Paenibacillus polymyxa HX-140, isolated from the rhizosphere soil of rape, to control Fusarium wilt of cucumber seedlings caused by Fusarium oxysporum f. sp. cucumerinum.The aim of this study is to evaluate the efficacy of the strain 6 107 and 108 cfu/mL of HX-140 bacterial suspension reduced the incidence of Fusarium wilt of cucumber seedling by 19.5, 41.1, and 50.9%, respectively.Strain HX-140 was able to produce protease, cellulase, β-1,3-glucanase and antifungal volatile organic compounds. An in vitro dual culture test showed that strain HX-140 exhibited broad spectrum antifungal activity against soil-borne plant pathogenic fungi. Strain HX-140 also reduced the infection of Fusarium wilt of cucumber seedlings by 55.6% in a greenhouse pot experiment. A field plot experiment confirmed the biocontrol effects and further revealed that antifungal activity was positively correlated with inoculum size by the root-irrigation method. Here, inoculums at 10P. polymyxa HX-140 has significant potential in the control of Fusarium wilt and possibly other fungal diseases of cucumber.Taken together, our results suggest that The online version contains supplementary material available at 10.1186/s12866-021-02131-3. Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FOC) is one of the most destructive soil-borne diseases that causes large economic losses on a worldwide scale [Fusarium genus have an extremely broad host range including cotton, tomato, banana, strawberry, potato, capsicum, beans, peas, chickpea, and melons, and are among the most harmful soil-borne pathogens in crop production systems. They usually infect plants through the roots, causing damping-off, root rot, and vascular wilt [Cucumber is an economically important and popular vegetable crop. Vascular wilt of cucumber caused by de scale . Pathogelar wilt , 44.Fusarium spp. However, their extensive usage has not only caused negative environmental and toxicological impact but has also jeopardized human and animal health [Chemical control using fungicides has been the most common control strategy for managing diseases caused by l health . Furtherl health , 28. TheBacillus subtilis SQR9 was found to control Fusarium wilt of cucumber by colonizing plant roots and producing lipopeptides (fengycin and bacillomycin) that were effective against F. oxysporum [B. velezensis RC 218 reduced disease severity of Fusarium head blight (FHB) caused by Fusarium graminearum and reduced the accumulation of the associated mycotoxins [Paenibacillus Alvei K165 exhibited significantly lower disease severity and lower relative AUDPC (Area Under the Disease Progress Curve) values than those of un-treated, diseased plants [Streptomyces griseorubens E44G was found to possess chitinolytic activity and was capable of decreasing the severity of Fusarium wilt of tomato and increasing the growth and yield of tomato [Trichoderma asperellum that exhibited high levels of chitinase and β-1,3-glucanase activities strongly inhibited the mycelia growth of F. oxysporum f. sp. Lycopersici [Biological control is an ecologically friendly, sustainable, and safe method for the control of plant infections. Biocontrol is a reliable alternative to the use of chemical fungicides . Specifixysporum . B. velecotoxins . “Amfissd plants . Streptof tomato . Lastly,opersici .Paenibacillus polymyxa has shown strong inhibitory effects against pathogenic nematodes, oomycetes, and fungi, including F. oxysporum. This biocontrol agent was reported to produce antifungal peptides, volatiles, proteins, and other antimicrobial substances that inhibit fungal pathogen mycelial growth. Several studies have revealed multiple mechanisms of anti-fungal activities, including competition for nutrients, the production of antifungal compounds such as antibiotics, extracellular enzymes, and organic volatiles [In the context of the current study, the bacterium olatiles  and the P. polymyxa isolate HX-140) with antagonistic effects on FOC and other phytopathogenic fungi was identified, and its potential mechanism and control effect of Fusarium wilt disease were also explored.In this study, an antagonistic bacterium . In this paper, HX-140 was taken as an example to study the biological control mechanism and the effect of HX-140 on cucumber Fusarium wilt control , this strain was revealed to be most similar to ist Fig. . Gram stPaenibacillus (P. polymyxa ATCC 842T), strain HX-140 exhibited almost identical physiological characteristics, except for a variation in nitrate reduction. Strain HX-140 and P. polymyxa ATCC 842T are both positive for growing in 3% NaCl (w/v), anaerobic growth, starch hydrolysis, casein hydrolysis, the VP (Voges-Proskauer) test, and acid production using galactose, L-arabinose, D-fructose, and D-xylose. However, HX-140 was negative for indole production and unable to use citrate  was significantly lower than those seedlings with only F. oxysporum (CK2). As expected, the healthiest seedlings were in the negative control with only sterile water (CK1). The infection of Fusarium wilt in cucumber seedlings was significantly (P < 0.05) reduced to 35%, and the relative control efficacy was 55.6% after treatment with P. polymyxa HX-140  of HX-140 bacterial suspension were evaluated and shown to be 19.5, 41.1, and 50.9%, respectively. The control efficacy increased as the concentration of HX-140 increased. Likely influenced by a diversity of biotic and abiotic factors in the field, the control efficacy of strain HX-140 in the field was lower than that in the greenhouse pot culture. However, all three treatments  showed lower disease incidence and lower disease index than those of the control group. These results suggest that P. polymyxa HX-140 had a significant suppressive effect on Fusarium wilt of cucumber seedlings in the field with a history of the disease.Within the field experiment, a small proportion of cucumber seedlings exhibited symptoms of wilting before the inoculation of the HX-140 bacterial suspension. The field site was partitioned into small plots all with similar rates of infected plants and similar disease indices. These small plots were randomized to receive four treatments: CK, T1, T2 and T3 treatment Table . FourteeP. polymyxa HX-140 on F. oxysporum was tested in vitro prior to a greenhouse pot experiment, and P. polymyxa HX-140 exhibited antagonistic activity against the tested pathogenic fungi. As determined by the greenhouse pot and the field experiments, P. polymyxa HX-140 significantly decreased the wilt disease of cucumber seedlings caused by FOC. Specifically, biocontrol efficacies of strain HX-140 in the greenhouse pot and field experiments were 55.6 and 50.9%, respectively, similar to or higher than several previously reported candidate biocontrol agents. For example, the biocontrol efficacies of Streptomyces albospinus CT205 were reported to be 8.7 and 51.9% in 2011 and 2012, respectively [In this study, the antifungal activity of ectively . In theiectively . The higectively . RegardlP. polymyxa strain HX-140 was selected due to its high antifungal activity against F. oxysporum. In addition, P. polymyxa HX-140 displayed a broad spectrum of antifungal activities against six other pathogens. P. polymyxa was previously designated to the genus Bacillus and reclassified to the new genus Paenibacillus in 1994 [P. polymyxa strains have been reported as highly effective biocontrol agents for controlling soil borne fungal diseases [C. gloeosporioides and C. acutatum in harvested apples were suppressed by 83.6 and 79%, respectively, after treatment with P. polymyxa APEC128 [P. polymyxa SC09–21 produced cellulase and protease to control P. capsici, and the gene expression levels of pathogenesis-related (PR) proteins, including CaPR4 and CaChi2, were also enhanced after treatment with the SC09–21 strain [P. polymyxa in the control of F. oxysporum. For example, P. polymyxa NSY50 can significantly up-regulate the expression level of defense related genes PR1 and PR5 in cucumber roots at the early stages upon challenge with FOC [P. polymyxa WLY78 can strongly inhibit fusarium wilt of cucumber [P. polymyxa strains were able to inhibit the growth of F. oxysporum f. sp. albedinis for a long time in vitro [P. polymyxa SQR-21 systemically affects root exudates of watermelon to decrease the conidial germination of F. oxysporum f.sp. niveum [P. polymyxa antagonizes the plant pathogenic fungus F. oxysporum in liquid medium by means of an interaction process in which the presence of living bacteria is a prerequisite for continuous suppression of fungal growth [In our study,  in 1994 . P. polydiseases . For exa APEC128 . P. poly1 strain . There hwith FOC , P. polycucumber , NSY50 a. niveum . P. polyl growth .P. polymyxa HX-140 against Fusarium spp. P. polymyxa HX-140 exhibited strong protease, cellulase and β-1,3-glucanase activities and produced antifungal VOCs. The fungal cell wall is a dynamic and complex structure. For most fungi, the core of the cell wall is a branched β-1,3 and β-1,6 glucan that is linked to chitin via a β-1,4 linkage. The composition of the cell wall varies markedly between different species of fungi [P. polymyxa HX-140 inhibited the mycelial growth of FOC in vitro, which indicates that they may play an important role in the inhibition of soil-borne diseases caused by FOC. The antifungal activity of VOCs has often been an understudied component of research on biocontrol agents, including those of P. polymyxa strains [We analyzed the potential contributors to the antifungal activities of of fungi , 6, 24. of fungi , 32, whi strains . VOCs pr strains , 21, 43.P. polymyxa HX-140 against F. oxysporum f. sp. cucumerinum, the causative agent of fusarium wilt on cucumber. Our in vitro analyses showed the antagonistic effect of HX-140 on pathogen growth, including its potent enzymatic activities against fungal pathogens. HX-140 was shown to reduce the incidence of cucumber wilt disease in the greenhouse. Furthermore, the bacterium reduced the incidence of cucumber wilt disease by 50.9% in the field, consistent with its significant application potential in the field. The potential molecular contributors to its antifungal activity as identified here also lay a foundation for further studies of the genes and biochemical pathways of HX-140 to improve its antagonistic efficiency and commercial application in the field.This study focused on the isolation, identification and characterization of the antagonist activity of the bacterium Fusarium oxysporum, Fusarium lini, Colletotrichum lini, Colletotrichum gloeosporioides, Sclerotinia sclerotiorum, Phytophthora capsici, and Rhizoctonia solani) used in the study were provided by the Key Laboratory of Biology and Processing of Bast Fiber Crops, Ministry of Agriculture, Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences. and stored at 4 °C using potato dextrose agar (PDA) medium.The fungal strains  medium and incubated at 28 °C for 3 days. Three replicates were performed for each dilution. Bacterial isolates were sub-cultured and purified as single colonies. Representative colonies of different sizes and morphologies were stored on NA slants at 4 °C.F. oxysporum f. sp. cucumerinum, the plates were incubated at 28 °C in the dark and the inhibitory effect on growth was assessed after 9 days. The bacterial isolate with the strongest antagonistic effect was selected for further study.All bacterial isolates were tested for their ability to inhibit fungal growth using the technique of dual culture analysis , 45. ThrFusarium oxysporum activity, was obtained from the screening test. In order to taxonomically identify isolate HX-140, its total genomic DNA was extracted using the Bacteria Genomic DNA Kit , following the manufacturer’s instructions. The 16S rDNA was amplified using the universal bacterial primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′) [Isolate HX-140, a strain with the highest anti-ACTT-3′) , as descACTT-3′) . The ampACTT-3′)  with MolThe morphological characteristics of strain HX-140 on the NA plate were recorded after incubation at 28 °C for 3 days.Physiological tests were performed following the Systematic Determinative Manual of General Bacteria . Gram stTo assess the potential antifungal effects of HX-140, antagonistic activity against seven phytopathogenic fungi was assessed on PDA plates by dual culture analysis as described in a previous study . Three rDetermination of the production of protease, chitinase, cellulase and β-1,3-glucanase.3 2 g, K2HPO4 1 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, FeSO4·7H2O 0.01 g, and agar 16 g in 1000 ml distilled water at pH 6.8, supplemented with 1% CMC–Na salt as the carbon source [The production of protease by HX-140 was analyzed using the spot inoculation technique . The chin source . The actn source , 32. PetF. oxysporum mycelia was placed in the center of the PDA medium on the bottom dish. The dish containing the mycelial plug was inverted over the bacterial plate. The dishes were sealed with parafilm. All tests were carried out in triplicate and the experiment was repeated twice. The plates were incubated for 9 days in the dark at 28 °C, and the antagonism was determined by comparing the diameters of F. oxysporum colonies in the test groups with the control groups.The production of volatile organic compounds (VOCs) by strain HX-140 was determined using the sealed plate method . BrieflyCucumber seeds (No. 4 Jinchun) were surface-sterilized by immersion in 0.1% mercuric chloride for 60 s, rinsed in sterile water 3 times, and then stored at 4 °C overnight to induce germination. Seeds were germinated in a Petri plate incubated with a piece of moist filter paper in the dark at 28 °C and then sown into the 9 × 9-hole seedling pots and covered with approximately 1 cm of sterilized substrate (121 °C for 20 min) , 46. CucF. oxysporum mycelia disk was picked from the margin of the dish and inoculated in PDB (Potato Dextrose Broth) liquid medium. The liquid culture was incubated at 28 °C for 48 h on a rotary shaker at 180 r/min and then diluted to 108 cfu/mL using PDB for backup. Actively growing cells of strain HX-140 were incubated in Lysogeny broth (LB) medium at 28 °C for 24 h at 180 r/min and then centrifuged at 12000 r/min for 5 min. The bacteria precipitate was washed three times with sterile water and then diluted to 108 cfu/mL for downstream use.An actively growing 8 cfu/g microorganisms . Equal volumes of Fusarium liquid culture and HX-140 suspension were mixed with the nutrient soil evenly in the treatment group (T). Control group 1 (CK1) was treated with an equal volume of sterile water, and the control group 2 (CK2) was treated with equal volumes of Fusarium liquid culture and sterile water. All treatments were kept at 28 °C for 2 days to ensure that the Fusarium pathogen (in T and CK2 treatments) and strain HX-140 (in T) antagonist colonized the soil.The pot experiment was carried out in greenhouse using soil inoculated with 10For each treatment, twenty cucumber seedlings in the two-leaf stage were planted in the treated nutrient soil. Before planting, their roots were mechanically damaged to help initiate infection. Three replicates were maintained in each treatment and the experiment was repeated twice. The disease severity was investigated 20 days after transplantation of cucumber seedling.Level 0-Plant was healthy with no illness;Level 1-Leaves with slight wilting;Level 2-Stems and leaves slightly wilted with necrotic spots on the leaves;Level 3-Plants moderately wilted with necrotic spots on leaves and stems;Level 4-Plants died.Disease severity of cucumber seedlings was divided into five classes and recorded using a disease index ranging from 0 to 4 as follows:The disease index and biocontrol efficacy were calculated according to the following formulas :\docume2, divided into subplots of 1.4 × 1.6 m. The chosen field for this experiment was planted with cucumber for more than 3 years, and the incidence of cucumber wilt was over 70%. We used a complete randomized block design with three replicates for each treatment. Each sub-plot was planted with 56 seedlings. The experiment was repeated twice from June to November 2018.The field experiments were carried out on an experimental farm of the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, in Yuanjiang Country , Hunan Province, China. The total field plot size was ~ 200 m6 cfu/mL of the HX-140 bacterial suspension (T1); (iii) a treatment group inoculated with 107 cfu/mL of the HX-140 bacterial suspension (T2); and (iv) a treatment group inoculated with 108 cfu/mL of the HX-140 bacterial suspension (T3). When the seedlings had 1–2 leaves, 20 mL of HX-140 bacterial suspension was used to drench their roots. The incidence of cucumber wilt among the different treatments was investigated before HX-140 inoculation and 14 days after inoculation.The field trial included four treatments: (i) control group with no inoculation (CK); (ii) a treatment group inoculated with 10The disease severity of cucumber seedlings was recorded using a disease index ranging from 0 to 4, identical to the greenhouse pot experiment, and the control efficacy was calculated using the following formulas , 41:\doP < 0.05 as the level of significance.Statistical analysis was performed using Microsoft Excel 2010  and DPS 7.05 software . Mean values were compared using Duncan’s multiple range test with Additional file 1: Figure S1 Gram staining of strain HX-140. Bar = 20 μm.


Giardia duodenalis is a major gastrointestinal parasite found globally in both humans and animals. This work examined the occurrence of G. duodenalis in coypus (Myocastor coypus) in China. Multi-locus analysis was conducted to evaluate the level of genetic variation and the potential zoonotic role of the isolates. In total, 308 fecal samples were collected from seven farms in China and subjected to PCR screening to reveal G. duodenalis. Notably, G. duodenalis was detected in 38 (12.3%) specimens from assemblages A (n = 2) and B (n = 36). Positive samples were further characterized by PCR and nucleotide sequencing of the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) genes. Multi-locus genotyping yielded 10 novel multi-locus genotypes (MLGs) . Based on the generated phylogenetic tree, AI–novel 1 clustered more closely with MLG AI-2. Furthermore, within the assemblage B phylogenetic analysis, the novel assemblage B MLGs were identified as BIV and clustered in the MLG BIV branch. This is the first report of G. duodenalis in coypus in China. The presence of zoonotic genotypes and subtypes of G. duodenalis in coypus suggests that these animals can transmit human giardiasis. Giardia duodenalis  is a flagellate protozoan parasite recognized as a significant global contributor to diarrheal disease, affecting humans, domestic animals, and wildlife across the globe [G. duodenalis infections are asymptomatic; however, in rare cases, some patients may experience severe gastrointestinal disturbances for several weeks [G. duodenalis utilizes the fecal–oral route for lifecycle maintenance, projections indicate that this parasite causes ~28.2 million foodborne disease cases [Giardia 11th of 24 food-borne parasites [he globe ,2. The mal weeks . As G. dse cases ,5. BasedG. duodenalis transmission in humans and animals [tpi; glutamate dehydrogenase, gdh and beta giardin, bg) have been developed to create a multi-locus genotyping (MLG) tool for subtyping assemblages A, B, and E and to explore the population genetic characterizations of G. duodenalis [The current wide use of genotyping tools has immensely improved our understanding of  animals ,6. At le animals . Moreoveodenalis . Subsequodenalis .G. duodenalis have been conducted for humans, non-human primates, ruminants, companion animals, domestic animals, wildlife, and in the environment in China [Myocastor coypus) is a large, amphibious rodent native to South America, which has become invasive in Europe and other parts of the world except Oceania and Antarctica [G. duodenalis in coypus globally. Only two studies in Italy and the USA reported Giardia spp. prevalence in coypus, but neither identified the species [G. duodenalis in coypus in China and assess its zoonotic potential based on MLG analysis.In recent years, studies on the epidemiology of in China . Howevertarctica . Coypus  species ,13. ThusG. duodenalis was present in 38 samples  , followed by Ganzhou , Chengdu , Laibin , Yongzhou , Kaifeng , and Anyang   gene. Of the 308 samples, 5–16.2%) . Each ex, 1/101) .p = 0.023) associated with the 3–6-month-old group and an OR of 5.47  associated with the >6-month-old group.By age, the highest infection rate was reported in the 3–6-month-old group , followed by >6-month-old group , and <3-month-old group  . Furthertpi, gdh, and bg loci  and B (35), were identified based on sequence analysis of the SSU rRNA,  bg loci . Notablyectively . tpi sequences, 3 and 22 belonged to assemblages A and B, respectively. Sequences from all isolates from assemblage A exhibited two single-nucleotide polymorphisms (G155T and G186A) relative to the MN174855 sequence. Within the assemblage B isolates, three subtypes were formed, which were designated as B1 (n = 17), B2 (n = 3), and B3 (n = 2) for convenience. Of note, B1, B2, and B3 sequences were identical to MH644772, KM977653, and HM140711, respectively. At the gdh locus, 1 and 10 samples were found positive and identified as assemblages A and B, respectively. The one assemblage A sequence was identical to MN174853. Among the assemblage B sequences, B1 (n = 5), B2 (n = 3), and B3 (n = 2) sequences were identical to KM977648, MK952603, and MK982476, respectively. Sequence analysis demonstrated high genetic diversity in assemblage B at the bg locus. Ultimately, five subtypes (B1–B5) were formed in the 11 assemblage B sequences. The subtypes B1 (n = 4), B2 (n = 2), B3 (n = 3), B4 (n = 1), and B5 (n = 1) exhibited consistency with MT487587, MK982544, MN174847, MT487587, and KY696837, respectively. Moreover, one assemblage A sequence was identical to MN704938.Of the Collectively, sequence data sets from the three loci were available from 10 isolates. Multi-locus genotyping yielded 10 novel MLGs (one MLG for assemblage A and nine MLGs for assemblage B) . The sinG. duodenalis is very commonly found in humans and domestic animals, as revealed by numerous prevalence studies across the globe [G. duodenalis has been widely studied in livestock, which revealed its transmission dynamics and zoonotic significance in these animals. However, the parasite has not been extensively investigated in rodents; therefore, very little knowledge on the distribution, genetic diversity, and zoonotic potential of Giardia spp. in these animal hosts has been published [G. duodenalis infections in rodents in Australia, Belgium, China, Croatia, Germany, Malaysia, Poland, Romania, Spain, Sweden, and the USA . To the best of our knowledge, this is the first molecular study of G. duodenalis infections in coypus, except for two studies in Italy and the USA that assessed Giardia spp. infection in coypus with the prevalence of 0% (0/153) and 73.3% (22/30), respectively, using an immunoenzymatic assay [he globe ,10,14. Tublished ,7,15,16. the USA  have preic assay .Giardia species are considered valid, including G. duodenalis, Giardia agilis, Giardia ardeae, Giardia psittaci, Giardia muris, Giardia microti, Giardia peramelis, and Giardia cricetidarum [Giardia spp. have been detected in rodents, including G. muris and G. microti [G. duodenalis has been reported in rodents of several genera, including Chinchillas (Chinchilla lanigera), Asian house rats (Rattus tanezumi), brown rats (Rattus norvegicus), house mice (Mus musculus), chipmunks (Eutamias asiaticus), and bamboo rats (Rhizomys sinensis) [G. duodenalis were found in rodents, including A, B, C, D, E, F, and G  ,19,26,30F, and G . Both zomblage A ,36. We amblage A ,37. Mixemblage A ,15. Consgdh, bg, and tpi loci represents a more informative approach for genotyping and elucidating the characteristics of G. duodenalis in different hosts, in addition to its zoonotic potential [Giardia isolates mainly belong to sub-assemblage AII but also AI, whereas animals harbor AI, AII, and AIII [G. duodenalis isolates from coypus were successfully sequenced at all three gene loci; the sequence analysis of which resulted in one MLG in assemblage A and nine MLGs in assemblage B. Based on the phylogenetic analysis, the one MLG in assemblage A (AI–novel 1) was clustered with sub-assemblage AI-2, whereas the nine remaining MLGs in assemblage B (BIV–novel 1 to 7) were clustered with sub-assemblage BIV. Sub-assemblage AI was previously reported in various hosts in China, including humans, cattle, goats, sheep, dogs, cats, pigs, chinchillas, and chipmunks [G. duodenalis assemblage AI was revealed by epidemiological data showing a highly significant association between Giardia infection of schoolchildren and the presence of Giardia-positive dogs in the same household in Mexico [G. duodenalis in handlers/workers on farms should be initiated to address the zoonotic potential of G. duodenalis in coypus.In recent studies, the MLG tool based on sequence analysis of the otential ,15,19. Thipmunks ,10. Othehipmunks ,10. Addin Mexico . TherefoThis study was performed with strict adherence to the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. The research protocol was reviewed and approved by the Research Ethics Committee of Tarim University . Farm owners gave permission before we commenced fecal sample collection.A total of 308 fecal samples were collected during autumn, winter, and spring of 2018 and 2019 from seven farms in Hebei Province (Baoding), Henan Province (Anyang and Kaifeng), Sichuan Province (Chengdu), Hunan Province (Yongzhou), Jiangxi Province (Ganzhou) and Guangxi Zhuang Autonomous Region (Laibin) in China . The bre® Stool DNA Kit  following the protocol stipulated by the manufacturer. The quantity of nucleic acid in samples was photometrically estimated at OD260 and stored at −20 °C for subsequent molecular analysis.The genomic DNA sample was extracted from approximately 200 mg of each fecal specimen using a commercial E.Z.N.AG. duodenalis infection [G. duodenalis-positive samples were further analyzed via nested PCRs of gene loci tpi, gdh, and bg . The sequence assembly and editing were performed with the DNAstar Lasergene Editseq 7.1.0. Multiple-sequence alignment analysis was employed on the obtained and GenBank reference sequences using software ClustalX 2.1 to ascertain the genotypes and subtypes of G. duodenalis. We concatenated sequences for each positive isolate to obtain a multi-locus sequence  in accordance with a previous report [Bi-directional sequencing of all the secondary PCR products from SSU rRNA, s report . Phylogep value < 0.05, the differences were appraised as significant.The differences in infection rates among locations and age groups were compared with the χ2 test performed in SPSS 18. At the level of Here, the representative nucleotide sequences of this study were deposited in the GenBank database under the following accession numbers: MW322754–MW322755, MW321613–MW321626.G. duodenalis infections in coypus in China based on MLG analysis. Multi-locus genotyping yielded 10 novel MLGs, including one assemblage A MLG (AI–novel 1) and nine assemblage B MLGs (BIV–novel 1 to 7). Of note, the presence of zoonotic assemblages and sub-assemblages of G. duodenalis in coypus suggests the potential contribution of these animals to human giardiasis transmission.This work presents the first report of


Recent evidence indicates that p13 may influence the cell’s innate immune response to viral infection and the infected cell phenotype. Association of the HTLV-1 transcriptional activator, Tax, with p13 increases p13’s stability, leads to its partial co-localization with Tax in nuclear speckles, and reduces the ability of Tax to interact with the transcription cofactor CBP/p300. Comparison of p13 sequences isolated from HTLV-1-infected individuals revealed a small number of amino acid variations in the domains controlling the subcellular localization of the protein. Disruptive mutations of p13 were found in samples obtained from asymptomatic patients with low proviral load. p13 sequences of HTLV-1 subtype C isolates from indigenous Australian patients showed a high degree of identity among each other, with all samples containing a pattern of 5 amino acids that distinguished them from other subtypes. Further characterization of p13’s functional properties and sequence variants may lead to a deeper understanding of the impact of p13 as a contributor to the clinical manifestations of HTLV-1 infection.Human T cell leukemia virus type-1 (HTLV-1) was the first retrovirus found to cause cancer in humans, but the mechanisms that drive the development of leukemia and other diseases associated with HTLV-1 infection remain to be fully understood. This review describes the functional properties of p13, an 87-amino acid protein coded by HTLV-1 open reading frame II ( Th. Th31]. The C-terminal portion of p13 (and p30) is rich in prolines, including those arranged in a sequence resembling P-x-x-P (PPII) helical motifs that interact with SH3 domain-containing proteins such as the Src family kinases (SFKs). Using in vitro binding and kinase assays, Tibaldi et al. confirmed that p13 is able to associate with SFKs Src, Fyn, Fgr, and Lyn . This inThe role of autophagy in recycling damaged or unneeded organelles has gained increased attention in recent years. Mitophagy is a mitochondria-specific subtype of this recycling mechanism that can be induced by different factors, including viral infection . MitophaStudies on the interplay between p13 and Tax  indicateHTLV-1 is classified in the deltaretrovirus genus, together with the related HTLV-2, 3, and 4 orthologues that infect nonhuman primates as simian T cell leukemia virus (STLVs); and bovine leukemia virus (BLV). Due to their shared molecular features, the human and simian viruses are collectively referred to as primate T-lymphotropic viruses (PTLVs).orf codes for a protein named p28 that, despite limited sequence identity, shares several functional properties with HTLV-1 p30 . TwHTLV-1 C, present mainly in Australia and Melanesia, has recently gained attention due to it high prevalence (in up to 40% of adults) in indigenous populations living in remote regions of central Australia . Unlike gag, pol, pro, and env are more highly conserved compared to the pX region, suggesting that the X-region proteins might contribute to the different clinical manifestations observed in infected individuals [Pairwise sequence comparisons at the nucleotide and amino acid levels between HTLV-1A and HTLV-1C show that the structural genes, ividuals . To gainividuals –85. SequOverall, this analysis confirms similar amino acid sequences and conservation of the principal domains described for HTLV-1A p13, including the MTS, the transmembrane region, the putative NLS region and the P-x-x-P SH3 binding motif. It is notable that the MTS is perfectly matched among all of the sequences. Interestingly, three of the ‘Australian signature’ residues are located in the putative NLS region, and the last signature residue is a carboxy-terminal proline, which would add a third potential P-x-x-P motif.Whether these changes affect the function of p13 and contribute to the distinct pathogenic properties of the HTLV-1C clade found in indigenous Australian patients  should bMany studies have shed light on the expression, intracellular trafficking, and function of p13, but the function of this protein in the context of the complete viral genome remains elusive. Given the importance of mitochondria in anti-viral innate immunity, it will also be important to discover if, by targeting mitochondria, p13 may hamper the host cell’s anti-viral responses. Notably, several other human tumor viruses also code for mitochondrial proteins, with diverse effects on mitochondrial function and the viral life cycle .+ cells [In vivo study of a molecular clone of HTLV-1 without changing the start codon of HBZ is essential for determining the true importance of p13 in HTLV-1 infection. Galli et al. showed the possibility of utilizing humanized mice for studying the p12 protein of HTLV-1 in the whole virus using infected primary CD4+ cells . This mo


Inositol pyrophosphates (PP-InsPs) comprise an important group of intracellular, diffusible cellular signals that a wide range of biological processes throughout the yeast, plant, and animal kingdoms. It has been difficult to gain a molecular-level mechanistic understanding of the actions of these molecules, due to their highly phosphorylated nature, their low levels, and their rapid metabolic turnover. More recently, these obstacles to success are being surmounted by the chemical synthesis of a number of insightful PP-InsP analogs. This review will describe these analogs and will indicate the important chemical and biological information gained by using them. Human Iin vitro , but it 8 back to InsP6 in animal cells  methyl (DEACM) to the 5-β-phosphate  binding site for the signaling phospholipid, PtdInsP3, which normally drives AKT translocation from the cytoplasm to plasma membrane, where it encounters upstream activating protein kinases. In their experiments, Jessen and colleagues uncaged 5-InsP7 after their AKTPH construct had already been relocated to the plasma membrane by prior addition of growth factors [PH was observed after 5 min, and nearly completed by 15 min. The length of this time frame could indicate that it depends upon 5-InsP7 metabolism to 1,5-InsP8, but that seems unlikely because the latter is a significantly weaker AKTPH ligand [7 to AKTPH that is already associated with PtdInsP3. Indeed, even in vitro, 5-InsP7 is less potent at competing for the AKTPH – bound PtdInsP3 if the lipid is prebound to the protein [Jessen and colleagues produced caged 5-InsPhosphate A. The mopothesis ,66 that  factors . A releaH ligand . Perhaps7 was made cell-permeable by attaching acetoxybenzyl groups to the monophosphates and DEACM was added to the 5-β-phosphate (7 was found to block Ca2+ oscillations in the mouse pancreatic insulinoma (MIN6) cell-line, after a delay of 15 min [8, its photorelease instantly stopped Ca2+ oscillations [7 phosphorylation to 1,5-InsP8. It can be anticipated that inhibition of Ca2+ signaling in β-cells by 1,5-InsP8 would inhibit exocytic insulin secretion [Another approach to delivering PP-InsPs into cells is to use analogues that are inherently cell-permeant. This can be accomplished by masking the negative charges with lipophilic groups that are naturally removed once the compound has entered the cell; the application of this method to an inositol phosphate (InsP) was first described in 1994 . Acetoxyhosphate B. Cell lf 15 min . In a sullations . Thus, iecretion , but thi8, which, theoretically at least, could promote exocytosis, through PP-InsP’s interaction with synaptotagmin-like protein 4 (Slp-4)/granuphilin [6 for plasma-membrane associated PtdIns2 binding to granuphilin’s C2A and C2B binding domains [8 was shown in Jessen’s study to also bind to each of these two domains in vitro . Moreov,5-InsP8 . That is7 (FAM-5-InsP7) [Recently, Jessen’s group synthesized a fluorescein-tagged analog of 5-InsP5-InsP7) ,53. This5-InsP7) .7 from the vesicles, predominantly into the cytoplasm, by using the tools of confocal microscopy and flow cytometry [We have used this new analog to validate our liposome delivery protocols ,53 by exytometry ,53.7-βS reagent could be delivered into cells using the nanoparticle delivery systems described above. These and other technical innovations will increase the opportunities available to those working in this unique field of signal transduction.Based on the successes described above, we can confidently predict that further, dramatic developments in our understanding of PP-InsP biology will result as additional synthetic analogs provide information from inside intact cells and eventually in vivo. Two developments in particular appear close to fruition. First, photocage technology has improved to the point that the toxic effects of UV-mediated uncaging can be avoided by using photosensitive moieties that respond to visible light while retaining good water solubility . Second,


Streptomyces due to their dual function in signal detection and gene expression control through DNA-binding in the promoter region of their target genes. In Streptomyces ambofaciens the alp biosynthetic gene cluster includes the signaling molecule receptor AlpZ that negatively regulates through a complex regulatory cascade the expression of key genes involved in the kinamycin antibiotic production until its cognate ligand, a yet unidentified signaling molecule, prompts its release from target promoters. Here we use an original molecular dynamics method to evaluate the DNA-binding properties of AlpZ to its target DNA sequence and the impact the signaling molecule has on the interaction. It is the first time this approach is used to characterize a regulator from the γ-butyrolactone receptor family. The observed KD in the nanomolar range indicates that AlpZ-DNA constitute a particularly stable complex. The signaling molecule ably disturbs this binding while kinamycin has no effect on the activity of AlpZ. Regulator size was determined and found to be considerably large regarding protein sequence, indicating that AlpZ regulates gene expression by binding the DNA as a homodimer, and structural modeling comparison with closely related γ-butyrolactone receptors supports this conclusion.Signaling molecule receptors play a central role in quorum sensing and in the coordination onset of specialized metabolite biosynthesis in  Streptomyces, ubiquitous filamentous Gram-positive bacteria characterized for their complex life cycle, linear chromosome and their ability to produce a wide variety of specialized metabolites including antibiotics, herbicides, immunosuppresssants and anticancer agents, display a different profile of signaling molecules.Bacterial quorum sensing is a well-established phenomenon by which bacteria produce and sense chemical signals to communicate with neighboring cells. These diffusible signaling molecules are able to trigger a plethora of activities such as genetic exchange, antibiotic production, motility, virulence, biofilm formation, among others. The signaling molecule accumulates extracellularly during cell growth and when the concentration reaches a certain threshold it is detected by a receptor that elicits a downstream signal transduction cascade, triggering a specific gene expression program. An array of signaling molecules are used for cellular communication. For example, the acylhomoserine lactones (AHL) are the representatives and most studied chemical signals in Gram-negative bacteria, while Firmicutes mainly use peptides as signaling molecules . StreptoThe vast majority of signaling molecules produced by Streptomycetes and identified to date are classified into three major groups, γ-butyrolactones (GBL) , 2-alkylStreptomyces, and the first bacterial signaling molecule to be discovered was the A-factor of Streptomyces griseus  and five regulatory genes. Recent reports indicate that the cluster size could be extended to include a 24 gene region containing 6 genes responsible for the synthesis of the diazo functional group  family . AfsA-like enzymes have been shown to be key in both γ-butyrolactones and AHFCA synthesis (The type II polyketide synthase (PKS) ucidated , 2011. Sucidated  and for al group . Of the ) family , and alpectively . Pseudo-ectively , the poscterized , 2011 ancterized . Followioduction . At this resumes , 2011. Aeratures , but ratynthesis .Streptomyces  was used as a model of the known recognized sequences in the gene cluster  measurements. As the experimental set-up uses dsoverhangs, the movements of the biochip’s nanolevers with the dsoverhangs used for the analysis have increased friction and lower dynamic response (DR) compared to those of the nanolevers alone. It is therefore necessary to first determine the relative size of the dsoverhang . By comparing the switching dynamics of the dsoverhang to those of the reference nanolevers, the nanoAREV was shown to have 3.65 nm in relative size   . AlpZ anor A3(2) , furtheror A3(2)  predicten method  to model fradiae , showing fradiae . This reS. ambofaciens signaling molecule is able to affect the DNA binding by AlpZ, effectively modulating the regulation of kinamycin biosynthesis . This strain is also deficient in kinamycin production [loci alpIABCD deleted, in which the alpABC locus encodes the minimal PKS  and signaling molecule containing extract to observe its effects on the association, followed by a conformational analysis . As in eStreptomyces, such as JadR2 , or rather the induction of structural conformational changes on the regulator hindering its DNA-binding capability. Based on previous studies, the latter hypothesis seems more probable, as TetR regulators appear to shift between mutually exclusive conformational states, either binding DNA or their cognate ligand of choice , unlike bioticus  and the bioticus . Howeveradomycin .afsA-like gene identified in the proximity of the alp cluster has been shown to contain an ARE motif (unpublished). It would be interesting to explore the role of AlpZ in the expression regulation of the afsA-like gene. One could envision that the AlpZ-AREafsA-like complex would activate gene expression for a positive feed-back loop of induction of production of signaling molecule. Even though the nature of this signaling molecule continues to elude us, this work clearly demonstrates that the response of AlpZ to the extract of S. ambofaciens is in no way linked to kinamycin and broadens the knowledge on AlpZ regulatory role, possibly paving the way for similar autoregulator receptors. Efforts are underway for the purification and identification of the S. ambofaciens signaling molecule. The discovery of new signaling molecules in Streptomyces is impaired by the fact that these compounds are typically produced in very small amounts. Moreover, purification scale can be highly variable, avenolide in S. avermitilis required 2000 L of fermentation broth whereas S. coelicolor furans were identified using only 40 squared Petri dishes . Binding assays were performed with AlpZ protein, purified as described previously (4)2SO4, 1 mM DTT, 0.2% Tween 20, 30 mM KCl) containing 50 μg/ml poly(dI-C) in a 20 μl final volume. When necessary, S. ambofaciens ΔΔalpWΔΔalpID extracts (1 μl) were added after the incubation period and incubated for further 10 min to verify the presence of signaling molecule. Binding reactions were analyzed with 5% native PAGE and run in 0.5X TBE buffer. DNA was then transferred onto a positively charged nylon membrane (Amersham Hybon-N+) by electroblotting, then fixed by UV crosslinking, detected with anti-digoxigenin antibodies, and developed by chemiluminescence with the CDP-StarTM reagent (Roche Applied Science).DNA binding assays were performed by EMSA as described previously . Brieflyeviously . Labeled® DRX instrument and MPC-48-2-R1-S biochips  were used to characterize the binding kinetics and protein size changes . For the analysis of protein affinity to the dsoverhang, 150 μl of AlpZ at a range of concentrations were injected with a flow rate of 30 μl/min, and dissociation was measured with running buffer at a flow rate of 30 μl/min over 120 min to 4 h. Assays to assess the effect of signaling molecule on the binding of AlpZ to DNA started with a preparation step with dsoverhang  and 150 μl of AlpZ  at 50nM with a flow rate of 30 μl/min without dissociation, followed by a sandwich type of capture injecting 140 μl of signaling molecule containing extract  at different concentrations (dilutions performed in PE40) with a flow rate of 20 μl/min. All assays used dsoverhangs at 500 nM and were performed at 30°C, combining both association/dissociation and size measurements and used PE40 as running buffer. A regeneration solution  was used to remove the ligand after each assay.Specific dsDNA sequences coupled with ssDNA overhang regions complementary to the ssDNA sequences tethered to the biochip (nanolevers) called dsoverhangs, were designed. To prepare the dsoverhangs 2 μM of the two comprising ssDNA sequences (listed in kON and kOFF) and dissociation constants (KD) were determined using real-time measurements of the switching dynamics. Protein size was estimated by comparing the switching dynamics of bound protein with those of bare DNA and with the lollipop biophysical model  monitoring the absorbance at 420 nm as described previously .All datasets generated for this study are included in the article/CV and LH performed the experiments. CV, J-MG, and BA analyzed the data. CV and BA conceived the experiments and wrote the manuscript. BA supervised the project and obtained the funding. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The long reads produced by third generation sequencing technologies have significantly boosted the results of genome assembly but still, genome-wide assemblies solely based on read data cannot be produced. Thus, for example, optical mapping data has been used to further improve genome assemblies but it has mostly been applied in a post-processing stage after contig assembly.OpticalKermit which directly integrates genome wide optical maps into contig assembly. We show how genome wide optical maps can be used to localize reads on the genome and then we adapt the Kermit method, which originally incorporated genetic linkage maps to the miniasm assembler, to use this information in contig assembly. Our experimental results show that incorporating genome wide optical maps to the contig assembly of miniasm increases NGA50 while the number of misassemblies decreases or stays the same. Furthermore, when compared to the Canu assembler, OpticalKermit produces an assembly with almost three times higher NGA50 with a lower number of misassemblies on real A. thaliana reads.We propose OpticalKermit successfully incorporates optical mapping data directly to contig assembly of eukaryotic genomes. Our results show that this is a promising approach to improve the contiguity of genome assemblies. Nevertheless, assemblies produced solely on read data are not complete and typically contain orders of magnitudes more contigs than the sequenced organism has chromosomes. To further improve these assemblies, several long-range technologies such as optical mapping, genetic linkage maps, and Hi-C based analysis have been developed  is greater than i is chosen as the starting fragment i.e. the starting color. Similarly, the first fragment m, for which the sum of the lengths of the contig fragments  is greater than j, is used as the end fragment i.e. the end color. As Kermit only uses the start and end colors of a read, this coloring scheme suffices.For each alignment, the start and end positions in a contig are known, and they are used to determine which colors of the contig are used to color the read. For example, suppose the aligning section in the contig starts from position i and ends at position j. Additionally, suppose we also know that there is a sequence of length a in the beginning of the aligning section of the read, and a sequence of length b at the end of aligning section of the read. The question is, would it be beneficial to adjust the starting position of the aligning section in the contig to start at i−a and to end at j+b to possibly extend read colors. Figure We deliberated if the beginning and end parts of the reads that are left outside of the aligning section should be used to extend the coloring beyond the aligning section in the contig. For example, suppose the alignment in the contig starts again at position After the reads are colored, some of the available colors in the contigs can be completely unused by the reads. In other words, none of the read alignments overlapped with the fragments corresponding to the missing colors. To be clear, colors between the start and end colors of a read are also considered as used. It is possible that two reads are in reality physically close to each other, even though there is a color gap between them due to inaccurate alignments for example. This might affect the performance of Kermit negatively, so we decided to shift the colors so that there were no missing colors.s·k term in the colors was left untouched. We ran experiments with and without this adjustment to determine its effects, and the results suggest that readjusting the colors is beneficial.As a simplified example, suppose all the reads used colors {1,2,4,7,8,10}, while the colors appearing in the reference were {1,2,3,4,5,6,7,8,9,10}. These read colors would then be mapped to consecutive colors {1,2,3,4,5,6} in their respective order; 1→1, 2→2, 4→3, 7→4, 8→5, 10→6. Then the already colored reads would be recolored according to these mappings. They would still keep their respective order, but the gaps caused by unused colors get removed. Colors were only shifted within a single chromosome’s colors i.e. the OPTICALKERMIT assembly pipeline uses the Kermit assembler [After all the data is gathered and processed to get the corrected and colored reads, we can start assembling post-coloring contigs. Our ssembler  to do thssembler , the difKermit starts by building an overlap graph with the help of minimap2  alignmenvi to vertex vi+1 means the corresponding sequences overlap, and can be merged together to be used as a part of a unitig. Some of the connections are bound to be erroneous, which can be detected with the help of the colors.As with miniasm, a unitig is again defined as a maximal non-branching path in the overlap graph. However, with the vertex color information, we can alter the paths that are used to build the unitig. An edge from vertex vi,vi+1) exists in the graph, but the color number of vertex vi was larger than that of vertex vi+1. The color information suggests that vi should appear after vi+1, but the edge says they are connected together in the reverse order. We trust the coloring information acquired from the optical maps more than the overlap edge, and it is removed completely from the graph. Even if the order of the colors is correct, their distance can cause suspicion. If the colors are very far apart, we can deduce the vertices should not be connected with an edge, and such edges are also removed from the graph. These kind of contradicting edges are called inconsistent edges.Suppose an edge  is an edge such that at least one of the colors of vertex vi+1 is equal to or exactly one greater than at least one of the colors in vertex vi. By default Kermit only allows the colors to differ by at most one which is the option we used, but this restriction can be adjusted by the user. All edges that do not follow this requirement are discarded. This way we cannot take a path with a huge gap or where some of the sequences are in the wrong order.A Some of the reads might be left uncolored due to difficulties and uncertainties during the coloring step. An edge that is connected to an uncolored vertex would be automatically removed, but Kermit alleviates this problem of uncolored vertices by allowing the colors to propagate. An uncolored vertex is assigned all colors of the vertices that are reachable from it by traveling only through uncolored vertices. There is a limit on how far the colors should propagate, since allowing the colors to flow as far as possible would most likely lead to incorrect colorings. By default Kermit allows the colors to propagate through five vertices, but this can again be changed to the user’s liking. If propagated colors have some missing colors between them, the vertex is deleted completely from the graph because the propagated coloring is not coherent which makes us suspicious of its correctness. We experimented with the default option and the no propagation option, and the results can be seen in Table rainbow paths. A rainbow path is like a normal path, but all the colors must appear in increasing order and only once. Kermit loosens this condition by allowing consecutive vertices to use the same color on a path. The removal of all inconsistent edges guarantees that every path we find is also a rainbow path. Kermit outputs these maximal non-branching rainbow path unitigs as our final assembly product.After inconsistent edges are removed, Kermit starts looking for the unitigs. Instead of finding all maximal non-branching paths, we automatically find all maximal non-branching


Alcohol is the most widely used addictive substance. Severe alcohol abuse is diagnosed as “alcohol use disorder” (AUD). A common and harmful drinking pattern is binge drinking that elevates a person’s blood alcohol concentration to ≥ 0.08%. Such drinking may be an early indicator of AUD. Opioid misuse and dependence have become worldwide crises. Patterned consumption of various opioids can develop into opioid use disorder (OUD). An intertwined epidemic exists between opioid abuse, alcohol addiction, and binge drinking. Currently, studies on the interaction of AUD and OUD are limited and the underlying mechanisms linking these disorders remains unclear. We reviewed studies on AUD and OUD and utilized Ingenuity Pathway Analysis (IPA) to identify mechanisms of AUD and OUD interaction and potential gene targets for therapeutic agents. According to IPA Canonical Pathways Analysis, Gamma-aminobutyric Acid (GABA) Receptor Signaling, Neuroinflammation Signaling Pathway, Opioid Signaling Pathway and Dopamine-DARPP32 Feedback in cAMP Signaling are potential contributors to the interaction of AUD and OUD. The effects of drinking depend on the volume consumed, the concentration of ethanol by volume, and the drinking pattern. –4. Alcohvs. 5.3 million) ) –34.3.Among the 235 genes related to AUD and the 131 genes related OUD, there were 65 genes related to both disorders. The relations of the 65 genes to AUD  Receptor Signaling, Neuroinflammation Signaling, Opioid Signaling, dopamine-DARPP32 feedback in cAMP signaling. Circadian Rhythm Signalling, nNOS Signaling in Neurons and Glutamate Receptor Signalling are predicted to be involved in AUD and OUD interaction in IPA. Literatures reported that GABA Receptor Signalling and Opioid Signalling have been reported to be related to alcohol and opioid addiction; Glutamate Receptor Signalling has been reported to be related to alcohol and opioid addiction and tolerance. It is found that Dopamine-DARPP32 feedback in cAMP signalling cross-talks with GABA Receptor Signaling and that nNOS Signaling in Neurons has cross-talk with Glutamate Receptor Signalling. In addition, cross-talk and regulation between GABA Receptor Signaling and Glutamate Receptor Signalling have been reported. Involvement of Neuroinflammation Signaling and Circadian Rhythm Signalling is unclear.Alcohol exerts effects on the reward system through both the opioid and dopaminergic systems . GABA Revia GABA Receptor Signalling.Glutamate is the most abundant excitatory neurotransmitter in the mammalian nervous system, and acts via glutamate receptors. Both alcohol and mu-opioid are well-known to affect glutamate receptor function. Alcohol is known to inhibit glutamate, particularly at the N-methyl-d-aspartate (NMDA) glutamate receptor –48. And OPRM1 polymorphisms are found to be associated with alcoholism treatment and risk [Oprm1 polymorphism and alcohol dependence may be explained by different physiological responses to alcohol by persons with the various genotypes. People with the 118G variant have a stronger association between their desire to drink and subsequent drinking than people lacking the variant, an effect attenuated by naltrexone [Opioid receptor genes have been studied frequently in relation to both alcohol and opioid addiction –58. It iand risk . Adolescand risk , 67. Theltrexone . It is lltrexone . And croltrexone , 69.Perhaps surprisingly, the inflammation and immune response also is associated with the rewards of alcohol and opioid addiction . NeuroinCyclic AMP-regulated phosphoprotein (DARPP-32) is a key actor of this integration in the GABAergic medium-size spiny neurons, particularly in response to dopamine , 75. IncCircadian rhythm is a roughly 24-hour periodicity in the physiological processes in various organisms, which is endogenously maintained by a series of molecular mechanisms. The circadian rhythm, however, can be moderated by external environment, such as light and temperature. Limited studies have reported effects of alcohol and opioid use on circadian rhythm. Baird et al reported that circadian rhythm of body temperature changed following alcohol treatment , 78. Bin2+ through interaction with CALM and Calcineurin. In the brain, NO synthesis is regulated through stimulation of NMDAR by glutamate [2+-dependent manner [Nitric oxide (NO) is formed endogenously by a family of enzymes known as NO synthases (NOS) Neuronal nitric oxide synthase (nNOS) is a family of enzymes producing Nitric oxide (NO), expressed in specific neurons of the Central Nervous System (CNS) . nNOS islutamate , 84. NOSlutamate , 86, andlutamate  Meanwhilt manner . Direct 5.In summary, this review studied AUD and OUD, and predicts potential targets in AUD and OUD interaction based on a bioinformatics tool, IPA. Multiple pathways could contribute to the interaction. According to IPA Canonical Pathways Analysis, GABA Receptor Signaling, Neuroinflammation Signaling Pathway, Opioid Signaling Pathway and Dopamine-DARPP32 Feedback in cAMP Signaling are potential pathways contributing to interaction of AUD and OUD. Our review provides a possible strategy for studying disease interaction and mining drug targets.


The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a neoplasm of CD4+CD25+ T cells that occurs in 2–5% of infected individuals after decades of asymptomatic latent infection. Multiple HTLV-1-encoded regulatory proteins, including Tax and HTLV-1 basic leucine zipper factor (HBZ), play key roles in viral persistence and latency. The HTLV-1 Tax oncoprotein interacts with a plethora of host cellular proteins to regulate viral gene expression and also promote the aberrant activation of signaling pathways such as NF-κB to drive clonal proliferation and survival of T cells bearing the HTLV-1 provirus. Tax undergoes various post-translational modifications such as phosphorylation and ubiquitination that regulate its function and subcellular localization. Tax shuttles in different subcellular compartments for the activation of anti-apoptotic genes and deregulates the cell cycle with the induction of DNA damage for the accumulation of genomic instability that can result in cellular immortalization and malignant transformation. However, Tax is highly immunogenic and therefore HTLV-1 has evolved numerous strategies to tightly regulate Tax expression while maintaining the pool of anti-apoptotic genes through HBZ. In this review, we summarize the key findings on the oncogenic mechanisms used by Tax that set the stage for the development of ATLL, and the strategies used by HTLV-1 to tightly regulate Tax expression for immune evasion and viral persistence.  Retroviridae and genus deltaretrovirus. This genus also includes three additional HTLV members, HTLV-2, -3, and -4 [The human T-cell lymphotropic virus type 1 (HTLV-1) is the first oncogenic retrovirus discovered in humans in the early 1980s by two independent research groups in Japan and America . HTLV-1 , and -4 . HTLV-1 , and -4 ,7. Althode novo infection) and clonal proliferation or mitotic expansion of infected cells while limiting its replication [HTLV-1 transmission occurs by three different routes: vertical transmission from a carrier mother to her infant through breast-feeding, horizontal transmission of infected lymphocytes through sexual contact, and parenteral  ,10,11,12lication ,16,17. Alication . The cur+ T lymphocytes harbor the vast majority (>90%) of the HTLV-1 viral load in vivo [The HTLV-1 virion is enveloped, ~100 nm in diameter, and carries two identical strands of genomic RNA inside a protein capsid. The viral capsid also contains functional protease (pro), integrase (IN), and reverse transcriptase (RT) enzymes. CD4 in vivo . However in vivo ,21. HTLV in vivo , and cel in vivo ,25,26,27 in vivo ,29. The cis-acting promoter elements that drive the transcription of viral genes such as the structural protein Gag , Pro (Protease), polymerase (Pol) and Env from unspliced/singly spliced mRNA [HTLV-1 entry into cells depends on sequential interactions with three host components. The HTLV-1 viral envelope (Env) is thought to initially interact with heparin sulfate proteoglycans (HSPGs) followed by the formation of a complex with neuropilin-1 (NRP1) and glucose transporter-1 (GLUT1), which results in fusion ,31,32,33ced mRNA . The regced mRNA ,36,37,38ced mRNA . The matced mRNA ,41. The ced mRNA . During Tax, Rex, p21, p12, p13 and p30 are all encoded by various open reading frames (ORFs) in the pX region located in the 3’ end of the genome [p12, p13 and p30 play significant roles in establishing and maintaining viral persistence, whereas Rex regulates post-transcriptional viral gene expression and increases the stability of viral RNA for the latency phase of the viral life cycle [trans-activator protein Tax is expressed from a doubly spliced mRNA transcript and is a multifunctional protein that triggers a plethora of events, such as the activation of cell signaling pathways that drive proliferation and survival of T cells that can promote their immortalization and transformation [The HTLV-1 regulatory genes e genome ,44,45. Tfe cycle ,47,48,49fe cycle ,51. HBZ fe cycle . Of notefe cycle . The HTLormation ,55. Howein vivo by the generation of different Tax transgenic mouse strains, which develop distinct tumors and/or inflammatory lesions depending on the promoter used to drive Tax expression [The oncogenic function of Tax has been demonstrated pression ,57,58,59pression , immortapression  and indupression . Moreovepression ,63,64. Tpression . Tax propression , and thepression . Similarpression .trans-activation of the viral promoter [Tax is a critical regulator of viral gene expression and recruits host transcription factors such as cyclic AMP (cAMP)-response element-binding protein (CREB) and the coactivators, CREB binding protein (CBP) and p300 to the LTR for promoter . Tax forpromoter ,71. Intepromoter ,73, and promoter . Moreovepromoter . Tax alspromoter . Furtherpromoter . TogetheTax controls virus replication and persistence by regulating its localization in different compartments of cells, including the cytosol, nucleus, Golgi apparatus, and endoplasmic reticulum (ER) via different sequences including a nuclear localization sequence (NLS) and nuclear export sequence (NES) ,80,81,82NF-κB is an evolutionarily conserved transcription factor family with diverse physiological roles including cell proliferation, apoptosis, oncogenesis, development and the innate and adaptive immune response. NF-κB family members consist of p65 (RelA), c-Rel, RelB, p50/p105 and p52/p100 . NF-κB dNF-κB signaling is normally transiently activated by acute stimuli and terminated as a result of induction by negative feedback inhibitors. However, oncogenic viruses have evolved various strategies to manipulate NF-κB signaling to establish long-term persistence within their hosts, and inhibition of NF-κB signaling impairs the proliferation and survival of virus-infected cells ,92,93. CTax also chronically activates the noncanonical NF-κB pathway, which contributes to cell survival and oncogenesis. Surprisingly, in contrast to TNFR superfamily members which require only IKKα, Tax requires both IKKα and NEMO to activate the noncanonical NF-κB pathway via formation of a complex that also contains p100 . Tax phyTax interacts with a host of signaling proteins to impart persistent NF-κB activation. Tax has been reported to interact with and activate the kinase activity of TGF-β-activating kinase 1 (TAK1) through TAB2 (TAK1 binding protein 2) . Tax alsTax relies on a number of mechanisms and binding proteins to ensure persistent NF-κB signaling including cross-talk between canonical and non-canonical pathways, the establishment of feed-forward signaling loops and inactivation of negative regulators. Tax-mediated activation of the noncanonical pathway suppresses the expression of the WWOX (WW domain containing oxidoreductase) tumor suppressor gene, which inhibits canonical NF-κB signaling . Tax-IKKtrans-activation, and interaction with host proteins to modulate their function in order to enhance viral persistence and spread. Tax is subject to multiple PTMs including ubiquitination, SUMOylation, acetylation and phosphorylation, which influence Tax protein-protein interactions and trafficking to different compartments of the cell, and ultimately modulate distinct Tax functions. It is likely there is cross-talk or interdependence among the different PTMs of Tax; however, the chronological order of these modifications essential for NF-κB activation and the enzymes involved in the different modifications are not fully realized. Like other viruses, HTLV-1 hijacks the host cellular machinery to promote PTMs of viral, as well as cellular proteins to enhance viral replication and persistence, and immune evasion. These PTMs can also influence signaling pathways contributing to uncontrolled cell proliferation and transformation. Tax PTMs regulate its cellular localization, Mutagenesis studies have revealed that Tax undergoes ubiquitination at multiple lysines including K263, K280 and K284, which modulates its stability, cellular trafficking and NF-κB signaling ,130. Alttrans-activation in the nucleus [SUMOylation refers to the reversible covalent attachment of a SUMO  moiety to target proteins executed by sequential enzymatic reactions catalyzed by E1, E2 and E3 enzymes, analogous to ubiquitination. Tax has been shown to be SUMOylated at K280 and K284, sites which can also be ubiquitinated . The mod nucleus , another nucleus .Tax is a phosphoprotein which can be phosphorylated at serines 300 and 301 (S300 and S301). Tax phosphorylation promotes its localization to nuclear bodies and activation of gene expression by the ATF/CREB and NF-κB pathways . Tax hasTax has also been shown to be acetylated at lysine 346 (K346) and phosphorylation of Tax may serve as a prerequisite event for its acetylation . Tax aceIn cancer cells, there is a loss of balance between cell division and cell death due to dysregulated signaling pathways. Programmed cell death or apoptosis is tightly regulated by pro- and anti-apoptotic factors, and evasion of apoptosis can lead to the accumulation of genetic abnormalities and malignant transformation. Like other oncogenic viruses, HTLV-1 specifically targets apoptosis signaling mechanisms in order to suppress apoptosis for the uncontrolled proliferation of virally-infected cells which can eventually result in tumorigenesis. The evasion of apoptosis by cancer cells can be broadly categorized into two groups: (1) an imbalance between pro- and anti-apoptotic proteins and (2) impaired caspase signaling and compromised death receptor signaling .Bim gene by inhibiting the function of the FoxO3a transcription factor [Tax modulates the expression of numerous genes regulating cell survival . For exan factor . Togethetrans-activation by CBP/p300 sequestration [TP53 gene is not mutated in all ATLL patients, but rather ~30% of ATLLs exhibit p53 mutations [Tax also targets tumor suppressors to enhance cell survival and sustained cell proliferation. Tax inhibits the tumor suppressor p53 through either CREB or NF-κB pathways to induce immortalization of HTLV-1-infected T cells ,163. Taxstration ,166. Sinutations ,168. Ras proteins are small GTPases that regulate cell differentiation, proliferation, and survival, and when aberrantly expressed or dysregulated contribute to oncogenesis. Tax targets and induces the expression of Ras family members to protect cells from apoptosis . Tax alsThe survival and proliferation of HTLV-1-infected T cells also depends on Tax-driven dysregulation of cytokines . Tax proThe caspase family of proteases induces apoptosis and are categorized as initiators  or effectors  based on their function. Tax drives the expression of the caspase inhibitor XIAP through activation of NF-κB signaling, and XIAP inactivates caspases-3, -7 and -9 for the inhibition of apoptosis ,181,182.The highly regulated multi-step process of cell cycle regulation and DNA repair safeguards the errorless inheritance of genetic material; however, oncogenic viruses such as HTLV-1 modulate the function of cell cycle regulatory proteins including cyclin/CDK complexes as well as key DNA repair enzymes, resulting in increased error frequency and uncontrolled cell proliferation . Tax indwaf1/cip1, which binds and inactivates CDK2 to mediate cell cycle arrest in response to various cellular stresses to prevent progression to the S phase of the cell cycle until DNA repair is completed [waf1/cip1 and CDK2 in a p53-independent manner in HTLV-1-infected T cells to sequester p21waf1/cip1 from cyclin E/CDK2, possibly as a mechanism for the active cyclin-CDK complex to phosphorylate Rb proteins and accelerate the progression through the G1/S checkpoint [The tumor suppressor protein p53 also regulates G1 to S transition through the expression of cell cycle control proteins such as p21ompleted . Howevereckpoint ,203. Fineckpoint . Thus, vSimilarly, S phase or DNA replication is tightly regulated by cyclin A/CDK2 which phosphorylates the pre-replication protein complex to restrict a single round of replication . Intereswaf1/cip1 interacts with endogenous proliferating cell nuclear antigen (PCNA) to prevent DNA replication while allowing PCNA-dependent DNA repair or nucleotide excision repair (NER). Notably, Tax increases PCNA gene expression to overcome the p21waf1/cip1-mediated replication block and to sustain DNA replication by impairing NER, thus promoting genomic instability by elevating the frequency of mutations [The ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-Related) kinases regulate checkpoint activation in response to DNA damage . ATM andutations ,216,217.utations . Double-utations . MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of various genes by the inhibition of protein translation or degradation of target mRNA transcripts upon binding to their untranslated regions (UTRs). The regulation of miRNA expression has garnered much attention due to their crucial regulatory roles in several pathophysiological processes, including cancer . GeneralSeveral studies have described a dysregulation of miRNA expression in HTLV-1-infected T cell lines, ATLL cell lines, and ATLL patient samples suggesting potential roles in ATLL pathogenesis ,227,228.waf1/cip1 and p27-mediated cellular senescence through the hyper-activation of NF-κB signaling [Several oncogenic viruses have evolved to counteract host immune signaling by diverse strategies . A key signaling ,236. Taxignaling . Given tignaling , HTLV-1 ignaling ,241,242.trans-activation of the viral LTR by competitive binding to CBP/300 [HBZ RNA may increase Tax expression indirectly through the downregulation of p30, thus highlighting the complexity and cross-regulation between different HTLV-1-encoded regulatory genes [BATF3 locus and establishing an auto-regulatory loop; genetic ablation of either BATF3 or IRF4 in ATLL cells induces a profound cell-cycle arrest, possibly due to the downregulation of the target gene MYC, even in the absence of Tax [Tax is also tightly regulated by other HTLV-1-encoded genes. HTLV-1 p30 inhibits Tax expression by sequestering doubly spliced viral RNA in the nucleus and also blocks Tax  CBP/300 ,244. Sim CBP/300 . Howeverry genes . Interese of Tax . Thus, HPLCG1, PRKCB and CARD11) in the T-cell receptor (TCR) signaling pathway likely also contribute to persistent NF-κB activation in the absence of Tax [Myc, Ccr4 and Birc3 and form a feed-forward loop driving cell survival in ATLL [As discussed earlier, Tax protein levels can be downregulated by host E3 ligases such as PDLIM2 which inhibits Tax expression through ubiquitin-mediated degradation in the nuclear matrix . Howevere of Tax . Interes in ATLL .Anti-sense RNAs containing the HTLV-1 LTR region, and RNA from the Rex-responsive element in the LTR have been proposed to regulate NF-κB activation by stimulating pattern recognition receptors (PRRs) such as RNA-dependent protein kinase (PKR) and 2’5’-oligoadenylate synthetase, an interferon stimulated gene (ISG), in Tax-negative ATLL cells ,252. IntHTLV-1 has evolved multiple strategies to exploit and disrupt the host cellular machinery for its long-term persistence that can ultimately cause the immortalization and transformation of infected T cells. In this review, we have highlighted the mechanisms of HTLV-1 Tax manipulation and subversion of key cellular pathways and checkpoints and discussed the functional roles of Tax PTMs in the regulation of its protein-protein interactions and subcellular localization. Tax expression is also tightly regulated at the transcriptional and post-translational levels by other HTLV-1 regulatory proteins as well as host factors that optimize viral immune evasion and persistence. Although recent studies have identified stress stimuli and host factors  that can regulate Tax expression and the reactivation of latent HTLV-1, there are likely additional pathways and mechanisms important for HTLV-1 latency. Ubiquitination (mono-and poly-) represents a versatile PTM and critical regulatory mechanism for Tax shuttling between cytoplasmic and nuclear compartments as well as chronic NF-κB activation and oncogenicity. Ubiquitination of host proteins is also key for HTLV-1 persistence as histone H2A monoubiquitination plays a critical role in silencing of the HTLV-1 provirus . HoweverIn addition to Tax, HBZ is a key viral factor important for HTLV-1 persistence and oncogenesis. There exists a complex and cooperative interplay between Tax and HBZ that maintains the fragile equilibrium of NF-κB signaling for driving clonal expansion that is balanced by the inhibition of senescence. This optimal level of signal transduction fine-tuned by Tax-HBZ is likely critical for malignant transformation of infected T cells. Further elucidation of the cross-talk between Tax and HBZ is needed to understand the oncogenic mechanisms of HTLV-1. Another interesting avenue of exploration is to understand how Tax protects and maintains the entire pool of ATLL cells despite its low expression in a negligible fraction of infected cells . Togethe


Danhong injection (DHI), which is one of the most well-known Traditional Chinese Medicine (TCM) injections, widely used to treat unstable angina (UA). However, its underlying pharmacological mechanisms need to be further clarified.In the present study, network pharmacology was adopted. Firstly, the relative compounds were obtained by a wide-scaled literatures-mining and potential targets of these compounds by target fishing were collected. Then, we built the UA target database by DisGeNET, DigSee, TTD, OMIM. Based on data, protein-protein interaction (PPI) analysis, GO and KEGG pathway enrichment analysis were performed and screen the hub targets by topology. Furthermore, evaluation of the binding potential of key targets and compounds through molecular docking.The results showed that 12 ingredients of DHI and 27 putative known therapeutic targets were picked out. By systematic analysis, identified 4 hub targets  mainly involved in the complex treating effects associated with coagulation and hemostasis, cell membrane region, platelet alpha granule, NF-kappa B signaling pathway and TNF signaling pathway.The results of this study preliminarily explained the potential targets and signaling pathways of DHI in the treatment of UA, which may help to laid a good foundation for experimental research and further clinical application. According to American Heart Association statistics, cardiovascular and cerebrovascular diseases are the leading causes of death, imposing immense health, financial and emotional burdens on the world , 2. UA, Salvia miltiorrhiza  and Carthami Flos  [TCM is a clinically proven medical practice that has been in existence for thousands of years –14. DHI eraceae) –17. DHI eraceae) , 19. Eveeraceae) .Network pharmacology with systematic and holistic characteristics has become a promising method to explain the complex interactions between herbs and diseases at the system level –23. To shttps://www.ncbi.nlm.nih.gov/pubmed/), and China National Knowledge Infrastructure Database  were applied. Furthermore, the PubChem [https://pubchem.ncbi.nlm.nih.gov/), and ChemDraw [http://www.chemdraw.com.cn/) was used to find Canonical simplified molecular input line entry specification (SMILES) information of the compounds.To collect the chemical ingredients of DHI, PubMed ( PubChem  (https:/ChemDraw  (http://http://prediction.charite.de/), SwissTargetPrediction [http://www.swisstargetprediction.ch/) and BATMAN-TCM [http://bionet.ncpsb.org/batman-tcm/) for potential targets related to DHI compounds. What’s more, the UniProt [http://www.uniprot.org/) was utilized to convert the protein name of the DHI bioactive ingredients to the gene names. The search results were filtered to retain only studies conducted on “Homo sapiens (Human)” so that the names can be standardized and data duplicates deleted.We searched the SuperPred  (http://ediction  (http://TMAN-TCM  (http:// UniProt  (http://http://210.107.182.61/geneSearch/) [http://www.disgenet.org/search) [https://omim.org/) [https://db.idrblab.org/ttd/) [Homo sapiens (Human)”. The intersection between the UA-related human gene and the target gene of the active compound was retained for further analysis.Upload “Unstable angina”, “UA”, and “Unstable angina Pectori” as search terms to the DigSee (Search/) , DisGeNE/search) , OMIM (him.org/)  and Therrg/ttd/) . In addihttps://string-db.org/) as a central protein, which stores information about protein interactions. Only “Homo sapiens” proteins with the confidence score higher than 0.7 were picked out.The names of putative UA/compound targets were submitted to STRING 11.0 database  (https:/https://cytoscape.org/) were employed to construct six visualization networks as follows: (1) DHI compound-predicted target network; (2) UA-associated target network; (3) Compound- DHI/UA putative therapeutic target network; (4) DHI-UA PPI network; (5) Module analysis network; (6) Drug- key compounds- hub targets-pathways network. The “degree” is regarded as the number of edges connected to it [To characterize the therapeutic mechanisms of DHI against UA from a network target perspective, the Cytoscape 3.7.1   algorithms can find dense regions of interaction in PPI networks based on complex connection data . In the http://geneontology.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (https://www.genome.jp/kegg/) [To evaluate the role of potential core targets by bioinformatic annotation, the R 3.6.1 software with the Bioconductor package was manipulated, including Gene Ontology (GO) knowledgebase (p/kegg/) –46.https://www.rcsb.org/) [Homo sapiens. Based on the above conditions, a total of 11 core target protein PDB IDs were gathered. At the same time, the Notepad++ (https://notepad-plus-plus.org/) and AutoDockTools were applied to not only remove water molecules and pro-ligand small molecules, but also hydrogenate and charge. Finally, molecular docking calculations were performed using Autodock Vina 1.1.2 [https://pymol.org/2/) software were wielded to visualize the docking results [Initially, AutoDockTools 1.5.6 was employed to set the number of rotatable bonds for 12 small molecule compounds obtained . Subsequsb.org/) . The scrna 1.1.2 . The PyM results , 51.After deleting duplicate data, a total of 12 major compounds were collected as candidate compounds  of the identified DHI-UA PPI network and 3 core modules to gain insights into the cellular component (CC), molecular function (MF) and biological processes (BP) that are affected in UA   Fig.  and S5. In this paper, four potential targets with five corresponding compounds were simulated by molecular docking, and the docking results were analyzed. Using Pymol software, these five compounds were observed to enter the active pocket of the protein Fig. . Taking p-value and q-value will be analyzed as an important pathway  for a long time –56. AlthBased on module and network topology analysis, the four potential hub targets were found: TNF, TLR4, NFKB1 and SERPINE1. The studies of Biasucci et al. and Huang et al. showed that patients with UA have significantly increased proinflammatory cytokines compared to healthy individuals , 58. Notp-value< 0.05 and q-value< 0.05), we recognized 78 UA-related signaling pathways, NF-kappa B, TNF, complement and coagulation cascades, and toll-like receptor signaling pathway et al. Accordingly, these pathways may be involved in the progress of UA. Based on p-value and q-value, we choose NF-kappa B signaling pathway as most candidate signal for further study. Furthermore, nuclear factor kappa-B (NF-κB), which is a key transcription factor, may play a pivotal role in plaque instability by promoting the cascade expression of procoagulant genes and the regulation of pro-inflammatory genes in response to various stimuli [To understand potential biological mechanism of DHI against UA, GO and KEGG functional enrichment analysis were applied. Through the KEGG pathway analysis  were highly connected with regulation of coagulation, hemostasis, cell membrane region, platelet alpha granule, peptidase activity and cofactor binding. Therefore, the results suggest that DHI treats UA by participating in these BP, CC and MF.Molecular docking analysis simulation provided a visual interpretation of the interaction between key compounds and their potential protein targets. For example, rosmarinic acid small molecule mainly forms 9 hydrogen bonds with GLN-102, PRO-100, SER-99, TYR-115 and ARG-103 residues on TNF. Rosmarinic acid, which is considered one of the most important polyphenols, has several pharmacological effects: anti-oxidant, inhibition of oxidative stress, anti-inflammatory, anti-cancer and immunomodulatory , 78. BesVia the method of network pharmacology and molecular docking, 12 ingredients of DHI and 27 putative known therapeutic targets were collected and explored the underlying mechanism of DHI in treatment of UA. Then, DHI exerted treatment effects on UA by regulating 4 hub targets: TNF, TLR4, NFKB1 and SERPINE1. Based on the results of GO and KEGG pathway enrichment analysis, we found that these hub targets ameliorated UA by participating in regulating coagulation, hemostasis, peptidase activity, signal transduction and immune system. In conclusion, the results of the study preliminarily predicted the related underlying mechanism of DHI against UA, proving that the characteristics of multi-target synergy. However, animal experiments, molecular biological experiments and clinical investigations should be performed to verify the mechanism of DHI against UA in future studies.Additional file 1 Supplementary 1. Table S1. The information of all ingredients of DHI.Additional file 2 Supplementary 2. Table S2. The information of GO enrichment analysis of DHI-UA PPI network.Additional file 3 Supplementary 3. Table S3. The information of GO enrichment analysis for each cluster.Additional file 4 Supplementary 4. Table S4. The information of KEGG pathway analysis of DHI-UA PPI network.Additional file 5 Supplementary 5. Table S5. The information of KEGG pathway enrichment analysis for each cluster.