactiae infection compared to control fish (27% vs. 24%), administration at 16% produced a higher survival rate (38%) [S. agalactiae challenge [Five days of feeding tilapia (control) . No signz et al.  fed tilae et al.  found 73urvival) . Fish feurvival) . While ste (38%) . Intereshallenge . These dhallenge .Zataria multiflora) were inhibitory towards clinical strains of S. iniae obtained from diseased rainbow trout [S. iniae obtained from diseased rainbow trout were exposed to sub-MICs of Shirazi thyme essential oil, a dose-dependent reduction was exhibited in the hemolytic activity of the bacterial supernatant containing streptolysin S, and the transcription of the sagA gene was significantly downregulated in the treated bacterial isolates, suggesting that the essential oil may be used as an alternative to the antibiotic treatment of S. iniae infection. The anti-S. iniae activity of the ethanolic extract of Shirazi thyme was significantly stronger than other tested plants including peppermint (Mentha piperita), savory (Satureja khuzistanica) and chamomile/camomile (Matricaria recutica) and was comparable to erythromycin [Both essential oil and ethanolic extract of Shirazi thyme  was more inhibitory than the ethanolic extract towards S. iniae activity in vitro [Thymus vulgaris) was inhibitory to S. agalactiae [Origanum vulgare) and eucalypts  revealed equal MIC and minimum bactericidal concentration (MBC) of 6.25 μL/mL [The essential oil of thyme  fry fed with 1.0% garden thyme for 45 days exhibited a significantly higher survival rate than control fish after challenge with S. iniae infection [S. iniae infection. The inhibitory activity of garden thyme to streptococcal infection could be in part due to citraconic anhydride, 1,8-cineole and thymol. For instance, thymol and citraconic anhydride are well-known antibacterial substances [Mozambique tilapia  exhibited moderate anti-S. iniae activity in vitro, with equal MIC and MBC values < 2 mg/mL [Cuminum cyminum) as a feed additive at 0.5–2.0% was efficacious against S. iniae infection, providing 62.5–89.5% survival in treated fish vs. 40% survival in control fish. The highest survival achieved in the fish fed 1% cumin, with no adverse side effect on the fish growth [S. iniae challenge, while there was no difference in survival between fish fed 0.5% (45%) and control fish (43%). As fish fed with 2% cumin powder had a lower survival (61%) than those fed 1% , there appears to be an optimum for the effective control of streptococcosis. Such anti-S. iniae activity by cumin may be in part due to substances such as limonene, α–pinene and 1,8–cineole, as these compounds can improve the antioxidant activity and immune responses [Ethanolic extract of black cumin  revealed high activity against both S. iniae and S. agalactiae strains, with the lowest MICs of 39 μg/mL and 31.2 µg/mL, respectively [Satureja montana), obtained by the decoction method, however, exhibited moderate antagonistic activity towards S. dysgalactiae species. These findings were supported by the chemical analysis of the savory essential oil, which contained carvacrol (up to 62.3%), thymol (up to 40.6%) and terpinene (up to 28.3%). The ethanolic extract of savory (Satureja khuzistanica) has also been shown to be inhibitory to S. iniae [The essential oil of Bakhtiari savory (ectively . AqueousS. iniae .Aloe vera) exhibited low inhibitory activity against S. iniae strains originally isolated from diseased rainbow trout [S. iniae effect of its ethanolic extract was comparable with erythromycin [Aloe arborescens), however, showed no anti-S. uberis activity [S. iniae challenge, compared to untreated fish [ at 1% or 1.5% produced a significant increase in disease resistance  with S. iniae challenge, compared to control fish  [S. iniae demonstrated higher survival (about 67–73%) than control fish (about 50%). No significant differences were seen between aloe-treated fish and fish treated with erythromycin at 80 mg/kg body weight, with no toxic effects for the fish at any concentration of the herb.Essential oil of aloe  . The finurvival) . In a suurvival) , a ten-dSalvia tomentosa) was moderately inhibitory to S. agalactiae [S. verticillate) [S. agalactiae was inhibited by lilac sage , balsamic sage (weak with water and methanol extracts) and Jerusalem sage (Phlomis pungens) (moderate with water and weak with methanol extract) [S. officinalis) extract at 0.5 to 1.5% in rainbow trout led to increased disease resistance against S. iniae challenge  at 1.5% and reduced resistance at lower dosages (about 55%), compared to <50% survival in control fish, with this effect possibly be due to the enhancement of fish immune responses, including increased leukocyte population, IgM level, lysozyme and complement activity [S. iniae activity of an ethanolic extract of garden sage  was comparable with erythromycin [S. iniae were orally treated with 0.5 to 1.5% of the plant extract for ten days, they had only slightly better survival than control fish and significantly lower survival than fish treated with erythromycin (80 mg/kg b.w.). The aqueous extract of balsamic sage  . In addiextract) . One-monactivity . The in hromycin , but wheMyrtus communis) extract revealed relatively high anti-S. iniae activity [Rhodomyrtus tomentos) exhibited a strong inhibitory effect against both S. iniae and S. agalactiae, with the lowest MIC of 7.8 μg/mL, much higher than oxytetracycline MIC (0.1 μg/mL) [S. agalactiae. Pre-treatment of S. agalactiae culture with the extract at sub-MIC (0.25 MIC) did not change the susceptibility of bacterial cells to the lysozyme compared to untreated cells, but the pre-treated bacteria were more sensitive to H2O2 exposure; the population of bacteria pre-treated with the extract at 0.25 MIC and 0.125 MIC decreased from 7–8 log10 CFU/ ml to an undetectable level (<2 log10 CFU/mL) within 1–2 h, while untreated cells were able to survive under H2O2 conditions [S. agalactiae with 0.25 MIC of rose myrtle leaf extract caused a significant reduction in the mortality (10%) of tilapia in an intraperitoneal challenge with the bacterium compared to 45–50% mortality in controls [The essential oil of myrtle  . The planditions . Furthercontrols . Syzygium aromaticum = Eugenia caryophyllus) was inhibitory against S. agalactiae [S. iniae and S. parauberis [S. iniae isolates indicates a greater bactericidal tendency from clove essential oil, whereas eugenol had more bacteriostatic activity at lower concentrations [S. iniae.While clove oil  demonstrated ameliorating inhibitory activity against S. iniae compared to their essential oil counterparts [The use of nano-emulsions of essential oils obtained from Tasmanian blue gum  essential oil was shown to be a promising inhibitor against isolates of S. iniae and S. parauberis, mainly due to bacteriostatic activity . One strnterpart , suggestOliveria decumbens) revealed inhibition against S. iniae, with the lowest and the highest MICs measured for the essential oil and aromatic water compounds, respectively [S. iniae, while the use of aromatic water at 0.125% or a combination of aromatic water and essential oil at 0.1% revealed slightly higher survival (64.28%). The survival rates of all treated fish were, however, higher than control fish. The antibacterial activity of this plant could be due to the high concentration (up to 52%) of bioactive substances such as carvacrol [Essential oil, ethanolic extract and aromatic water of Moshkoorak (ectively . Nile tiarvacrol .(Allium sativum) was strongly inhibitory towards S. iniae and S. agalactiae strains [O. niloticus) with garlic powder at 0.5 g and 1 g/100 g diet revealed significantly higher survival (54% at 0.5 g inclusion and 84% at 1.0 g inclusion) than control fish (10%) following a challenge infection with S. iniae [S. iniae infection.The ethanolic extract of garlic  strains ,167, andS. iniae . The incCinnamomum tamala) was inhibitory to S. iniae strains [Cinnamomum verum) showed high anti-S. agalactiae activity [S. uberis were sensitive to both the essential oil and aqueous extract of cinnamon (Cinnamomum cassia), possibly due to the presence of compounds such as cinnamaldehyde, eugenol, cinnamic acid, witherhin, mucilage and diterpenes [The ethanolic extract of Indian bay leaf  showed 60% and 68.7% survival, respectively, in a S. agalactiae challenge [Cinnamomum camphora) revealed significantly higher survival (80%) against S. agalactiae infection than control fish  [The seven-day feeding of tilapia with extract and powder of cinnamon  .Spatholobus suberectus) and streptomycin sulfate displayed antibacterial synergism against S. agalactiae in an agar well diffusion assay [S. alaglactiae infection in fish.There are very scarce data on the efficacy of medicinal plants in combination with antibiotics against streptococcal infections in fish. A combination of extract of Milletia dielsiana (on assay . Nile tiPimenta dioica) to Mozambique tilapia fry revealed that the use of allspice at 10 g/kg feed not only provided the best growth performance but also the highest protection  against S. iniae challenge [A-50-day oral administration of various dosages of allspice  exhibited inhibitory effects against S. uberis, with a best index value of 4.22 [Zingiber officinale) at 1% to olive flounder for 8 weeks demonstrated a significantly higher survival rate following S. inae infection  compared to control fish  [S. agalactiae [Water and acetone extract preparations of ginger bush  . This imurvival) , feedingalactiae .Origanum vulgare) demonstrated improved inhibitory activity against S. iniae compared to the essential oil counterpart [S. iniae activity could be in part due to carvacrol, a monoterpenic phenol, one the major antibacterial compounds found in oregano.The nano-emulsion of the essential oil of oregano  revealed a relatively high anti-S. iniae activity, according to Ghasemi Pirbalouti et al. [S. uberis isolates with identical MIC and MBC values [Using disk diffusion or micro-dilution methods, an ethanol extract of pomegranate  present in cruciferous vegetables were inhibitory against oraphane , and S. S. iniae .Ecklonia cava) and its ethyl acetate soluble fractions were better inhibitors than the E. stolonifera extract towards S. iniae and S. parauberis strains [E. cava by Kim et al. [n-hexane soluble fraction of the extract showed the highest anti-S. parauberis activity, with MICs ranging from 256 to 1024 μg/mL [S. parauberis isolates, suggesting a synergistic antibacterial activity.The methanolic extract of marine brown alga  and olive (Olea europaea) exhibited equal anti-S. iniae activity [Matricaria recutica) was more active than peppermint (Mentha piperita) [Quercus branti) and liquorice (Glycyrrhiza glabra) and essential oils of hogweed , tarhana herb (Echinophora platyloba), celeriac (Kelussia odoratissima) and lolopashmak (Stachys lavandulifolia) exhibited anti-S. iniae activity, although the essential oil of tarhana herb and the ethanol extract of oak showed the highest inhibitory activity [Mentha pulegium) was inhibitory against several clinical isolates of S. iniae recovered from diseased rainbow trout [Azadirachta sp.) leaves and turmeric rhizome (Curcuma longa) were effective against S. iniae with an identical MIC of 1000 μg/mL that could be due to bioactive ingredients such as saponins, tannins, flavonoids and polysterols, which are present in both herbs [Phyllanthus emblica), bohera leaves  and arjun (Arjun coomaraswamy) were inhibitory to S. iniae strains [Urtica dioica) at 3% supplementation for 8 weeks showed enhanced antagonistic activity against S. iniae compared to mucus from control fish [Cyprinus carpio koi) fed ferula (Ferula asafo etida) powder at 20 and 25 g/kg in the diet for nine weeks exhibited anti-S. iniae activity [Carthamus tinctorius) ethanolic extract (100 mg/kg body weight) significantly increased anti-S. iniae activity compared to control fish [S. iniae activity was seen in the sera of fish treated with a higher dosage (200 mg/kg body weight). Among forty natural Korean plants assessed by Kang et al. [S. iniae and S. parauberis strains, with the strongest inhibition with extracts of Jipsinnamul (Agrimonia pilosa), Dokhwal , N, Nadosongipul (Phtheirospermum japonicum), and Sumbadi (Dystaenia takesimana) providing the greatest anti-S. parauberis activity. Ethanolic extract of Korean blackberry (Rubus coreanus) inhibited the growth of S. iniae and S. parauberis at various concentrations (12.5–100 g/mL), with the extent of inhibition being dose-dependent [Cymbopogon flexuosus) exhibited strong activity against S. iniae and S. parauberis strains [Abies holophylla), Japanese black pine (Pinus thunbergii), southern Japanese hemlock (Tsuga sieboldii), and pitch × loblolly hybrid pine (Pinus rigitaeda) exhibited activity against S. parauberis, with Japanese black pine and southern Japanese hemlock oils showing the highest and the lowest actively, respectively [Ethanolic extracts of skullcaps  , probabliperita) ,164,165.activity . The essow trout . All aquth herbs . Ethanol strains . Skin murol fish . In a suactivity . In bothrol fish , but no g et al. , only thependent . The autectively .Trigonella foenum graecum) for 45 days exhibited 69% survival against S. iniae challenge, compared to 39% survival in control fish [Citrus sinensis) at 0.1 to 0.5% induced 46.67 to 58.33% survival compared to 13.3% in control fish, probably due to an enhancement of fish immune-physiological functions [S. iniae challenge compared to control fish. This may have been due in part to monoglycerides, lauric acid and monolaurin, which have been identified as components of coconut oil with antibacterial and immunostimulatory effects [Mozambique tilapia fed fenugreek  and blueberry (Cyanococcus) each at 1% to olive flounder resulted in significantly higher disease resistance, 66.7–76.7% survival, respectively, against S. iniae infection, compared to 5% survival in control fish [Echinacea purpurea)  revealed higher survival compared to control fish only in fish fed a 1.5 g/kg diet, after challenge with S. iniae infection [Eichhornia crassipes) to the rainbow trout diets significantly enhanced fish survival following S. iniae infection, with the highest survival obtained in fish fed 1% of the plant [Eight-week dietary supplementation of yacon  leaves were inhibitory towards S. agalactiae strains, but with different degrees of inhibitory activity [S. agalactiae infection, but in vivo studies need to confirm this. The aqueous extract of green chiretta (Andrographis paniculate) displayed anti-S. agalactiae activity and feeding leaf powder or dried aqueous extract to tilapia decreased fish mortality after challenge with S. agalactiae [Five flavonoids  obtained from lemon guava  including P. macrostomum, P. dubium, P. argemone, P. bracteatum, P. armeniacus microstigma and P. chelidonium folium exhibited anti-S. agalactiae effects, with ethanolic and methanolic extracts having greater efficacy than aqueous extracts [P. argemone showed the highest inhibitory effect, followed by the root, aerial parts and flowers of P. chelidonium folium. In addition, the results revealed that the isokinolin alkaloids of the examined plants possessed an antimicrobial effect [Anredera diffusa) was inhibitory to S. agalactiae strains [S. agalactiae activity was obtained with an ethanolic extract of everlasting (Helichrysum plicatum) [Nuphar lutea) with various solvents and the ethanolic extract of lydian broom (Genista lydia) displayed a moderate anti-S. agalactiae activity, but only a weak inhibitory effect was seen with extracts of European white-water lily  [Various extracts from aerial (leaf- stem), flower and root parts of six species of poppy plants  . Furtherea alba) .Cestrum auriculatum), rhatany (Krameria triandra) and sauco (Sambucus peruviana) [Hibiscus sabdariffa Linn) and alcoholic extracts of koon (Cassia fistula Linn) and banana (Musca saientum Linn) inhibited the growth of S. agalactiae strains [Asteracantha longifolia), crowfoot grass (Dactyloctenium indicum) and Indian borage (Trichodesma indicum) were inhibitory to S. agalactiae, with methanolic extractions showing a better inhibition [Ethanolic extracts of orange cestrum (ruviana) , both aq strains . Rosellehibition .Piper nigrum), curry leaf (Murraya koenigii), onion (Allium cepa) and Vietnamese coriander (Persicaria odorata), a methanolic extract of curry leaf was the most inhibitory towards S. agalactiae strains [Moringa oleifera) were more inhibitory towards S. agalactiae biotype II than garden apple (Aegle marmelos) [Toona sinenses) also exhibited activity against biotype II, while the ethanolic extract of the neem (margosa) tree (Azadirachta indica) showed moderate inhibitory activity [Among black pepper ( strains . Aqueousarmelos) . Chlorofactivity , and theactivity .Cratoxylum formosum) at 0.5–1.5% in feed enhanced the innate immune responses of tilapia and significantly increased the survival (44–90%) of fish against S. agalactiae challenge compared to control fish (18%), with significantly higher survival at higher dosages [Sophora flavescens) exhibited survival rates of 47.8 to 79.9% compared to about 20% survival in control fish after challenge with S. agalactiae [Carica papaya) seed and asthma weed (Euphorbia hirta) exhibited 70% survival against S. agalactiae challenge [Excoecaria agallocha) leaf at 20–50 mg/kg in the diet led to a significantly higher survival rate (40–63%) after challenge with S. agalactiae compared to control fish  [The oral use of the aqueous extract of mampat  induced an enhancement in fish immune responses  and increased disease resistance against S. agalactiae challenge [Crataegus hupehensis), dong quai  at equal ratios produced 70% survival vs. 35% survival in control fish following challenge with S. agalactiae, possibly due to enhancement in immune responses  [Aristolochia debilis) (fruit) and Chinese ginseng (Panax ginseng) (leaf) exhibited the highest therapeutic efficacy  in a S. agalactiae challenge [Eight weeks of feeding tilapia with an extract of Assam tea  . Among thallenge .S. dysgalactiae, with the strongest inhibition shown by trans-cinnamaldehyde [Portulaca oleracea) and common dandelion (Taraxacum mongolicum) exhibited higher inhibition of S. dysagalactiae strains than the aqueous extracts of the same plants [Withania somnifera), colocynth/bitter (Citrullus colocynthis) and black pepper (Piper nigrum) at various concentrations inhibited the growth of S. dysgalactiae strains, with the level of activity being dose-dependent [S. dysgalactiae was challenged with billygoat weed (Ageratum conyzoides), betel (Piper betle) and turmeric (Curcuma domestica), all herbs inhibited growth of the bacterium even at high bacterial concentrations [Plant-derived compounds including thymol, carvacrol, eugenol and trans-cinnamaldehyde were inhibitory towards aldehyde . The ethe plants . Methanoependent . The inhependent . When S.trations .S. uberis than for other streptococcal species in fish. As S. uberis is a zoonotic pathogen, the in vitro data discussed here are for isolates recovered from terrestrial animals. Four plant-derived compounds, including trans-cinnamaldehyde (TC), eugenol, carvacrol and thymol, were inhibitory to S. uberis isolates, with TC exhibiting better inhibition than the others [Usnea barbata) exhibited stronger anti-S. uberis activity than ethanolic extracts [S. uberis than ampicillin, erythromycin and penicillin [Minthostachys verticillate) essential oil and limonene were antagonistic to S. uberis strains, with limonene having a lower MIC value [Acacia nilotica) leaves [Crassula multicava) [S. uberis strains. The inhibitory activity of thorny acacia was dose-dependent and could be in part due to the bioactive compounds, including the carbohydrates, glycosides, phytosterols, phenols, saponins and flavonoids that are major constituents of this plant [There are fewer data available for phototherapeutic efficacy towards e others . Bioactiextracts . The extnicillin . Both peIC value . The aqu) leaves  and fairlticava)  were inhis plant .S. iniae and S. agalactiae has been assessed in vitro, but less attention has been paid to the correlation of in vitro data with in vivo clinical efficacy. Most in vivo studies have used only two susceptible fish species, tilapia and rainbow trout, and have usually found variable increased clinical efficacy against infections. The efficacy of phytotherapy depends to a substantial extent on the form of application, such as powder, essential oils or extracts. In addition, the method of inclusion, dose, duration of feeding, level of supplementation and the size of the host and species all affect the bioavailability of plants and plant products for the host species and, thereby, immunity and resistance against infection. In the case of the dietary usage of phytotherapy, it must be considered that pelleting heat can affect the functionality and the actual dose of the active ingredients. Thus, future studies are required to guarantee the safe and full efficacy of phytotherapy when included in aquafeed. Further, the mode of action and the functionality of phytotherapeutic products in the entire body needs more clarification using modern techniques . Although medicinal plants have been shown to have beneficial effects on human health and have a therapeutic effect against disease agents and on various organs of the body due to their active ingredients, their toxicity evaluation in the host is crucial before being formulated for use in aquaculture.Streptococcosis causes massive mortality and economic loss in the global aquaculture sector. Traditionally, chemotherapeutic drugs and antibiotics have been applied for reducing the impacts of streptococcal infections on aquatic organism, but this may have serious adverse effects on aquatic ecosystems, wildlife and humans. Medicinal plants and their extracts may provide more environmentally beneficial alternatives to antibiotics for the treatment and control of streptococcal infections in aquaculture. The efficacy of a large numbers of plant species against


Aspergillus oryzae is a well-known producer of KA, and its KA biosynthesis gene cluster has been identified. In this study, we showed that nearly all section Flavi aspergilli except for A. avenaceus had complete KA gene clusters, and only one Penicillium species, P. nordicum, contained a partial KA gene cluster. Phylogenetic inference based on KA gene cluster sequences consistently grouped section Flavi aspergilli into clades as prior studies. The Zn(II)2Cys6 zinc cluster regulator KojR transcriptionally activated clustered genes of kojA and kojT in Aspergillus flavus. This was evidenced by the time-course expression of both genes in kojR-overexpressing strains whose kojR expression was driven by a heterologous Aspergillus nidulans gpdA promoter or a homologous A. flavus gpiA promoter. Using sequences from the kojA and kojT promoter regions of section Flavi aspergilli for motif analyses, we identified a consensus KojR-binding motif to be an 11-bp palindromic sequence of 5′-CGRCTWAGYCG-3′ . A CRISPR/Cas9-mediated gene-targeting technique showed that the motif sequence, 5′-CGACTTTGCCG-3′, in the kojA promoter was critical for KA biosynthesis in A. flavus. Our findings may facilitate strain improvement and benefit future kojic acid production.Kojic acid (KA) is a fungal metabolite and has a variety of applications in the cosmetics and food industries.  Aspergillus and Penicillium [Aspergillus oryzae grown on steamed rice used in oriental food fermentation [Kojic acid (KA) is a secondary metabolite produced by fungi, including many species of icillium . It was entation ,3. The centation . KA is aentation  and its entation . In the entation .KA has been on the market for over sixty-five years. Charles Pfizer and Company, Brooklyn, New York, USA, was the first company to announce its attempt to manufacture this product . Though 14C-labeled isotope tracer studies have proven that KA is directly synthesized from glucose. A biosynthetic scheme for KA formation, which requires at most two or three enzymes, was then proposed [kojA-kojR-kojT, in the A. oryzae genome that are responsible for KA biosynthesis. These genes encode an oxidoreductase, a transcriptional activator, and a transporter, respectively [Despite being a secondary metabolite with an all-carbon backbone, KA is quite different from those in the polyketide category. Forming units and precursors for the biosynthesis of polyketides are breakdowns of primary metabolites, such as acetates. proposed . Howeverproposed . With thectively ,17.Aspergillus and Penicillium species, examined their gene organization structures, and delineated phylogenetic relationships among Aspergillus section Flavi species based on KA gene cluster sequences. We then explored the transactivation mechanism of the Aspergillus flavus KojR regulator on the expression of kojA and kojT using a heterologous gpdA promoter and a homologous gpiA promoter to drive kojR expression. Lastly, we identified a consensus KojR-binding motif in the promoter regions of section Flavi aspergilli to be an 11-bp palindromic sequence of 5′-CGRCTWAGYCG-3′ . Using a CRISPR/Cas9-mediated gene-targeting approach, we showed that the motif sequence, 5′-CGACTTTGCCG-3′, in the A. flavus kojA promoter was critical for KA biosynthesis.In this study, we surveyed the presence of KA gene clusters in A. flavus was grown on V8 agar medium, which contains 5% V8 vegetable juice  and 2% agar, pH 5.2. Kojic acid medium (KAM) for KA production [2HPO4·3H2O, 0.05% MgSO4·7H2O, and 2% glucose (pH 6.0). A potato dextrose agar medium  supplemented with 1 mM ferric ion also was used for the examination of KA production. All fungal cultures were grown at 30 °C in the dark.For spore production, oduction  was modiA. flavus NRRL3357 was retrieved from the National Center for Biotechnology Information (NCBI) genome database (https://www.ncbi.nlm.nih.gov/genome/). Analyses of the genome sequence was performed via CoGe , an online platform for sequence comparison and retrieval. The extracted complete KA gene cluster sequence of NRRL3357, derived from comparisons to A. oryzae KA gene cluster sequences  (https://www.genome.jp/kegg/), was used as the alignment template for BlastN genome assemblies of other aspergilli available in NCBI , previously known as AFLA_096040, AFLA_096050, and AFLA_096060, are now updated as AFLA_009845, AFLA_009846, and AFLA_009847.The genome sequence of  in NCBI . Alignedhttp://darlinglab.org/mauve/mauve.html) for multiple genome alignments [Aspergillus species  [The online system Mauve (ignments  was used species . Aligninny.html) .A. flavus strains deficient in the NHEJ pathway have been described in detail [kojR were amplified by PCR using AccuPrimeTM Pfx Taq polymerase  with primers listed in pyrG-containing plasmid, pPG28. The resulting deletion vector was linearized with SacI and SphI prior to polyethylene glycol-CaCl2 mediated fungal transformation [A. flavus SRRC1709 (=CA14PTS∆pyrG), was pyrithiamine-sensitive and uracil auxotrophic. All kojR deletion mutants were confirmed by diagnostic PCR and verified for their inability to produce KA. A deletion mutant, ∆kojR#4, was used for subsequent genetic complementation experiments.Gene deletion procedures via double-crossover recombination in n detail . This weormation . The recA. nidulans glyceraldehyde-3-phosphate dehydrogenase gene  promoter and trpC terminator and the A. oryzae pyrithiamine resistance gene (ptrA) as the selection marker [http://www.aspergillusgenome.org/) revealed that only the upstream 1.0 kb (579170 to 580140) is the gpdA promoter region, which is consistent with the previous report [gpdA promoter but contains the phosphate transporter gene, AN8040, was removed by HindIII and XhoI digestion followed by self-ligation. This derived cloning vector was named pHgpdA-trpC-PTR in this study. A full-length A. flavus genomic kojR coding sequence was PCR amplified with primers kojR-OE and kojR-STOP , an orthologue of Saccharomyces cerevisiae ecm33, is a highly expressed gene [gpdA promoter of pHgpdA-trpC-PTR to give pHgpiA-trpC-PTR. The vector had a unique NotI site and a unique SmaI site. Lastly, the full-length A. flavus kojR coding sequence was PCR amplified and cloned. The complementation vectors were individually transformed into the ∆kojR#4 mutant. Three KA-producing transformants from the A. nidulans gpdA promoter set  and the A. flavus gpiA promoter set  were selected based on visual inspection showing high levels of KA production  at a flow rate of 1.0 mL/min. The mobile phase was MeOH:0.1% H3PO4 (25:75), isocratic. The detection of KA was based on UV absorption at 265 nm. A linear standard curve constructed from standard KA concentrations was used to determine the amounts of KA in the filtrate samples.Spores of the six 4 of the kojR-overexpressing and the KuPG control strains, prepared from V8 medium plates, were inoculated, respectively, into 1.0 mL PDB in 2-mL microfuge tubes. The cultures were grown stationary at 30 °C for two days. Mycelia were then harvested and disrupted with ZR bashing beads using a Mini-Beadbeater-8 cell disrupter . Genomic DNA was purified using a ZR Fungal/Bacterial DNA MiniPrep kit . The concentration of genomic DNA was determined with a Thermo Scientific™ NanoDrop™ 2000 spectrophotometer. qPCR was used to determine the copy numbers of overexpression strains using 20 ng of DNA as templates. Three technical replicates were used for each sample. The primers were kojR-F: AATACCGACGATTCCGGTCG and kojR-R: TTTCCTCTTGCGCAGTTTGC.Spores of 1.0 × 106 spores. At 48 and 72 h, mycelia were harvested, rinsed with sterilized distilled water, and ground with a mortar and pestle under liquid nitrogen. Total RNA was extracted with a Direct-Zol RNA Miniprep Kit  and followed by DNase I treatment. qRT-PCR was carried out in a 20 μL reaction volume with the LuminoCT SYBR Green qPCR ReadyMix  and the reverse transcriptase enzyme mix  in an Applied Biosystems StepOneTM thermal cycler. The amplification conditions were as follows: an initial step of 48 °C for 30 min for reverse transcription reactions was followed by forty cycles, each consisting of 95 °C for 5 s and 60 °C for 20 s. Four technical replicates were used for each sample. Gene-specific primers used were listed in −ΔΔCt method.Cultures for qRT-PCR analyses were grown on duplicate Petri dishes (100 × 15 mm), each containing 25 mL PDB and 1.0 × 10http://meme-suite.org/tools/meme) [kojA and kojT promoters of the twenty-three aspergilli in the section Flavi. Sequences of around 370 and 380 nucleotides in the respective 5’-untranslated regions of kojA and kojT were extracted from genome assemblies   was usedsemblies  and uploPstI and KpnI sites of pAsp-AMA-gpdA-ptr (Addgene plasmid #191016) to give the resulting gene-targeting vectors. The target sequences in kojA and kojT for the CRISPR/Cas9 complexes were GGTGGAATGAGCGGCAAAGTCGG and AAGCCATTCAGCGGCTAAGTCGG, respectively (motif sequences are underlined). To construct a sgRNA expression cassette, two target-specific DNA fragments were first generated by PCR with primer sets of U6-F-P/kojA_R or kojT_R and kojA_F or kojT_F/U6-R-K, respectively  [Sequencing was performed to reveal defects in MA, USA) . The PCRkojA-kojR-kojT in over 100 Aspergillus species, whose genome sequences are publicly available at NCBI. The sequence of the A. flavus NRRL3357 KA gene cluster (CP044621.1: 4359976 to 4365751), which resides on chromosome five, was the alignment template. We found that twenty-three known species contained the complete KA gene cluster (Flavi section (“section” is a taxonomic rank in-between genus and species). Phylogenetic inference based on variations in conserved sites in the KA gene clusters showed that they were grouped into six clades  were in one clade, well separated from A. parasiticus, A. novoparasitcus, A. sojae, A. arachidicola and A. transmontanensis, which were in another clade. The phylogeny also showed that A. oryzae was closer to A. aflatoxiformans than to S-morphotype A. flavus, and it was adequately separated from A. minisclerotigenes. All known aflatoxin producers, A. flavus, A. pseudonomius, A. aflatoxiformans, A. parasiticus, A. novoparasiticus, A. arachidicola, A. minisclerotigenes, A. transmontanensis, A. nomiae, A. luteovirescens, and A. sergii had complete KA gene clusters. We investigated the presence of a complete KA gene cluster, that is,  cluster . They exx clades . A. flavkojR homolog (An09g05060) in Aspergillus niger, which encodes a protein with 63% amino acid sequence identity to A. oryzae and A. flavus KojR proteins, has been reported [kojT homolog with 78% amino acid sequence identity. However, we did not find homologous genes encoding the KojA oxidoreductase next to or adjacent to An09g05060. Therefore, A. niger contained only a partial KA gene cluster. We identified homologous proteins from other twenty-one aspergilli, including A. avenaceus, a section Flavi species  as an alignment template, we obtained the same results. The Penicillium KojR and KojT amino acid sequences were 50–56% and 70–73% identical to those of the homologs of A. steynii and A. tanneri, respectively). However, many of the originally annotated Penicillium KojR proteins varied greatly in their size, and some were mis-annotated. We found that only kojR and kojT of P. nordicum were organized as a partial gene cluster  in the gpdA promoter set contained more than one copy of kojR. In contrast, the strains of the gpiA set contained two to three copies of kojR 2Cys6 zinc cluster domains, CETCKLRKRKCDGHEPCTYCLRYEYQC . To identify possible consensus KojR-binding site(s), we examined sequences in the kojA and kojT promoter regions of twenty-four aspergilli, including A. flavus L- and S-morphotype. kojA and kojT promoter sequences of A. flavus shared various degrees of nucleotide sequence identity with those of the other twenty-two aspergilli, which ranged from 63.9% to 100% and 60.5% to 99.5%, respectively. We next performed motif analysis using MEME on sequences from half of the intergenic regions of kojA and kojR (370 bps upstream of kojA) and in the complete inter-gene regions of kojR and kojT (383 bps)  are present on the positive strands in the kojT promoters (relative to the ATG codon). In other words, putative motif sequences were found on both strands when viewed because of their palindromic nature.The alignment of zinc-cluster domains in section 383 bps) . We obta383 bps) . All putkojA and kojT promoters in KA biosynthesis, we carried out site mutation analyses in A. flavus using a CRISPR/Cas9-based genome editing technique. This technology is well-known for generating small deletions in targeted sequences. We analyzed twenty and thirty-four primary transformants of the kojA and kojT sets, respectively, for KA production by transferring them onto KAM plates. For the kojA set, no transformants were produced for KA. For the kojT set, twenty-three produced KA but eleven did not. PCR examination of the fifty-four transformants with location-specific primers (kojA set and seventeen transformants from the kojT set (fifteen KA producers and two KA non-producers) gave positive PCR products. Subsequent sequencing of these PCR products showed that the KA-nonproducing kojA transformants contained six types of sequence defects, which consisted of five types of deletions and one type of insertion. These defects disrupted various parts of the putative KojR-binding motif in the kojA promoter  that disrupted the putative KojR-binding motif in the kojT promoter. kojT start site (404 bps) deleted  Cohn (ATCC® 12892™), turned out to be misidentified. It is most likely a strain of A. parasiticus or a closely related species. Its genome size (41.2 kb), which is 10% larger than those of A. oryzae RIB40 and A. flavus, also supports this notion. Misclassification of Apergillus species has been a concern, especially in the genomic era [Aspergillus species by multi-locus sequence typing has been a conventional approach. The current study shows that using SNPs in the KA gene clusters is an equally valid method, and it can distinguish a misidentified species from correctly named aspergilli.A list of thirty-three accepted species and their synonyms in ublished . Most reentified . Our ana members . Therefoomic era . ClassifFlavi aspergilli has been reported. However, not all Flavi species can produce KA, for example, A. coremiiformis and A. avenaceus [A. coremiiformis was 20% smaller than the average  identified in 2010 is likely an incomplete gene cluster. More than one enzyme is required for KA formation [kojA gene product, an oxidoreductase, should be directly involved in the conversion of the glucose precursor or intermediate metabolites. However, neither kojR nor kojT, which encode a Zn(II)2Cys6 zinc cluster regulator and a transporter protein (efflux pump), respectively, possess enzymatic activities. Therefore, we cannot exclude the possibility that the inability of A. coremiiformis to produce KA is due to defects in another yet-to-be-identified gene(s). KA production by most sections venaceus ,29. The  average . Nonetheme clade . We notivariable , which mormation . The kojA. niger has been reported as being unable to produce KA [A. avenaceus to produce KA is probably due to not having a complete KA gene cluster. Other aspergilli not belonging to section Flavi but containing partial KA gene clusters nd yeast ,35,36. Tnd yeast . In the antially . In thisrestored . Howeverand kojT . This imae study , KA was  enzymes , may havA. oryzae kojA and kojT was proposed to be responsible for the regulation of kojA and kojT [A. oryzae KA gene cluster showed that this heptanucleotide sequence resided on different strands, that is, on the negative strand upstream of kojA but on the positive strand upstream of kojT (kojA and kojT might not be coordinately regulated by KojR. The proposed A. oryzae sequence (bold) was a part of 5′-AGTCGCGGCAAGG-3′on the positive strand upstream of kojR . Zn(II)2Cys6 type regulators like KojR bind as a dimer to both DNA strands [kojR coding sequence start codon, ATG, was approximately twice that from the motif site to the kojA start codon  in their kojA promoters 2Cys6-type proteins act as pathway-specific regulators [A. nidulans and A. flavus/A. parasiticus, respectively, have been characterized; and their binding motifs are 11-bp palindromic sequences [A. flavus KojR has the same binding capacity. Despite the initial characterization of KojR-binding sites in the present study, the final proof of the identity of the KojR-binding motif, including its actual length, will have to come from direct evidence of protein-DNA interaction, such as the use of ChIP technology. Future characterization of those yet-to-be-identified structural genes that are directly involved in KA formation will shed light on genuine KojR-binding sequences in the KA biosynthesis gene promoters.The CRISPR/Cas9 technology could be used as a complementary method to the ChIP-seq method for identifying DNA-binding motifs or the electrophoretic mobility shift assay (EMSA) for detecting protein-DNA interactions ,42. In toduction A. Hence,romoters A. It wasoduction B. Howevei et al. , the kojgulators . Three, equences ,44,45. A


CohesinDB includes 2043 epigenomics, transcriptomics and 3D genomics datasets from 530 studies involving 176 cell types. By integrating these large-scale data, CohesinDB summarizes three types of ‘cohesin objects’: 751 590 cohesin binding sites, 957 868 cohesin-related chromatin loops and 2 229 500 cohesin-related CRMs. Each cohesin object is annotated with locus, cell type, classification, function, 3D genomics and cis-regulatory information. CohesinDB features a user-friendly interface for browsing, searching, analyzing, visualizing and downloading the desired information. CohesinDB contributes a valuable resource for all researchers studying cohesin, epigenomics, transcriptional regulation and chromatin organization.Cohesin is a multifunctional protein responsible for transcriptional regulation and chromatin organization. Cohesin binds to chromatin at tens of thousands of distinct sites in a conserved or tissue-specific manner, whereas the function of cohesin varies greatly depending on the epigenetic properties of specific chromatin loci. Cohesin also extensively mediates cis-regulatory modules (CRMs) and chromatin loops. Even though next-generation sequencing technologies have provided a wealth of information on different aspects of cohesin, the integration and exploration of the resultant massive cohesin datasets are not straightforward. Here, we present CohesinDB ( Cohesin is an essential protein complex that encircles chromatin within its ring-shaped structure . While tWithin the past few decades, advances in next-generation sequencing (NGS) technologies have enabled the genome-wide analysis of cohesin from different aspects. However, the integration and exploration of the massive cohesin datasets are not straightforward. First, the chromatin binding sites of cohesin are highly diverse. Unlike typical transcription factors (TFs) or CTCF, cohesin does not show any binding motifs, impeding the computational prediction of cohesin sites. Cohesin can bind to chromatin at tens of thousands of distinct sites in a conserved or tissue-specific manner ,12, wherhttps://cohesindb.iqb.u-tokyo.ac.jp), a comprehensive multiomics cohesin database in human cells that integrates 2,043 ChIP-seq, ChIA-PET, Hi-C, Hi-ChIP, RNA-seq and microarray datasets from 530 studies (176 cell types). CohesinDB offers a sophisticated interface for browsing, searching, visualizing and analyzing cohesin datasets and three types of ‘cohesin objects’: cohesin binding, cohesin-related chromatin loops and CRMs. We examined the reliability of the cohesin information in CohesinDB by comparing it with known knowledge. We also compared CohesinDB with related databases. CohesinDB contributes a valuable resource not only for cohesin research but also for research on various genomic events, including chromatin folding, enhancer activity, CRMs, epigenetics, transcriptional regulation, and human diseases.Here, we present CohesinDB . RNA-seq reads were mapped with STAR version 2.7.3  were aligned with Bowtie2 version 2.4.1 , followeon 2.7.3 , and genon 2.7.3 . Differeon 2.7.3 . All of Only high-quality ChIP-seq data, defined according to a normalized strand coefficient (NSC) > 1.2 and number of peaks > 1000, were retained to construct cohesin binding information. After excluding indirect factors , we used 550 ChIP-seq datasets to generate the universal binding sites of cohesin. In total, we obtained 34 534 315 independent cohesin sites. We then retained cohesin sites identified in more than two ChIP-seq samples to increase the reliability of binding sites. Two sites at a distance of <200 bp were merged to avoid clustered small peaks. Finally, we obtained 751 590 cohesin binding sites (coverage 27.93% whole genome), each of which was assigned a unique ID, for the construction of CohesinDB.https://www.ebi.ac.uk/gwas/) and somatic mutations from the COSMIC project (https://cancer.sanger.ac.uk/cosmic). (vi) Hyperlinks to the associated cohesin loop objects and cohesin CRM objects are provided. (vii) Selected cohesin sites, related cohesin loops, and target genes of related cohesin CRMs are visualized in a genome browser.CohesinDB provides comprehensive annotations for each cohesin binding site . (i) Basundaries , chromatundaries  and A/B undaries . (iv) Thundaries , chromatundaries , co-bounundaries , and tarundaries   <0.05. The loops obtained from Hi-C and Hi-ChIP were called by using HiCCUPS  with the2-fold change| >1 and FDR <0.01. Only DEGs identified in more than two studies were retained, to obtain a more reliable gene list. Then, cohesin sites less than two megabases away from the promoter of a DEG were considered regulatory sites, providing pairings between genomic loci and genes. The second type of evidence comes from cohesin-mediated loops. The pairing of cohesin sites with genes was identified based on cohesin-mediated loops that connect a cohesin site to the promoter of a gene. Cohesin sites directly located at promoters were also included in the cohesin-loop evidence. To obtain ‘double-evidenced’ CRMs, only pairs of cohesin sites with genes that appeared in both the cohesin-DEG and cohesin-loop evidence were retained. Finally, we obtained a total of 2 229 500 cohesin-related CRMs involving 15 203 target genes. Cell type information on the involved cohesin binding sites, chromatin loops and DEGs was recorded for each CRM.Cohesin-related CRMs are defined based on the pairing of cohesin sites with target genes, together with epigenomics and chromatin structural features. In CohesinDB, two types of evidence are used to identify cohesin-related CRMs. The first type, cohesin-DEGs evidence, comes from transcriptomic data in the context of cohesin depletion. Cohesin DEGs  were filtered according to a |Loghttps://gtexportal.org) and the cell-type specificity of the regulatory cohesin sites ; (iv) cohesin-DEG information, including whether the gene is a cohesin DEG, the cell types involved, the original studies of cohesin-DEGs, the number of studies involved, and the cohesin subunits involved; (v) cohesin-loop information, including hyperlinks to chromatin loop objects, the loop type, the cell types involved, the original studies of the loops and the cohesin subunits involved and (vi) visualization of selected gene, related cohesin sites and cohesin loops.CohesinDB assigned an ID to each gene and annotated  each genhttps://djangoproject.com) version 3.0.14. All data in CohesinDB were organized and queried by using MySQL (https://mysql.com) version 8.0.28. The front-end interface was implemented by using HTML, CSS, jQuery and JavaScript. Bootstrap (https://getbootstrap.com) was used to achieve a responsive web design. CohesinDB was deployed using Ubuntu 20.04 through Nginx web server (http://nginx.org) version 1.18.0. CohesinDB is available online at https://cohesindb.iqb.u-tokyo.ac.jp without any registration or login required. CohesinDB can be used in mainstream web browsers, including Google Chrome, Mozilla Firefox, Safari and Microsoft Edge, with JavaScript enabled. The CohesinDB Django project and the data processing script are available as open-source resources at https://github.com/wangjk321/CohesinDB_public.CohesinDB was written by using Python3 with Django framework (https://cohesindb.iqb.u-tokyo.ac.jp/help/).The contents of CohesinDB consist of four components: processed files obtained from raw sequencing data, and three types of objects concerning cohesin-binding sites, cohesin-related loops and cohesin-related CRMs . Using tThe ‘Browse’ page has a drop-down list that provides browsing and searching functions for three types of cohesin objects: cohesin-binding sites, cohesin-related loops and CRMs Figure . For eacBased on the CRMs and chromatin loops in CohesinDB, we designed an analysis page with three modules Figure . The firThe second module, ‘predict target gene’, takes a set of genomic regions as input and then discovers genes regulated by the input regions as well as the KEGG pathway enrichment of the discovered genes Figure . The outThe third module, ‘loop discovery’, takes a file containing genomic regions as input and then outputs chromatin loops anchored to the input region as well as the other end of the chromatin loops Figure . Users cUsers can also specify the cell type of interest when analyzing CRMs and chromatin loops. All three modules can be tested with a ‘Run example’ button. Given the extensive involvement of cohesin in transcriptional regulation and chromatin organization, these analyses can be used for a wide range of purposes.https://genome.ucsc.edu/). Visualizations in CohesinDB can be downloaded as PDF files.CohesinDB prepares visualizations of not only each cohesin object Figure  but alsoAll processed data from the 2043 samples are listed on the ‘Data’ page Figure . This pahttps://cohesindb.iqb.u-tokyo.ac.jp/pipeline/#maintain). The web server, web domain and SSL certificate of CohesinDB are provided by an academic institution, and we anticipate that CohesinDB will be maintained for a long time.The contents of CohesinDB will be maintained and updated in two ways: through a Django admin page, where we can manually modify the information of each dataset and cohesin object, or a programmable pipeline, through which we can process new datasets and update all contents . This is reasonable because cohesin plays context-specific roles in transcriptional regulation (P < 10−17), indicating a tendency for cell-type specific cohesin to regulate tissue-specific genes. Indeed, conserved cohesin and cell-type-specific cohesin exhibit a significant difference (P < 10−37) in the tissue specificity of the target genes . Taken together, the statistics and examinations above support the richness and reliability of the multiomics information contained in CohesinDB.Since the discovery of SMC  protein family in 1997 , the undNAR database issue, 2013) is a database designed for CTCF binding sites. Although some cohesin co-localizes with CTCF, many reports have shown that cohesin is different from CTCF .CohesinDB can be accessed online at gkac795_Supplemental_FileClick here for additional data file.


Helicobacter pylori (H. pylori) is one of the globally recognized causative factors of gastric cancer (GC). Currently, no definite therapy and drugs for H. pylori-related GC have been widely acknowledged although H. pylori infection could be eradicated in early stage. Inflammation and immune response are spontaneous essential stages during H. pylori infection. H pylori may mediate immune escape by affecting inflammation and immune response, leading to gastric carcinogenesis. As an important component of transcriptome, non-coding RNAs (ncRNAs) have been proven to play crucial roles in the genesis and development of H. pylori-induced GC. This review briefly described the effects of ncRNAs on H. pylori-related GC from the perspective of inflammation and immune response, as well as their association with inflammatory reaction and immune microenvironment. We aim to explore the potential of ncRNAs as markers for the early diagnosis, prognosis, and treatment of H. pylori-related GC. The ncRNAs involved in H. pylori-related GC may all hold promise as novel therapeutic targets for immunotherapy. Helicobacter pylori (H. pylori) has been well-accepted as one of the multiple pathogenic factors of GC. It has been listed as a primary carcinogen by the International Agency for Cancer  lymphoma may also develop in some patients  is one of the leading malignancies in terms of morbidity and mortality worldwide, which has attracted extensive attention among global health problems . Its higr Cancer , 3. Chropatients –7. HowevH. pylori-related GC, the virulence factors of H. pylori associated with immune response mainly consist of cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA), and H. pylori neutrophil-activating protein (HP-NAP), etc.  in human body could activate the NF-κB pathway and promote the chemotaxis of immune cells such as neutrophils and dendritic cells to release large amounts of inflammatory factors attempting to phagocytose H. pylori. However, the specific virulence factors of H. pylori could protect themselves from phagocytosis initiated by innate immune response. Consequently, the large quantity of inflammatory factors have to accumulate at H. pylori colonization sites and cause a long-term inflammation in epithelium, leading to chronic gastritis and even GC (As a key element of pathogenic factors for P), etc. . Among togenesis . CagA caresponse , 10. Vac. pylori . Unlike . pylori , 13. The even GC .H. pylori to escape from the eradication by immune system and colonize in gastric mucosa making carcinogenic effects. After H. pylori infection, innate immunity is first activated to release pro-inflammatory factors and immune cells intending to phagocytose and eliminate H. pylori, then local immune microenvironment is altered. Once the changes fail to resist the pathogenic effects of virulence factors, H. pylori could colonize in gastric mucosa, break through the barrier and survive in the deep site of stomach chronically, resulting in gastritis, gastric ulcer and even GC over time. Therefore, inflammation and immune response are essential parts during the initiation of H. pylori-related GC.The unique virulence factors enable H. pylori-related GC have focused on non-coding RNAs (ncRNAs). NcRNAs consist of microRNAs (miRNAs), long ncRNAs (lncRNAs), circular RNAs (circRNAs), small nucleolar RNAs (snoRNAs), and piRNAs  of target genes  with their 5′ ends . MiRNAs cascades . The strsistance . Therefosistance –28.H. pylori could induce miR-155 in gastric epithelial cells and gastric mucosa, which has been widely reported to damage H. pylori by triggering immune response , inhibiting cell apoptosis caused by DNA damage , Fas-associated death domain (FADD) as well as Sma- and Mad-related protein 2 (SMAD2), which negatively regulated the release of IL-8 and growth-related oncogene-α (GRO-α). Except for miR-155, myeloid differentiation protein 88 (MyD88) was also observed to be involved in the negative regulation of H. pylori-induced inflammatory reaction as a target gene of miR-155 , a tumor suppressor protein with ATP enzymatic activity, to promote GC proliferation and migration. Therefore, H. pylori might participate in CagA-mediated biological effects in GC cells via the NF-κB/miR-223-3p/ARID1A axis  promoting cell proliferation. Accordingly, Gao et al. believed that miR-218 could be considered as a therapeutic target for H. pylori-related GC. The inhibition of NF-κB by increasing miR-218 expression level artificially might become a kind of therapy to prevent the progression from precancerous lesion to cancer . Further research revealed that gastric SLFN4+ cells infected with H. pylori might induce miR-130b and exhibit a T cell suppressive phenotype resulting in H. pylori-induced gastric metaplasia, tumor formation and growth  regulates H. pylori-induced inflammatory response by inhibiting miR-21 expression and the methylation level of target gene programmed cell death 4 (PDCD4)  . Furtherhylation . JARID1By for GC .H. pylori-related GC. H. pylori up-regulates the immune receptor CD300E by down-regulating miR-4270 to enhance the pro-inflammatory potential and impair the sterilization ability of macrophages  and the migration of GC cells was inhibited, suggesting that lncPVT1 might activate immune function to affect GC caused by H. pylori  patients treated with endoscopic submucosal dissection (ESD), the expression of miR-942 and miR-510 was suppressed while their target genes CD44 and CDH1 were increased in the group with high circFNDC3B expression when compared with the low expression group, which contributed to a higher recurrence rate in EGC patients consequently (H. pylori infection (H. pylori can upregulate circMAN1A2 expression in GC cells, and circMAN1A2 may promote proliferation, migration and invasion of H. pylori-induced GC by sponging miR-1236-3p to increase MTA2 expression (However, the association of circRNAs with h miRNAs . H. pyloequently . CD44 wanfection . Earliernfection . Additiopression .H. pylori-related GC, further investigations are needed to support circRNAs in serving as diagnostic biomarkers and therapeutic targets for H. pylori-related GC.In spite of the previous research on circRNAs associated with H. pylori-related GC although the known mechanisms are limited. H. pylori was suggested to be delivered into host cells by two snoRNAs enriched in outer membrane vesicles of bacteria including sR-2509025 and sR-989262, reduce LPS stimulation and inhibit IL-8 secretion, thereby mediating immune escape (H. pylori-related GC worth further exploration (Except for the highly studied miRNAs, lncRNAs, and circRNAs, ncRNAs also contain snoRNAs and piRNAs. The research on them could also be conducive to deeply understanding the association of ncRNAs with e escape . The assloration .H. pylori with ncRNAs has been extensively studied. NcRNAs have been clarified as a key link from persistent infection of H. pylori to GC and to make profound impacts despite the specific mechanisms to be confirmed. The regulation of ncRNAs on the immune microenvironment of body after H. pylori infection could be a therapeutic target for H. pylori in the future. The present review elaborated the bridge role of ncRNAs between H. pylori and GC from the perspective of immune inflammatory response, indicating that ncRNAs held promise as biomarkers for the early diagnosis, prognosis, and treatment of H. pylori-related GC. However, relevant exact molecular mechanisms need to be verified and more clinical data should be involved as additional evidence to the prognosis study, aiming to improve the survival and life quality of H. pylori-related GC patients.NcRNAs play critical roles in transcriptome with regulatory function in all aspects of physiological process, pathological process and disease progression. As one of the important pathogenic factors of GC, the association of B-GW, Y-KL, and ZL conceived and designed this study and revised the manuscript. A-RL and L-YJ collected the data for the review. A-RL and Z-WY wrote the manuscript. All authors contributed to the article and approved the submitted version.


Bifidobacterium strains have been sequenced, delivering valuable insights into their genetic makeup. However, bifidobacterial genomes have not yet been systematically mined for genes associated with stress response functions and their regulation. In this work, a list of 76 genes related to stress response in bifidobacteria was compiled from previous studies. The prevalence of the genes was evaluated among the genome sequences of 171 Bifidobacterium strains. Although genes of the protein quality control and DNA repair systems appeared to be highly conserved, genome-wide in silico screening for consensus sequences of putative regulators suggested that the regulation of these systems differs among phylogenetic groups. Homologs of multiple oxidative stress-associated genes are shared across species, albeit at low sequence similarity. Bee isolates were confirmed to harbor unique genetic features linked to oxygen tolerance. Moreover, most studied Bifidobacterium adolescentis and all Bifidobacterium angulatum strains lacked a set of reactive oxygen species-detoxifying enzymes, which might explain their high sensitivity to oxygen. Furthermore, the presence of some putative transcriptional regulators of stress responses was found to vary across species and strains, indicating that different regulation strategies of stress-associated gene transcription contribute to the diverse stress tolerance. The presented stress response gene profiles of Bifidobacterium strains provide a valuable knowledge base for guiding future studies by enabling hypothesis generation and the identification of key genes for further analyses.Over the last decade, the genomes of several IMPORTANCE Bifidobacteria are Gram-positive bacteria that naturally inhabit diverse ecological niches, including the gastrointestinal tract of humans and animals. Strains of the genus Bifidobacterium are widely used as probiotics, since they have been associated with health benefits. In the course of their production and administration, probiotic bifidobacteria are exposed to several stressors that can challenge their survival. The stress tolerance of probiotic bifidobacteria is, therefore, an important selection criterion for their commercial application, since strains must maintain their viability to exert their beneficial health effects. As the ability to cope with stressors varies among Bifidobacterium strains, comprehensive understanding of the underlying stress physiology is required for enabling knowledge-driven strain selection and optimization of industrial-scale production processes. With the single exception of the glutamate/gamma-aminobutyrate antiporter GadC, genes encoding transporters were not included in the study due to the difficulty of computationally determining the substrate specificity based solely on their protein sequence. All stress-associated genes included in this study are listed in Table S1 in the supplemental material.In this study, the prevalence of stress-associated genes in bifidobacterial genomes was investigated to gain new insight into the diverse stress physiology of the genus. A list of 76 genes that have been previously implicated in stress responses of Bifidobacterium strains (Table S2), representing 22 species . In the following sections, each category of stress-associated genes will be discussed along with the current knowledge on the studied genes and the homologs identified in this study. When very low protein sequence similarity was detected across homologs, the sequences of the homologs were more thoroughly compared through multiple-sequence alignment.To investigate the correlation between the presence of stress-associated genes and actual stress tolerance of Different stresses, such as oxidative and heat stress, have a detrimental effect on macromolecules, including DNA and proteins, making the PQC system and DNA repair systems crucial for a cell’s stress resistance , 46. So-B. breve UCC2003 exposed to heat, osmotic, and organic solvent stress  in B. pullorum subsp. gallinarum CACC 514 (only representative of B. pullorum species) . Moreover, all strains belonging to the B. adolescentis group, B. angulatum, B. indicum LMG 11587 (only representative of B. indicum species), and B. pullorum subsp. gallinarum CACC 514 . The lack of sufficient knowledge on the function of CspA and UspA in bifidobacteria makes the biological implication of the absence of these genes ambiguous. In general, cold shock proteins might serve as RNA chaperones under stress conditions   or in soCACC 514 , and somnditions , and uninditions . In B. bt stress , 31, whe was not . Interes UCC2003 , and a g species . Althoug species .Streptomyces, contain several copies of clpC  and HrcA (heat regulation at controlling inverted repeat of chaperone expression [CIRCE]) as well as the transcriptional activator ClgR (Clp gene regulator)  . The proc groups  with difc groups .B. breve UCC2003 in all three strains, except for the absence of a CspA homolog in B. adolescentis ATCC 15703. As in B. breve UCC2003, an HspR-associated inverted repeat (HAIR) motif was detected twice upstream of the dnaK operon in B. longum subsp. longum NCC2705 and B. adolescentis ATCC 15703 , with a tyrosine inserted into the adenine-rich extension. The presence of a single HAIR motif upstream of the dnaK operon was described for B. animalis subsp. lactis DSM 10140 , but not in B. animalis subsp. lactis BB-12. In B. adolescentis ATCC 15703, the ClgR motif was only detected upstream of the clpC gene and the clpP operon . Hsp20 was found to be strongly induced upon multiple stresses, including heat, high osmolality, hydrogen peroxide (H2O2), and starvation, and was proposed as a suitable biomarker for stress . In contrast to the previous hypothesis that Hsp20 is exclusive for isolates from the human intestine    , includiSM 16992 . Since aolerance , it can olerance . Interesolerance . Thereforculosis . To incrBifidobacterium strains, but the molecular basis of this variation is not fully understood , including H2O2, superoxide anions (O2·−), and hydroxyl radicals (OH·). In contrast to aerobic bacteria, O2-sensitive bacteria lack an efficient system to detoxify ROS. For the elimination of oxidative stress, the interplay of O2-scavenging and ROS-detoxifying enzymes is critical to avoid accumulation of H2O2, which is produced by some of these enzymes and decomposed by others.As for other stressors, the sensitivity to oxidative stress varies significantly among derstood , 58. The2 tolerances of Bifidobacterium strains, most of the 34 investigated genes that are related to oxidative stress were not conserved across species  , succinate dehydrogenase subunits (SdhA and SdhB), and all subunits of F1F0-ATPase in all bee isolates included in the analysis. Homologs of all these proteins were further found in B. subtile KCTC 3272  , which mironment . Previoupiration , which hpiration . In agreCTC 3272 , an isolCTC 3272 .2 consumption was previously only detected in B. asteroides PRL2011 but not in B. coryneforme LMG 18911, B. indicum LMG 11587, and B. actinocoloniiforme DSM 22766  and their homologs in other species  was highly conserved across genomes of the investigated species  and the delta subunit (part of the stator) of F1F0-ATPase  negative and catalase negative. However, genes predicted to encode SOD and catalase have been identified in the bee isolates, e.g., B. xylocopae subsp. nov. XV2 and B. asteroides PRL2011 (B. asteroides DSM 20089 (B. xylocopae XV2 (B. asteroides and B. actinocoloniiforme DSM 22766 (the only representative of B. actinocoloniiforme species) were found to have a catalase homolog . This strain has been reported to have low SOD activity and to be hypersensitive to O2 (B. longum subsp. infantis ATCC 15697 is significantly shorter than the SOD homologs in bee isolates (132 versus 206 amino acids). The putative SOD homolog was included in our homology search. Homologs with high sequence similarity to the putative SOD gene were detected in a subset of B. breve strains and B. longum subsp. infantis strains as well as in four bee isolates, in the latter with only 50% protein sequence identity . It remains to be confirmed if the gene actually encodes an SOD that might negatively influence the O2 tolerance in strains lacking a sufficient H2O2 detoxification system.Even though homologs of SOD from  sources –70. Spon homolog . Nonetheve to O2 , 69, 70.B. tibiigranuli TMW 2.2057 identified genes, including pyruvate oxidase, glutathione peroxidase, and the peroxide stress protein YaaA, that potentially contribute to O2 tolerance  to the reference in members of the B. pseudolongum and B. asteroides group  was induced in the O NCC2705 , 27. Oures group .B. longum subsp. longum NCC2705 as well as from 33 additional B. longum strains, B. coryneforme LMG18911, and B. indicum LMG 11587 (Data Set S1). In line with this, in Lactococcus lactis, a protein originally annotated as HemN was previously found to lack coproporphyrinogen III oxidase activity and was instead assigned to the protein HemW family with a putative function as a heme chaperone (The role of HemN (a key enzyme in heme biosynthesis) in the oxidative stress response remains to be understood . In conthaperone .Bifidobacterium strains, its protein sequence in B. longum subsp. longum NCC2705, B. animalis subsp. lactis BB-12, and B. asteroides PRL2011 was scanned for conserved amino acid motifs specific to the HemW family to investigate if HemN in bifidobacteria also belongs to the HemW family. All HemW-specific motifs were detected in the gene products of the three strains H oxidases have been characterized in Bifidobacterium strains: a b-type dihydroorotate dehydrogenase (DHOD) from the O2-sensitive B. bifidum ATCC 29521 and an NADPH oxidase (NPOX) from the O2-hypersensitive B. longum subsp. infantis ATCC 15697 . Overall, 94% and 89% of the analyzed strains possessed PyrK, PyrDb, and NPOX homologs . Therefore, it can be concluded that the sole presence of these genes is not indicative of O2 sensitivity. It seems more likely that the expression level of H2O2-forming NAD(P)H oxidases  that is similar to the active-site motif of peroxiredoxins (The lowest sequence similarity (<60%) was detected for NPOX homologs in strains of the redoxins . Thus, tB. longum subsp. longum NCC2705, described to possess relatively high intrinsic H2O2 tolerance . It is possible that the absence of the H2O2-forming activity of NPOX reduces the burden on the strain’s H2O2-detoxifying enzymes from intracellularly formed H2O2 and free capacity for the detoxification of extracellular H2O2. Therefore, it should be assessed if the strains lacking a homolog of NPOX show higher O2 tolerance than other strains of the species.Only olerance , and a f2O2 formation is the l-aspartate oxidase, which catalyzes the first step of de novo NAD biosynthesis by oxidizing aspartate to iminoaspartate. It has been suggested that both fumarate and O2 can serve as electron acceptors for l-aspartate oxidase, while using the latter results in the formation of H2O2  has been purified and characterized from a Bifidobacterium strain. However, an NADH oxidase upregulated in the O2-tolerant B. animalis subsp. lactis IPLA4549 upon oxidative stress was suggested to contribute to detoxification of O2 to H2O  in the active site is determinative of the four-electron reduction of O2 to H2O in the NADH oxidase  and showed similarity in the surrounding amino acid sequence. Thus, the NADH oxidase of all Bifidobacterium strains would be expected to have H2O-forming activity instead of H2O2-forming activity, as was previously suggested for B. longum subsp. longum NCC2705 , B. dentium, B. lemurum, B. subtile, and some strains of B. adolescentis, including B. adolescentis ATCC 15703, which is also known to be highly O2 sensitive (Table S3), lacked both genes  . In addition, species of the B. asteroides group and B. subtile lacked a BCP homolog , first detected in oxidase)  and indue stress , may conver H2O2 , 80, but species . The ana strains  and some homolog .B. animalis and B. pseudolongum strains shows low sequence identity to the query gene   . Besides-xxxx-C) .2+ and H2O2 that could otherwise react in Fenton reactions, resulting in OH· formation (2-sensitive strains of B. adolescentis and B. angulatum as well as relatively O2-tolerant strains of B. thermophilum (Table S3). In contrast, multiple B. longum and B. breve strains were found to possess a second copy of Dps.Additional ROS detoxification enzymes detected in bifidobacteria included the DNA-binding protein from starved cells (Dps) and the peptide-methionine sulfoxide reductase (MsrA/B). Dps protects cells from oxidative damage through binding genomic DNA and by sequestering free Feormation . MsrAB rormation . Strainsormation , includiB. dentium, B. choerinum, and B. pullorum subsp. gallinarum and isolates from lemurs. Due to the various O2 sensitivities of strains lacking a Dps and MsrAB homolog, their sole absence appears not to be indicative of O2 sensitivity.All Dps-negative strains also lacked a gene encoding MsrAB . MsrAB was further absent from nine additional strains of 2. Instead, the results indicated that the lack of multiple ROS-detoxifying genes, as observed for B. angulatum and some B. adolescentis strains, might cause exceptionally high sensitivity to O2. Strains of B. dentium, which have not been described as being particularly O2-sensitive, lacked the same ROS-detoxifying enzymes as B. angulatum and some B. adolescentis strains except for Dps . Gene regulation, the ratio of different enzymes involved in the ROS-detoxification system , glutamate-cysteine ligase (GCL), and aminopeptidase PepP, were found to be highly conserved . PPK1 isCM 12489 . PolyphoCM 12489 . GCL and strains , 11, 83.B. dentium Bd1  decarboxylase and formyl-CoA transferase are responsible for the detoxification of oxalate, a strong acid present in plant-based food. The expression of both enzymes was found to be induced upon acid stress in the acid-tolerant tium Bd1 . Our gen strains . Strains strains , may conBifidobacterium strains were proposed to contribute to pH homeostasis upon acid stress due to proton scavenging, including the glutamate decarboxylase (GadB) pathway. GadB catalyzes the conversion of glutamate to gamma-aminobutyrate (GABA), consuming an intracellular proton, while a glutamate/gamma-aminobutyrate antiporter (GadC) transports GABA out of the cell and exchanges it with another glutamate .Enzymes involved in amino acid metabolism in lutamate . Genes ed stress . Homolog strains . The abiis study . A B. loproducer , but nonB. aquikefiri CCUG 67145T and B. tibiigranuli TMW 2.2057T   , 61. ThuB. angulatum (47%). In agreement with their natural environment, no BSH homolog was detected in isolates from the digestive tract of bees or isolates from lemurs. Interestingly, lemurs lack the ability to produce glycine-conjugated bile salts  ; however, the biological significance of this remains unknown.Bile salt hydrolases (BSHs), which catalyze the deconjugation of primary and secondary bile salts, have been extensively studied in the context of bile salt resistance of bifidobacteria. However, it remains unclear if and how the activity of BSHs contributes to higher bile stress resistance , 91. Sev92–le salts , which ale salts , 94, 98 le salts , 100. ThB. breve NCFB 2258 has previously been suggested to contribute to tolerance to butanol , B. longum BORI and CCUG 30698, and B. animalis subsp. lactis ATCC 27673, which has already been described as having a genetic content distinct from that of other strains of the species  RNA polymerase sigma factor RpoE, in their genomes (RpoE_1 in B. breve UCC2003 (253 amino acids), resulting in a query coverage between 71% and 79% (Data Set S1). Alternative sigma factors are known to control specialized regulons, e.g., regulons of stress response genes  . Moreovese genes . However strains , 50, 103of Hsp20 . Howeveraneously . Moreoveaneously .B. breve and B. longum and in isolates from bees, including B. asteroides, B. coryneforme, and B. indicum  is close to the gene encoding the putative SOD (Blon_1406), so it is conceivable that RpoE plays a role in transcriptional regulation of the putative SOD. In general, the presence of RpoE in all strains and a second copy in some strains indicates an important role of the sigma factor in transcriptional regulation.A second copy of RpoE was identified in 36 strains from Bifidobacterium strains . Homologs with very low identity (43% to 50%) were detected in B. subtile and B. pullorum subsp. gallinarum , suggesting that these WhiB-like proteins differ in function compared to the protein in B. longum subsp. longum NCC2705. Homologs of two additional WhiB-like proteins of the well-characterized bee isolate B. asteroides PRL2011 across strains of this study were assessed (Whib-like protein_2 and Whib-like protein_3). Whereas high-identity homologs were found for one of the genes in strains of various phylogenetic groups, the second appeared to be present only in B. asteroides and B. thermophilum . Moreover, the results confirmed that strains of  homolog . In addi homolog . Homolog NCC2705 , 35 wereBifidobacterium species as well as some variations among strains of the same species, e.g., B. adolescentis. While genes encoding molecular players of the PQC and DNA repair systems were found to be highly conserved across species, the regulatory mechanisms of these systems might differ between phylogenetic groups. The presence/absence pattern of genes linked to oxidative stress response could not fully explain variations in the O2 tolerance across species, suggesting that their expression level upon oxidative stress influences O2 sensitivity. However, the absence of several ROS-detoxifying enzymes in strains of B. angulatum and B. adolescentis most likely contributes to their O2 sensitivity. Species-dependent genes associated with acid stress response might be a result of adaptation to natural habitats. Similarly, the presence of a gene encoding a bile salt hydrolase in human and mammalian isolates appeared highly linked to the presence of bile salts in their natural habitat. Moreover, the distribution of putative regulators of various stress responses varied across species, indicating that the involvement of different sets of regulators contributes to the highly diverse stress physiology of Bifidobacterium species.Our analysis revealed multiple differences in the stress-associated genetic makeup of different Bifidobacterium strains as well as the identification of the biological function of stress-induced genes is required to obtain stronger correlations between the presence of stress-associated genes and stress tolerance of different strains.In conclusion, mining bifidobacterial genomes for homologs of genes associated with various stress responses and their regulatory elements provided new insights into the molecular mechanisms underlying their diverse stress physiology. The obtained results lay out the foundation for hypothesis generation in future studies. However, systematic assessment of the effect of various stressors on growth and survival of sequenced Bifidobacterium strain in which it was proposed to be involved in stress responses. Moreover, NCBI GenBank was searched for bifidobacterial genes annotated as superoxide dismutase and l-aspartate oxidase.Previous studies on stress response in bifidobacteria were consulted to identify representative genes that have been implicated in the stress response of bifidobacteria  2–, 64, 65.Bifidobacterium strains were retrieved from the NCBI RefSeq database, resulting in 171 genomes representing 22 out of 54 currently recognized Bifidobacterium species (https://github.com/kblin/ncbi-genome-download) with the arguments “–genera Bifidobacterium –section refseq –assembly-levels complete.” The RefSeq genomes include structural and functional annotations computed through NCBI’s PGAP.All publicly available complete genome sequences of  species  61). Al. AlBifidBifidobacterium strains were searched for homologs of the representative stress-associated gene products using the DIAMOND BLASTp algorithm (v2.0.9.147)  as an outgroup. Bifidobacterium species were assigned to the previously suggested phylogenetic groups, including B. adolescentis, B. asteroides, B. boum, B. longum, B. pseudolongum, and B. pullorum, except for the strains of six species that have not yet been unambiguously assigned to any phylogenetic group (The proteomes of the 171 0.9.147) . For eacic group , 40.B. animalis subsp. lactis BB-12, B. adolescentis ATCC 15703, and B. longum subsp. longum NCC2705 were screened for the presence of previously proposed consensus sequences of the three transcriptional regulators HspR, HrcA, and ClgR  was set to 0.0001, and the q-value  was set to 0.01 .”B. longum subsp. longum NCC2705, B. animalis subsp. lactis BB-12, and B. asteroides PRL2011 were compared to the protein sequence of HemW in Lactococcus lactis through multiple-sequence alignment and were subsequently screened for conserved amino acid residues specific for HemW-like proteins, including (i) His134, (ii) H184VxxYxLxLE, (iii) Y234ExS, and (iv) H248NxxYW (The protein sequences of HemN in 248NxxYW .We declare that all the data supporting the work are available within the paper and its supplemental material.


Here, we reported a panel of rare diseases involving bilateral lower extremities with similar imaging patterns on  Although frequent deformities are encountered in pediatric orthopedic departments, diseases involving bilateral lower extremities are relatively rare in clinical practice. Clinical and radiological phenotypic characterizations are the baseline tools of diagnosis. However, the expanding availability of bone scintigraphy may help the physicians to better understand these diseases. In diseases such as hyperparathyroidism, typical imaging characteristics with hypercalcemia and hypophosphatemia usually enable a definitive diagnosis. Hypertrophic pulmonary osteoarthropathy (HPO) can likely be diagnosed based on clinical symptoms  and radiographic findings . However, the imaging features of many other diseases may be disappointingly nonspecific, and the possible diagnostic considerations initially may seem extensive. In such cases, the use of a systematic approach can help narrow down the differential diagnosis.Diseases involving bilateral lower extremities may be classified in one of the following general diagnostic categories: metabolic bone disorders, haematological systemic disorders, genetic developmental diseases, tumors, trauma, autoimmune diseases, and inflammatory lesions. Although these categories comprise a plethora of possible diagnoses, an orderly list of differential considerations may be constructed by considering the patient’s age, laboratory examination, salient imaging features, and clinical manifestations.The current article is based on our experience with diseases involving bilateral lower extremities. It is not intended as a comprehensive review, but rather as an overview, with emphases on lesions that are more common or relatively more common and on diagnoses that may be suggested by bone scintigraphy and radiologic imaging.Ethical review and approval were waived for this study due to its retrospective nature. Informed consent has been waived for most patients except for those whose medical images were used in the current study. Bone SPECT was acquired 3–3.5 h after intravenous administration of radiotracer technetium G-6PD deficiency is one of the most common human enzyme defects that causes many biochemical and clinical phenotypes, including neonatal jaundice, chronic haemolysis and acute haemolytic anaemia . Howeverreported . TherefoPatient 1 was a 17-year-old man who complained of fatigue, back and lower limbs’ pain for three years. Spleen volume and liver volume of the patient were found to be significantly enlarged upon abdominal computed tomography (CT) A. To expe joints B. Furtheiotracer C–E. Nota9mTc-MDP F–H. Consiagnosis .Hematologic abnormalities of G-6PD deficiency are exceedingly common. Besides ingestion of fava beans, drugs and infection affecting oxidative stress of red blood cells can also trigger manifestations of G-6PD deficiency . This pafication , but thefication . The mosG-6PD deficiency needs to be differentiated from Gaucher’s disease (GD), where the latter is the most common lysosomal storage disease resulting from accumulation of undegraded glucosylceramide in the reticuloendothelial system of the bone marrow, spleen and liver . AlthougPatient 2 was a 22-year-old female admitted to our hospital due to suffering from left thigh pain and swelling after an accidental crash. CT scanning revealed altered bone formation, resorption as well as pathologic fracture of the left femur . MRI shoOne week  and one nomegaly . The infnomegaly . Notablynomegaly F. Replacnomegaly .Giuliano et al. reported that  disease ,7,8. Howd images  10]. Th. Th99mTcOsteonecrosis has a large number of etiologies and multifocal osteonecrosis is an uncommon entity usually seen in the clinical setting of corticosteroid administration, connective tissue disorders, dysbarism, hemoglobin patties, arteritis/vasculitis, pancreatitis, Gaucher’s disease, pregnancy and alcohol abuse . We repoA 54-year-old female (Patient 3) was admitted to our hospital for evaluation of arthralgia. After initial negative laboratory tests, the patient was recommended to have a ral head A. 99mTc-truction B–D. Althtruction A,B, substruction C, sagitttruction D, sagitttruction E and cortruction F T2-weigEarly diagnosis of steroid-induced osteonecrosis is of great importance to prevent irreversible bone and joint destruction. It is difficult to judge whether or not increased activity on a plain necrosis . Althougnecrosis . If patinecrosis ,14,15.Progressive diaphyseal dysplasia, or Camurati–Engelmann disease (CED), is a rare autosomal dominant skeletal dysplasia caused by mutations in the transforming growth factor-b1 (TGF  1) gene . TGF β 1A reported case (Patient 4) presented at 30 years of age with complaints of pain over both lower limbs and increasing difficulty in walking. Laboratory studies showed that serum calcium, phosphorus and PTH were within the normal range. Alkaline phosphatase was elevated . Her CTX-I was elevated as well , with decreased hemoglobin  and increased blood sedimentation rate. A w cavity B–E. The w cavity F,G. MRI w cavity A and hetw cavity B,C. On dw cavity D. The onCamurati first suggested the familial nature of PDD in 1922, and subsequently, Engelmann reported a single case with muscular wasting and marked bone involvement in 1929 . The typay films ,19,20,21Erdheim–Chester disease (ECD) is a rare non–Langerhans cell histiocytosis characterized by infiltration of tissues by foamy histiocytes . By JuneThe first case reported (Patient 5) was a 51-year-old male patient who suffered from renal failure in 2011 and skin pigmentation of the anterior chest wall in 2012. Since 2013, the patient has suffered from increasing bone pain of the lower extremities. A timely l tibiae . The patl tibiae . Final pWe have encountered another two ECD cases and both patients suffered from orbital and sphenoid bone involvement and subsequent visual extinction. A remities A. Abnormremities B. The paremities C and X-rremities D. X-ray remities E. The muremities A–C. In aremities D. Craniaremities E,F and eremities G. The saremities A. The saremities B. The garemities C. Coronaremities D. Microsremities E. Immunoremities F and CD1remities G and negremities H. PositiThe third patient (Patient 7) was a 56-year-old female who complained of interrupted fevering and paining of bilateral knee joints since 2014. The patient had Sjogren’s syndrome and usually took hydroxychloroquine and prednisone when the disease was active. The patient also suffered from diabetes and anxious depression for 3 years. Because of increasing pain and stiffness of the knee joints and right finger joints, she was admitted to department of osteoporosis in our hospital for further evaluation. The results of extensive serum laboratory analyses were otherwise unremarkable. Plain film showed abnormal density of sella turcica and basalis , sclerosses. MIP  and fusiw cavity . From a ECD is a rare disease first described by Jacob Erdheim and William Chester in 1930. The disease is characterized by tissue infiltration by foamy histiocytes, typically in the long bone marrow, but also in numerous other organs . Common ne scans ,29,30,31t in ECD . Previout in ECD ,33. HereLangerhans cell tumors are generally classified into Langerhans cell histiocytosis (LCH) and Langerhans cell sarcoma (LCS), and while the former is a benign entity the latter is a rare and malignant tumor of Langerhans cells. Up to now, there are only 66 LCS cases reported in the literature . Given iThe last patient (Patient 8) was a 47-year-old female patient initially presenting with lower leg pain. Previously, she had received surgery on the right tibia because of suspicious chronic osteomyelitis. A  humerus A. Subseqx of 4.5 B–D. Our x of 4.5 E, and pox of 4.5 F, S-100 x of 4.5 G, Langerx of 4.5 H proteinLCS can present de novo or progress from antecedent LCH. Skin, lymph nodes, spleen, liver, bone marrow, thymus, lung, and kidney have been frequently involved . It is nBesides those rare diseases involving metaphyses/extremities of lower extremities, fibrous dysplasia, hyperparathyroidism and other metabolic bone diseases are of relatively higher incidence and may present similar bone scan patterns. Fibrous dysplasia (FD), which accounts for about 2–3% of bone-derived tumors, is a benign bone lesion characterized by the replacement of normal bone structure with abnormal fibro-osseous connective tissue . It has Hyperparathyroidism, especially primary hyperparathyroidism (PHPT), is a frequent and potentially debilitating endocrine disorder. In up to 85% of clinical cases, PHPT is characterized by the autonomous overproduction of parathyroid hormone (PTH) by a solitary parathyroid adenoma . We prevTH level . Therefotaplasia , gastrictaplasia ,48,49, itaplasia , prostattaplasia ,52, breataplasia . In someHere, we show that when symmetrical uptake of radiotracer appears on