Lactobacillus is the most frequently isolated microorganism from the healthy human vagina, this includes Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii. These vaginal lactobacilli have been touted to prevent invasion of pathogens by keeping their population in check. However, the disruption of vaginal ecosystem contributes to the overgrowth of pathogens which causes complicated vaginal infections such as bacterial vaginosis (BV), sexually transmitted infections (STIs), and vulvovaginal candidiasis (VVC). Predisposing factors such as menses, pregnancy, sexual practice, uncontrolled usage of antibiotics, and vaginal douching can alter the microbial community. Therefore, the composition of vaginal microbiota serves an important role in determining vagina health. Owing to their Generally Recognised as Safe (GRAS) status, lactobacilli have been widely utilised as one of the alternatives besides conventional antimicrobial treatment against vaginal pathogens for the prevention of chronic vaginitis and the restoration of vaginal ecosystem. In addition, the effectiveness of Lactobacillus as prophylaxis has also been well-founded in long-term administration. This review aimed to highlight the beneficial effects of lactobacilli derivatives (i.e. surface-active molecules) with anti-biofilm, antioxidant, pathogen-inhibition, and immunomodulation activities in developing remedies for vaginal infections. We also discuss the current challenges in the implementation of the use of lactobacilli derivatives in promotion of human health. In the current review, we intend to provide insights for the development of lactobacilli derivatives as a complementary or alternative medicine to conventional probiotic therapy in vaginal health.Human vagina is colonised by a diverse array of microorganisms that make up the normal microbiota and mycobiota.  Human Microbiome Project (HMP) and Integrative HMP (iHMP) were funded by the National Institutes of Health (NIH). They are interdisciplinary effort that engaged in human microbiome profiling for gut, vaginal, oral, and skin communities , 2. BothCandida species are likely to exist as commensals in the mucous layer of vagina and they form part of the complex vaginal ecosystem with other bacteria ., 10.L. cL. iners in CST III and CST IV were associated with higher baseline in pro-inflammatory factors such as macrophage migration inhibitory factor (MIF), interleukin-1α, interleukin 18, and tumour necrosis factor-alpha (TNF-α) which are responsible for the activation of inflammatory responses in vagina  that resulted in vaginal immune-toxicity . Ts (SAMs) . Lactobaids (TA) , 186. Thids (TA) . This coids (TA) . As LactN-acetyl-glucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) side chains that form the cell surface of Gram-positive bacteria such as Lactobacillus and bifidobacteria [Lactobacillus is surrounded by PG network and other biopolymers, namely teichoic acids (TA), S-layer proteins, and polysaccharides [Peptidoglycan (PG) is a biopolymer which comprises glycan strands connected by bacteria , 189. Gecharides .N. gonorrhoeae is able to suppress the host Th-1-driven adaptive immune response by inhibiting the production of interleukin-12 (IL-12) [Lactobacillus PG demonstrates outstanding immunomodulatory activity in improving host innate immune responses. For instance, Lactobacillus casei PG was able to induce the production of IL-12 by mice peritoneal macrophages through toll-like receptors 2 (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) [L. plantarum CAU1055 PG demonstrated the ability to ameliorate nitric oxide-induced inflammation in RAW264.7 murine macrophages through the inhibition of nitric oxide (NO) synthase, cyclooxygenase-2 (COX-2), and cytokines (TNF-α and interleukin-6) [L. acidophilus has been reported to significantly reduce the NO synthase and COX-2 levels on LPS-induced RAW 264.7 macrophages as well [L. crispatus PG was able to stimulate the expression of CD207 of Langerhans cells (antigen-presenting dendritic cells on vagina) and significantly reduced the expression of receptors for HIV entry [L. brevis PG also exhibited strong antiviral activity against genital herpes simplex virus-2 (HSV-2) [L. brevis PG was unaffected by heat- and protease-treatment, and it still inhibited HSV-2 replication significantly in a concentration-dependent manner.In general, STI pathogen such as  (IL-12) . In view (IL-12) . At the 2 (NOD2) . Moreoveeukin-6) . In a si as well . On top IV entry . The balIV entry . The pot (HSV-2) . Accordi (HSV-2) , the antLactobacillus PG is usually decorated with teichoic acids (TA) or lipoteichoic acids (LTA) [Lactobacillus [Lactobacillus plantarum LTA hampered the formation of S. mutans biofilms on hydroxyapatite discs via the attenuation of the sucrose decomposition [L. plantarum LTA significantly inhibited E. faecalis biofilm formation and preformed biofilm on human dentin slices suggesting that LTA can be employed as a preventive and therapeutic measures for E. faecalis infection [L. plantarum LTA inhibited polymicrobial biofilm consists of Actinomyces naeslundii, Lactobacillus salivarius, E. faecalis, and S. mutans on dentin slices [Lactobacillus LTA also possesses immunomodulatory activity. For instance, Lactobacillus johnsonii La1 and Lactobacillus acidophilus La10 LTAs ameliorated the overstimulation of pro-inflammatory cytokines production such as TNF-α, IL-8, and interleukin-5 (ENA-78) in intestinal epithelial cells, in the presence of lipopolysaccharides (LPS) or Gram-negative bacteria [L. plantarum K8 also modulated inflammatory cytokines (TNF-α and IL-10) production in LPS-challenged THP-1 cells [ds (LTA) . Lipoteids (LTA) , 200. Tobacillus . The erabacillus . Lactobaposition . Moreovenfection . Also, Ln slices . Other tbacteria . Accordi-1 cells . Patient-1 cells , 206. ThBacillus cereus [Lactobacillus EPS is regulated by the culture conditions and nutrient compositions during fermentation [L. rhamnosus EPS was significantly increased by 40 to 50% following 48 h of co-fermentation with S. cerevisiae, owing to the upregulation of EPS operons expression that enhanced amino acid biosynthesis, carbohydrate metabolism, and fatty acid metabolisms [Lactobacillus pentosus EPS was strongly affected by different carbon sources used for the fermentation process such as glucose which enhance the viscosity of EPS for a better thickening effect in milk production [L. plantarum EPS was observed in de Man, Rogosa and Sharpe (MRS) medium supplemented with glucose as carbon source [Lactobacillus EPS. As proof, MRS supplemented with sucrose significantly increased the EPS production of L. plantarum LPC-1 [L. plantarum LPC-1 when glucose is used as the carbon sources as compared to sucrose [Lactobacillus strains and carbon source) resulted in distinct rheological properties of EPS which can influence the EPS functional activity.Bacteria form tightly-linked polymers on the cell surface and release them to the environment as exopolysaccharides (EPS) (loosely unattached slime) , 207. Bys cereus . The proentation , 215. Foabolisms . Besidesoduction . Similarn source . Differeum LPC-1 . However sucrose . CollectLactobacillus EPS has the potential to confer health benefits to human as it has been reported to possess anti-atherosclerotic, anticancer, antioxidant, antiviral, anti-yeast, immunomodulatory, and prebiotic properties [Lactobacillus crispatus L1 EPS strongly enhanced the ability of vaginal epithelial VK2 cell to produce human defensin-2 protein  and it also reduced the adhesion of C. albicans (48%) by competitive exclusion [L. rhamnosus GG EPS and multiple Candida species, as demonstrated by the significant reduction of the adhesion of C. albicans (30%) and C. glabrata (25%) on VK2 and human bronchial Calu-3 cell line, respectively [C. albicans pathogenesis and immunopathogenesis [L. rhamnosus GG inhibited hyphal formation in C. albicans by 40% further corroborates the potential anti-Candida properties of Lactobacillus EPS. Bacterial vaginosis is characterised by the presence of polymicrobial biofilm on the vaginal epithelia [Lactobacillus plantarum WLPL04 EPS was reported to significantly reduce the adhesion of E. coli, P. aeruginosa, S. aureus, and Salmonella typhimurium on HT-29 cell line [The unique physiochemical properties of operties –224. Humoperties . Correspxclusion . Likewisogenesis . A studypithelia . Lactobaell line , which mLactobacillus plantarum C88 EPS was also shown to demonstrate high antioxidant effects by reducing MDA level and raising SOD activity in a dose dependent manner [L. helveticus KLDS1.8701 significantly improved the antioxidant activity of mice liver towards oxidative stress through the reduction of SOD activity [Lactobacillus EPS also demonstrated the ability to ameliorate degraded mucin barrier in cell. For instance, L. plantarum EPS promotes mucosal healing and protection by increasing the mucin MUC2 gene expression, tight junction protein expression and goblet cell differentiation in mouse colon [The occurrence of BV has been associated to the formation of high oxidative stress  production and low superoxide dismutase (SOD) activity) and degradation of mucin in the vaginal milieu , 230. Tht manner . Besidesactivity . Apart fse colon , 234. Inse colon .Lactobacillus brevis ED25 was demonstrated to increase the shelf life of probiotic L. rhamnosus GG in food and also elevated the viability of L. rhamnosus GG [The prebiotic properties of EPS have been actively explored. Generally, prebiotics can be retrieved from natural sources to serve as an energy source for epithelial cells and stimulate the growth of beneficial bacteria in the gut . The gennosus GG . Polysacnosus GG . ExtrapoLactobacillus BS remains understudied [Lactobacillus is dependent on the choice of fermentative carbon sources. Carbon sources originated from sugarcane and glycerol increased the yield of BS effectively compared to MRS broth [Biosurfactants (BS), also known as bio-emulsifier are amphipathic active compounds mostly synthesised by microorganisms . These arstudied . Mouafo RS broth .Starmerella bombicola [C. albicans biofilm, as well as disrupt the preformed C. albicans biofilms [C. albicans as shown by the absence of C. albicans hyphae and biofilm complex networks after treatment. As for BS produced by Lactobacillus, it was recently reported that BS from L. acidophilus ATCC 4356, Lactobacillus debrueckii ATCC 9645, and Lactobacillus paracasei 11 significantly reduced the biofilm formation of vaginal pathogen C. albicans by 40 to 50% [L. brevis CV8LAC was reported to effectively inhibit 24 h-, 48 h-, and 72 h-C. albicans biofilm formation on silicone elastomeric discs (~ 90%) [C. albicans biofilm, BS from L. jensenii P6A and L. gasseri P65 also exhibited potent antimicrobial and anti-biofilm activities against multiple urogenital pathogens such as E. coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, and Enterobacter aerogenes [L. paracasei BS inhibited Streptococcus pyogenes, Staphylococcus epidermidis, E. coli, P. aeruginosa, S. aureus, and S. agalactiae [L. paracasei BS is still highly stable following pH alkalisation (pH 6 to 10) and heat treatment (60 °C). On top of that, the extraction of crude L. paracasei BS via acidic preparation also greatly enhanced the antimicrobial activity [L. gasseri 33323 BS demonstrated anti-adhesion activity against sexually-transmitted pathogen Neisseria gonorrhoeae through the blocking of fibronectin, an extracellular matrix component on the epithelial cells. Biosurfactant from L. crispatus also significantly inhibited N. gonnorrhoeae growth (> 50%) following incubation at two timepoints, i.e. 7 min and 60 min [L. helveticus 27170 BS against S. aureus was associated with the disruption of autoinducer-2 signaling (quorum sensing molecule) in S. aureus [L. acidophilus BS was able to reduce E. coli, S. aureus, P. vulgaris, B. subtilis, and P. putida biofilms on medical implant polydimethyl siloxane (PDMS) surface through anti-adhesion mechanism [L. crispatus BC1 also demonstrated significant in vitro anti-adhesion activity against C. albicans through exclusion mechanism on human cervical cancer HeLa cell line and in vivo immunomodulatory activity by reducing the leukocyte influx  caused by C. albicans in mice [Lactobacillus BS involves adhesion interruption rather than killing of the invading pathogens.Biosurfactants have been reported to exhibit anti-adhesion and antimicrobial characteristics by altering the surface chemistry for microbial attachment . Sophoroombicola . This cobiofilms . Astonisbiofilms  also fou0 to 50% . Also, B (~ 90%) . Other terogenes . Besidesalactiae . Gudiña alactiae  found thactivity . Multiplactivity , L. gassd 60 min . Jiang e. aureus . Besidesechanism . Recentl in mice . Based oLactobacillus SAMs could benefit human, implementation their use still remain obscure and challenging. One such challenge would be the cost required for their production. As their extraction is often hindered by low yields, optimisation of growth medium composition and extraction methods are essential [L. acidophilus EPS production [L. acidophilus EPS by using MRS-nutrient broth culture medium was able to reduce by 30% (USD 7.5/kg/L culture) as compared to MRS only (USD 11.0/kg/L culture). Apart from that, statistical design is also one of the salient approaches that can be utilised to optimise media composition for the maximum yield of lactobacilli SAMs. The most widely used statistical designs for the optimisation of media suitable for SAMs (e.g. biosurfactant) production are factorial design and Response Surface Methodologies (RSM) [L. rhamnosus by modifying single- and multi-factor-at-a-time (i.e. type of carbon and nitrogen sources) through statistical modelling [L. plantarum [Lactobacillus could be improved by using genetically engineered SAMs-producing Lactobacillus strain. For instance, Li et al. [L. casei LC2W EPS by 46%. However, the usage of genetically engineered lactobacilli may engender safety concerns among public. The potential risk and safety issue can be addressed by careful experimental inspection before the administration [Lactobacillus SAMs effectiveness to prevent vaginal infection remains understudied. Nonetheless, allogeneic immune response makes the reconstitution of healthy vaginal microbiota to a Lactobacillus-dominated microenvironment challenging. Thus, a personalized approach in treating vaginal infections is required to proclaim the beneficial effect of Lactobacillus [Lactobacillus SAMs in order to solve the production bottleneck and to be used as the potential treatment for human vaginal infections.Although there are substantial evidences that ssential , 254. Inssential . Therefossential . The utissential . For insoduction . Accordioduction , the totes (RSM) . In factlantarum . In briei et al.  have shostration . While abacillus . BesidesLactobacillus), low diversity of anaerobic bacteria, and a balanced vaginal immune system (e.g. pro-inflammatory and anti-inflammatory cytokines). The host predisposing and genetic factors have been proven to alter vaginal microbial composition. Thus, disrupted vaginal ecosystem often results in diseased state of CST and symptomatic vaginitis. Meanwhile, Lactobacillus have shown to possess potential health benefits in immunomodulation and restoration of healthy microflora in gut and vagina. Despite rare Lactobacillus bacteraemia reported in immunocompromised patients, their beneficial effects in reducing recurrence rate of vaginal infection and preventing vaginally-acquired infections are well-founded. Therefore, the development of other potential treatments from probiotics should be invested to position the promising benefits of probiotics in immunocompromised patients.The presence of vaginal microbiota and mycobiota in human vagina shape the healthy and diseased state of vaginal ecosystem. Over the past decade, vaginal microbiome profiling has been extensively studied. Numerous studies reported that healthy vaginal CSTs are usually dominated by LAB  could be utilised as a prevention for vaginal infection via restoration of indigenous microbiota and their anti-biofilm capability. The successful restoration of Lactobacillus-dominated composition in BV patients was reported with lower recurrence rate along with the significant decrease in BV-related bacteria such as Gardnerella, Prevotella, Megasphaera, Coriobacteriaceae, and Atopobium [Lactobacillus SAMs to significantly inhibit the in vitro growth of vaginal pathogens, further studies should be directed on their mechanisms in in vivo model. This will be a valuable tool to facilitate the understanding of role of lactobacilli and derivatives in modulating mucosal barrier of vagina against invading pathogens. The new evidence in the understanding of potential lactobacilli SAMs and their respective mechanistic knowledge will greatly promote the development of prebiotics and antimicrobial agents, aiming on the prevention and treatment of vaginal diseases such as BV, STIs and VVC.The utilisation of topobium . The ben


Phytophthora infestans and the biotrophic Plasmopara halstedii demonstrated distinct expression profiles between life stages. In addition to providing detailed comparative information on the CYPs in multiple oomycetes, this study identified candidate CYP effectors that could be the foundation for future studies of virulence.Cyclophilins (CYPs), a highly-conserved family of proteins, belong to a subgroup of immunophilins. Ubiquitous in eukaryotes and prokaryotes, CYPs have peptidyl-prolyl cis–trans isomerase (PPIase) activity and have been implicated as virulence factors in plant pathogenesis by oomycetes. We identified 16 CYP orthogroups from 21 diverse oomycetes. Each species was found to encode 15 to 35 CYP genes. Three of these orthogroups contained proteins with signal peptides at the N-terminal end, suggesting a role in secretion. Multidomain analysis revealed five conserved motifs of the CYP domain of oomycetes shared with other eukaryotic PPIases. Expression analysis of CYP proteins in different asexual life stages of the hemibiotrophic  Cyclophilins (CYPs), a highly-conserved protein family, are ubiquitous in eukaryotes and prokaryotes. CYPs belong to the peptidyl-prolyl cis–trans isomerase (PPIase) family that catalyze the trans–cis isomerization of peptide bonds with proline residues, a rate-limiting step in many protein folding processes ,2,3. As Cryphonectria parasitica and appressorium-forming Magnaporthe grisea, mutant Δcyp1 strains were less virulent on their respective hosts [M. grisea [In mammals and plants, CYPs, FK506 binding proteins, and parvulins are the three immunophilin protein superfamilies. Immunophilins have been demonstrated to bind to cyclosporin A (CsA), an immunosuppressant molecule of fungal origin ,15,16. Tve hosts ,20. Phen. grisea . These dPhytophthora spp. [Phytophthora sojae, the PsAvr3b effector is delivered into host cells, where it is activated by direct interaction with the host cyclophilin GmCYP1; this activation is required for both virulence and avirulence activities [In the oomycetes, CYPs have previously only been cataloged in ora spp. , and virtivities ,22,23.Phytophthora infestans and Plasmopara halstedii were further characterized by analyzing transcriptional data obtained during asexual development. This analysis revealed 16 distinct CYP orthogroups, of which half were ubiquitous in all oomycetes; the number of CYPs annotated in each species varied from 15 to 35. This study provides a deeper understanding of the prevalence and possible functions of CYPs in oomycetes.This study identified oomycete candidate effectors by annotating cyclophilins from 21 diverse oomycete species including nine genera across four families . These oomycetes present widely divergent life styles, including saprotrophs, necrotrophs, hemibiotrophs, and biotrophs, with broad or narrow host ranges of plants or animals. Evidence of CYP gene expression in Albugo spp., an oomycete genus that adapted to biotrophy independently from the downy mildews [The number of CYPs identified for each oomycete varied between species . These p mildews . Manual Drosophila melanogaster) [Phytophthora spp.: a FK506-binding protein (FKBP) immunophilin domain, tetracopeptide repeat (TPR), glutaredoxin (GRX), RNA recognition (RRM), modified DNA-binding ring-finger (U-box), and WD40 repeat domains [Cyclophilins in oomycetes were classified into two major categories: single-domain proteins (five orthogroups) and multi-domain proteins  . Investi domains . This st domains ,28,29,30 domains ,32. This domains ,35,36,37There was no strong evidence for secretion signals of all proteins in a single orthogroup, though a few proteins were implicated as being secreted . This doThe CYP domains of oomycete species ranged from 121 to 259 residues in length. The consensus sequence for nine of the orthogroups contained five motif blocks  that werP. infestans and P. sojae CYP sequence as the sample; Albugo species assemblies do not contain oomcCYP14 and oomcCYP15 cyclophilins. Additionally, two oomcCYP00 clades (oomcCYP00-iv and oomcCYP00-v) were not detected from these species  . OomcCYP species . Like doancestor , meaningancestor .Phytophthora proteins as queries. Phylogenetic trees were constructed from diverse oomycete, plant, and fungal CYP sequences. In most cases, oomycete orthogroups clustered together, away from plant and fungal CYP sequences , ranked by percent identity, were added to the alignments, using equences . This waal BLAST  of P. inomcCYP13 . In addiomcCYP13 . AdditioomcCYP13 . For theproteins . Only twproteins . Therefoproteins .P. infestans and the biotrophic P. halstedii  and the spores time point were almost inverts of each other, except for oomcCYP00-i and oomcCYP07  had low expression levels during infection, but higher expression in spores, the opposite of what was observed in P. infestans. The difference between P. halstedii and P. infestans suggests that many cyclophilins may have opposite roles in the life-cycle for these two oomycetes.The analysis of CYP expression during omcCYP07 b, indica similar b. These  similar , indicatP. halstedii were poorly expressed in the mycelia of P. infestans, consistent with a non-critical role in infection. These proteins were commonly expressed in later stages of infection, including sporangia formation and cleavage. This expression pattern coincided with the necrotic stage of P. infestans infection, which was absent in downy mildews. The expression profile of these genes in P. infestans may be part of a transcription-level molecular signature for the onset of the hemibiotrophic phase. The absence of these genes in the genome of P. halstedii possibly reflects the lack of a selective pressure to maintain them during the evolution of its biotrophic life style.The proteins absent in the biotrophic Oomycete genomes and annotations were downloaded from their respective sources . InterPrhttps://www.qiagenbioinformatics.com; https://secure.clcbio.com/helpspot/index.php?pg=kb.page&id=78). Conserved amino acid motifs were identified using the MEME v5.0.5 suite (http://meme-suite.org/) [P. infestans sequences were queried against the NCBI nucleotide (nt) database to independently identify cyclophilins of plants (taxid: 3193), animals (taxid: 33208), fungi (taxid: 4751), Rhizaria (taxid: 543769), Alveolata (taxid: 33630), and stramenopiles (taxid: 33634), using taxid numbers to reduce the database size. An additional search of stramenopiles was conducted excluding oomycetes (taxid: 4762). The top 10 non-redundant plant and fungal hits were aligned with each oomycete orthogroup. A maximum likelihood protein tree was produced using RAxML v8.2.9, with 1000 bootstraps and a GAMMA substitution model [P. infestans assembly to infer support for orthology.Phylogenetics was used to investigate the evolutionary relationships among the oomycete CYPs. Coordinates for the CYP domains were obtained from searches against the NCBI conserved domain database  and Intete.org/)  with defon model . Alignmeon model . ReciproP. infestans and P. halstedii (SRR5179148 to SRR5179157 and ERR583683 to ERR583685) were used to investigate the transcription of CYPs at distinct asexual life stages. Reads were mapped to their respective assembly using STAR v2.6.0c (-quantMode GeneCounts) [P. infestans and P. halstedii were generated in RStudio using tidyverse, ggplot2 [Previously published transcriptome data of eCounts) , and theeCounts) . Heatmap ggplot2 , gplots  ggplot2 , and edg ggplot2 .P. infestans and the biotroph P. halstedii was detected at different times throughout the course of infection. The differential expression of CYPs during an infection cycle in these oomycetes is consistent with CYPs playing diverse functions including, but not exclusively, pathogenicity.We conducted a comprehensive sequence analysis of the CYPs encoded in the genome assemblies of 23 oomycetes, from 21 species. The oomycete CYPs were clustered into 16 orthogroups, largely supported by phylogenetic analysis of the CYP domains. Six CYP orthogroups included proteins that formed bigrams with a diverse range of domains indicative of a wide diversity of functions, which may include virulence. Significantly, the CYP-FBKP bigram (oomcCYP03) was found to be unique to stramenopiles and alveolates, and was not detected in Rhizaria, Plantae, or Opisthokonta. The function of these proteins is yet to be elucidated. Variable transcription of every CYP encoded by the hemibiotroph


Lignosus rhinocerus (Tiger Milk Mushroom) is used by indigenous communities in Malaysia as a traditional medicine to treat various diseases. The sclerotium of L. rhinocerus has been reported to have medicinal properties, including various bioactivities such as neuritogenic, anti-inflammatory, and anticancer effects. This study aims to investigate the neuroprotective activities of L. rhinocerus sclerotial extracts. Human embryonic stem cell (hESC)-derived neural lineages exposed to the synthetic glucocorticoid, dexamethasone (DEX), were used as the in vitro models. Excess glucocorticoids have been shown to adversely affect fetal brain development and impair differentiation of neural progenitor cells. Screening of different L. rhinocerus sclerotial extracts and DEX on the hESC-derived neural lineages was conducted using cell viability and neurite outgrowth assays. The neuroprotective effects of L. rhinocerus sclerotial extracts against DEX were further evaluated using apoptosis assays and Western blot analysis. Hot aqueous and methanol extracts of L. rhinocerus sclerotium promoted neurite outgrowth of hESC-derived neural stem cells (NSCs) with negligible cytotoxicity. Treatment with DEX decreased viability of NSCs by inducing apoptosis. Coincubation of L. rhinocerus methanol extract with DEX attenuated the DEX-induced apoptosis and reduction in phospho-Akt (pAkt) level in NSCs. These results suggest the involvement of Akt signaling in the neuroprotection of L. rhinocerus methanol extract against DEX-induced apoptosis in NSCs. Methanol extract of L. rhinocerus sclerotium exhibited potential neuroprotective activities against DEX-induced toxicity in hESC-derived NSCs. This study thus validates the use of human stem cell-derived neural lineages as potential in vitro models for screening of natural products with neuroprotective properties.In the biomedical field, there is growing interest in using human stem cell-derived neurons as Post hoc Bonferroni test revealed that the L. rhinocerus HA extract contained significantly higher carbohydrate content (P < 0.001) and significantly lower protein level (P < 0.01) than the CA, RT, and ME extracts. There were no significant differences between carbohydrate, protein, and phenolic contents of L. rhinocerus CA extract versus RT extract. On the other hand, the L. rhinocerus ME extract contained significantly higher phenolic content than all the aqueous extracts (P < 0.01).The yields (based on dry weight) and chemical compositions  were com L. rhinocerus (1 - 1000 μg/ml) were screened for their effect on the viability of the in vitro models  = 2.462, P < 0.05; L. rhinocerus ME: F  = 3.251, P < 0.05; L. rhinocerus CA: F  = 62.51, P < 0.0001; L. rhinocerus RT: F  = 51.62, P < 0.0001]. Both L. rhinocerus HA extract at all concentrations tested . These results indicated that L. rhinocerus HA and ME extracts (at concentrations up to 500 μg/ml) are noncytotoxic to NSCs, MNs, and differentiated SH-SY5Y cells, whereas L. rhinocerus CA and RT extracts are cytotoxic.The sclerotial extracts ofo models . Two-ways tested  and ME ed  was conducted to compare the effects of heat-treated extracts on the viability of the in vitro models  = 0.3872, P > 0.05] on the viability of the in vitro models  = 8.564, P < 0.01; boiled CA: F  = 15.46, P < 0.001; boiled RT: F  = 24.35, P < 0.0001]. Both boiled L. rhinocerus CA  of all in vitro models, whereas boiled L. rhinocerus ME extract significantly increased the viability of differentiated SH-SY5Y cells only (P < 0.001)  could reverse their cytotoxic effects on the in vitro models.Further boiling of thes Figure 2. Two-was Figure 2A, whereA Figure 2C and RTA Figure 2D extrac L. rhinocerus HA and ME extracts  were further tested for neurite outgrowth assay using hESC-derived NSCs (P < 0.05) compared to the negative control. One-way ANOVA revealed that L. rhinocerus HA and ME extracts had significant effects on the neurite outgrowth of NSCs . The L. rhinocerus HA extract at 10 μg/ml (μg/ml (P < 0.05). The percentage of neurite-bearing cells in NSCs treated with 10 μg/ml L. rhinocerus HA extract or 5 μg/ml L. rhinocerus ME extract was also comparable to that of B-27 treatment (positive control).Both noncytotoxicved NSCs . As the 10 μg/ml  and ME el . Neurite extension of twice the length of the cell body diameter was observed along with the expression of TUJ-1 in NSCs treated with L. rhinocerus HA and ME extracts ( L. rhinocerus HA (10 μg/ml) and ME (5 μg/ml) extracts stimulated maximal neurite outgrowth in NSCs and suggested that both extracts potentially contain neuroactive compounds that promote neuritogenesis. Therefore, L. rhinocerus HA and ME extracts were further tested in neuroprotective assays against DEX-induced effects on hESC-derived neural lineages.Immunostaining and fluorescence microscopy were performed to examine TUJ-1 expression in NSCs treated with 10 extracts . Taken tμM) for its effects on the viability of the in vitro models was conducted  = 202.3, P < 0.0001; MNs: F  = 3.320, P < 0.05; differentiated SH-SY5Y cells: F  = 8.918, P < 0.001]. Post hoc Bonferroni test showed that DEX significantly reduced viability of NSCs  at all concentrations tested in a dose-dependent manner  at high concentrations   . No sign1000 μM) . These rμM) for its effects on proliferation of NSCs was carried out to examine whether the DEX-induced reduction in cell viability was related to decreased proliferation. One-way ANOVA demonstrated that DEX had a significant effect on the proliferation of NSCs . High concentrations of DEX  caused significant decreases in the proliferation of NSCs (P < 0.01)  . Post hoμM) on the neurite outgrowth of NSCs were investigated (P < 0.01) compared to the negative control. One-way ANOVA showed that there was a significant effect of treatment on the neurite outgrowth of NSCs . Treatment with DEX (1 and 10 μM) resulted in significantly lower percentage of neurite-bearing cells in NSCs (P < 0.01) compared to the positive control (μM) had no significant effect on the percentage of neurite-bearing cells compared to the negative control. In addition, immunostaining and fluorescence microscopy of TUJ-1 expression in NSCs treated with DEX showed less neurite extension compared to NSCs treated with B-27 supplement  were cotreated with L. rhinocerus HA (10 and 100 μg/ml) or ME (5 and 50 μg/ml) extracts  = 14.30, P < 0.0001; DEX: F  = 90.41, P < 0.0001], whereas L. rhinocerus HA extract had no significant effect on the viability of NSCs . Two-way ANOVA also demonstrated no significant interaction effects between L. rhinocerus HA or ME extracts and DEX. DEX significantly decreased viability of NSCs in a dose-dependent manner , whereas treatment with 50 μg/ml L. rhinocerus ME extract significantly increased viability of NSCs compared to the negative control (P < 0.05). Coincubation of L. rhinocerus HA (10 and 100 μg/ml) extract with DEX  did not significantly increase viability of NSCs (μg/ml L. rhinocerus ME extract with 1 μM DEX significantly increased viability of NSCs (P < 0.01)  . These r L. rhinocerus ME extract against DEX-induced apoptosis in NSCs was investigated using Annexin V flow cytometry analysis  = 4.749, P < 0.01]. Post hoc Bonferroni test showed that 100 μM DEX significantly increased apoptosis of NSCs compared to that of the negative control (P < 0.05), whereas cotreatment with 5 μg/ml L. rhinocerus ME extract and 100 μM DEX significantly decreased the apoptosis of NSCs (P < 0.05). Therefore, these results suggested that cotreatment with L. rhinocerus ME extract can potentially provide neuroprotection by attenuating DEX-induced apoptosis in NSCs.The neuroprotective effect ofanalysis . Two-way L. rhinocerus ME extract against the apoptotic effects of 100 μM DEX were further confirmed using Hoechst staining ( L. rhinocerus ME extract and DEX or etoposide (positive control) showed that nuclei of the negative control cells appeared to be round or elongated with overall similar level of blue fluorescence, whereas the nuclei of apoptotic cells were brighter and smaller in size (chromatin condensation) or fragmented  = 3.575, P < 0.05]. Post hoc Bonferroni test showed that 10 μM etoposide or 100 μM DEX significantly increased the percentage of apoptotic nuclei in NSCs (P < 0.001), whereas cotreatment with L. rhinocerus ME extract (5 or 50 μg/ml) and DEX or etoposide significantly decreased the percentage of apoptotic nuclei in NSCs . These results demonstrated that etoposide and DEX treatments induced apoptosis in NSCs while cotreatment with L. rhinocerus ME extract attenuated the apoptotic effects of etoposide and DEX on NSCs.The neuroprotective effects ofstaining . Hoechstagmented . Two-way L. rhinocerus ME extract and DEX  = 13.26, P < 0.001; DEX: F  = 10.61, P < 0.001], but there was no significant interaction effect between the L. rhinocerus ME extract and DEX. Treatment of 100 μM DEX decreased the expression level of pAkt compared to the negative control, whereas cotreatment with 5 μg/ml L. rhinocerus ME extract and 100 μM DEX significantly increased the pAkt expression level in NSCs (P < 0.05)  in NSCs cotreated with and DEX . Two-way < 0.05) . Taken t L. rhinocerus sclerotium have different chemical compositions and bioactivities. Among the extracts, L. rhinocerus HA extract had high carbohydrate content, CA and RT extracts had high protein contents, and ME extract had high phenolic content. Both L. rhinocerus HA and ME extracts were found to have negligible cytotoxicity and exhibited neuritogenic properties in promoting neurite outgrowth in hESC-derived NSCs. By contrast, both L. rhinocerus CA and RT extracts displayed strong cytotoxic effects on all in vitro models, and heat treatment of both extracts abolished the cytotoxicity. In addition, DEX at high concentrations was found to decrease NSC proliferation, but had no effect on the neurite outgrowth. Furthermore, our study demonstrated that DEX exerted adverse effects on the viability of NSCs by inducing apoptosis and nuclear morphological changes, such as chromatin condensation and nuclear fragmentation. On the other hand, the L. rhinocerus ME extract exhibited potential neuroprotective activities against GC-induced cell death in NSCs. Cotreatment with L. rhinocerus ME extract and DEX in NSCs attenuated DEX-induced apoptosis, reduced apoptotic cell nuclei morphology, and reversed DEX-induced decrease in pAkt expression.This study demonstrated that the aqueous and methanol extracts of L. rhinocerus sclerotium gave the highest yield, followed by CA and RT extractions, whereas ME extraction had the lowest yield. Previous findings reported similar trends in the yields of hot aqueous, cold aqueous, and methanol extracts of L. rhinocerus cultivar TM02 sclerotium powder [ L. rhinocerus cultivar TM02 contains a higher proportion of water-soluble compounds. Hot aqueous extraction is the traditional and most common method of preparation used by native communities in Malaysia. The sclerotium of L. rhinocerus is typically boiled with other herbs to produce a decoction that can boost weak constitution and treat various diseases [ L. rhinocerus HA extract. The application of heat in the hot aqueous extraction method helps to degrade the chitinous cell wall of L. rhinocerus and increases the solubility of polysaccharides such as glucans [ L. rhinocerus ME extract showed the highest phenolic content and the lowest carbohydrate level among the sclerotial extracts. Mushrooms normally consist of high phenolic content which are secondary metabolites that exert strong antioxidant effects [ L. rhinocerus sclerotium utilized aqueous methanol (80% methanol and 20% water), which can broaden the range of extracted compounds, particularly components with high polarity such as phenolic compounds [ L. rhinocerus sclerotium, but they are insoluble in methanol during methanol extraction. However, simpler components such as amino acids, sugars, and peptides are still soluble in aqueous methanol [The HA extraction ofm powder , 32. Aqudiseases . This ho glucans . Additio glucans . Similarompounds . Carbohymethanol . L. rhinocerus hot aqueous and methanol sclerotial extracts has been demonstrated in both cancer and normal cell lines [ L. rhinocerus sclerotium were reported in several human cancer cell lines including lung, breast, prostate, and colorectal carcinoma cells [ L. rhinocerus cold aqueous extract on normal cell lines [ L. rhinocerus CA and RT extracts exhibited nonselective cytotoxicity against normal and cancer human neuronal cell lines. The cytotoxic agents in both L. rhinocerus CA and RT extracts have been postulated to be heat-sensitive compounds, as demonstrated by the loss of cytotoxicity with heat treatment and the negligible cytotoxicity of L. rhinocerus HA extract extracted at high temperature. A previous study also demonstrated the loss of cytotoxicity of cold aqueous sclerotial extract of L. rhinocerus after being heated to 80 and 100°C [ L. rhinocerus cold aqueous extract were found to be potentially cytotoxic as proteins denatured at temperatures higher than 60°C [ L. rhinocerus cold aqueous extract were reported to have selective cytotoxicity towards human breast cancer cells and were subsequently isolated and identified as subtilisin-like serine protease [ L. rhinocerus CA and RT extracts that exhibited cytotoxic effects on all in vitro models used in this study.The negligible cytotoxicity ofll lines , 32, 33.ma cells , 32, 34.ll lines , 34. In nd 100°C . The heahan 60°C . Furtherprotease . Taken t L. rhinocerus HA (10 μg/ml) and ME (5 μg/ml) extracts at low concentrations were able to promote maximal neurite outgrowth in NSCs. Similarly, the hot aqueous extract of L. rhinocerus sclerotium has been reported to stimulate neurite outgrowth in the brain, retinal, and spinal cord cells from chick embryos as well as mouse neuroblastoma (N2a) and rat pheochromocytoma (PC-12) cells [ L. rhinocerus sclerotium have also been shown to induce neurite extension in PC-12 cells [ L. rhinocerus sclerotial extract was possibly attributed to the presence of polysaccharides or triterpenoids [ Sarcodon scabrosus and hericenones isolated from ethanolic extract of Hericium erinaceus [ L. rhinocerus was reported to have high carbohydrate content including polysaccharides such as β-glucans, which are bioactive components with anticancer and immunomodulatory activities [ L. rhinocerus aqueous sclerotial extracts and genome sequencing of L. rhinocerus sclerotium (cultivar TM02) demonstrated many bioactive secondary metabolites, such as triterpenes, terpenoids, phenolic, and alkaloids [ L. rhinocerus HA and ME extracts exhibited neurite stimulatory activity in hESCs-derived NSCs. Our results suggest that the neuroactive compounds present in the L. rhinocerus sclerotial extracts could have potential in promoting neurite outgrowth and differentiation of neural progenitor cells into mature neurons. The neuritogenic property of L. rhinocerus sclerotial extracts demonstrated in our study could be linked to the presence of high carbohydrate levels in the HA extract and high phenolic contents in the ME extract. This implies that the neuroactive polysaccharides and/or secondary metabolites in the L. rhinocerus sclerotial extracts could be used to stimulate neurite outgrowth in NSCs. However, the potential neuroactive compounds present in the L. rhinocerus sclerotial extracts need to be further explored and elucidated.Our study found that2) cells , 36, 37.rpenoids . Secondarinaceus , 39. Scltivities . Furtherlkaloids . In our  in vitro and in vivo studies reported that treatment with DEX (1 and 5 μM) lowered proliferation and viability of NSCs in rat embryonic and adult brains, indicating the detrimental effects of GC on brain development [μM DEX in hNPCs was shown to downregulate Wnt signaling, which is involved in maintenance and proliferation of various types of stem cells [μM DEX did not affect the neurite outgrowth or the number of neurons in both hESC-derived neural cells (hN2) and primary rat cortical neural cells [μM DEX was demonstrated to restrict the number and length of neurites in SH-SY5Y cells [ in vitro models used.In this study, DEX at high concentrations was found to reduce the proliferation of NSCs. In contrast to our findings, previous studies reported that DEX at much lower concentrations had adverse effects on cell proliferation of animal- and human-based models. Bothelopment , 42–44. elopment . Additioelopment , 45. Treem cells . Possiblem cells , 43, 44.al cells . However5Y cells . It was 5Y cells . Taken t L. rhinocerus extracts have been reported including anti-inflammatory, anticancer, neuritogenic, and immunomodulatory activities [ L. rhinocerus extracts against GC-induced toxicity in neuronal cells. Interestingly, in our study, the L. rhinocerus ME extract was observed to have potential neuroprotective effects in NSCs by attenuating DEX-induced apoptosis and cell nuclei apoptotic morphology. Several studies have described that extracts from other medicinal mushroom  had antiapoptotic effects in neuronal cells [ L. rhinocerus sclerotium have been shown to possess antioxidant activities [ L. rhinocerus ME extract suggest the presence of antioxidant activity. Therefore, the neuroprotective effects of the L. rhinocerus ME extract against DEX-induced apoptosis in NSCs could potentially be attributed to antioxidants in the extract. The underlying mechanisms of the antioxidant properties of the L. rhinocerus ME extract in relation to its neuroprotective activity on the DEX-induced effects in neuronal cells need to be further explored.Our study showed that DEX induced apoptosis as demonstrated by nuclear condensation and fragmentation in NSCs. Apoptosis is programmed cell death that is characterized by morphological modifications such as cell shrinkage and chromatin condensation and biochemical features such as DNA fragmentation and by the expression of cell surface markers such as phosphatidylserine . It was tivities , little al cells –59. The al cells . In addial cells , 61. Mettivities , 62. Our L. rhinocerus ME extract attenuated this DEX-induced decrease in pAkt expression. The increase in the pAkt expression level in NSCs could be linked to the decrease in percentage of apoptosis observed in our study. This suggests that DEX may promote apoptosis in NSCs by suppressing Akt activation whereas L. rhinocerus ME extract can potentially exert neuroprotective effects against DEX-induced apoptosis by stimulating Akt phosphorylation. Akt, also known as protein kinase B, is a serine/threonine protein kinase which serves as the main mediator of survival signaling generated via activation of the PI3K pathway [ L. rhinocerus ME extract could potentially promote Akt activation and enhance neuronal resistance to DEX-induced apoptosis in NSCs. However, further research is required to validate the involvement of Akt signaling in the neuroprotective effects of L. rhinocerus.Our study also demonstrated that DEX decreased the expression level of pAkt in NSCs, and coincubation with the pathway . Akt act pathway , 64. It  pathway . Moreove pathway . On the  pathway –70. Take L. rhinocerus ME extract using the human-derived NSCs, which have higher resemblance of the human neural physiology compared to the animal-derived neuronal models used in previous studies [ L. rhinocerus ME extract contains compounds with potential neuroprotective activity against DEX-induced toxicity in NSCs.Induction of apoptosis by DEX in NSCs may have critical clinical significance, as treatment using synthetic GCs can cause loss of neural progenitor cells and reduction of specific neuronal populations in children, leading to adverse effects on certain cognitive or motor skills . It was  studies , 15, 37. L. rhinocerus sclerotium had opposing effects on the viability of the in vitro models. Hot aqueous and methanol extracts of L. rhinocerus sclerotium were found to be noncytotoxic in hESC-derived NSCs, MNs, and differentiated SH-SY5Y cells, whereas cold aqueous and room temperature aqueous extracts had strong cytotoxic effects. In addition, hot aqueous and methanol extracts of L. rhinocerus sclerotium exhibited neuritogenic property by stimulating neurite outgrowth in NSCs. Elevated levels of the synthetic glucocorticoid, DEX, decreased viability of NSCs by promoting apoptosis and inducing chromatin condensation and nuclear fragmentation, whereas methanol extract of L. rhinocerus sclerotium attenuated this DEX-induced apoptosis in NSCs. The L. rhinocerus methanol extract was also shown to attenuate the DEX-induced reduction in phospho-Akt level in NSCs, suggesting the involvement of PI3K/Akt signaling pathway in the neuroprotective activity of L. rhinocerus. Our study demonstrated the potential of using hESC-derived neuronal cells as in vitro models for the screening of neuroprotective activities of natural products, which could be applied in searching for neuroactive compounds and in elucidating the underlying mechanisms of their neuroprotective activities.Our study found that different extracts of


CHAT) gene mutation is characterized by episodic apnoea. To date, 52 cases of CMS caused by CHAT gene mutations have been reported. Here, we report a neonate with the third hemizygous mutation [a 4.9 Mb deletion [10q11.22–10q11.23 (chr10: 46123781–51028772)] containing the whole CHAT gene and c.1976A>T (p.Gln659Leu in the CHAT gene)]. The c.1976A>T (p.Gln659Leu) variant had not been reported in the ExAC or gnomAD databases and was predicted to be pathogenic. The alignment of amino acid sequences revealed that glutamine at codon 659 is highly conserved in different species and causes structural changes in the substrate-binding site. Our female patient with neonate-onset CMS presented with apnoea, dyspnoea, feeding difficulties, weak crying, and seizure-like episodes, and her respiration was ventilator dependent. The prostigmine test was positive. This case may help to further elucidate clinical features and treatment methods in neonate-onset CMS caused by CHAT gene mutations.Congenital myasthenic syndrome (CMS) is a neuromuscular transmission disorder caused by mutations in genes encoding neuromuscular junction proteins. CMS due to choline acetyltransferase ( Congenital myasthenic syndromes (CMSs) are rare genetically heterogeneous diseases caused by abnormal signal transduction due to mutations in genes coding for proteins expressed at the neuromuscular junction (NMJ). According to the site of mutation, CMS can be divided into presynaptic, synaptic or postsynaptic . IndividCHAT gene mutations are presynaptic types. They account for approximately 5% of CMS probands (CHAT gene is located on the long arm of human chromosome 10 (10q11.23) and encodes the biosynthetic enzyme of the neurotransmitter acetylcholine, namely, choline acetyltransferase. CHAT gene mutations can result in deficient resynthesis of acetylcholine following its reuptake at the nerve terminal (CHAT gene mutations have been identified as related to CMS (http://www.hgmd.cf.ac.uk/ac/dipx.php). Mutations located near the active-site tunnel, impairing substrate binding, result in more severe phenotypic effects in patients with CHAT-related CMS  × 1, included 91 genes. Only five were associated with abnormal phenotypes in the Online Mendelian Inheritance in Man (OMIM®) database: CHAT (choline acetyltransferase), ERCC6 (chromatin remodeling factor), SLC18A3 , GDF2 (growth differentiation factor 2), and RBP3 (retinol binding protein 3). Whole exome sequencing showed a hemizygous mutation in CHAT , which likely accounts for the patient's phenotype. The clinical features and treatment of this neonate were discussed below.Here, we report a third case of hemizygous mutation. A neonate with CMS carried a 4.9 Mb deletion and a c.1976A>T (p.Gln659Leu) mutation in the The proband was a 13-day-old female, the second child of healthy non-consanguineous parents. She was born as the first of fraternal twins at 37 weeks via cesarean section due to reduced heart rate of the twins. Her birth weight was 2.4 kg (<10th p). Her older brother and the other neonate twin were both normal, and there was no relevant medical family history. After birth, she required a laryngeal mask to pressurize the oxygen. The treatment relieved her cyanosis. She was then immediately transferred to the local neonatal intensive care unit where she was intubated and ventilated. She was also treated with anti-infectious and excitatory respiration center drugs . However, the effect was poor. She failed to extubate after multiple attempts due to carbon dioxide retention. The patient was hospitalized in our neonatal intensive care unit for dyspnoea for 13 days with failure to extubate for 1 day.The patient had a full-term appearance, a weight of 2.35 kg, poor response to physical examinations, weak crying, labored breathing, shortness of breath, and a positive three-concave sign. Both lungs were clear on auditory percussion. Neither obvious wet nor dry rattling was heard. The heart rate was strong, and no obvious murmurs were heard in the precordial region. There was no abdominal guarding upon palpation, and abdominal rumbling was weak. There was reduced movement of the limbs, decreased muscle tone and absent primitive reflexes.9/L, erythrocyte count 3.61 × 1012/L, hemoglobin 113.0 g/L, platelet count 390 × 109/L, percentage of neutrophils 55.8%, and percentage of lymphocytes 32.8%. The levels of inflammatory markers were as follows: procalcitonin 0.390 ng/mL and C-reactive protein 7.60 mg/L. The N-terminal pro b-type natriuretic peptide level was 1,334.00 pg/mL. The prostigmine test was positive. The fungal glucan level was 405.05 pg/mL. The myocardial enzyme levels were as follows: creatine kinase 70.00 U/L and creatine kinase isoenzyme 20.10 U/L. The chest plain film indicated a double-lung texture growing thick and heavy, and CT images showed an increasing density of patchy shadows with blurry margins, which was consistent with the manifestations of pneumonia  intramuscular injection, which relieved the dyspnoea and improved her feeding. We then orally administered pyridostigmine (5 mg/kg) to replace the intramuscular injection, which controlled her dyspnoea. We did not observe any side effects from these drugs. Later, she was weaned successfully from mechanical ventilation and could gradually breathe independent of oxygen therapy. However, episodes of dyspnoea accompanied by cyanosis and decreased oxygen saturation occurred upon reducing the dosage of pyridostigmine. We failed to make a definitive diagnosis at the initial stage of treatment due to rejection of genetic testing by the family members. She was transferred to a hospital with better equipment and treatment capacity, according to the parent's wishes, where she died.CHAT gene , which is inherited from her father . Two milliliters of peripheral blood was extracted from the proband and her parents. The blood was sent to Cipher Gene LLC, and the following tests were conducted. Whole exome sequencing was performed on the proband and her parents using the Illumina NovaSeq 6000 platform. Agilent SSELXT Human All Exon V6 was used to capture and construct the library, and a paired-end sequencing strategy was used. CNV-Seq was performed on the proband using the Illumina NovaSeq 6000. A TruSeq Library Construction Kit was used to capture and construct the library, and a paired-end sequencing strategy was used. CNV-Seq indicated a 4.9 Mb deletion [10q11.22-10q11.23 (chr10: 46123781-51028772)], which contained the whole HAT gene . The whor father .CHAT-related CMS.Our patient presented with dyspnoea, ventilator dependence, and apnoea. The prostigmine test was positive. Pyridostigmine relieved dyspnoea in our patient, and she weaned successfully from mechanical ventilation and could gradually breathe independent of oxygen therapy. The clinical course of the neonate and whole exome sequencing indicated CHAT-related CMS have been reported to date (CHAT (p.Gln659Leu). The copy number variation of the 4.9 Mb deletion in this study and its highly overlapping deletion fragment could not be found in the Genetic Variation Database. Highly overlapping pathogenic or suspected pathogenic CNVs were found in the Decipher patient database (https://decipher.sanger.ac.uk/syndromes#syndromes/overview (CHAT (http://exac.broadinstitute.org/) or gnomAD databases (http://gnomad.broadinstitute.org/about) and was predicted to be pathogenic by multiple prediction software programs . The alignment of amino acid sequences revealed that glutamine at codon 659 is highly conserved in different species (https://www.ebi.ac.uk/Tools/msa/), which suggests p.Gln659Leu is likely pathogenic (CHAT protein of human were constructed by the homologous modeling database SWISS-MODEL (http://swissmodel.expasy.org/). Residual changes between the wild-type and mutant CHAT proteins were mapped to the model by PyMol v1.3 software. This mutation causes a structural change in the substrate-binding site .QZ conceived and designed the work. YZ drafted the manuscript and collected the data. XC contributed to the conception of the work and played an important role in interpreting the clinical data. CL contributed to the manuscript revision. ML contributed to figure preparation. FM contributed to the adjustment of the structure of the manuscript. ZS contributed to figure preparation. WC contributed to table preparation. JZ contributed to data collection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clostridioides difficile-colonized patients. We included 102 C. difficile non-colonized (group I), 93 C. difficile colonized subjects (group II), and 89 diarrhea patients with C. difficile (group III). Chao1 index for alpha diversity and principal coordinate analysis was performed for beta diversity using QIIME. The mean relative abundance in each group was compared at the phylum and genus levels. Fecal calprotectin was measured using EliA calprotectin (Thermo Fisher Scientific). Group II showed significantly lower levels of Sutterella, Blautia, Ruminococcus, Faecalibacterium, Bilophila, and Ruminococcaceae and higher levels of Enterobacteriaceae compared to group I . Toxigenic C. difficile colonized subjects showed significantly lower levels of Prevotella, Phascolarctobacterium, Succinivibrio, Blautia, and higher levels of Bacteroides. The level of fecal calprotectin in group III was significantly higher than those in group I and group II (p < 0.001 for both). These data could be valuable in understanding C. difficile colonization process and the microbiota and inflammatory markers could be further studied to differentiate colonization from CDI.In this study, we aimed to evaluate the composition of the intestinal microbiota and level of fecal calprotectin in  Clostridioides difficile, previously known as Clostridium difficile, is an anaerobic Gram-positive, spore-forming bacterium and a common cause of antibiotic-associated nosocomial diarrhea in the industrialized world [C. difficile infection (CDI) has been shown in previous studies using various techniques from culture-based methods to high throughput sequencing [C. difficile in patients with significant diarrhea, defined as unexplained and new-onset (≥3 unformed stools in 24 h) [C. difficile or its toxins or toxin gene. Many assays cannot differentiate between colonization and infection. This causes a false classification of C. difficile-colonized patients with diarrhea as CDI [C. difficile-colonized subjects is sparse and there are no data on the comparison of toxigenic and non-toxigenic C. difficile colonized subjects.ed world ,2. Aftered world ,4,5,6,7.quencing ,9,10,11.in 24 h) . Unfortua as CDI ,14. Meana as CDI ,16. Altha as CDI ,17. To tC. difficile colonized subjects and compare them with those in non-colonized control and patients with significant diarrhea. We also compared the differences between toxigenic and non-toxigenic C. difficile colonized subjects.The present study aimed to evaluate the composition of the intestinal microbiota and inflammatory status in C. difficile colonization in 812 residual fecal samples from individuals who visited our center for a general health examination. Korean adults (>30 years old) were included and subjects with obesity (body mass index > 25), other ethnicity or history of recent hospitalization were excluded. C. difficile colonization was firstly assessed by glutamate dehydrogenase (GDH) testing using VIDAS C. difficile GDH on the VIDAS instrument  according to the manufacturer’s instructions. Among the screened samples, 93 (11.4%) were GDH-positive. We defined “C. difficile colonization” as positive GDH in the absence of CDI symptoms [C. difficile non-colonized) and 93 GDH-positive samples  for further study. Toxin status was determined by tcdB gene real-time PCR . The Xpert C. difficile is an automated, real-time multiplex PCR assay, which detects the genes for tcdB, binary toxin (cdt), and a point mutation at position 117 associated with hypervirulent 027/NAP1/BI strains. A maximum valid Ct is 37 for tcdB and cdt according to the manufacturer’s instructions. Among the 93 C. difficile-colonized subjects (group II), 13 (14%) were tcdB gene positive. In addition, we collected 89 GDH-positive samples from hospitalized patients with significant diarrhea (≥3 unformed stools in 24 h) [This study was approved by the Institutional Review Board of the Konkuk University Medical Center, Seoul, Korea . In addition to this, all methods were performed in accordance with the relevant guidelines and regulations. From March 2018 to June 2019, we screened for symptoms . We incltcdB gene-negative and 69 tcdB gene-positive samples . A total of 284 samples were analyzed and the characteristics of each group are summarized in They included 20 ® XP beads , sequencing libraries were prepared using an Ion Plus Fragment Library Kit and Ion Xpress™ Barcode Adapters . Prepared libraries were quantified using a High Sensitivity DNA kit on an Agilent 2100 Bioanalyzer . Template preparation and sequencing were performed using the Ion Chef™ System and Ion S5™ XL system with Ion 530™ Chip Kit . After filtering out low quality and polyclonal reads, and trimming any adaptor sequences at the 3′ end, the sequencing data were exported as FASTQ files and were processed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline 1.9.1 [http://qiime.org/home_static/dataFiles.html).DNA extraction from a stool sample was performed using a QIAamp DNA stool mini kit  and bacterial 16S rRNA genes were amplified using an Ion 16S™ Metagenomics Kit  according to the manufacturer’s protocol. The kit included 2 primer tubes with 3 primer sets that amplify the hypervariable regions of 16S rRNA . After PCR amplicons were purified using Agencourt AMPurene 1.9.1 . After qAlpha and beta diversity measures were calculated by QIIME . Alpha dAfter thawing, fecal samples were extracted using EliA Stool Extraction Kit . The extraction tubes were pre-filled with 750 μL of EliA Calprotectin Extraction Buffer. Fecal calprotectin was measured using EliA calprotectin on the Phadia 250 system . The wells of the EliA Calprotectin were coated with monoclonal antibodies against calprotectin, and the calprotectin levels were quantified by fluoroenzyme immunoassay. Phadia 250 measures calprotectin concentrations in ng/mL, and by using a conversion factor given by the lot-specific code of the EliA Calprotectin Well, the results were automatically converted to mg/kg.t-test or the Mann–Whitney U test, and that between categorical variables was analyzed using the chi-squared test, Fisher’s exact test, or the McNemar test. The Kruskal–Wallis test and one-way analysis of variance (ANOVA) were used to assess the differences between groups. Permutational multivariate analysis of variance (PERMANOVA) between groups was performed using QIIME. Statistical analysis was performed using MedCalc Statistical Software  and IBM SPSS Statistics 22.0 . p values less than 0.05 were considered statistically significant.The difference between the continuous variables was analyzed using Student’s C. difficile-non colonized), group II (C. difficile-colonized), and group III (diarrhea with C. difficile). The distribution of the Chao1 indexes in each group is presented in p < 0.001 for both). The median Chao1 index between group I and group II was not significantly different (p = 0.797). The median Chao1 index between subjects with non-toxigenic and toxigenic C. difficile was not significantly different in group II and III .We evaluated the differences in alpha diversity between group I (p = 0.001 by PERMANOVA), while group I and II could not be separated (p = 0.057 by PERMANOVA). In weighted UniFrac analysis, all three groups clustered separately  (C. difficile clustered separately both in unweighted and weighted UniFrac matrix (both p = 0.001). In group III, subjects with non-toxigenic and toxigenic C. difficile clustered separately only in unweighted UniFrac matrix (p = 0.001 in unweighted and 0.721 in weighted UniFrac matrix).PCoA using unweighted and weighted UniFrac matrix was performed to evaluate the beta diversity between each group. In unweighted UniFrac analysis, group I vs. III and group II vs. III clustered separately (both ctively) . In grouBacteroidetes  and significantly higher levels of Proteobacteria compared to group I (p < 0.001). Comparing subjects with non-toxigenic and toxigenic strains in group II, those with toxigenic C. difficile colonized subjects showed significantly lower levels of Firmicutes (p < 0.001). In group III, there was no significant difference in composition between patients infected with non-toxigenic and toxigenic C. difficile strains  compared with those in groups I and II. Group III also showed a significantly higher mean relative abundance of Parabacteroides, Serratia, Veillonella, Enterococcus, and an unassigned genus of Enterobacteriaceae. Group II showed a significantly lower abundance of Sutterella, Blautia, Ruminococcus, Faecalibacterium, Bilophila, and an unassigned genus of Ruminococcaceae and higher abundance of an unassigned genus of Enterobacteriaceae compared with those in group I . Moreover, toxigenic C. difficile colonized subjects showed a significantly lower abundance of Prevotella, Phascolarctobacterium, Succinivibrio, Blautia, and an unassigned genus of Ruminococcaceae, and higher abundance of Bacteroides and an unassigned genus of Enterobacteriaceae compared with non-toxigenic C. difficile colonized subjects . In group III, there was no significant difference between patients with non-toxigenic and toxigenic C. difficile . There was no significant difference between group I and group II (p = 0.256). The fecal calprotectin levels were not significantly different between individuals with non-toxigenic and toxigenic C. difficile in both group II and group III .We compared fecal calprotectin levels in groups I, II, and III and its distribution is presented in C. difficile among healthy individuals ranges from 1.8 to 15% [C. difficile colonization among the screened population were 11.4% and the toxigenic portion among colonized C. difficile was relatively low (14%), which was lower than our previous studies performed in the same institution using the same method [The asymptomatic colonization rate of toxigenic 8 to 15% ,21,22. He method . This die method .C. difficile) compared with those in group I (C. difficile non-colonized) and group II (C. difficile colonized). Previous studies also showed decreased alpha diversity in CDI or antibiotic exposed group compared with the control [C. difficile colonization itself in healthy individuals does not seem to affect species diversity. The present study also showed that the alpha diversity in group II and group III was not affected by toxigenic status.As expected, the alpha diversity index (Chao1), as shown in  control ,24. As d control ,4,25. InC. difficile colonization , the composition and abundance of microbiota were changed in C. difficile colonization. Regarding toxin status, subjects with non-toxigenic and toxigenic C. difficile clustered separately both in unweighted and weighted UniFrac matrix, indicating that the intestinal microbiota is further changed distinctly upon colonization of toxigenic C. difficile strains compared to non-toxigenic strains. In patients of group III, non-toxigenic and toxigenic C. difficile also clustered separately, but significant difference was observed only in the unweighted UniFrac matrix. Disruption of intestinal microbiota composition occurs in many hospitalized patients, which could weaken the difference between those colonized with non-toxigenic and toxigenic strains.In PCoA analysis, group III clustered separately from group I and II by unweighted and weighted UniFrac analysis, which was consistent with previous studies ,24. ThisBacteroidetes and Proteobacteria, respectively, at the phylum level, and lower relative abundance of many genera, including butyrate-producing organisms and health-promoting bacteria [Parabacteroides or Enterococcus, which reflect a blooming phenomenon by reduced ecological niche competition [C. difficile, suggesting again that this disruption of microbiota was not specific to patients with toxigenic C. difficile.Our study showed different patterns of the composition of fecal microbiota in patients with diarrhea, as shown previously ,26. Specbacteria ,27. Incrpetition ,28,29, wC. difficile colonization and a few studies only described a very limited number of subjects [C. difficile colonization, including those due to the toxigenic status of the strains. Group II (C. difficile colonized) did not show significant differences compared with non-colonized subjects (group I) at the phylum level and the results at phylum levels were the sum of the changes of many genera. At the genus level, group II showed a significantly lower abundance of many genera and a higher abundance of Enterobacteriaceae compared to group I. This pattern of change was partly similar to that observed in CDI or hospitalized patients, indicating that the genus compositions of C. difficile colonized subjects lies between non-colonized and hospitalized patients. Moreover, toxigenic C. difficile-colonized subjects showed significant changes, such as a lower abundance of Prevotella, Phascolarctobacterium, Succinivibrio, Blautia, and an unassigned genus of Ruminococcaceae and higher abundance of Bacteroides and an unassigned genus of Enterobacteriaceae than non-toxigenic C. difficile-colonized subjects. These features were closer to the patterns observed in hospitalized patients with diarrhea. We could not find a significant difference in hospitalized patients with diarrhea based on the presence or absence of toxigenic C. difficile.There is very limited data on the composition of intestinal microbiota in subjects ,14,30,31C. difficile-colonized subjects, regardless of toxin status, had comparable fecal calprotectin levels to those of non-colonized subjects and significantly lower levels than in diarrhea patients with C. difficile. The C. difficile-colonized subjects seemed to show a non-inflammatory status, although they showed changes in the composition of intestinal microbiota (beta diversity). The diarrhea patients had significantly higher fecal calprotectin levels, reflecting in high inflammatory status, and the toxin status did not show significant differences in calprotectin levels, indicating the non-specific increase of this marker, as suggested by previous studies [Fecal calprotectin is a 36.5 kDa molecule derived from the cytoplasm of neutrophils, mononuclear cells, and squamous epithelial cells and reflects the inflammatory status of the intestine . The pro studies ,35,36.This study had several limitations. The cause-effect relationship between specific alterations of the microbiota and clinical status or colonization could not be elucidated in this study design. Many covariates such as age or food intake habit could also affect the intestinal microbiota composition . The medC. difficile. We found significant differences in the composition of intestinal microbiota in apparently healthy C. difficile colonized subjects in comparison to C. difficile non-colonized subjects. Despite the significant difference in C. difficile colonization such as lower levels of Sutterella, Blautia, Ruminococcus, Faecalibacterium, Bilophila, and Ruminococcaceae and higher levels of Enterobacteriaceae, inflammatory status and alpha diversity were not significantly different between C. difficile colonized and non-colonized subjects. Further investigation of the intestinal microbiota and immunological studies could provide some insight into the conditions that allow for colonization and protect against disease progression.There were significant alterations in the alpha and beta diversity in intestinal microbiota in hospitalized patients with diarrhea, but these alterations were not significantly affected by the toxin status of


Hydra species have been employed as model organisms since the 18th century. Introduction of transgenic and knock-down technologies made them ideal experimental systems for studying cellular and molecular mechanisms involved in regeneration, body-axis formation, senescence, symbiosis, and holobiosis. In order to provide an important reference for genetic studies, the Hydra magnipapillata genome (species name has been changed to H. vulgaris) was sequenced a decade ago (H. vulgaris belongs to the non-symbiotic brown hydra lineage, the green hydra, Hydra viridissima, harbors algal symbionts and belongs to an early diverging clade that separated from the common ancestor of brown and green hydra lineages at least 100 million years ago (H. viridissima and endosymbiotic unicellular green algae of the genus Chlorella have been a subject of interest for decades, genomic information about green hydras was nonexistent. Here we report a draft 280-Mbp genome assembly for Hydra viridissima strain A99, with a scaffold N50 of 1.1 Mbp. The H. viridissima genome contains an estimated 21,476 protein-coding genes. Comparative analysis of Pfam domains and orthologous proteins highlights characteristic features of H. viridissima, such as diversification of innate immunity genes that are important for host-symbiont interactions. Thus, the H. viridissima assembly provides an important hydrozoan genome reference that will facilitate symbiosis research and better comparisons of metazoan genome architectures.Various  Hydra are relatively simple. Hydra possess only a polyp stage, while other medusozoans exhibit alternation of polyp and medusa stages.The Cnidaria is an evolutionarily ancient and well-defined phylum, characterized by the possession of nematocytes . AlthougHydra has been an experimental model for studying cellular and molecular mechanisms underlying the formation of the body axis (Hydra magnipapillata (now classified as H. vulgaris) genome was sequenced in 2010 (https://research.nhgri.nih.gov/hydra/).With its simple body structure and easy laboratory cultivation, ody axis , regenerody axis . IntroduH. vulgaris belongs to the non-symbiotic brown hydra lineage, the green hydra, Hydra viridissima, establishes a mutualistic relationship with microalgae and exchanges metabolites with its symbionts , genomes of representative species in each subgroup of cnidarians have been sequenced, including another hydrozoan, Clytia hemisphaerica -1,1-dimethylurea), as described previously  and digested with 100 mg/L Proteinase K. Genomic DNA was extracted using the standard phenol-chloroform method with 100 mg/L RNaseA treatment. The quantity of DNA was determined using a NanoDrop , and the quality of high molecular-weight DNA was checked using agarose gel electrophoresis.In paired-end library preparations for genome sequencing, genomic DNA was fragmented with a Focused-ultrasonicator M220 . A paired-end library (average insert size: 540 bp) and mate-pair libraries  were prepared using Illumina TruSeq DNA LT Sample Prep Kits and Nextera Mate Pair Sample Preparation Kits , following the manufacturers’ protocols. These libraries were quantified by Real-Time PCR  and quality checked using capillary electrophoresis on a Bioanalyzer. Genome sequencing was performed using the Illumina Miseq system with 600-cycle chemistry (2 × 300 bp). Genome sequencing statistics is shown in Table S1A.Total RNA was extracted from about 1000 polyps in six different conditions  using Trizol reagent (Thermo Fisher Scientific) and an RNeasy Mini kit . The quantity of RNA was determined with a NanoDrop (Thermo Fisher Scientific). Quality of total RNA was checked with a BioAnalyzer . For mRNA-seq, libraries were produced using an Illumina TruSeq Stranded mRNA Sample Prep Kit and were sequenced on HiSeq 2000 instruments using 2 × 150-cycle chemistry. mRNA-sequencing statistics are shown in Table S1B.Hydra viridissima transcriptome and raw RNAseq reads against the genome assembly. Mapping and gene structure annotation were performed using the PASA pipeline v2.01 and were used to train AUGUSTUS software , and subsequent scaffolding was performed with SSPACE (Hydra viridissima genome size was estimated from k-mer distribution frequencies using the GenomeScope web tool (http://qb.cshl.edu/genomescope/).Genome size was estimated from raw paired-end reads by k-mer distribution analysis. Jellyfish v2.0.0 was used to count k-mers and their frequencies . The HydHydra viridissima were identified de novo with RepeatScout version 1.0.5 (http://www.repeatmasker.org/RepeatModeler) and RepeatMasker version 4.0.6 (http://www.repeatmasker.org). Repetitive elements were filtered by length and occurrence so that only sequences longer than 50 bp and present more than 10 times in the genome were retained. The resulting sets of repetitive elements were annotated using BLASTN and BLASTX searches against RepeatMasker.lib  and RepeatPeps.lib  bundled with RepeatMasker version 4.0.6. The results of both searches were combined, and BLASTX results were given priority in cases where BLASTN and BLASTX searches gave conflicting results.Repetitive elements in the draft genome assembly of H. viridissima genes among cnidarians, protein sequences were obtained from Hydra 2.0 web portal (https://research.nhgri.nih.gov/hydra/) and the Compagen server (http://www.compagen.org) for Hydra vulgaris (H. magnipapillata) strain 105, from JGI (https://genome.jgi.doe.gov/Nemve1/Nemve1.home.html) for Nematostella vectensis (http://marimba.obs-vlfr.fr/organism/Clytia/hemisphaerica) for Clytia hemisphaerica  for Acropora digitifera  and BUSCO values than the originally published assembly (https://research.nhgri.nih.gov/hydra/) are shown side by side in For comparative analysis of http://homeodb.zoo.ox.ac.uk/families.get?og=All)  were performed using HMMER . To clas?og=All) .http://tree.bio.ed.ac.uk/software/figtree/). Gene/protein IDs used for phylogenetic analysis are shown in the trees .For phylogenetic analysis, multiple alignments were produced with CLUSTALX (2.1) with gap trimming . Phylogehttps://marinegenomics.oist.jp/hydraviridissima_A99). A genome browser was established for assembled sequences using the JBrowse 1.12.3  and Hydra (B) are in Table S4. Gene IDs of ANTP genes in Hydra viridissima A99 are in Table S5. Supplemental material available at figshare: https://doi.org/10.25387/g3.12911426.This whole-genome shotgun sequencing project has been deposited at DDBJ/ENA/GenBank under BioSample ID SAMN09635813 and BioProject ID: PRJNA480404. RNA-seq reads have been deposited at SRA of NCBI (SRX6792700-SRX6792705). Genome sequences, gene models, and a genome browser are also accessible at the website of the OIST Marine Genomics Unit Genome Project  based on paired-end sequence data . This indicates that we achieved more than 90-fold sequence coverage of the genome. On the other hand, the total length of the genome sequence assembly reached 284,265,305 bp. That is, the total assembly closely matched the estimated genome size.The genome of H. viridissima has an AT-rich genome similar to that of H. vulgaris (25.4%). Using 67,339,858,036 nucleotides of RNA-sequence data (Table S1), we predicted gene models. The genome was estimated to contain 21,476 protein-coding genes  . Thus, polyps originally collected from the wild had a high level of heterozygosity. Repetitive sequences constituted 37.5% of the genome and the gap rate was 16.8% of the genome . The GC ng genes . We did ectively . Compareer genes . The BUSgene set . Comparileteness .H. viridissima mitochondrial genome. The mitochondrial genome of H. viridissima strain A99 was linear, as reported by Hydra vulgaris, mitochondrial genome is composed of two linear molecules , contained almost the entire H. vulgaris, 41% in Clytia, 45% in Aurelia, and 37% in Morbakka (H. viridissima (37.5%) , genomes of medusozoans and hydrozoans have comparatively high levels of repetitive sequences, 60% in Morbakka . This wa (37.5%) . DNA tra (37.5%) . On the H. viridissima and H. vulgaris, it became apparent that DNA transposons  occupy a similar portion of both Hydra genomes  and has fewer repetitive elements (41%) than H. vulgaris, the ancestral Hydra genome was likely rather compact, with fewer retrotransposons. Molecular and evolutionary mechanisms involved in the insertion of LINE components in the H. vulgaris genome will be a subject of future studies in relation to diversification and speciation within the Hydra clade.In comparing repetitive elements between  genomes . In addiH. viridissima genome. We found approximately 4,500 different Pfam domains in this species (Table S3), a number comparable to those of other cnidarians. To identify the domains that are enriched in the H. viridissima genome, we counted the number of genes with each Pfam domain in cnidarian genomes, and selected the domains of which number are ≥2x higher in the green hydra genome than those of non-symbiotic cnidarians and show significant difference based on Chi-Square test   . Then wee top 10 . NACHT/Nidarians .H. viridissima genome . On the other hand, in Nod-like receptors of basal metazoans, not only LRR, but also tetratricopeptide repeats (TPR), WD40 repeats, and ankyrin repeats (Ank) are found as repeat domains. We previously showed that Acropora has all 4 types of Nod-like receptors, and that those with LRR are the most common , which are not found in H. vulgaris.In t common   of the Nod-like receptors most abundant in green hydras and corals are different. This indicates that species-specific adaptations to the environment and particular symbionts occurred independently in these lineages.As mentioned above, diverse pattern-recognition receptor-related genes are found in both H. viridissima and others present in both H. viridissima and H. vulgaris. This was done using the same criteria as above, that is, that the number of domains is ≥2x higher than those in other cnidarians and that the difference is significant by Chi-Square test . . .H. viridissima and H. vulgaris possess many genes encoding domains that function in DNA binding. For example, genes containing transposase-related domain  and DNA-binding motif  were overrepresented in both H. viridissima and H. vulgaris  were most abundant, with 105 members, although the abundance of this family has been noted in other cnidarian genomes (H. viridissima are discussed in the next section). A similar analysis of putative signaling molecule genes showed that the H. viridissima genome contains 16 fibroblast growth factor (FGF)-like domain genes, 11 transforming growth factor-beta (TGB-β) genes, and 10 Wnt genes , PRD, LIM, POU, PROS, SINE, TALE, CERS, or ZF using bi-directional BLAST searches against sequences of homeodomains in other animals, using HomeoDB  apparently contain the most homeodomain-containing genes, while scyphozoans (Aurelia) and cubozoans (Morbakka) have intermediate numbers, and hydrozoan genomes contain the fewest. CUT class genes are not found in medusozoan genomes and all cnidarian genomes lack PROS and ZF class genes altogether. In addition, NKL genes are less abundant in Hydra and HOXL genes are less abundant in hydrozoans generally, than in other cnidarians.In the hpaerica . Among cH.viridissima and H. vulgaris possess the same ANTP genes (Hydra species, although the body size of H. viridissima is smaller. As previously reported (Gsh and Meox are present in Hydra, whereas Xlox and Cdx are missing, unlike other medusozoans (Hox1 and Hox9-14, but lack Hox2, Evx, Gbx, Mnx, unlike anthozoans. Medusozoans have lost many NKL genes, Nedx, Hlx, Mnx, Msx, and Lbx compared to anthozoans. In addition, Dbx, Hlx, Nk3, Nk6 and Nk7 are not found in hydrozoans, nor are Nk5, Exm or Msxlx in Hydra (Nematostella expresses Gbx, Hlx, Nk3 and Nk6 in the pharyngeal or mesenteric region (Gbx in pharyngeal endoderm (Hlx and Nk6 in pharyngeal ectoderm, and Nk3 in nutrient-storing somatic gonads in mesentery (Hydra. Loss of these genes reflects the simplification of body structure in the Hydra lineage.usozoans . On the in Hydra . In addiin Hydra , suggestH. viridissima, which is one the most basal species in the genus Hydra and the only species with symbiotic algae. Compared to H. vulgaris, H. viridissima has a compact genome one-third the size and with 36.5% fewer genes (H. viridissima genome has fewer repetitive sequences. RNA transposons, in particular, are almost absent (H. viridissima is quite similar to that in H. vulgaris (Hydra lineage (Hydra and the absence of jellyfish and planula stages. In addition, we found diverse innate immunity genes in the H. viridissima genome that are also observed in corals (H. viridissima genome presented here provides a Hydra genome comparable in quality to those of other cnidarians, including medusozoans and anthozoans, which will hopefully facilitate further studies of cnidarian genes, genomes, and genetics to understand basal metazoan evolution and strategies to support algal symbiosis in cnidarians.In this study, we report the first genome assembly of er genes . In addit absent . On the vulgaris , reflect lineage , which in corals , indicat


Coptis is one of the most important medicinal plant genera in eastern Asia. To better understand the evolution of this genus, the complete chloroplast genome of C. chinensis var. brevisepala was obtained by next-generation sequencing. The plastome of C. chinensis var. brevisepala is 155,426 bp in length, and consists of large  and small  single-copy regions, separated by pair of inverted repeat regions . It harbours 111 unique genes, including 78 protein-coding genes, 29 transfer RNA genes, and four ribosomal RNA genes. Rps19 and ycf1 were pseudogenized due to incomplete duplication in IR regions. The nucleotide composition is asymmetric  with an overall G + C content of 38.2%. The phylogeny of Ranunculaceae based on 75 CDSs of 27 taxa showed that Ranunculoideae is paraphyletic and thus needs redefinition. Coptis (Ranunculaceae) is one of the most important and well-known plant genera in eastern Asia. Dried rhizomes of Coptis plants are utilized for Rhizoma Coptidis (RC), a traditional Chinese medicine famous for its functions of clearing heat, dispelling dampness, and purging fire toxins , due to habitat destruction and long-term over-harvesting  was deposited at the Herbarium of Zhejiang University (HZU). We extracted total DNA from ca. 3mg of the silica-gel dried leaf tissue using DNA Plantzol Reagent , following the manufacturer’s protocol. Then, next-generation sequencing was conducted on the Illumina Hiseq Platform . The complete cp genome was assembled via NOVOPlasty  as a reference. The annotation was performed using Geneious 11.0.4 . After all, clean reads were re-mapped to the draft genome and yielded the cp genome sequence of C. chinensis var. brevisepala. The complete chloroplast genome sequence was deposited in GenBank (MH509384).Leaf samples were collected from China, Zhejiang Province, Hangzhou City, Lin’an District, Qingliangfeng Botanical Garden. Voucher herbarium specimen  and two inverted repeat regions . The nucleotide composition is asymmetric  with an overall G + C content of 38.2%. It contained 111 unique genes, including 78 protein-coding genes, 29 transfer RNA genes, and four ribosomal RNA genes. Sixteen genes are duplicated in the IR regions, including all four rRNA genes, seven tRNA genes, and five protein-coding genes. Each IR region contained seven tRNA genes . rps19 and ycf1 were pseudogenized due to incomplete duplication in IR regions.The complete plastome of Berberis amurensis, Berberidaceae), using both maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was implemented in RAxML-HPC v.8.2.10 (Stamatakis Coptis, C. chinensis var. brevisepala is sister to C. chinensis. Overall, our data will largely enrich the genetic information of Coptis and facilitate future studies on its conservation genetics.The phylogeny of Ranunculaceae were reconstructed based on 75 CDSs of 26 Ranunculaceae species and one outgroup taxa (