Dothistroma septosporum, which is a widely spread threat to northern European forests. We sampled two Pinus sylvestris sites in Estonia and two in Norway in order to analyse the relations between the abundance of D. septosporum and overall fungal richness, specific fungal species composition, time of season, needle age and position in the canopy. In both countries, the overall species richness of fungi was highest in autumn, showing a trend of increase with needle age. The overall species richness in the second-year needles in Estonia and third-year needles in Norway was similar, suggesting that a critical colonization threshold for needle shed in P. sylvestris is breached earlier in Estonia than in Norway. The fungal species richness in P. sylvestris needles was largely affected by Lophodermium conigenum. Especially in older needles, the relative abundance of L. conigenum was significantly higher in spring compared to summer or autumn. The timing of recruitment and colonization mechanisms of different foliage endophytes are shortly discussed.The relationship between the ecological success of needle pathogens of forest trees and species richness of co-inhabiting endophytic fungi is poorly understood. One of the most dangerous foliar pathogens of pine is  Fungal diversity in the foliage of conifers is affected by many biotic and abiotic factors, e.g., tree health, vegetation , latitude, climate and microclimate, needle age, position of needles in tree crown, etc. ,2,3. TheChrysomyxa spp. [Meria laricis are able to infect needles of their host trees only during the first four weeks after needle emergence. Most needle endophytes can infect needles of all age classes, with the susceptibility of the needles and the frequency of colonization being considered to increase with needle age. Johnson & Whitney [Picea mariana (Mill.) Britton, Sterns & Poggenburg) needles by endophytic fungi increased from 4% in the current-year-needles to 90% in 3-year-old needles. In addition, there are examples of first-year needles of Sitka spruce (Picea sitchensis (Bong.) Carr.) and Pinus strobus L. having an extremely low amount of endophytes in the genera Lophodermium and Hormonema in comparison to older needles [During the last decade, it has become evident that it can be difficult to differentiate endophytes from necrotrophs due to certain species having the ability to switch between feeding modes depending on the growth conditions and health status of host species . As Siebyxa spp.  and the  Whitney  showed t needles ,17. Pinus sylvestris L. needles [P. sylvestris needles showed an increase along the north to south gradient in Fennoscandia [Pinus nigra J.F Arnold) was similar across sites and tree cohorts or needle location within the canopy (shade needles compared to the light needles) but differed significantly among forest patches and trees of different age. Previous studies using either fungal culturing or 454 sequencing of the ITS2 region have shown that sampling time and geographical location can be the most common drivers of fungal diversity and richness of  needles ,2. Overaoscandia ,2 and froscandia . Taudieroscandia  concludeP. sylvestris in northern Europe, e.g., Dothistroma septosporum (Dorogin) M. Morelet and Lophodermium seditiosum Chevall. Dothistroma needle blight (DNB) is a dangerous foliar disease of pine caused by two species, Dothistorma septosporum and Dothistroma pini Hulbary [D. septosporum is more widely distributed across Europe, D. pini is present only in the southern and central parts of the continent [Pinus spp. [There are several needle pathogens threatening  Hulbary ,19. WhilDothistroma septosporum was first described in 1911 in Russia [Pinus nigra in 2006 and a year after on the native P. sylvestris. By 2008, D. septosporum was recorded across the country on P. sylvestris [D. septosporum was first discovered in 2009 in the Troms county [Dothistroma species in the world. In Norway, the main damage caused by D. septosporum has been observed on trees growing along rivers in valley bottoms, predominantly on second and third-year needles [D. septosporum infection than current -year needles, whereas no correlation between disease severity and tree vicinity to waterbodies is recorded [P. sylvestris [n Russia ,21. Sincn Russia . In Estolvestris . In Norws county , this rerecorded . It is klvestris ,26.Lophodermium Chevall. is a well-studied fungal genus in the family Rhytismataceae . Species within this genus are ecologically diverse even though they exploit a common habitat, i.e., pine needles [Lophodermium species have been historically the most often isolated species from needles of Pinus, Abies and Picea [Lophodermium are known to inhabit coniferous trees and shrubs and most of them are primary saprotrophs that are responsible for early decomposition of shed needles [L. seditiosum, is considered a major pathogen [Lophodermium seditiosum is known to infect pine needles in summer and early autumn, and the infected needles are shed from trees in the following spring [P. sylvestris is propagated [P. sylvestris, particularly in forest nurseries and young plantations [L. seditiosum and D. septosporum) have been influenced by changes in fungal species composition, forest management activities, distribution of susceptible host species, global climate change or by a combination of all these factors [ needles . Lophodend Picea . More th needles . Only onpathogen . Lophodeg spring ,31. Thisg spring , is commntations . It is a factors ,34.P. sylvestris in northern Europe to test the following hypotheses: (1) Fungal species richness depends on the site temperature sum, sampling season, needle canopy position, tree health and needle age; (2) Dothistroma septosporum and Lophodermium species are more abundant in needles from lower parts of the canopies compared to upper parts; (3) Owing to species-specific variation in sporulation regimes, the relative abundance of the dominant fungal species peaks at different times of the season.We used PacBio third-generation sequencing to analyze fungal species richness and composition in the needles of Vaccinium site type [P. sylvestris-dominated stand, with a pine age of 5–20 years and height of 1–8 m. Pinus sylvestris is present as both canopy forming and understory tree — the sampled DNB- symptomatic trees and healthy trees were growing in the understorey. The Konguta site is a pure P. sylvestris stand with 5–10- year-old (1–3 m) canopy-forming saplings. The Norwegian sites were located in Gransherrad  and Engerdal N61.7460, E11.9754, 544 m.a.s.l.), with effective temperature sums of 1590 °C and 895 °C, respectively, in 2014. The Gransherrad site was a pure stand of 3–6 m tall P. sylvestris, very dry with patches of understory vegetation dominated by Calluna. The Engerdal site was dominated by 3- to 12-m-tall P. sylvestris. The site was very heterogeneous, with boggy areas dominated by Sphagnum and dry areas characterized by Vaccinium in the ground vegetation.The Estonian sites represented the ite type , situateP. sylvestris trees with DNB symptoms and two trees with no foliage symptoms were selected and marked, this totaling eight healthy and eight diseased trees. The presence of typical DNB symptoms (red bands on needles) throughout the canopy was estimated visually during each sampling time. If no red bands were detected, the tree was considered healthy. From each sampled tree, the first- (needle-age-class 1) and second-year needles (needle-age-class 2) were sampled at each time point. In the case that third-year needles (needles of age-class 3) were present, these were also sampled. In the Estonian sampling sites, no third-year needles were retained in 2014. Although needles were sampled essentially from three different needle-age classes, the age of these needles within these classes differed depending on sampling time. In the spring 2015 (April), first-year needles were 11 months old, whereas in the summer (July) and autumn (September) 2014 first-year needles were 2 and 5 months old, respectively. Second-year needles were 23 months old in the spring 2015, and 14 and 17 months old in the summer and autumn 2014, respectively. Third-year needles were 26 and 29 months old in the summer and autumn 2014, respectively, and 35 months old in the spring 2015. From the 16 trees selected for sampling, the needles were collected separately from one randomly chosen alive branch from the third branch whorl, counting from the top of the tree canopy, and from one randomly chosen alive branch from the third branch whorl, counting from the base of the living canopy. This was done to consider potential qualitative or quantitative differences in fungal communities within tree crown. From each tree, three random needle pairs per needle-age class were sampled, cut into 0.5–1 cm pieces, placed into 2 mL Eppendorf tubes and stored in −20 °C for future analyses. The sampling scheme is illustrated in Sampling was performed in summer (July) and autumn (October) of 2014 and in spring (April) of 2015 at the four above-mentioned sites in Estonia and Norway. In each site, two The needle tissue disruption was performed in a Retsch MM400 homogenizer  using metal beads (2.5 mm), and DNA extraction was carried out using GeneJET Genomic DNA purification kit , following the manufacturer’s instructions. For identification of fungi, the rDNA gene region ITS1-5.8S-ITS2 was amplified using the ITS1catta  and ITS4Conventional PCR was carried out according to Agan et al. . The ampD. septosporum and D. pini, a 99% sequence similarity cut-off is needed. The OTUs were taxonomically identified based on representative sequences using Pipecraft against UNITE v.7 database [D. pini or Lophodermium sp. were also manually checked against UNITE v.7 to reduce the risk of false identification. Bioinformatics was undertaken using the programs mothur , UCHIME database . OTUs wedatabase . All OTUD. septosporum and Lophodermium spp. were tested using linear mixed models. Species richness and sequence read percentages of species were log-transformed prior to analyses.Richness of OTUs and rarefaction analyses were performed using PAST v3.25 . StatistTo consider differences in fungal communities in relation to the experimental factors  and their interactions, we used PRIMER v6 and PERMANOVA+ ,50. The Lophodermium conigenum , Phacidiaceae sp. , Capnodiales sp. (6.6%) and Dothistroma septosporum (6.4%). Dothistroma septosporum had two different OTUs in this dataset, the less abundant variant accounted for 0.1 % of all sequences in the dataset. There were considerable differences among the 20 most common fungal species between Estonia and Norway  (mean ± SE) fungal OTUs. On average, the Estonian sites Haabsaare and Konguta had 15.9 (±1.63) and 7.6 (±1.19) OTUs per sample, respectively, while the Norwegian sites Engerdal and Gransherrad had 5.8 (±0.62) and 8.9 (±1.28) OTUs per sample, respectively.Needles of D. septosporum increased from 4.3% in spring to 11.0% by summer and declined in autumn (3.7%). The relative abundance of L. conigenum was highest in spring (37.1%) and declined in summer (11.4%) and autumn (7.3%). The needle pathogen Lophodermium seditiosum was detected only in the Estonian samples at low abundance: 0.3% of all sequences.There were marked differences in the relative abundance of phyla, orders, dominant species, and overall richness across sampling times . The relP. sylvestris needles were country and sampling site , species richness being significantly higher in Estonia than in Norway . A high sequence read percentage of L. conigenum  was accompanied with lower general species richness. Needle age, presence of D. septosporum symptoms on tree and needle location in canopy did not have any significant (p > 0.05) relationship with overall fungal species richness in P. sylvestris needles. The two-year-old needles in Norway had significantly lower species richness when compared to needles of the same age in Estonia . While the needles were retained longer in the sampled trees in Norway compared to Estonia, the overall species richness of two-year-old needles in Estonia and three-year-old needles in Norway were similar (p > 0.05). The constructed full model across two countries and four sampling areas showed that significant predictors of general species richness in n Norway . Species2.114 = 2.45; R2adj = 0.305; p < 0.05; 2.114 = 24.4; R2adj = 0.201; p < 0.001; 2.83 = 8.42; R2adj = 0.657; p < 0.05; As there were significant differences in fungal species richness between countries, we constructed separate models to determine the influencers of species richness within each country. We found that the significant drivers of species richness were sampling season  higher in spring when compared to summer and autumn. Conversely, the abundance of D. septosporum was low in spring at all four sites. In three sites , D. septosporum showed its highest abundance in summer and a decline in autumn  and that of D. septosporum . Second-year and third-year needles harboured significantly  but not with that of L. conigenum. At all sampling sites, the older needles also harboured a considerably higher percentage of L. conigenum in comparison to current-year needles  in Estonia and S. polyspora, Capnodiales sp. and D. septosporum  in Norway  with the relative abundance of D. septosporum: needles in the upper part of canopy had significantly lower sequence read percentages of D. septosporum than needles in the lower part in Norway. A similar trend was observed in Estonia, although it was not significant (p = 0.08). Relative abundance of L. conigenum was significantly lower in the upper-canopy needles than lower-canopy needles in both countries .The interaction of country and needle position within the canopy was associated (Fp = 0.002), followed by needle location in the canopy and needle age . Sampling date explained 8.3% of the species composition (p = 0.01). Fungal species composition was also significantly (p = 0.004) different between healthy and diseased (DNB symptoms) trees. The interaction location x sampling date also had a significant effect on fungal species composition (p = 0.02). Other interactions with significant effect were sampling location x tree health (p = 0.02), sampling location x age of needles (p = 0.01) and needle location in canopy x tree health (p = 0.01), but these explained < 1% of the variation.According to permutational ANOVA, the biggest influencer of species composition was sampling site  differences in species composition between the sampling sites, sampling dates and needle age classes.CAP analysis  showed sLophodermium conigenum had no negative interactions, but 31 positive interactions. Dothistroma septosporum, however, had two statistically significant negative associations, with Chaetothyriales sp. (p = 0.008) and Malassezia restricta (p = 0.03), and 29 positive interactions . The results showed that L. conigenum had a significant negative association with fungal richness of pine needles in summer  and autumn  but not in spring even though the percentage of L. conigenum peaked at this time. The negative association of L. conigenum with the overall fungal species richness was significant in second-year needles of P. sylvestris in Estonia  and in second- and third-year needles in Norway . Dothistroma septosporum had no significant association with species richness for any of the sampling dates, sites or needle-age classes (p > 0.05).In order to consider the relations between relative abundance of L. conigenum, Phacidiaceae sp., Capnodiales sp. and D. septosporum prevailed. The genus Lophodermium is especially diverse on the needles of pine and these species mostly function as endophytes and saprobes [L. seditiosum. PacBio sequencing of 190 needle samples from four sampling sites revealed 261 OTUs, of which saprobes ,52, exceLophodermium conigenum was also one of the three most abundant species on 1-year-old needles of Corsican black pine in Corsica in a study based on metabarcoding of the ITS2 rDNA region, being present in all three of the investigated sites, although with variable abundance [Pinus heldreichii H. Christ harboured L. conigenum as the second most prevalent species in six different sites in Montenegro mountains [L. conigenum was primarily associated with trees in good health, being one of the most dominant species in sites with moderate growth conditions and healthy trees, but with low abundance in areas where beetle attacks and necrotic lesions (agent unknown) on needles occurred. It is noteworthy that Lazarevic and Menkis [L. conigenum was the most frequently isolated fungal OTU from symptomless needles of P. sylvestris in Finland [L. conigenum was more abundant on symptomatic needles of P. sylvestris in northern Sweden. However, the roles of L. conigenum in pine phylloplane remain to be clarified—but it seems to be an endophyte, detected in high frequency in recent mycobiome studies of different Pinus species in Europe, regardless of geographical location. L. conigenum has been proposed to act as an antagonist against L. seditiosum or L. pinastri s. str. [bundance . In anotountains . These ad Menkis  pursued  s. str. . D. septosporum has been seldom found [P. sylvestris foliage studies was performed during 2006–2008, at a time when D. septosporum was probably not widely spread in the region. In Finland and Sweden, the pathogen was first recorded in 2007 [D. septosporum was relatively abundant across the entire dataset, representing 6.4% of all sequences, and found on both visually healthy and symptomatic trees. Species co-occurrence analysis showed that abundance of D. septosporum was positively associated with that of L. conigenum. Further studies, including dual culturing, are needed to assess the nature of this relationship.om found  or not fom found ,2,3. In  in 2007 ,55. In op < 0.01) positive association with overall fungal species richness, but it should be noted that inclusion of more sites would be needed to test whether this represents a true gradient or stochastic variation only. In Norway, where also 3-year-old needles could be sampled, needle age showed a positive relation with species richness (p < 0.001). The fungal species richness was significantly (p < 0.001) higher in second-year needles in Estonia compared to needles of the same age in Norway. In the frame of critical colonization threshold [P. sylvestris in Estonia, which in turn can trigger earlier needle senescence compared to Norway  and Gransherrad (3-year-old-needles) sites especially in autumn . An autumn increase in fungal species richness in the foliage of different deciduous tree species has been reported by several previous studies ,57,58, wtumn see . For Eng needles . After tg bodies . It is rtosporum , appear elopment . The auty period . HoweverD. septosporum on the attached needles was high in spring, but during the summer almost all the needles with conidiomata were shed [D. septosporum in pine needles was highest in summer (p < 0.05), followed by spring and autumn (except in Konguta site). As D. septosporum life cycle includes a substantial epiphytic period during which an extensive web of hyphae is formed on the needle surface and persists for several weeks in the case of P. radiata [P. sylvestris in northern Europe. During this epiphytic period, no symptomatic red bands appear on needles, which can explain the sequencing-based detection of D. septosporum in asymptomatic needles in our study. To determine the length of the epiphytic phase of D. septosporum in the case of P. sylvestris needles in northern Europe, further studies are needed. Millberg et al. [D. septosporum may involve an up to12-month-long latent period before symptoms and conidiomata are formed, the length of this time depending on site and local abiotic conditions. DNB infection occurs mainly in the spring and early summer with the aid of rain-dispersed conidia produced by conidiomata formed on infected needles . The firere shed . Yet, re radiata , it is rg et al.  concludeP. sylvestris canopies suffer more by D. septosporum than needles in the upper part [D. septosporum. Relative abundance of D. septosporum was significantly (p < 0.05) lower in needles in the upper part of the crown, compared to the lower part. The percentage of D. septosporum was significantly higher in second- and third-year needles compared to first-year needles. In 2-month-old needles (2014), the relative abundance of D. septosporum was 3.2% and that of L. conigenum 4.5%. Needle age was positively related to relative abundance of D. septosporum but not to that of L. conigenum. Generally, needles situated in the lower part of per part . We alsoLophodermium conigenum and D. septosporum differed in their seasonal pattern. Lophodermium conigenum was extremely abundant in older spring needles in both countries, showing significantly higher relative abundance compared to younger spring needles or older summer or autumn needles  on lower part of the canopy compared to the upper part where the needles are exposed to harsher environmental conditions, e.g., the frequent, repeated, rapid shifts in humidity, temperature and UV radiation. Such conditions favor fungi that have specific traits to inhabit foliage surfaces. Extremophilic species such as A. pullulans (de Bary) G. Arnaud and foliicolous yeasts use production of an extracellular polysaccharide matrix that is thought to facilitate survival and growth in oligotrophic extreme environments. In yeasts, these capsules secure attachment, provide protection against desiccation and allow efficient rehydration following periods of drought, and bind nutrients, thus contributing to higher growth rates of encapsulated yeast cells versus non-capsulated variants on nutrient-poor media [Different epiphytes and endophytes have distinct sporulation regimes and possibly prefer tissues with different age indicating temporal complementarity in niche recruitment. Similar to findings from needles of larch  and firsosporium . As withor media . Lophodermium seditiosum is known as a causative agent of pine needle-cast epidemics that can last up to 2 years. The low relative abundance of this species in the present study would indicate that the environmental conditions during the study period were not favourable for this pathogen [L. seditiosum has been found in both healthy and diseased needles, the former probably representing latent infections [L. seditiosum sequences were found in current study in both DNB symptomatic and asymptomatic trees. Of endophytic Lophodermium species [L. pinastri s. str. and L. conigenum displayed a higher relative abundance in diseased needles in Sweden compared to our study [L. pinastri s. str. was relatively abundant as well and, similarly to Sweden, it showed higher relative abundance on DNB symptomatic trees (2.8%) compared to visibly healthy trees (0.6%), even though the difference was not statistically significant (p > 0.05). pathogen . In Swed species , L. pinaPicea abies trees, but they assessed the overall health condition of the tree and did not consider specific diseases or symptoms separately. PERMANOVA analysis showed that species composition was mostly influenced by sampling site, which may relate to site-specific differences in climate, understorey vegetation ,58 and fL. conigenum was negatively associated (p < 0.01) with overall fungal species richness in both countries, but this relationship disappeared as overall fungal species richness increased, e.g., in summer or autumn, and it may thus represent a stochastic relation. In different needle-age classes, it became evident that L. conigenum had a significant positive relationship with overall fungal species richness on second-yearneedles in Estonia and on second- and third-year needles in Norway. The relative abundance of D. septosporum showed no significant relation with overall fungal species richness in any of the four sampling sites, or between the three sampling dates or needle-age classes. The fungal community composition did not differ between DNB-symptomatic and DNB-free P. sylvestris needles. This provides support to the hypothesis that D. septosporum has been co-evolving a long time with P. sylvestris and its associated mycobiota [The relative abundance of ycobiota ,70. D. septosporum and D. pini, at the most commonly used 97% sequence similarity threshold and even at the 98% sequence similarity threshold. The issue was resolved after raising the similarity threshold up to 99%. In our study, all D. septosporum sequences could be reliably identified as D. pini. In addition to Dothistroma species, we noticed that similar clustering and mislabelling issues concern also Lophodermium species, e.g., L. seditiosum and L. pinastri. At the 97% similarity threshold these species were clustered together as L. pinastri. We used a 99% sequence similarity threshold to resolve this issue. We also manually BLAST-ed all identified D. pini, Dothistroma sp., L. pinastri, L. seditiosum and Lophodermium sp. OTU reference sequences against the UNITE database [As Loit et al.  observeddatabase  to reducD. septosporum and L. conigenum had higher relative abundance in the lower parts of tree canopies. DNB symptomatic and healthy trees had a similar relative abundance of D. septosporum. The relative abundance of D. septosporum peaked in summer, while that of L. conigenum was highest in spring, reflecting that different dominant species in P. sylvestris needles can have different sporulation regimes and niche recruitment times. Relative abundance of L. conigenum had a significant negative relation with overall species richness in two- and three-year-old P. sylvestris needles in spring. In contrast, relative abundance of D. septosporum had no relation with overall species richness of pine needle mycobiome. Fungal species composition was mostly influenced by sampling site, needle age, needle location in canopy and presence of D. septosporum symptoms.We conclude that species richness on pine needles increased from spring to autumn and with needle age. Species richness is possibly affected also by temperature sum but examination of a larger number of sites is needed to test this hypothesis. Additionally, fungal species richness was significantly higher in the needles situated in the lower part of tree canopies. It is noteworthy that species richness in second-year needles in Estonia and third-year needles in Norway was statistically similar, suggesting that a critical colonization threshold for pine needle shed is reached in Estonia a year earlier than in Norway, possibly due to warmer climate that stimulates microbial growth. Needle-age class also had a significant positive relationship with species richness. Both


Candida species, Candida albicans is the most virulent and acts as the main causative fungus of oral candidiasis. Novel treatment modalities are needed because of emergent drug resistance and frequent candidiasis recurrence. Here, we evaluated the ability of Lacticaseibacillus rhamnosus L8020, isolated from healthy and caries-free volunteers, to prevent against the onset of oral candidiasis in a mouse model. Mice were infected with C. albicans, in the presence or absence of L. rhamnosus L8020. The mice were treated with antibiotics and corticosteroid to disrupt the oral microbiota and induce immunosuppression. We demonstrated that oral consumption of L. rhamnosus L8020 by C. albicans-infected mice abolished the pseudomembranous region of the mouse tongue; it also suppressed changes in the expression levels of pattern recognition receptor and chemokine genes. Our results suggest that L. rhamnosus L8020 has protective or therapeutic potential against oral candidiasis, which supports the potential use of this probiotic strain for oral health management.Oral candidiasis presents with multiple clinical manifestations. Among known pathogenic  Candida albicans is the most virulent known pathogenic Candida species and the most common causative fungus of oral candidiasis. Importantly, C. albicans is an opportunistic fungal pathogen of humans, which colonizes the skin and mucosal surfaces of most healthy people in an asymptomatic manner [C. albicans on mucosal surfaces, thereby resulting in the development of disease [C. albicans in patients with oral candidiasis are 15–71% in denture wearers and 80–95% in human immunodeficiency virus-infected individuals [C. albicans involves its ability to switch between yeast and hyphal morphologies; C. albicans attached to host surfaces can switch to an invasive hyphal and filamentous morphology, facilitating invasion of the epithelium [Oral candidiasis presents with various clinical manifestations, including the classic white thrush lesion that develops into pseudomembranes resembling milk curds . Candidac manner . However disease . The detividuals . The corProbiotics have been defined as “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” by the Food and Agriculture Organization/World Health Organization ; this deLactobacillus spp. have a long history of safe usage as probiotics, and over 200 species have been classified within the Lactobacillus genus [Lactobacillus spp.  can diminish the influence of C. albicans through the release of anti-fungal molecules and/or regulation of the immune system [Lactobacillus was proposed to undergo reclassification into 25 genera, on the basis of whole genome sequence analyses [us genus . Some Lae system ,12. Receanalyses . FollowiLactobacillus spp. from healthy volunteers who had no caries or caries-treatment experience. Among the 42 isolates, L. rhamnosus L8020  showed tivation . These p disease .L. rhamnosus L8020 for preventing oral candidiasis in vivo in a mouse model of C. albicans infection. ICR mice, a typical outbred stock, have more variability in genetic characteristics and faster growth rate than inbred mice; disease models using ICR mice are widely used in studies for probiotics [C. albicans infection.In this study, we investigated the efficacy of obiotics ,16,17,18L. rhamnosus L8020, previously isolated from healthy and caries-free volunteers  [C. albicans GDH18 (ATCC MYA-274), an oral isolate obtained from the routine microbiology services of the Glasgow Dental Hospital and School , was used for infection in this study. L. rhamnosus L8020 was preincubated in Lactobacillus de Man, Rogosa and Sharpe broth  for 24 h at 37 °C. C. albicans GDH18 was cultured aerobically in Sabouraud dextrose broth  at 37 °C [at 37 °C .C. albicans GDH18 infection and drinking water (control group); no C. albicans GDH18 infection and L. rhamnosus L8020 consumption (L8020 group); C. albicans GDH18 infection and drinking water (C. a GDH18 group); and C. albicans GDH18 infection with L. rhamnosus L8020 consumption (C. a GDH18 + L8020 group).Animal care and experiments were performed in accordance with the guidelines of institutional authorities and approved by the Ethics Committee for Animal Experiments at Hiroshima University . Six-week-old female ICR mice were purchased from Charles River Laboratories Japan . The mice were acclimatized to the experimental facility for 1 week to aid in stress relief. Twenty-eight mice were randomly divided into four groups of seven animals each: no C. albicans GDH18 infection; they then received 100 mg/kg prednisolone  subcutaneously, beginning at 24 h before C. albicans GDH18 infection. Beginning at 24 h before infection, mice were also freely administered 1.0 × 106 cells/mL of L. rhamnosus L8020 via drinking water; this was continued until the end of the experimental period. Mice were anesthetized using a combination of 0.3 mg/kg medetomidine, 4 mg/kg midazolam, and 5 mg/kg butorphanol; they were then orally infected with 1.0 × 109 cells/mL of C. albicans GDH18 by means of rubbing C. albicans-impregnated cotton swabs inside all parts of the mouth, followed by C. albicans-impregnated cotton swab placement on the dorsum of tongue for 90 min. The mice were euthanized at 48 h after C. albicans infection Total RNA was isolated from palatal gingival tissues using TRIzol reagent . First-strand cDNA was synthesized from 100 ng of total RNA using ReverTra Ace qPCR RT Master Mix . cDNA samples were mixed with Rotor-Gene Probe PCR Master Mix  for real-time quantitative reverse transcriptase polymerase chain reaction analyses; the analyses were performed using a Rotor-Gene 6000 (Qiagen). β-actin was used as an internal control to correct for amplification variability caused by differences in starting total RNA concentrations. The polymerase chain reaction primers and probes used in this study are shown in p-values < 0.05 were considered statistically significant.Statistical analyses were performed using IBM SPSS Statistics software, version 27.0 . One-way analysis of variance and Tukey’s multiple comparison test were used to analyze differences among groups; C. albicans GDH18 showed clinical symptoms of oral candidiasis  2, TLR4, Dectin-1, Dectin-2, CCL2, and CXCL1/KC were analyzed in mouse palatal gingival tissues  have been reported to exhibit anti-fungal activity in vivo [L. rhamnosus.This study demonstrated that treatment with the probiotic strain  strains . Further strains ,25,26,27 in vivo ,22,27. TL. rhamnosus L8020 demonstrates good adhesion to gingival epithelial-like cells; this adhesion ability is superior to the adhesion demonstrated by Lactobacillus spp. isolated from other organs and environments [L. rhamnosus L8020 is expected to have a beneficial effect in protecting against C. albicans infection through the colonization of mucosal surfaces, which prevents expansion of the pseudomembranous candidiasis region on the tongue.Colonization of the mucosal surface is an important anti-infection probiotic mechanism . We prevronments . Thus, LC. albicans in a sustained manner for up to 72 h after infection, whereas the TLR4 mRNA expression in oral tissues peaks at 6 h and then gradually declines until 48 h after infection [C. albicans recognition and the subsequent induction of anti-fungal signaling pathways [C. albicans infection [C. albicans. Phagocytic cells  are important components of the immunological response to oral candidiasis. Notably, neutrophils are essential for controlling and eliminating fungal infection; they can kill C. albicans through both intracellular and extracellular mechanisms. Similarly, macrophage activation leads to the release of several key cytokines that are important for host protection against C. albicans infection. Previous analyses of the oral mucosa in mice revealed the induction of chemokines that participate in neutrophil recruitment  and macrophage chemotaxis  after oral C. albicans infection [L. rhamnosus L8020 prevented and/or reduced the C. albicans-related induction of PRR and chemokine gene expression during the experimental period. Notably, few genetic changes were caused by the presence of L. rhamnosus L8020 in oral tissues. Although in vitro microarray results using mouse gingival epithelial cells showed that more than 400 changes in gene expression occurred within 1 h of co-culture with L. rhamnosus L8020, only 60 changes remained after 4 h of co-culture [L. rhamnosus L8020, alterations in gene expression had already occurred and a stable state had been achieved in oral tissues.The immunological response to oral candidiasis is orchestrated by oral epithelial cells and their secreted proteins, as well as phagocytic cells and components of the adaptive immune system. Oral epithelial cells sense the early stages of an infection and activate the p-MKP1/c-Fos pathway as “danger response” to alert the host that colonizing yeast have begun to transition to virulent hyphae . Followinfection . Among tpathways ,31. Exprnfection . These fnfection ,33,34,35-culture . These rL. rhamnosus L8020 in a mouse model of oral candidiasis. Specifically, L. rhamnosus L8020 abolished the pseudomembranous region of the mouse tongue following infection by C. albicans; this tissue alteration was accompanied by the suppression of changes in PRR and chemokine gene expression levels. Our results suggest that L. rhamnosus L8020 has protective or therapeutic potential with respect to oral candidiasis, which supports its use as a potential probiotic for improved oral health management. Further proof of concept studies should be conducted in humans to verify the mechanism by which L. rhamnosus L8020 acts as a probiotic, thereby providing robust evidence to support its use in clinical applications.In summary, we have demonstrated the in vivo efficacy of L. rhamnosus L8020.Prof. Hiroki Nikawa has patents for


Helicobacter pylori (H. pylori) infection worldwide has driven the search for a new therapeutic candidate. Recently, sitafloxacin, a novel 4-quinolone agent, has emerged as a new therapeutic option for H. pylori eradication, in Japan. However, data on its efficacy for H. pylori eradication in Korea are limited. Therefore, we aimed to investigate the therapeutic potential of sitafloxacin as a first-line treatment for patients with Helicobacter infection through gastric tissue culture-based studies. Materials and Methods: We prospectively enrolled treatment-naïve patients with H. pylori infection who visited the Gil Medical Center between March 2015 and March 2018. After obtaining written informed consent from patients, a total of 121 H. pylori strains were collected. We tested the susceptibility of these strains to sitafloxacin, and other antibiotics for Helicobacter eradication, including clarithromycin (CLR), metronidazole (MTZ), amoxicillin (AMX), tetracycline (TET), levofloxacin (LEV), and ciprofloxacin (CIP) using the agar dilution technique. The minimum inhibitory concentration (MIC) of these antibiotics against H. pylori strains were determined. Results: None of the H. pylori strains obtained were resistant to sitafloxacin , while other conventional eradication drugs including CLR, MTZ, AMX, and TET showed 24.8% (n = 30), 30.6% (n = 37), 5.0% (n = 6), and 0.8% (n = 1) resistance, respectively. Compared to the resistance rates of other quinolones , sitafloxacin showed the best antibiotic performance against Helicobacter strains . Furthermore, sitafloxacin also inhibited the growth of 14 H. pylori strains (12.4%), which were resistant to both of clarithromycin, and metronidazole, and 27 strains (22.3%) with multidrug resistance. Conclusions: Sitafloxacin might be a new promising candidate for Helicobacter eradication where antibiotic resistance for Helicobacter is an emerging medical burden, such as in Korea.Background: Increased prevalence of antibiotic resistance to  Helicobacter pylori(H. pylori) infections has increased worldwide, leading to a global medical burden  and CIP ), sitafloxacin showed the best antibiotic performance against Helicobacter strains , with statistical significance (p < 0.001)  C.Furthermore, sitafloxacin also inhibited the growth of 14 strains (12.4%), which were co-resistant to CLR and MTZ, and 27 strains (22.3%) with multidrug resistance activity (resistance to >2 drugs).H. pylori infection through gastric tissue culture-based studies using H. pylori strains obtained from 121 patients with H. pylori infection in Korea. According to our study results, sitafloxacin showed a 0% antibiotic resistance rate for H. pylori, and successfully acted against all 14 strains of multidrug-resistant H. pylori (resistant to ≥2 drugs). To overcome bacterial factor-oriented treatment failure, along with antibiotic sensitivity tests, new therapeutic targets and other novel drugs can be used as part of the Helicobacter eradication strategy. The results of this study showed the possibility of sitafloxacin as a new drug candidate for regimens in H. pylori infection in Korea, where fluoroquinolone resistance has increased over the years.In this study, we investigated the in vitro activity of sitafloxacin, a new-generation quinolone drug, in the treatment of Helicobacter eradication as a rescue regimen or as an alternative first-line treatment for patients with penicillin allergies [H. pylori eradication practice is as follows. Conventional quinolone series drugs, especially levofloxacin and moxifloxacin, act as bacteriostatic agents against H. pylori strains, and are components of a second-line or third-line rescue therapy [Helicobacter drugs such as clarithromycin and metronidazole, quinolone series drugs have also shown increased antibiotic resistance annually due to increased consumption of these drugs in respiratory tract infections, urinary tract infections, and other infections other than H. pylori infections [H. pylori to clarithromycin, metronidazole, and levofloxacin has reached alarming levels of up to 46%, even though it varied among countries [H. pylori infection significantly increased from 4.5% in 2003 to 62.2% in 2018 [Sitafloxacin, a novel fourth generation quinolone, effectively acts against a broad spectrum of gram-negative and gram-positive bacteria (including anaerobes), as well as atypical pathogens . Sitaflollergies ,20,21,22 therapy ,24. Howefections ,26. Thesfections ,28,29. Aountries . ReferriHelicobacter medication, there is limited data on its efficacy in Helicobacter eradication elsewhere, including in Korea. We searched the PubMed platform to find research articles  between January 2010 and January 2021 and found only two articles published outside Japan. First, Miftahussurur et al. reported the in vitro activity of sitafloxacin in 98 H. pylori strains from Nepal and Bangladesh after performing a whole-genome mutation analysis. According to the study results, 95.2% of H. pylori strains from Nepal and 98.2% of strains from Bangladesh showed high susceptibility to sitafloxacin. Korea, Nepal, and Bangladesh are classified as regions with a high prevalence of H. pylori infection , and high antibiotic resistance, including that of clarithromycin, metronidazole, and levofloxacin , resulting in eradication failure [H. pylori infection using 63 H. pylori strains from Dominican patients [H. pylori resistance to levofloxacin and metronidazole. Similar to our study results, they also revealed that all collected strains were susceptible to sitafloxacin. Despite the limited number of studies, three studies performed outside Japan, including our current research, reported that sitafloxacin showed excellent in vitro antibiotic activity against H. pylori infection.Sitafloxacin is not yet approved for global use; however, it was recently used in Japan and Thailand. Even though sitafloxacin has been used in Japan as an anti- failure . Second,patients . In the Helicobacter eradication regimen, including primary or rescue therapy. Sitafloxacin-based triple regimens usually consist of sitafloxacin (100 mg bid)/proton pump inhibitor (PPI) along with MTX (250 mg bid) or AMX (750 mg bid or 500 mg qid or 250 mg bid). With a variety of combinations for sitafloxacin-based triple regimens, several researchers investigated its efficacy in Helicobacter eradication as compared to other conventional regimens as first line or rescue regimen. To verify sitafloxacin as the first line regimen, Sugimoto et al.  investigated the efficacy of sitafloxacin containing a triple regimen (sitafloxacin 100 mg bid + PPI + MTX 250 mg bid for 1 week) among 45 treatment naïve H. pylori infection patients, and they showed eradication success rate (ESR) of 100% in intention-to-treat analysis (ITT), and 92.7% in per protocol (PP) analysis [H. pylori infection is an emerging issue. Therefore, with these data, further randomized controlled studies to validate the efficacy of sitafloxacin as a rescue regimen in Korea should be conducted.In Japan, based on the stacked in vitro activity data of sitafloxacin from Sanchez et al. , severalanalysis . Matsuzaanalysis . Murakamanalysis . Accordianalysis . Mori etanalysis . Even moHelicobacter infection, which varies among nations for antibiotic sensitivity of H. pylori infection owing to different consumption of national antibiotic use, and H. pylori infection prevalence status; however, its efficacy should be validated outside Japan in H. pylori infection treatment. Further clinical studies using sitafloxacin outside Japan should be performed. Our study results pertaining to the in vitro activity of sitafloxacin against Korean H. pylori strains might serve as a basis for further clinical research in Korea.As mentioned above, even in Japan, several studies have investigated the therapeutic potential of sitafloxacin in H. pylori have been previously determined [H. pylori with mutations in the gyrA gene, one of the hotspots of the quinolone resistance-determining gene [gyrA genes of H. pylori strains encode DNA gyrase, an essential enzyme that maintains the helical structure of DNA, and is involved in DNA replication, recombination, and transcription [H. pylori strains have evolved to evade attachment from conventional quinolone series drugs though mutations in their gyrA gene [gyrA gene plays a pivotal role in nucleic acid synthesis, mutations in this gene may result in the resistance of H. pylori to conventional quinolone drugs [gyrA gene mutations, sitafloxacin especially showed activity at D91, but not at N87. If patients infected with H. pylori with gyrA mutation at N87 were diagnosed, a third-line regimen other than sitafloxacin should be prescribed [The possible underlying mechanisms of action of sitafloxacin against drug-resistant termined ,17,18,34cription . Conventcription . HoweveryrA gene . Therefone drugs ,22,36,37H. pylori infections who visited the Gil Medical Center, which is located in Incheon, Korea. Since the antibiotic resistance status of H. pylori differs among geographic regions and time, caution should be exercised when applying our results to other locations. Second, even though we included H. pylori strains from 121 patients and enrolled a relatively large number of strains to draw conclusions compared to those of previous studies, further multicenter studies are needed. Third, because antibiotic exposure history was dependent on patients’ recall, natural bias from recall might have affected our study results. Fourth, since our study aimed to investigate the antibiotic resistance status of H. pylori for sitafloxacin through an in vitro stomach tissue culture-based assay and results from antibiotic sensitivity tests, real-world clinical practice might differ, so further clinical studies using sitafloxacin as a Helicobacter eradication regimen for patients with H. pylori infection should be further considered. Our study results may then be the basis for further clinical research on this. Fifth, in this study, we focused on whether or not sitafloxacin, a novel antibiotic for the Korean general population, showed potential activities against H. pylori infection. In this regard, we did not further investigate the acquired double mutations in the gyrA gene at the A87 and D91 positions on the H. pylori species owing to time and cost issues. Further investigation should be guaranteed. Sixth, H. pylori can acquire double mutations in the gyrA gene at the A87 and D91 positions, which results in drug resistance against sitafloxacin [H. pylori also has traits of cross-resistance with other quinolone series drugs such as ciprofloxacin, careful clinical antibiotic use of sitafloxacin for appropriate indications, as well as the use of antibiotic sensitivity test–based regimens rather than empirical use, are still imperative to reduce the possibility of antibiotic resistance. Further studies should be guaranteed to investigate the efficacy of sitafloxacin in the presence of double mutations in the gyrA gene at the A87 and D91 positions of H. pylori strains.There are several limitations to this study. First, we enrolled patients with floxacin . FurtherH. pylori eradication showed that the in vitro efficacy of sitafloxacin was proven in Helicobacter pylori infection outside Japan, in Korean patients. Additional clinical studies regarding sitafloxacin against H. pylori infection should be conducted.Despite the aforementioned limitations, our data on the efficacy of sitafloxacin against This study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the ethics committee of the Gil Medical Center. The Institutional Review Board of Gil Medical Center reviewed the study protocol and ethics (GAIRB2016-329).Helicobacter infection, no previous history of Helicobacter eradication, and no history of antibiotic use within 2 years between June 2016 and March 2019. After obtaining written informed consent from patients who agreed to obtain stomach biopsy and tissue cultures for antibiotic sensitivity testing, a total of 121 H. pylori strains from all patients were collected.We prospectively enrolled patients in the Gil Medical Center with H. pylori from the stomach biopsy samples of 121 patients was performed as follows, the detailed process of which was illustrated in a previous publication by our group [2, 10% CO2, 85% N2) at 37 °C.The isolation of ur group . ImmediaH. pylori. To obtain H. pylori colonies, each H. pylori strain was stored in Brucella liquid medium  containing 15% glycerol at −70 °C.We used gram staining and biochemical methods to confirm whether the bacterial colonies from the cultures were indeed H. pylori strains using agar dilution methods with Mueller–Hinton agar  supplemented with 5% sheep blood in accordance with guidelines from the Clinical and Laboratory Standards Institute and recent review articles [H. pylori eradication, including the quinolone series , amoxicillin, tetracycline, rifabutin, and furazolidone, and the broth microdilution technique was used. Each antibiotic was diluted in a medium supplemented with 5% sheep blood  .We further tested the antibiotic susceptibility of the articles ,14. The 7 colony-forming units of H. pylori strains cultured in blood culture medium for 72 h were inoculated on Mueller–Hinton agar containing each targeted antibiotic. After cultivation and incubation of the cultures under micro-ventilation conditions  at 37 °C for 3 days, we observed the presence of bacterial colonies. Each experiment was performed in triplicate and repeated at least three times per strain.Afterwards, 1 × 10H. pylori strains in the agar dilution test using the broth microdilution technique. The cut-off values for H. pylori resistance against each targeted antibiotic were as follows: sitafloxacin (>1 μg/mL), clarithromycin (>1 μg/mL), amoxicillin (>0.5 μg/mL), tetracycline (>4 μg/mL), and other quinolone series, including levofloxacin (>1 μg/mL) and ciprofloxacin (>1 μg/mL).The minimum inhibitory concentration (MIC) was defined as the lowest concentration in which antibiotics completely inhibited the visible growth of H. pylori infections, especially among Korean patients. Our study results may serve as a basis for further multicenter, randomized controlled clinical studies.In conclusion, we showed the therapeutic potential of the novel fluoroquinolone drug sitafloxacin in the treatment of


Entamoeba is a genus of Amoebozoa that includes the intestine-colonizing pathogenic species Entamoeba histolytica. To understand the basis of gene regulation in E. histolytica from an evolutionary perspective, we have profiled the transcriptomes of its closely related species E. dispar, E. moshkovskii and E. invadens. Genome-wide identification of transcription start sites (TSS) and polyadenylation sites (PAS) revealed the similarities and differences of their gene regulatory sequences. In particular, we found the widespread initiation of antisense transcription from within the gene coding sequences is a common feature among all Entamoeba species. Interestingly, we observed the enrichment of antisense transcription in genes involved in several processes that are common to species infecting the human intestine, e.g., the metabolism of phospholipids. These results suggest a potentially conserved and compact gene regulatory system in Entamoeba. Entamoeba organisms are endobiotic amoebae colonizing species of animals. The persistence of at least seven species of Entamoeba depends on their ability to infect humans, mainly in the intestinal tract, where they divide and encyst [E. histolytica, the etiological agent of amebiasis. As a pathogenic parasite, E. histolytica varies between the commensal status seen in 90% of infected people and the virulent status that leads to intestinal invasion in the remaining 10% [Entamoeba are intestinal commensals and include E. dispar which has a life cycle similar to that of E. histolytica [E. histolytica and E. dispar together account for nearly 82% of Entamoeba infections in humans. Infections with E. dispar seems to be ~10 times more common than E. histolytica [Entamoeba infections correspond to other non-pathogenic species, E. coli (1.98%), E. hartmanni (0.96%), E. polecki (0.04%) and E. gingivalis (4.6%) [E. histolytica is E. moshkovskii, a free-living amoeba commonly found in sewage and known to infect humans, although its pathogenicity is not precisely determined [Entamoeba infections in humans seems to be caused by commensal species and these species feed on and evolve with Enterobacteriaceae. Another widely studied species is E. invadens, which is pathogenic in a wide range of reptiles [E. invadens is used as a model for the studying the formation of amoebic cysts, due to the possibility of completing its cell cycle under laboratory conditions.d encyst . There hd encyst ,3. One oning 10% . Other stolytica . The rems 4.6%) . Another% . Anothtermined . OverallE.histolytica strain HM1:IMSS [E. dispar strain SAW760 [E. moshkovskii strain Laredo [E. invadens strain IP-1 [E. invadens and E. moshkovskii genomes have unusually biased GC distributions [E. histolytica and E. dispar are closely related species, bearing 95% and 85% of nucleotide identity in their genic and intergenic regions, respectively [E. moshkovskii is slightly larger than that of E. histolytica, and the phylogenetic relationship between diverse isolates of E. moshkovskii indicates that their most recent common ancestor is 500 times more ancient than the ancestor of isolates from E. histolytica [E. invadens, is readily distinguishable from the other human infecting species, sharing only 74% and 62% of nucleotide identity in their genic and intergenic regions, respectively [E. moshkovskii and E. invadens, retrotransposons dominate in E. histolytica and E. dispar [E. histolytica has the most compact genome among the four species, with ~8300 coding genes in 20 Mb, compared to the E. invadens genome which bears 11,549 coding genes in 40 Mb [E. histolytica and E. invadens are similar while the intergenic regions in E. invadens are longer than those in E. histolytica (408 bp vs. 282 bp) [The genomes of HM1:IMSS ,11,12; En SAW760 , E. moshn Laredo  and E. iain IP-1 ,16 have ibutions . E. histectively ,18. The tolytica . The parectively ,16. Despectively ,20, ther. dispar . These t. dispar ,23. E. hin 40 Mb . The gen 282 bp) .E. invadens and E. histolytica [E. invadens and gene silencing in E. histolytica [E. histolytica, enabled genome-wide identification of transcription start sites (TSS) [E. histolytica. NAT transcription is abundant as roughly 25% of genes generate NATs in all environmental conditions tested [E. histolytica genome [E. dispar, E. moshkovskii and E. invadens have not yet been completely described.Transcriptomic analyses enabled the identification of gene promoter sequences in tolytica ,26,27,28tolytica ,30,31. Mes (TSS)  and polyes (TSS) ,34, as wes (TSS) , small Res (TSS)  and longes (TSS) . More rees (TSS) , which as tested . In addia genome . These cE. histolytica, E. dispar, E. moshkovskii and E. invadens. We analysed similarities and differences in transcriptional genomic determinants for mRNA and NATs and concluded that TSS for NATs occurs on the opposite strand within the coding sequence, although variations exist between the species. NATs precisely initiate at the stop codon in E. histolytica and E. dispar but appear to be dispersed inside the intergenic spacers in the two other species. Comparing the transcriptomic profiles of genes targeted by NATs in E. histolytica to E. dispar and E. moshkovskii, we discovered significant biological processes common to amoebic species infecting the human intestine, with lipid metabolism activities as the most important enriched functions, whereas the comparison with E. invadens highlighted vesicular trafficking and regulation of RNA transcription. Common gene products and functions associated with NATs identify robust candidates for monitoring NAT biogenesis in all four species of Entamoebidae.In this study, we aimed to study the conservation of gene regulatory sequences  and NAT transcription between Entamoeba histolytica strain HM-1: IMSS was isolated in 1967 from a biopsy of a rectal ulcer from an adult male with amoebic dysentery, Mexico City, Mexico. Entamoeba dispar strain SAW760 was isolated in 1979 from an asymptomatic adult male at the London School of Hygiene and Tropical Medicine, UK. The Laredo strain of Entamoeba moshkovskii was isolated from a resident of Laredo, TX, USA, who showed symptoms of diarrhoea. Entamoeba invadens strain IP-1 was isolated in Canada from a natural infection of a painted turtle Chrysemys picta. The HM-1: IMSS strain was a gift from Professor Ruy Perez Tamayo and Dr Alfonso Olivos . The three other strains are from the collection of Professor Graham Clark. All the strains were cultured in axenic media whose compositions have been described previously [E. histolytica strain HM-1: IMSS was cultured in TYI-S-33 medium at 37 °C; E. moshkovskii strain Laredo was cultured in LYI-S-2 medium at room temperature; E. dispar strain SAW760 was cultured in LYI-S-2 medium at 37 °C and E. invadens strain IP-1 was cultured in TYI-S-33 medium at 25 °C.eviously . E. hist6 trophozoites (with each culture performed in triplicate) using Trizol . The poly(A) fraction was purified from 10 to 100 µg of total RNA using Dynabeads according to the manufacturer’s instructions (Thermo Fisher Scientific ref. 28152103011150). Small RNAs were purified from 10 μg of total RNA loaded on a denaturing 15% TBE- Urea gel . More specifically, following ZR-small-RNA Ladder  size indications, a fragment of gel corresponding to the 15–35 nt region was excised with a scalpel and RNAs were extracted using the ZR small-RNA PAGE Recovery Kit  following manufacturer’s recommendations. Small RNAs were then treated with Tobacco Acid Pyrophosphatase  for 1 h and purified by RNA Clean and Concentrator-5  following the manufacturer’s instructions.Total RNA was extracted from approximately 1 × 10First, libraries were constructed using TruSeq Small RNA Sample Prep Kit  following the manufacturer’s instructions. Then, all the libraries were purified on a 5% TBE gel  and were quantified by Bioanalyzer DNA High Sensitivity Chips . Sequencing was performed on HiSeq-2000 (Illumina) in a multiplexed 51 + 7 nucleotide single-end read using a TruSeq SR Cluster kit v3 cBot HS  and a TruSeq SBS kit v3 HS 50 cycles . Finally, FASTQ sequence files were generated using CASAVA 1.8.2 (Illumina) and adapter sequences were trimmed using AlienTrimmer  , E. dispar , E. moshkovskii  and E. invadens  were downloaded from AmoebaDB v46 .The reference genome sequences and annotations of https://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA781395.The data are available in the SRA database . The remaining unique reads were then mapped to the corresponding reference genomes using Bowtie v. 0.12.7 [The small RNA data were processed as previously described . Briefly. 0.12.7  with maxn 2) and reads mapped to multiple locations (-m 50) were reported only once (-k 1). The alignments produced were sorted and indexed with SAMTools [The reads were aligned to the different reference genomes, using Bowtie v. 0.12.7  with theSAMTools . CoveragSAMTools ,46, focuSAMTools  was perfSAMTools . A TSS wn 2 -k 1 -m 50 -l 30. To avoid false positives due to sequencing errors, reads with low quality (<20) around PAS (5 nt upstream and downstream) were removed from the set. To discriminate real poly(A) tracks of true polyadenylation from poly(A) tracks of internal homopolymeric stretches on the mRNAs, false positives were discarded if they met the following criteria: (i) reads with ≥8 nt within 10 nt immediately upstream of the PAS are A’s, (ii) mapping with ≥5 ‘A’s immediately downstream of the PAS.First, reads with a stretch of five or more ‘A’s at their ends (or ‘T’s at their beginning) were selected for this analysis, as they potentially contain mRNA poly(A) tails. Redundant reads were removed and stretches of As at the ends were trimmed. Remaining reads with a minimum length of 18 nt were then mapped on the reference genome using Bowtie  with folPAS candidates within 12 nts from each other were then clustered together in PAS clusters and the position of the strongest coverage was defined as the peak. PAS with fewer than 2 reads of coverage at the peak were removed.The sequences immediately upstream and downstream of the gTSS, and asTSS (100 nt on each side), as well as the PAS of mRNA and NAT were used to scan for conserved motifs using DREME . The immhttps://amoebadb.org, accessed on 5 January 2022).The four species’ proteomes were used to compute orthologous groups of genes among all strains with OrthoFinder v. 2.3.8 , with thE. histolytica, using STAR v. 2.5.0 [Firstly, reads of each replicate were mapped to the reference genome of v. 2.5.0 ) with a v. 2.5.0  separatev. 2.5.0 . The DESv. 2.5.0  within tv. 2.5.0 . False dv. 2.5.0  and figuv. 2.5.0 .https://bioinfogp.cnb.csic.es/tools/venny/, accessed on 15 December 2021). Predicted proteins were retrieved using UniProt  using gene name and the Uniprot function. For annotation, predicted proteins of Entamoeba were assigned to the orthologous groups of the EggNOG database v. 5 [http://eggnog-mapper.embl.de/, accessed on 27 August 2021) according to their defined parameters . One-letter abbreviations for the functional categories’ correspondence are at https://www.ncbi.nlm.nih.gov/research/cog#, accessed on 27 August 2021. Further identification of protein classes and gene ontology enrichments corresponding to genes harbouring sense and antisense transcripts were performed with Amoeba DB tools: search for gene ID, data analysis for GO terms  and synteny search in the setting pre-configured table, orthologs and paralogs within VEuPathDB . For proteins related to lipid metabolism analysis we used UniProt  and STRING (https://string-db.org/ accessed on 28 December 2021) clustering methods.To combine gene datasets the Venn diagram approach was used . Previous studies have shown that NATs are shorter than the corresponding gene and more likely to map to the 3′ region in E. histolytica [E. histolytica, E. dispar and E. moshkovskii, while E. invadens displayed a uniform pattern of coverage.To compare the transcriptomes between the four species of ted RNAs . De novoted RNAs . In totated RNAs , Sheet 1ted RNAs . Interesntamoeba , Sheet 2tolytica . After dE. histolytica and E. invadens [Entamoeba species and investigated the correlation of the genome-wide distributions of small RNAs and NAT in each species. As formerly highlighted [E. histolytica and E. invadens, based on their size distribution , and in particular at the stop codon, which acts as the Initiator (Inr) motif at the TSS of NATs [Entamoeba species. Using the method previously described [E. histolytica , E. dispar , E. moshkovskii  and E. invadens . Antisense TSSs (asTSS), defined as TSSs around the stop codon (+ /−100 nt) on the antisense strand, were also mapped in E. histolytica , E. dispar , E. moshkovskii  and E. invadens   . While g9 genes) A. Indeedthe CDSs A. These e others B. IndeedE. histolytica and E. dispar display clear similarities in nucleotide proportion on both sense and antisense strands, including (i) an A-rich region around −80 to −20 nt, (ii) a T/A enriched region (at −30 nt) within this A-rich region (iii) a C/T enriched region around −10 nt, (iv) a YA motif around TSS  and ta-NAT (primary asTSS) were compared between the four species C. E. hisE. histolytica, and similarly, 4835 and 4015 in E. dispar; 5789 and 4609 in E. moshkovskii and 5825 and 2584 in E. invadens. The median distances of mRNA PAS and NAT PAS from the stop-codon are similar in the four species, at 20 to 24 nt and 417 to 494 nt, respectively  than in the others (from 1 to 26 nt after PAS). The cleavage sequence element (CSE), representing the 3 nucleotides around the PAS, is also well identified in all species but slightly less significantly enriched in E. invadens. These observations imply that the genomic determinants of polyadenylation are not only conserved between mRNA and NAT, but also conserved among the different species, with slight variations in E. invadens, probably reflecting its greater evolutionary distance to the other species.Analysis of sequence composition around the PAS shows clear similarities among the four species, for both mRNA and NAT B, exceptn = 5069), and 3565 of them exist as a unique copy in each species (E. histolytica and E. invadens showed a similar extent of NAT transcription (~46%), the extent is substantially higher in E. moshkovskii and E. dispar (65% and 72% respectively) . We identified a set of genes that are orthologous among all four species  , Sheet 4E. histolytica and E. dispar have closely related genomes, only a few genes were identified as species-specific , compared to E. moshkovskii and E. invadens which harboured large numbers of species-specific genes  (E. histolytica and E. dispar displayed a similar extent of NAT transcription (12% and 19% of genes respectively), compared to the greater extents in E. invadens and E. moshkovskii   , Sheet 7ctively) , Sheet 8n = 531) are associated with NAT in all four species  are annotated as “unknown functions” in AmoebaDB. To investigate the functional enrichment of genes in the coreNATcore, we annotated their potential functions based on orthology mapping using eggNOG-mapper [n = 63); signal transduction mechanisms and trafficking ; posttranslational modifications, protein turnover and chaperones ; DNA replication, repair, chromatin structure and chromosomes partition ; transport and metabolism of carbohydrates, lipids, coenzymes, amino acids and ions ; cytoskeleton ; and other COG categories (n = 24). In addition, 98 proteins have a recognizable domain but with unknown function . We also annotated the genes in the core-genome using eggNOG-mapper  that are common among E. histolytica strains under various environmental conditions (defined as E. histolytica core NAT) [n = 444) are present in that dataset of E. histolytica genes presenting NATs. Next, we examined in a Venn diagram the overlap between these E. histolytica core NATs and the inter-species coreNATcore defined above and 86 genes were found to be present in both datasets ,  from a bacterial genome. The six other genes correspond to long-chain-fatty-acid-CoA ligase EHI_029050 ; triosephosphate isomerase EHI_056480 (enzyme related to glycolysis); galactokinase EHI_094100 ; pyridoxal kinase EHI_126090 ; enolase EHI_130700 (enzyme related to glycolysis) and leucyl-tRNA synthetase EHI_161970 (ligation of L-leucine to tRNA). Apart from genes with GO term annotations, other proteins involved in known metabolic processes reinforced the above observations. For example, EHI_155520 encodes a protein carrying the C-terminal catalytic domain of glutamine synthetase involved in nitrogen assimilation. Three leucine-rich-repeat-containing proteins (EHI_023340 EHI_024640 EHI_148460), which were also acquired by the E. histolytica genome through LGT from bacterial genomes, are of unknown function. Another three proteins containing LIM zinc finger domains , several kinases and small GTPAses are expected to have signalling actions through the cytoskeleton. In addition, EHI_107280 encodes a V-type ATPase from V0 complex regulating cytoplasm pH, which is a homologue of EHI_078250 vacuolar protein sorting protein 26 of the amoebic retromer-like complex [E. histolytica) forms a cofactor for AAA-family ATPase CDC48 for proteasome-dependent processing of ubiquitinated ER-associated proteins [Entamoeba.Previously, we defined a set of NAT-associated genes (ore NAT) , includitively), . They matively), , Sheet 3tively), , Sheet 4 complex . The Ubiproteins . A zinc proteins . A 5′-3′Entamoeba genomes are largely incomplete, complicating comparisons of the transcriptomes between species. To investigate the conservation of NAT between species, we examined the expression of NAT in syntenic blocks based on the syntenic annotations in AmoebaDB. In the core-genome (n = 3565), 3496 and 926 genes from E. histolytica are syntenic with at least a second species and with all four species, respectively (n = 531), 524 are syntenic between at least two species and 141 genes are syntenic in all the four species  and non-overlapping pairs are also observed. The last map according to their forward or reverse transcription sense and positioned in a convergent, opposite or similar direction , we identified significantly differentially expressed E. histolytica mRNAs or NATs  compared to another species  and 1688 NATs (1240 up-regulated and 448 down-regulated) differentially expressed. E. moshkovskii showed a moderate extent of differential expression, with 1095 mRNAs (587 up-regulated and 508 down-regulated) and 1580 NATs (1282 up-regulated and 298 down-regulated) differentially expressed. E. dispar, which is phylogenetically closest to E. histolytica [We have previously profiled the gene expression changes in an colon . In line species . Among ttolytica , showed E. histolytica mRNAs is related to NATs. Interestingly, we found the up-regulated, but not the down-regulated, E. histolytica mRNAs are significantly associated with the presence of NATs  or down-regulated  for both mRNA and NATs expressions, and discordant pairs with up-regulated mRNA and down-regulated NAT  or down-regulated mRNA and up-regulated NAT . The significant associations of the inter-species differential expression mRNAs and NATs suggest the potential regulatory functions of NATs, or the potential linkage between biogenesis of NATs and mRNAs.We also explored whether the inter-species differential expression of E. histolytica NAT-associated genes with differentially expressed NATs between species. To avoid over-sophisticated interpretations, here we considered only syntenic genes. In the core-genome (n = 3565), there are 3450, 3108 and 911 E. histolytica genes are syntenic with E. dispar, E. moshkovskii and E. invadens, respectively , followed by E. dispar with 1016 NATs (736 up-regulated and 280 down-regulated) and then E. invadens with 447 NATs (346 up-regulated and 101 down-regulated)  . Among tE. dispar, among the 736 E. histolytica genes with up-regulated NATs, the significantly enriched processes are linked to lipid metabolism with 27 genes including the phosphatases necessary for the dephosphorylation of phospholipids ; 31 genes are involved in small GTPases signal regulation, 13 genes are necessary for ubiquitin-dependent protein catabolic process, 3 genes are involved in DNA damage response, 3 genes are linked to protein export from the nucleus and 2 genes are implicated in RNA decapping including methylguanosine-cap decapping, which inactivates translation initiation and promotes 5′-to-3′ decay of mRNA  and nuclear export of proteins (n = 3). In addition, diverse global metabolic pathways were found (n = 94); as were response to stimuli (n = 92), where small GTPases signal pathways are represented by 29 genes, and DNA damage , while eight genes were involved in the metabolism of RNA   and protein dephosphorylation (n = 2). Specific to E. moshkovskii NAT down regulation there was supplemental protein dephosphorylation (n = 5), chromatin organization (n = 4), secretion by cells (n = 3), the transcription factor TFIID subunit (n = 1) and cAMP signalling involving adenylate cyclase activity (n = 1).In comparison with  ligase) . The 265. dispar , includiE. invadens, the 346 E. histolytica genes with up-regulated NATs can be annotated with 43 GO terms (n = 17); protein modification (n = 11), including endocytosis  or protein translocation . Organonitrogen compound catabolic process (n = 10) which, in addition to ubiquitination process and proteasome degradation, also showed MTA/SHA nucleosidase, the transcription elongation factor B and RAD23 which plays a central role both in proteasomal degradation of misfolded proteins and DNA repair; two genes involved in gene silencing including the transcription regulator silent information regulator 2 (EHI_ 151300) and the NAD-dependent deacetylase (EHI_120280); two genes implicated in positive regulation of transcription: the transcription elongation factor SPT4 (EHI_148350) and the mediator of RNA polymerase II transcription subunit 7, (EHI_170300); four genes encoding factors necessary for tRNA modification including queuine tRNA-ribosyltransferase; the Elp1 subunit of the elongation protein complex, required for tRNA modification; and tRNA pseudouridine synthase. The other GO terms common to the other species includes eight GO terms shared with E. dispar, five shared with E. moshkovskii and two shared with both amoebae species containing few genes . For the 101 down-regulated NATs the enrichment of GO terms identifies few genes. These are related to protein ubiquitination (n = 3), protein maturation (n = 2) and vesicular retrograde transport (n = 2) (E. moshkovskii is the presence of adenylate cyclase but there is no GO term uniquely in common with E. dispar.In comparison with the relatively distant GO terms . These i (n = 2) . Shared E. histolytica compared to those of E. dispar and E. moshkovskii rank lipid metabolism activities as the most important enriched function specific to the species infecting humans , which networking by STRING clearly distinguished the most significant gene clusters  that are associated with NATs in at least two species, and are involved in cell motility, nuclear structure, cell cycle, gene transcription and transport and nucleotide metabolism, coenzymes and amino acids metabolism. Among them, 86 genes are commonly associated with NATs in all four species, representing promising loci for further studies of NAT biogenesis. Differential expression analyses of these mRNA/NAT pairs in the core-genome revealed the interdependence between mRNA and NAT expression  and in the biogenesis of glycosylphosphatidylinositol (GPI), which is a moiety that anchors cell surface proteins to membranes, as well as kinases/phosphatases involved in the renewal of phospholipids. Multiple phospholipids play an essential role in vesicular membrane trafficking and pathogenicity of E. histolytica [E. histolytica and regulate vesicular trafficking [Interestingly, a substantial number of genes involved in lipid metabolism are associated with NAT up-regulation in tolytica ,68. Thesfficking . For exaE. histolytica and E. moshkovskii genes involving DNA damage and repair is particularly interesting as previous studies suggested that DNA recombination and allele reassortment should be more important than point mutations in E. histolytica genome [E. moshkovskii strains is much greater than that demonstrated for E. histolytica strains [Entamoeba spp. This highly speculative consequence of NAT differential expression is expected to occur through regulating the expression of Mre11 (or other enzymes involved in DNA repair) rather than through a global mechanism dependent on antisense transcription, because the high abundance of NAT observed in this work does not correlate with the relatively low rate of recombination in Entamoeba species.In addition, the differential expression of NATs between a genome . In cont strains . One mayEntamoeba, we are far from understanding their biogenesis and potential roles in gene regulation. In many cases, we do not observe positive correlation between NATs and mRNA expression , and these discordant and concordant relationships between mRNA/NAT pairs suggest that there could be diverse mechanisms underlying their biogenesis or their potential regulatory roles in gene expression. Data from other eukaryotes indicate that NATs may regulate gene expression at the post-transcriptional or translational levels including modifying epigenetic markers [Plasmodium, NATs are associated with ~24% of coding genes, with a strong clustering towards the 3′ end of CDSs, and for some genes NAT transcript levels correlate with mRNA levels of neighbouring genes [Toxoplasma gondii, NATs are associated with ~21% of coding genes [Giardia lamblia, ~20% of sequenced cDNAs are NATs [T. gondii gene expression at the post-transcriptional level [E. histolytica and E. invadens [Entamoeba drives gene expression.Although we demonstrated the existence of NAT and the interdependence of mRNA and NAT expression  in the f markers  or speci markers . In adding genes . In Toxong genes . In Giarare NATs . Althougare NATs , currental level . In addial level  and it hal level . Interesinvadens ,80. Much


The prognosis of gliomas is poor and there are limited therapeutic approaches. Immunotherapy has become a promising treatment for gliomas. Here, we explored the expression pattern of Lck/yes-related protein tyrosine kinase (LYN) in gliomas and assessed its value as an immunotherapy biomarker. Transcriptional data was mined from two publicly available datasets, TCGA and CGGA, and used to investigate the correlation between LYN and clinical characteristics including patient prognosis, somatic mutation, and immune infiltrating features in gliomas. Besides, the correlation between LYN and classical immune checkpoint molecules was explored. Glioma samples obtained from the Xiangya Hospital cohort were used for immunohistochemistry staining. High expression level of LYN was observed in advanced gliomas and other cancer types, which predicted a worse prognosis. LYN stratified patients’ survival in the Xiangya cohort and was also significantly associated with infiltrating immune cell types and inflammatory activities in the tumor microenvironment. LYN was involved in tumor mutation, correlated with the regulation of oncogenic genes, and also showed a significant positive correlation with PD-L1. LYN can be a potential diagnostic marker and immunotherapy marker in gliomas. Gliomas are the most malignant tumors. The latest world health organization (WHO) category defines grade 2 and grade 3 glioma as diffuse lower-grade glioma (LGG), and grade 4 gliomas as glioblastoma (GBM). Although the classical treatment options of surgery and adjuvant chemoradiotherapy are reported, the median overall survival rate of glioma patients is still less than 10 years . BesidesGene markers are becoming increasingly attractive in tumor research, including predicting tumor progression and treatment efficiency, reducing the recurrence rate, and prolonging patients’ survival . Immune As a member of protein tyrosine kinases, LYN critically regulates essential cellular processes including cell growth and cell differentiation. LYN is found predominantly in myeloid cells, B lymphocytes, and cell types outside of the hematopoietic compartment . StudiesIn this study, we speculated that LYN may be another promising biomarker for immunotherapy in glioma patients. Using transcriptomic data from two datasets, we investigated the expression level, clinical features, and functional annotation of LYN. Notably, we found the co-expression pattern of LYN and PD-L1 in glioma microenvironment. LYN could also significantly predict the anti-PD-1 and anti-CTLA-4 immunotherapy responses.LYN expression in GBM samples was higher than that in LGG samples . In thisn = 40) from the Xiangya hospital cohort , HEL (human erythroleukemia cells), U-698 (human B cell lymphoma), HDLM-2 (human Hodgkin lymphoma), and Karpas-707 (human myeloma) cell lineages were relatively high based on Cancer Cell Line Encyclopedia (CCLE) . Likewisp = 0.16), CGGAarray (p = 0.55), and GSE108474 (p = 0.65) was not statistically significant, this could be attributed to the insufficient samples volume in these three datasets as the tendency in survival difference was evident that high LYN expression could predict worse survival. In the pan-cancer analysis, LYN indicated worse OS in thymoma (THYM), uveal melanoma (UVM), uterine carcinosarcoma (UCS), acute myeloid leukemia (LAML), kidney renal papillary cell carcinoma (KIRP), uterine corpus endometrial carcinoma (UCEC), liver hepatocellular carcinoma (LIHC), mesothelioma (MESO), breast invasive carcinoma (BRCA), ovarian serous pancreatic adenocarcinoma (PAAD), and testicular germ cell tumors (TGCT)  than patients with low LYN expression in TCGA and CGGA . Moreoves (TGCT) . We furts (TGCT) .p = 0.0454, p = 0.1062, respectively; p < 0.001, respectively; p < 0.001, p = 0.1533, respectively; p = 0.0074, p < 0.001, respectively; p = 0.0331, p < 0.001, respectively; p = 0.0961, p < 0.001, respectively; p = 0.1377, p = 0.0735, respectively; p < 0.001, p = 0.2452, respectively; p < 0.001, respectively; p < 0.001, respectively; In TCGA, high LYN expression was associated with reduced survival in glioma patients regarding different IDH statuses . A significant correlation for genes in the brown module is illustrated in the plots of module membership and gene significance . GO analysis revealed that neutrophil activation and chemotaxis, negative regulation of T cell proliferation and activation, and chemokine-mediated signaling pathways were the most related gene functions associated with the high expression of LYN . Three CGGA cohorts, including mRNAseq_693 (693 glioma samples), mRNAseq_325 (325 glioma samples), and CGGAarray (300 glioma samples), were downloaded from CGGA (http://www.cgga.org.cn/) and included in this study. CGGA325 and CGGA693 were combined as CGGA dataset after removing batch effect using the R package sva. 414 glioma samples from GSE108474 dataset were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Fragments per kilobase million (FPKM) values of gene matrix were transformed into transcripts per kilobase million (TPM) values. RNA-seq data of tumor localization in patients with GBM was downloaded from the Ivy Glioblastoma Atlas Project . A total of 8,295 normal samples were collected from GTEX (http://commonfund.nih.gov/GTEx/) and TCGA databases. Single-cell sequencing dataset was downloaded from GSE138794 (https://www.ncbi.nlm.nih.gov/) database. Eight scRNA sequencing samples of GBM were included in the analysis. Tumor tissues were from glioma patients underwent surgery in Xiangya Hospital, Central South University. Written informed consent was obtained from all patients.The transcriptome data of LYN was collected from glioma samples in TCGA and CGGA databases. 672 glioma samples were downloaded from TCGA (SE138794  in the GParaffin-embedded tissue sections were obtained from human gliomas (WHO grades II-IV) and used for IHC staining. 40 samples were used for LYN. The sections were incubated with the LYN primary antibody (Rabbit)  after blockage with 5% BSA. The antibody reaction was visualized after 3, 3′-diaminobenzidine (DAB) development, and sections were counterstained with hematoxylin.https://gatkforums.broadinstitute.org) was performed for enrichment of genetic alteration events. CIBERSORT algorithm was performed for quantifying the expression level of 22 immune cell types  of glioma samples were downloaded from TCGA. GISTIC 2.0  was identified after performing “FindVariableGenes”. “FindIntegrationAnchors” and “Integratedata” was applied to merge eight GBM samples of single-cell sequencing . PrincipR package WGCNA was applied to explore the LYN related genes. After performing the correlation analysis between LYN and the gene expression matrix in TCGA, 3923 LYN related genes (correlation efficient >0.4) were used as the input of WGCNA. A power of β = 8 and a scale-free R2 = 0.84 were determined and set as soft-threshold parameters for a scale-free topology network. The calculated scale-free distribution topological matrix determined the softConnectivity for constructing sample dendrogram. The labeledHeatmap was used for depicting the module-trait relationship. The internal gene significance of each module was calculated and visualized using verboseScatterplot. Genes within the identified module with the highest gene significance were extracted for GO and KEGG enrichment analysis.p-values < 0.05 were set as the criteria of statistically significant.The datasets were divided into high and low groups based on the median and cutoff expression level of LYN in pan-gliomas, LGG, and GBM, respectively. Kaplan-Meier survival curves were generated using R package survival. The oneway analysis of variance was used to determine the LYN expression levels among multiple groups. The Wilcoxon rank testing was applied for determining the LYN expression levels in relation to different pathological characteristics between two groups. R package pROC was used for generation of receiver operating characteristic (ROC) curves. The classifier of ROC curve was built based on IDH status (WT vs. mutant) and molecular subtypes . Pearson’s correlation coefficient was used for calculation of correlation coefficient. R version 3.6.3 was used for all statistical analyses. LYN was associated with malignancy of gliomas and served as a prognostic marker of glioma patients. Besides, LYN facilitated the establishment of an immune-suppressive and favorable glioma microenvironment. At single cell sequencing level, LYN was abundantly expressed by tumor associated macrophages and T cells. Furthermore, the robust relationship between LYN and PD-L1 indicated that LYN might be a potent immune checkpoint molecule in predicting immunotherapy response.


Sinocyclocheilus wenshanensis is a cyprinid fish species endemic to Southwestern China. In this study, we first sequenced and characterized the complete mitochondrial genome (mitogenome) of S. wenshanensis by next-generation sequencing method. The entire length of mitogenome is 16,595 base pairs (bp), containing 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a control region. Its gene arrangement pattern was identical to other previously reported Sinocyclocheilus fishes. The overall base composition is 31.12% A, 16.63% G, 25.45% T, and 26.80% C, with AT content of 56.57%. Phylogenetic analysis using mitogenome of 26 Cyprinidae fishes showed that S. wenshanensis are closely related to S. aluensis and S. oxycephalus. This work would provide molecular information fundamental to future phylogenetic analyses among Sinocyclocheilus species. Sinocyclocheilus wenshanensis belonging to the genus Sinocyclocheilus  is a freshwater fish species that distributes only in the karst landform areas within Wenshan, belonging to Red River drainage, located in Southeast Yunnan, China , Xigu Town, Wenshan, Yunnan Province, China. The specimen was preserved in 95% ethanol and deposited in the Zoological Specimen Museum of Yunnan University  under the voucher number YNUSW20160703016. Genomic DNA from muscle tissue was extracted by DNeasy Blood & Tissue Kit . The DNA library was prepared following Illumina’s instruction and sequenced on Illumina Miseq . It generated 3,506,072 clean reads with 699,718,556 base pairs (bp) in total. The complete mitogenome sequence of S. wenshanensis was assembled with A5-miseq v20150522 . The mitogenome sequence has 16,595 bp in length, consisting of 13 protein-coding genes (PCGs), 22 tRNA genes, two ribosomal RNA (rRNA) genes, and one control region. The gene organization and structure were consistent with those of previously reported mitogenomes of Sinocyclocheilus jii  available in GenBank database were used to perform phylogenetic analysis. The phylogenetic results (S. wenshanensis was closely related to Sinocyclocheilus aluensis (Cui et al. Sinocyclocheilus oxycephalus (Li et al. Sinocyclocheilus species were grouped as a monophyletic clade with strong supports. Phylogenetic analysis reveals Sinocyclocheilus form a solid monophyletic group, which was consistent with the traditional morphological classification and previous phylogeny studies based on partial mitochondrial genes or complete mitogenomes (Xiao et al. To determine the phylogenetic position of  results  revealedChunqing Li: analysis and interpretation of the data; investigation; drafting of the paper. Fang Hu: analysis and interpretation of the data; investigation. Junxian He: analysis and interpretation of the data; investigation. Xutong Li: formal analysis; methodology. Weixian Li: taxonomic identification. Hongfu Yang: sample collection. Shanyuan Chen: conception and design; funding acquisition; supervision; revising the paper critically for intellectual content. Heng Xiao: conception and design; funding acquisition; supervision; reviewing the final approval of the version to be published. All authors agree to be accountable for all aspects of the work.


Uncaria rhynchophylla (UR) and its predominant active phytochemicals alkaloids have been studied to treat AD. This study used a novel network pharmacology strategy to identify UR alkaloids against AD from the perspective of AD pathophysiological processes and identified the key alkaloids for specific pathological process. The analysis identified 10 alkaloids from UR based on high-performance liquid chromatography (HPLC) that corresponded to 127 targets correlated with amyloid-β (Aβ) pathology, tau pathology and Alzheimer disease pathway. Based on the number of targets correlated with AD pathophysiological processes, angustoline, angustidine, corynoxine and isocorynoxeine are highly likely to become key phytochemicals in AD treatment. Among the 127 targets, JUN, STAT3, MAPK3, CCND1, MMP2, MAPK8, GSK3B, JAK3, LCK, CCR5, CDK5 and GRIN2B were identified as core targets. Based on the pathological process of AD, angustoline, angustidine and isocorynoxeine were identified as the key UR alkaloids regulating Aβ production and corynoxine, isocorynoxeine, dihydrocorynatheine, isorhynchophylline and hirsutine were identified as key alkaloids that regulate tau phosphorylation. The findings of this study contribute to a more comprehensive understanding of the key alkaloids and mechanisms of UR in the treatment of AD, as well as provide candidate compounds for drug research and development for specific AD pathological processes.Presently, there is a lack of effective disease-modifying drugs for the treatment of Alzheimer’s disease (AD).  Alzheimer’s disease (AD) is the most common type of dementia characterized by extracellular amyloid-β (Aβ) plaques, intracellular aberrant hyperphosphorylated tau protein, neuronal synaptic dysfunction, and neuronal loss . ClinicaUncaria rhynchophylla (UR), named Gouteng in Chinese, is a traditional Chinese herb native to China, Japan and Vietnam that has been used for more than a thousand years. It is utilized to extinguish the wind, arrest convulsions, clear heat, and pacify the liver. Clinically, UR is widely used to treat dizziness, headaches, epilepsy, and hypertension  study . This avhttp://herb.ac.cn/) (www.swissadme.ch) (via Protox II webserver (https://tox-new.charite.de/protox_II/) (http://www.tcmip.cn/ETCM/) (www.ncbi.nlm.nih.gov/pubmed) was used to search the literature containing the content of UR alkaloids and the permeability of blood-brain barrier (BBB).A total of 10 main alkaloids of UR were obtained from a recent study based on HPLC . HERB da.ac.cn/)  was empl.ac.cn/) . Here, wadme.ch)  to evalutox_II/) . The Encn/ETCM/)  was utilhttps://pubchem.ncbi.nlm.nih.gov/) (http://www.swisstargetprediction.ch/) .Canonical SMILES of the main alkaloids in UR were extracted from PubChem database (ih.gov/) . The 2-dion.ch/) . Specifihttps://pathcards.genecards.org/)  (PathCards database (ds.org/)  is an inta.org/)  to screehttp://pantherdb.org/)  was used to graph results. Sankey diagrams were plotted using OriginPro 2021 software .The functional annotation and the classification of UR targets were performed using the Panther classification system (db.org/) . The UR https://string-db.org/) , and the top 10 targets ranked by degree were selected and identified as core targets.The PPI network of the target proteins was collected using the latest version of STRING database   and visudb.org/) . The orgp values were adjusted using a Benjamini-Hochberg (BH) approach. Statistical significance was denoted if adjust p value <0.05. Only GO terms in the category biological process were considered in this study. Top 20 GO biological processes and KEGG pathways sorted by the p value were visualized using an online tool (http://www.bioinformatics.com.cn/) (ClusterProfiler R package (version 4.0.2)  was usedcom.cn/) . We usedhttps://pubchem.ncbi.nlm.nih.gov/) (http://www.rcsb.org/) (http://www.lephar.com/software.htm) (https://www.ebi.ac.uk/thornton-srv/software/LIGPLOT/)  . The crysb.org/) . The LeDare.htm)  was applIGPLOT/) .in vivo absorption abilities of chemical compounds  is the most significantly enriched pathway, belongs to the Human Diseases category pathway, and is mainly associated with the production and clearance of Aβ and aberrant tau hyperphosphorylation. A PPI network of the 25 UR targets involved in pathways of neurodegeneration-multiple diseases (hsa05022) in KEGG pathway database is a collection of manually drawn pathway maps of molecular interactions. KEGG pathways mainly involved Alzheimer disease (hsa05010), pathways of neurodegeneration—multiple diseases (hsa05022), neuroactive ligand-receptor interaction (hsa04080), cAMP signaling pathway (hsa04024), apoptosis (hsa04210) and MAPK signaling pathway (hsa04010) . DetaileTo identify key alkaloids of UR against AD, pathview software was used to visualize the UR targets involved in the Alzheimer disease pathway and corresponding alkaloids . The redIt should be noted that dihydrocorynatheine interacts with GRIN2B, which forms NMDA receptor, and corynoxine interacts with CHRM3, which forms G protein-coupled receptor, thus affecting downstream calcium signaling pathway and cell death. It is well known that oligomeric forms of Aβ is an important inflammatory stimulus of neuronal cells. Angustoline and angustidine can interact with PTGS2, and corynoxine, isocorynoxeine, RHY, dihydrocorynatheine, IRN can interact with NOS2 to improve the inflammation induced by oligomeric Aβ. We further counted the number of times each UR alkaloids appeared in the Alzheimer disease pathway, and plotted its frequencies. Corynoxine and isocorynoxeine have the highest frequency (12 times), followed by angustoline (11 times), angustidine (11 times), RHY (9 times), dihydrocorynatheine (8 times) and IRN (8 times). The results above strongly suggest that a variety of UR alkaloids act on different important pathological processes of AD and play a key role in the treatment of AD.in vivo were not considered. Second, the current study is still static network analysis, which contradicts the dynamic changes of disease occurrence and development. Finally, chemical changes that occur during various drug manufacturing processes are not considered in this study.Here, key phytochemicals and potential mechanisms of UR alkaloids in the treatment of AD were analyzed from the perspective of AD pathophysiological processes. This study has several advantages over traditional network pharmacology approaches. First, this study adopted a novel strategy to identify UR alkaloids against AD from the perspective of AD pathophysiological processes and identified the key alkaloids for specific pathological process. This avoids the subjectivity of potential target selection for the disease and improves the accuracy of UR targets screening against AD. Second, the present study focused on alkaloids, the bioactive constituents of UR, and obtained 10 main alkaloids from a recent HPLC study . This prvia sequential cleaving by α-secretase, β-secretase, and γ-secretase. It is noteworthy that APP is widely expressed in many tissues except the brain, including the adrenal gland, kidney, heart, liver, spleen, pancreas, muscles, and various blood and endothelial cell , inducible NOS , and endothelial NOS . NOS1 is the most abundantly isoform expressed in the brain. NOS1 and NOS3 are constitutively expressed and dependent on calcium, whereas NOS2 is inducible and independent of calcium. NOS2 play an important role in neuroinflammation by generating nitric oxide (NO). In postmortem hippocampal tissue from AD brains, the transcript level of NOS2 was significantly upregulated compared with control brains . The losIn this study, we have applied a new strategy to uncover the key UR alkaloids against AD from the perspective of pathophysiological processes. Angustoline, angustidine and isocorynoxeine were identified as the key UR alkaloids regulating Aβ production and corynoxine, isocorynoxeine, dihydrocorynatheine, IRN and hirsutine were identified as key alkaloids that regulate tau phosphorylation. The findings of this study not only contribute to a more comprehensive understanding of the key alkaloids and mechanisms of UR in the treatment of AD, but also provide candidate compounds for drug research and development for specific AD pathological processes.