Her general, cardiovascular and respiratory examinations were all normal. A resting electrocardiogram (ECG) showed a normal sinus rhythm, PR interval 126 ms, prolonged QRS duration 138 ms, and a delta wave (pictured - lead II). Leads I, II, and aVF had inverted T waves. Ventricular pre-excitation, Wolff-Parkinson-White syndrome type B was diagnosed. Echocardiography was unremarkable. The nature of her condition was explained to her. Advice to avoid medications like verapamil and digoxin was offered along with a recommendation to avoid over-strenuous activity. Screening ECGs were advised for her immediate family. As she had been asymptomatic up to this point, cardiology follow-up was recommended for consideration of a prophylactic ablation procedure. This case illustrates the benefit of a high index of suspicion and the utility of electrocardiography in diagnosing a rare condition.A 25-year-old female presented for a medical consultation complaining of sudden central chest pain, pleuritic in nature. She had never experienced such pain before. She denied experiencing coughing, shortness of breath or other respiratory or cardiac symptoms. Notably her grandparents, now in their 80s, were apparently being treated for ischemic heart disease attributed to their old age. She occasionally drank alcohol < 20 units per week. She was not using tobacco. On examination, her body mass index was 24.8 kg/m


Further data dissection confirmed that concomitant bladder tumor is a risk factor of bladder recurrence  but not UTUC recurrence . Kidney-sparing surgery (KSS) , pathological T staging ≥ pT2  were significantly associated with UTUC recurrence. KSS does not affect bladder recurrence . A limitation is the retrospective nature of the present study analysis. Conclusions: The presence of concomitant bladder tumor does not increase risk of UTUC recurrence, but it results in an increased risk of bladder recurrence. KSS does not affect bladder recurrence and can still be considered in patients with concomitant bladder tumor.Objective: To evaluate the clinical outcomes of UTUC patients with or without concurrent bladder tumor. Design, Setting, and Participants: The Clinical Research Office of the Endourology Society-Urothelial Carcinomas of the Upper Tract (CROES-UTUC) Registry included 1134 UTUC patients with or without concurrent bladder tumor treated between 2014 and 2019. Results: In 218 (19.2%) cases, concurrent bladder tumor was present, while in 916 (80.8%) patients, no bladder cancer was found. In the multivariable Cox regression analysis, concomitant bladder tumor (hazard ratio (HR) 1.562, 95% confidence interval (CI) 0.954–2.560,  Urothelial carcinomas are the sixth most common tumors in developed countries . Most ofUTUC and bladder tumor have many differences not only in tumor pathogenesis and biological behavior, but also in diagnosis, treatment, and prognosis. Unfortunately, in 8–17% of newly onset UTUC cases, concomitant bladder cancer is found ,6,7,8. AThe Clinical Research Office of the Endourology Society Urothelial Carcinomas of the Upper Tract (CROES-UTUC) registry was established in 2014 . This isThe CROES-UTUC registry is a prospective, observational, international, multicenter, cohort study, which enrolled patients with suspected UTUC from 101 centers in 29 countries [Consecutive patients aged ≥18 years who had suspected UTUC undergoing any type of diagnostic or therapeutic surgical intervention were included. Patients with prior history of bladder cancer were not excluded. The study criteria were wide-ranging to provide comprehensive real-world data regarding the management and outcomes of patients with suspected UTUC.Clinical data on baseline characteristics, risk factors, clinical assessment, intervention received, and survival outcomes were recorded . Follow-The primary outcomes of this study were overall survival, cancer-specific survival, and recurrence-free survival. Patients were first dichotomized according to whether concurrent bladder tumor was present (concomitant bladder tumor and non-concomitant bladder tumor), and their baseline characteristics were compared.p < 0.05 was statistically significant. All statistical analyses were performed using SPSS version 25 .Chi-square tests were used for comparison of categorical variables. Kaplan–Meier analysis was performed for overall survival, cancer-specific survival, and recurrence-free survival; significance was determined by log-rank test. Multivariate Cox regression analyses on overall survival, cancer-specific survival and recurrence-free survival were performed. A A total of 1134 patients were included in this analysis, of which 218 patients (19.2%) had concomitant bladder tumor. p = 0.047), male gender (p = 0.010) and cigarette exposure (p = 0.047). By contrast, patients without bladder tumor were more likely to present with unifocal UTUC (p = 0.004) and received radical nephroureterectomy (p = 0.019).While ASA, CCI, pathological T staging and tumor grade showed no differences between the two groups, concomitant bladder tumor group had a high rate of age more than 70 (p < 0.001) (p = 0.042), concomitant bladder cancer demonstrated a trend of increasing recurrence   than concomitant bladder cancer patients (< 0.001) A. Althou= 0.076) .p < 0.001) (p = 0.812) while the kidney-sparing surgery (KSS) approach  and pathological T staging ≥ pT2  were independent risk factors (p = 0.020) that affected bladder recurrence, irrespective of which surgical approach was taken by patients   patients suffering from recurrence, 24 patients presented with upper urinary tract recurrence, 124 patients had bladder cancer recurrence, and 14 patients had recurrence of both. Interestingly, only bladder recurrence, instead of upper urinary tract recurrence, was increased when testing the effect of concomitant bladder tumor status (< 0.001) , which w factors . However= 0.315) .p = 0.767)  B. Multiv= 0.767) .p = 0.175) (p = 0.041)  B. Howeve= 0.041) .Our analyses revealed several remarkable findings. First, since concomitant bladder tumor is an independent risk factor, which increases bladder tumor recurrence in UTUC patients but does not increase upper urinary tract recurrence, urologists should pay more attention to those with concomitant bladder tumor patients during follow-ups, and regularly perform cystoscopy. Second, the upper urinary tract recurrence is associated with higher pathological staging and more conservative surgical approach in UTUC patients, which shows that surgeons should select proper treatment, considering the best interest of the patient. Third, regardless of the co-presence of bladder tumor, cancer-specific survival and overall survival depend more on UTUC tumor stage and patients’ age.To investigate the impact of concomitant bladder tumor, clinical and molecular differences between bladder cancer and UTUC must be deliberated. In this study, concomitant bladder tumor patients were different from non-concomitant bladder tumor patient’s cohort in terms of their baseline characteristics. Concomitant bladder tumor patients were older, presented more often in male gender and had a higher percentage of cigarette exposure and multifocal tumor but received RNU less often than patients without concomitant bladder tumors. These distribution characteristics might be the result of different biological behaviors and, as such, in line with previous studies . Balkan The impact of simultaneous bladder cancer on patient survival has been explored by several groups. Kuroiwa and colleagues collected data for 2668 nonmetastatic UTUC patients; 23.7% had previous or concomitant bladder cancer. The overall survival was short in patients with previous or concomitant bladder cancer . Zeng etFocusing on the impact on recurrence, Milojevic et al. concluded that prior bladder tumor history had no effect on non-bladder recurrence but was an independent predictor of intravesical recurrence . Pignot Apart from the concomitant situation, in our study, KSS is not a risk factor for intravesical recurrence. Therefore, the bladder is safe in well-selected UTUC patients under KSS. However, while concomitant bladder tumor is not a risk factor for upper urinary tract recurrence, KSS is an independent risk factor for UTUC recurrence, which shows that case selection is extremely crucial. The benefit from KSS for low-risk UTUC includes reduced morbidity associated with radical surgery . Consequp = 0.085) but not the upper urinary tract . Notably, multifocal status and concomitant bladder tumor are positively correlated. This probably represents some field change effect and underlying biology of the tumor. Chromecki et al. found that tumor multifocality was an independent prognosticator of cancer-specific mortality and disease progression in patients treated with RNU [In addition, multifocality is a risk factor for overall recurrence in our study, and seems to affect the intravesical recurrence (HR 1.559, 95% CI 0.941–2.581, with RNU . A high-with RNU . AccordiThe current study has multiple limitations. First, its retrospective design is an inherent limitation. Second, there could be missing data in our cohort that could be compensated for by the significant sample size. Third, it was a heterogenous cohort because of the real-world data collection, which we adjusted for using multivariable analysis. Despite these shortcomings, the strengths are also inherent, as they derive from the global cooperation practice and real-world data, which deliver valuable insights, qhich are needed for better UTUC management.The presence of a concomitant bladder tumor does not increase the risk of UTUC recurrence, but results in an increased risk of bladder recurrence. KSS does not affect bladder recurrence, and can still be considered in patients with concomitant bladder tumor.


Background: Existing studies have indicated that mitochondrial dysfunction may contribute to osteoarthritis (OA) development. However, the causal association between mitochondrial DNA (mtDNA) characterization and OA has not been extensively explored.Methods: Two-sample Mendelian randomization was performed to calculate the impact of mitochondrial genomic variations on overall OA as well as site-specific OA, with multiple analytical methods inverse variance weighted (IVW), weighted median (WM), MR-Egger and MR-robust adjusted profile score (MR-RAPS).Results: Genetically determined mitochondrial heteroplasmy (MtHz) and mtDNA abundance were not causally associated with overall OA. In site-specific OA analyses, the causal effect of mtDNA abundance on other OA sites, including hip, knee, thumb, hand, and finger, had not been discovered. There was a suggestively protective effect of MtHz on knee OA IVW OR = 0.632, 95% CI: 0.425–0.939, p-value = 0.023. No causal association between MtHz and other different OA phenotypes was found.Conclusion: MtHz shows potential to be a novel therapeutic target and biomarker on knee OA development. However, the variation of mtDNA abundance was measured from leukocyte in blood and the levels of MtHz were from saliva samples rather than cartilage or synovial tissues. Genotyping samples from synovial and cartilage can be a focus to further exploration. Osteoarthritis (OA) is a common joint disease with degenerative changes in articular cartilage and remains major impact on public health worldwide . AccordiMitochondria are complex multifunctional organelles involved in various cell functions, e.g., heat regulation, calcium homeostasis, reactive oxygen species (ROS) production, and proinflammatory cytokines production, the damage of which may affect chondrocyte health at an extent . They haIn a recent review, OA was recognized as a potential mitochondrial disease considering the impact of mitochondrial dysfunction on cartilage degradation . HoweverThe design of our research is displayed in p < 5 r2 < 0.001)  for MtHz and mtDNA abundance, all reach genome-wide significance (× 10–8) . Twenty < 0.001) . After L< 0.001) . The selSummary statistics data on overall OA and its specific sites, comprising knee, hip, spine, thumb, and hand OA, were obtained from the latest publicly available GWAS of OA. This GWAS covered nine populations with up to 826,690 participants  . The defTwo-sample MR analysis was executed in R software (R version 4.3.0) with the TwoSampleMR (version 0.5.6), MR-PRESSO (version 1.0) and mr. raps (version 0.2) R packages . The metp-values were derived based on 1,000 simulations (p < 0.05)  after Bonferroni correction.Several sensitivity analyses were applied to detect and correct for heterogeneity and pleiotropy. MR-PRESSO was conducted to detect the existence of horizontal pleiotropy and correct the causal estimate affected by possible pleiotropic outliers . Correspulations . And the < 0.05) . IVW met < 0.05) . Leave-o < 0.05) . As fivehttps://snipa.helmholtzmuenchen.de/snipa3/). The mean F-statistic was 36.357 which was above the threshold F value of 10. In total, there were 14 IVs for overall, knee, hip, thumb, and hand OA and 13 IVs for finger OA.Fifteen LD-independent genetic variants were taken for repeated MR analysis . We extrp > 0.05; MR-PRESSO global heterogeneity: p > 0.05). Cochran’s Q test did not detect heterogeneity in overall OA (p= 0.495 > 0.05).Results of the casual association between MtHz and OA are summarized in p > 0.05). The heterogeneity was tested by Cochran’s Q test and MR-PRESSO global heterogeneity test, providing no evidence about the existence of heterogeneity. The MR-Steiger results supported that these SNPs were more predictive of the exposure than of the outcome , finger (rs1065853), and hand OA (rs12451555). We searched their proxy SNPs on SNiPA and those whose proxy SNPs were absent on SNiPA were excluded. The mean F-statistic for IVs of mtDNA abundance was 93.380, which was above the threshold 10 . Finallyp = 0.108) and finger OA  in IVW model. The results are presented in p > 0.05; MR-PRESSO global heterogeneity: p > 0.05). Cochran’s Q test did not detect heterogeneity in above-mentioned outcome traits (p > 0.05). For the analysis of hip OA, rs16978036 were excluded from MR-PRESSO analysis. In presence of horizontal pleiotropy (p= 0.027) and absence of heterogeneity (p= 0.062), the nominally significant result of MR-Egger method was found . However, no evidence about causal relationship between the exposure and hip OA was observed with IVW method and MR-RAPS method  , thumb OA , hand OA  . The resScatter plots, forest plots and leave-on-out sensitivity analysis plots were displayed in The causal roles of MtHz and mtDNA abundance in OA pathogenesis were poorly studied in previous works. To our best knowledge, this is the first MR study to evaluate the causal association between mitochondrial genome traits and OA. Four MR methods were employed to estimate the causal association between mtDNA characterization and OA. The results of MR steiger test verified the causal direction of our research (MtHz and mtDNA abundance were exposure and OA were outcome). No effect of mtDNA abundance and MtHz on overall OA was detected. In subgroup analysis, a suggestively protective role of MtHz on knee OA was observed, but not on other sites. And we did not find that mtDNA abundance was causally associated with any site-specific OA.Although mitochondria is widely recognized as an important factor of OA development , the rol= 0.002) . Further= 0.002) . However= 0.002) . The stu= 0.002) . And the= 0.002) . Besides= 0.002) . In conj= 0.002) .However, the reason that MtHz showed diverse effects on different sites of OA remains unclear. So far, we did not find relevant research that investigated the relationship between MtHz and hand, finger, and thumb OA. A case-control study suggested that mitochondrial haplogroups was associated with hip OA  (OR = 0.p < 0.0001), whereas the results were not adjusted by sex and age . Furthermore, the variation of mtDNA abundance and the levels of MtHz varied in different tissues and cell types. And in original GWASs, mtDNA abundance was measured from leukocyte in blood and MtHz from saliva samples rather than cartilage or synovial tissues. Considering that OA is mainly characterized by progressive loss of cartilage and synovial hyperplasia, the application of data from blood samples and saliva samples would limit the explanation ability of our study to a certain extent (Our research applies MR methods to investigate causal relationships between mitochondrial genome characterization and OA. However, there are some limitations in our analysis. Firstly, we did not estimate causal effects of mitochondrial genome traits on OA stratified by gender. Mitochondrial genome are maternally inherited, and females may have lower MtHz than males from the perspective of mitochondrial inheritance . Therefon extent . ObservaIn conclusion, our MR analyses elucidated that MtHz is a suggestively protective factor of knee OA, implying that MtHz could be a genetically prediction factor and a therapeutic target in the development of knee OA. No causal association was found between mtDNA abundance and OA. Additional MR analyses are warranted to probe causal relationships between mitochondrial genome traits and OA stratified by gender. Moreover, GWAS covering more than one ethnic population are needed to detect the effect of characterization of mtDNA quantity and quality in different ethnic groups.


Polypodium) differs from that of free-living cnidarians and may be influenced by ecological and environmental factors. Phylogenetic analyses showed that toxin families of myxozoans and free-living cnidarians were clustered into different clades. Selection analyses showed that purifying selection was the dominant evolutionary pressure in toxins, while they were still influenced by episodic adaptive selection. This suggests that the potency or specificity of a particular toxin or species might increase. Overall, our findings provide a more comprehensive framework for understanding the diversity and evolution of Myxozoa venoms.Myxozoa is a speciose group of endoparasitic cnidarians that can cause severe ecological and economic effects. Their cnidarian affinity is affirmed by genetic relatedness and the presence of nematocysts, historically called “polar capsules”. Previous studies have revealed the presence of toxin-like proteins in myxozoans; however, the diversity and evolution of venom in Myxozoa are not fully understood. Here, we performed a comparative analysis using the newly sequenced transcriptomes of five Myxobolidae species as well as some public datasets. Toxin mining revealed that myxozoans have lost most of their toxin families, while most species retained Kunitz, M12B, and CRISP, which may play a role in endoparasitism. The venom composition of Endocnidozoa (Myxozoa +  Cnidarians  are among the earliest diverging extant venomous animals ,2. TheirPolypodium hydriforme [Myxozoans are obligate endoparasitic cnidarians that form Endocnidozoa with driforme ,8. They driforme ,10. Theidriforme . Unlike driforme . It is uKudoa iwatai genome and transcriptome did not detect venom proteins among the cnidarian-restricted genes [Ceratonova shasta, which found minimal evidence of proteins similar to the toxins [Myxobolus pendula identified 49 putative toxin proteins [Advances in omics technologies have enabled the investigation of the toxin components in several myxozoans at transcriptomic or proteomic level. Comparative analysis using the ed genes . This wae toxins . These sproteins . Venomicproteins ,16. ThesMyxobolus cerebralis can cause the well-known whirling disease [Myxobolus and Thelohanellus are also pathogenic to their hosts [Myxobolidae is a widespread and diverse component of Myxozoa. With some 1300 species currently described, they represent around 50% of the species diversity of myxozoans ,18,19. C disease . Some spir hosts ,22,23. Tir hosts . As a reThelohanellus kitauei, Myxobolus xiantaoensis, Myxobolus ampullicapsulatus, Myxobolus turpisrotundus, and Myxobolus honghuensis) and some representative cnidarians. In the present work, we aimed to (i) reveal the distribution patterns of venom in the Myxozoa through a customized bioinformatics pipeline; (ii) compare the venom gene profiles between myxozoans and free-living cnidarians; (iii) explore the evolution of myxozoan venoms using phylogenetic and selection pressure analyses.Here, we conducted venomic research on newly sequenced transcriptomes of five Myxobolidae species  of compressed raw reads were generated for five myxozoans using paired-end sequencing. The assemblies ranged from 23,995 to 174,559 unigenes with an N50 range from 381 to 1368. Overall completeness was evaluated by CEGMA and BUSCO .Nematostella vectensis, Hydra vulgaris, Chironex fleckeri, and Aurelia aurita).Using the customized bioinformatics pipeline depicted in + channel inhibitors, any Na+ channel inhibitors, SCRIP, ASIC, JFT, hydralysins, MACPF, or ATPase-like toxins. However, we found M12B in all species (n = 37), which is classed as a hemorrhagic toxin. Most of the myxozoans, except Tetracapsuloides bryosalmonae, possessed the type II K+ channel inhibitor Kunitz (n = 39). Kunitz is a dual function toxin that inhibits serine protease and blocks potassium channels [M. xiantaoensis, M. turpisrotundus, M. pendula, Sphaerospora molnari, and Buddenbrockia plumatellae. Interestingly, we found two actinoporin TLPs in M. pendula and S. molnari, a family of venom proteins usually reported in anthozoans and hydrozoans [The venom system of cnidarians is mainly composed of three toxin types: enzymes, pore-forming toxins, and neurotoxins ,26. As echannels . We alson = 124) with M. pendula and S. molnari contributing 62 and 28 respectively. Nearly all species had true venom lectin (n = 28) and CRISP (n = 39). Interestingly, we found many TCTP in myxozoans, which was an important part of the sea anemone defense system [M. pendula. TFT is a non-enzymatic peptide with multiple biological activities found in all snakes [Our survey also included toxin families from snakes, scorpions, spiders, cone snails, and insects. The most diverse type was snaclec .The venom composition is shown in M. squamalis, T. kitauei, H. salminicola, K. iwatai, and M. honghuensis [M. squamalis (11 vs. 9), M. honghuensis (19 vs. 12), T. kitauei (20 vs. 10), and H. salminicola (9 vs. 8). However, there were more TLPs found in the K. iwatai genome (n = 9 vs. 4 in transcriptome).Our analysis was mainly based on transcriptomes, but we also added five genomes  tWe have developed a phylogenetic framework to reveal the evolutionary patterns and interspecies differences of Myxozoa venoms. Seven toxin families were selected for in-depth analysis, including Kunitz, CRISP, actinoporin, peptidase S1, true venom lectin, M12B and PLA2. These families were selected because they were found in most species or had essential biological functions. These both were frequently studied in the cnidarian literature and other venomous lineages, allowing easier comparison.S. molnari clustered with the snake (A7X3V7), which is a serine protease inhibitor found in Philodryas olfersii [T. kitauei, K. iwatai, and spider (Q8T3S7) was supported by a relatively high bootstrap (86). Q8T3S7 was a putative insecticidal toxin that could inhibit trypsin [S. molnari clustered with cone snails (P0CY85 P0C1X2). They both specifically block voltage-activated potassium channels [Actinia tenebrosa (A0A6P8HC43), and three KU/Kunitz-BPTI domains in most myxozoans .Peptidase S1 is an enzyme that cleaves peptide bonds in proteins . The phyS. molnari and M. pendula were clustered with toadfish (Q66S03) [True venom lectins bind carbohydrates in snakes , exhibit(Q66S03) .Considering the obvious differences in phylogeny between free-living cnidarians and myxozoans, we also analyzed the selection pressures to further explore the evolutionary patterns of Myxozoa venoms. Using the codeml program in PAML v4.9  with NSIFurthermore, we used gene-specific and branch-specific tests implemented through HyPhy . First, Here, we sequenced and assembled the transcriptomes of five Myxobolidaes species, with BUSCO scores ranging from 57–78%. However, we do not think this is a defect in the data and will not affect the validity of our results. First, the fast evolutionary rates of myxozoans have led to underperformance in both BUSCO and CEGMA . SecondlOur results suggest that the venom composition of endocnidozoans differs from that of anthozoans and medusozoans. Hemorrhagic toxins were the most diverse type of toxin family in endocnidozoans. Anthozoans were rich in enzymes and medusozoans contained more cytolysins and neurotoxins. There are strong indications that various ecological and environmental factors  might influence venom composition in animals . We specWe noted some differences in venom-mining results at the transcriptomic and genomic levels. Overall, we found more TLPs in transcriptomes, suggesting that gene duplication may occur in some toxin families. Some toxin families were found in transcriptomes but not in genomes, we speculate that this may be caused by alternative splicing, which has been proved to influence transcriptional and post-transcriptional regulation of venom genes . ConsideEchinococcus granulosus and has been shown to interfere with host physiological processes during the initial stages of infection [Compared to free-living cnidarians, myxozoans have lost most of their toxin families. However, we can identify a small set of toxin families common to almost all myxozoans, such as Kunitz, CRISP, and M12B. These “core” toxin families may consist of proteins with an ancient and conserved role in nematocyst function [nfection . In addinfection . Metallonfection . To gain+ channel inhibitor. It protects the organisms from endogenous proteases, causes paralysis in prey, and defends against enemies [Kunitz-type venom is present in proteins from most venomous taxa and plays an important role in envenomation . In sea  enemies ,64. Mole enemies . We alsoMetalloproteases are important venom components of terrestrial animals . They haThe evolution of venom is usually accompanied by strong evidence of accelerated evolution and positive selection . This isCollectively, we provide a comprehensive view of Myxozoa venoms. We detected relatively few toxin-like proteins in myxozoans, suggesting that the reduction in myxozoan genome size includes reduced toxin diversity . The phyM. honghuensis was collected from infected allogynogenetic gibel carp Carassius auratus gibelio in Zoumaling Farm, Hubei Province, China on 29 July 2015. M. ampullicapsulatus was collected from infected allogynogenetic gibel carp C. auratus gibelio in Datonghu lake, Hubei Province, China on 12 May 2016. M. xiantaoensis was collected from infected yellow catfish Tachysurus fulvidraco in Xiantao, Hubei Province, China on 23 April 2016. M. turpisrotundus was collected from infected allogynogenetic gibel carp C. auratus gibelio in Wuhan, Hubei Province, China on 16 November 2015. T. kitauei was collected from infected common carp Cyprinus carpio in Liuerbao Town, Shenyang Province, China on 11 August 2015.Fish were held on ice before being killed with an overdose of MS-222 . From each fish, tissue containing one large cyst was homogenized by a manual glass tissue grinder and suspended in 0.1 M phosphate-buffered saline (PBS) with a pH 7.2 and then filtered through cotton gauze. Myxospores were separated from the filtrate by sucrose gradient centrifugation and Percoll gradient centrifugation, in turn. They were washed several times with distilled water and then examined microscopically to verify purity and identity. Purified myxospores were either placed into RNAlater (Sigma), frozen in liquid nitrogen, and finally stored at −80 °C, or immediately sent to nematocyst isolation as described below. Myxozoan identification was performed based on morphology and 18S sequencing ,79. Main® spectrophotometer , and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system  respectively. Libraries were prepared from purified mRNA using NEBNext® Ultra RNA kit (New England Biolab (NEB), Ipswich, MA, USA) following the manufacturer’s recommendations and sequenced as 2 × 125 paired-end (PE) runs with the Illumina HiSeq 2500 for M. honghuensis and T. kitauei; as 2 × 150 paired-end runs with the Illumina HiSeq 4000 for M. ampullicapsulatus, M. turpisrotundus, and M. xiantaoensis. Raw sequence data were cleaned and trimmed by removing adaptor and low-quality reads using Trimmomatic v0.33 [Total RNA was extracted from frozen preserved specimens using TRIzol 550 . The purity and integrity of RNA were assessed using NanoPhotometeric v0.33 . Filtereic v0.33  and clusic v0.33  and TGICic v0.33 . BUSCO vic v0.33  and CEGMic v0.33  were use−5). Only transcriptome sequences that exclusively matched host databases were removed. The retained transcriptome sequences were further blasted against a bacterial database using BLASTX . The positive hits were blasted to the NCBI nonredundant protein database (NR) using BLASTX . Only sequences that were annotated as “bacteria” were removed. For assessment and visualization of contamination in genomes, taxonomic assignment of each contig was carried out using Blobtools v1.0 [Here we used our recently developed method to construct a clean, efficient, and comprehensive protein reference that we denote as the customized comprehensive proteomic reference database (CCPRD) . Brieflols v1.0 . Sequenchttps://github.com/qingxiangguo/hercules, accessed on 5 April 2020). Furthermore, those transcripts that were translated neither by de novo nor homology-based method were translated into amino acid sequences using the Transeq script from the EMBOSS [For RNA-seq data, coding sequences were firstly predicted by TransDecoder  and Genee EMBOSS . Finally−5). All the candidate toxin proteins identified by RBBH were validated by blasting against the Tox-Prot UniProtKB/Swiss-Prot [−5). PFAM annotation by HMMER [We used a pipeline aimed at conservatively identifying toxins: a group of toxin sequences from cnidarian and other venomous animals was generated as seed for reciprocal best BLAST hits (RBBH) analysis to predict proteomes . Retrieved amino acid sequences were aligned with MAFFT v7.205 [We used TBtools v1.098669  to extraT v7.205  by the LT v7.205 . PhylogeT v7.205  and nodeWe first extracted the coding sequences of the different toxin families using TBLASTN, and then used MEGA-X  for sequ


Broiler leg diseases are a common abnormal bone metabolism issue that leads to poor leg health in growing poultry. Bone metabolism is a complicated regulatory process controlled by genetic, nutritional, feeding management, environmental, or other influencing factors. The gut microbiota constitutes the largest micro-ecosystem in animals and is closely related to many metabolic disorders, including bone disease, by affecting the absorption of nutrients and the barrier function of the gastrointestinal tract and regulating the immune system and even the brain–gut–bone axis. Recently, probiotic-based dietary supplementation has emerged as an emerging strategy to improve bone health in chickens by regulating bone metabolism based on the gut–bone axis. This review aims to summarize the regulatory mechanisms of probiotics in the gut microbiota on bone metabolism and to provide new insights for the prevention and treatment of bone diseases in broiler chickens. Bone diseases lead to poor leg health, which is one of the common problems in poultry growth. Leg diseases affect poultry athletic ability, causing lameness and paralysis, and limiting further improvement in poultry performance and the quality of related meat products ,2. The iThe reasons for the occurrence of leg diseases in broilers are complex, including genetics, nutrition, feeding management, various infectious diseases, and other influencing factors ,18,19,20The variation in the gut microbiota caused by antibiotics, pesticides, and other factors may reduce intestinal barrier function, thus affecting bone health ,23,24. TLactobacillus spp, Enterococcus spp, and Bacteroides spp .,76.57,76tivities . SCFAs ctivities . In additivities . Previoutivities . In additivities . Propiontivities . A studytivities . TherefoBile acids are the main organic components of bile and can be divided into primary and secondary bile acids according to their sources. Primary bile acids are synthesized directly from cholesterol in liver cells . During L. reuteri supplementation in ovariectomized (OVX) mice counteracted bone loss by suppressing the level of CD4+ T lymphocytes in the bone marrow and the expression of IL-6, tartrate-resistant acid phosphatase (TRAP), and RANKL [L. acidophilus was found to decrease levels of pro-inflammatory factors (TNF-α and IL-6) and increase levels of anti-inflammatory factors (IL-10) to alleviate arthritis [L. acidophilus to the broiler diet also reduced the expression of IL-6 and TNF-α [+ T cells (promoting osteoclastogenesis), TNF-α, and osteoclast precursor cells [As the largest immune organ in animals, the intestine is an important line of defense for the body against invasion by pathogenic microorganisms. Correspondingly, the gut microbiota plays a vital role in the regulation of bone metabolism through immune and inflammatory factors  29,91,9,991,92. nd RANKL . Similarnd RANKL ,98,99. Dnd RANKL ,96. Howend RANKL ,100,101.or cells . Howeveror cells . These fB. subtilis-based dietary supplementation has been reported to improve bone traits in broilers possibly by increasing the intestinal absorption of Ca and decreasing bone resorption through the inhibition of sympathetic nerve activity by the central serotonergic system [Sensory neurons, immune mediators, gut hormones, and other molecules derived from the gut microbiota transmit information between the gut and the brain, also known as the ‘gut–brain’ axis . 5-hydroc system . Thus, tL. reuteri affects osteoblastic activity by stimulating cytokine secretion in the T cells of mesenteric lymph nodes [Bifidobacterium longum in OVX rats resulted in an increase in femoral BMD associated with the high expression of BMP-2 [Osteoblasts are responsible for bone formation. Probiotics act on osteoblasts in multiple ways and are involved in bone homeostasis . L. reutph nodes . Wnt10b ph nodes . The stuph nodes . Bone moph nodes ,115,116.of BMP-2 .Propionibacterium(P.) freudenreichii enhanced bone mineralization by increasing the OPG/RANKL ratio and the level of BMP-2 [B. subtilis PB6 can increase the expression of OCN and BALP in broiler chickens, regulating tibial development [Osteoprotegerin (OPG), bone-specific alkaline phosphatase (BALP), and osteocalcin (OCN) are critical markers in bone formation and are often used as important indicators of bone health. OPG is mainly produced by osteoblasts or stromal cells, which can inhibit the formation and differentiation of osteoclasts and indirectly increase BMD. RANK exists on the surface of osteoclast precursors and osteoclasts and is a receptor for RANKL. The OPG/RANKL/RANK axis is a crucial signaling pathway in bone metabolism. OPG can bind to RANKL during osteoclast differentiation by inhibiting the binding of RANKL and RANK, blocking the differentiation of osteoclast precursors into osteoclasts . Yeom etof BMP-2 . BALP, aof BMP-2 . Supplemof BMP-2 . OCN is of BMP-2 . LGG preof BMP-2 . Furtherelopment . These sP. freudenreichii, L. reuteri, and LGG can reduce the formation of osteoclasts using TRAP staining [B. subtilis, indicating that B. subtilis can improve BMD by inhibiting the number and the activity of osteoclasts [Bifidobacterium longum inhibits bone resorption by reducing serum CTX levels in OVX rats [B. subtilis (1.0 × 106 spores/g feed) [Probiotics can affect bone metabolism by regulating bone resorption, which has a few biochemical markers, such as the TRAP, N-terminal peptide (NTX), and C-terminal peptide (CTX) of collagen type I (COLI) . TRAP isstaining ,96,126. staining . Takimoteoclasts . Serum COVX rats . Similar/g feed) . When br/g feed) . L. reuteri in OVX mice inhibited the bone marrow CD4+ T lymphocytes osteoclast-mediated bone resorption in vitro, preventing bone loss [L. rhamnosus attenuates bone loss in OVX mice by regulating Treg-Th17 cell balance, leading to an inhibition of RANKL-induced osteoclastogenesis [P. freudenreichii improves collagen-induced arthritis by inhibiting RANKL-induced osteoclast differentiation through the nuclear factor kappa-B (NF-κB) signaling pathway [B. subtilis can improve bone development in broilers by inhibiting inflammation at high ambient temperature, such as by reducing the TNF-α level [B. subtilis can be used as a dietary supplement to protect the bone health of laying hens by inhibiting gut and systemic pro-inflammatory factors and by increasing the OPG/RANKL ratio [In addition, probiotics also affect bone resorption by modulating immune cells and cytokines. Treatment with one loss . Lactobaone loss ,131. Cytone loss ,132. RANogenesis . P. freu pathway . TNF-α p pathway . IL-17 c pathway ,133,134. pathway . Yan et -α level . SimilarKL ratio . These rBacillus and Lactobacillus have been used as probiotics and are widely used in animal feeding. Bacillus spores can survive in harsh environments, such as high temperature, high pressure, and extreme pH, and can be used as a commercial animal feed additive to maintain animal health [Bacillus-DFM was found to help maintain gut microbiota balance in broilers and significantly increase tibial fracture strength and bone mineralization [B. subtilis, B. licheniformis, and B. cereus are commonly used commercial probiotic strains of Bacillus which promote the development of the tibia [B. subtilis to promote tibial development and gut microbiota balance, and this increased the total lactic acid bacteria count [B. subtilis ameliorates the reduction of tibial bone mass in chicks caused by Salmonella enteritidis infection [B. subtilis-based probiotics is conducive to broiler bone health. Probiotics affect bone metabolism by regulating the composition and function of the gut microbiota . Bacteril health ,135. Suplization . In addihe tibia ,136,137.ia count ,70. Addinfection . TherefoLactobacillus are important probiotics, including L. reuteri, L. rhamnosus, and L. acidophilus, and they affect BMD in animals and humans. Supplementation with L. reuteri 6475 can reduce bone loss in OVX mice by increasing BMD in the regulation of T cells in healthy male mice [Lactobacillus rhamnosus GG (LGG) affects the composition of the gut microbiota and the mechanical properties of long bones in immunodeficient mice [L. rhamnosus can also affect the development of broiler bone. A study has demonstrated that L. rhamnosus JYLR-005 prevented thiram-induced TD by maintaining the morphological structure of the chondrocytes and by improving broiler bone-related growth performance, including the weight, length, and mean diameter of the tibia [L. acidophilus inhibited bone loss by regulating Treg-Th17 cell balance and increasing BMD, bone heterogeneity, and femoral and tibial microarchitecture in OVX mice [L. acidophilus and L. plantarum, which promoted tibial development [The bacteria of the genus ale mice ,112,122.ent mice . In addient mice . Similarhe tibia . L. acidOVX mice . Moreoveelopment . TherefoProbiotics are widely used in animal feeding as a dietary supplement to maintain health. In fact, probiotics and gut microbiota influence the dynamic balance of bone formation and bone resorption through several pathways in different animal species. However, more explicit mechanisms are needed to further investigate the relationship between probiotics and gut microbiota in broiler bone health. Correspondingly, probiotics regulating the activity of osteoblasts and/or osteoclasts are more conducive to target the treatment and/or prevention of bone diseases in broilers, and provide a theoretical basis for the further exploration of efficient breeding methods of broilers. However, more studies have focused on the effect of probiotics on the skeletal development of broiler chickens, and there is a lack of thorough investigations on the treatment of bone diseases by regulating gut microbiota using probiotics. Furthermore, the gut microbiota is only one factor in the regulation of bone metabolism, and there may be more factors including age, diet, and physical condition involved in this process. Therefore, all these factors need to be further considered in depth by subsequent studies.


Gelsemium elegans (G. elegans) is a Chinese medicinal plant with substantial economic and feeding values. There is a lack of detailed studies on the mitochondrial genome of G. elegans. In this study, the mitochondrial genome of G. elegans was sequenced and assembled, and its substructure was investigated. The mitochondrial genome of G. elegans is represented by two circular chromosomes of 406,009 bp in length with 33 annotated protein-coding genes, 15 tRNA genes, and three rRNA genes. We detected 145 pairs of repeats and found that four pairs of repeats could mediate the homologous recombination into one major conformation and five minor conformations, and the presence of conformations was verified by PCR amplification and Sanger sequencing. A total of 124 SSRs were identified in the G. elegans mitochondrial genome. The homologous segments between the chloroplast and mitochondrial genomes accounted for 5.85% of the mitochondrial genome. We also predicted 477 RNA potential editing sites and found that the nad4 gene was edited 38 times, which was the most frequent occurrence. Taken together, the mitochondrial genome of G. elegans was assembled and annotated. We gained a more comprehensive understanding on the genome of this medicinal plant, which is vital for its effective utilization and genetic improvement, especially for cytoplasmic male sterility breeding and evolution analysis in G. elegans. Gelsemium elegans (Gardner and Champ.) Benth.  [Gelsemium in Loganiaceae. It is a medicinal plant with substantial economic and feeding value. The whole G. elegans plant can be used as medicine [G. elegans acts in boosting the immunity, deworming, and promoting appetite, improving livestock survival and growth rates [G. elegans additive feed have increased resistance, significant growth, and weight gain, as well as reduced meat ratio [G. elegans has immunomodulatory, neuropathic analgesic, anti-inflammatory, antianxiety, and antitumor effects [G. elegans, mainly including steroids, iridoids, and indole alkaloids [G. elegans and have received much attention in drug development [G. elegans [G. elegans secondary metabolites and functional gene mining. 427660) , also knth rates . Broiler effects ,6,7. Up lkaloids ,9. Indolelopment . With th elegans , which lhttps://www.ncbi.nlm.nih.gov/genome/browse#!/overview/) (accessed on 14 November 2022). The G. elegans mitochondrial genome was reported in 2019 [G. elegans mitochondrial genome is low, and the repetitive regions of this assembly are randomly selected; thus, the probability of genomic structural errors is high. Secondly, there is no annotation information. Thirdly, there is a lack of systematic research. The published mitochondrial genomes only report the length, GC content, and the number of genes in G. elegans mitochondria, but lack comprehensive information on mitochondrial genome codon preference, repetitive sequence, phylogeny, sequence migration, and RNA editing events. At present, the plant trait of cytoplasmic male sterility (CMS) in G. elegans has not yet been reported. CMS is determined by the mitochondrial genome and is associated with the pollen sterile phenotype [G. elegans is of great significance for its effective utilization and genetic improvement.Mitochondria are essential organelles in eukaryotic cells. The endosymbiosis theory postulates the production of mitochondria from an endosymbiotic alphaproteobacterium within an archaeal-derived host cell, which eventually evolved into organelles of eukaryotic cells . They ca in 2019 . Howeverhenotype . Mitochohenotype ,27,28. Ihenotype . Many inhenotype . TherefoG. elegans and investigated its genomic and structural features. The presence of its mitochondrial genomic substructure was explored through PCR experiments and nanopore reads. To better reveal the homologous fragments of the G. elegans mitochondrial and chloroplast genomes, we assembled its chloroplast genes using the same data set and completed the sequence similarity analysis. Moreover, the mitochondrial genome for RNA editing events was analyzed. This study further confirms the existence of multiple conformations in plant mitochondrial genomes, providing a theoretical basis for cytoplasmic male sterility breeding and plant evolution in higher plants.In this study, we sequenced, assembled, and annotated the mitochondrial genome of G. elegans mitochondrial genome was sequenced, assembled, and annotated. After resolving the repeat regions, the graph-based mitochondrial genome containing 37 nodes was established, as shown in G. elegans mitochondrial genome (NCBI Accession numbers: OP805353 and OP805354) has multiple “chromosomes” , 15 tRNA genes (five tRNAs were multiple copies), and three rRNA genes . Here, 82 SSRs were identified in chromosome 1 of the l genome , with mol genome . These aG. elegans mitochondrial genome. In chromosome 2, a total of five tandem repeats with a length ranging from 15 to 27 bp and the proportion of similarity of each unit from 84% to 96% were observed . On this basis, contig 1 and contig 8 of the independent circular chromosome minor conformation 1 and contig 7, contig 3, and contig 2 of the independent circular chromosome minor conformation 4 could form minor conformations 2 and 5 through repetitive sequence R2-mediated inversion. In addition to this, contig 4 and contig 3 of chromosomes could form the independent circular chromosome by repeating sequence R4. The entire mitochondrial genome of G. elegans formed three independent circular chromosomes. To sum up, the four pairs of repeats had mediated genome recombination, forming one major conformation and five minor conformations.According to the validation results, we could speculate the potential homologous recombination type of the l genome . The majG. elegans was compared with its plastid genome, and 37 matching points between the two organelle genomes were identified (petN and rps7) and six tRNA genes . These six tRNA sequences from the chloroplast and mitochondrial genomes remain similar, indicating that they may still function in the mitogenome.In the previous annotation of the organelle genome, it was found that there was extensive sequence migration between the plastid and mitochondria, and we called these mitochondrial plastid sequences (MTPTs). The mitochondrial genome sequence of entified . A totalG. elegans mitochondria, we constructed a phylogenetic tree  from nine species . Two mitochondrial genomes of Solanaceae species were set as outgroups. The topology of the mitochondrial DNA-based phylogeny coincided with the latest classification of the Angiosperm Phylogeny Group (APG). G. elegans belongs to the family Gelsemiaceae and is more closely related to R. stricta.To further explore the evolutionary relationships of tic tree B using tG. elegans mitochondria according to predictions from the online website PREP suit (http://prep.unl.edu/) (accessed on 6 July 2022). The setting standard was a cutoff value = 0.2. Under this criterion, a total of 477 potential RNA editing sites were identified on 33 mitochondrial PCGs , sequence arrangement, and repeat content . In thisomosomes . In contomosomes . Wu et aomosomes  found thomosomes . In the omosomes  with a shinensis  and Scutyunensis . Howeveryunensis . The mitomosomes . TherefoPrunus salicina mitogenome, three pairs of repetitive sequences were found to mediate the genome recombination into eight different conformations [I. batatas were shown to mediate the presence of seven conformations [S. tsinyunensis mitogenome could mediate two conformations [G. elegans mitochondrial genome contains abundant repetitive sequences, which may indicate frequent intermolecular recombination in the mitochondrial genome, with dynamically altered sequence and conformation during evolution. The four pairs of repetitive sequences identified in this study could mediate the formation of one major and five minor conformations in the G. elegans mitochondrial genome  of the mitogenome [G. elegans were complete tRNA genes . The samples were stored in the Fujian Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry University. High-quality genomic DNA was extracted using the CTAB method [Five fresh leaves in the same B method .The DNA library with an insert size of 300 bp was constructed using a Nextera DNA Flex Library Prep Kit  and sequenced using the Illumina NovaSeq platform . The libraries were prepared for Oxford Nanopore sequencing using the SQK-LSK109 ligation kit and the standard protocol. The purified library was loaded onto primed R9.4 Spot-On Flow Cells and sequenced using a PromethION sequencer  with 48 h runs at Wuhan Benagen Tech Solutions Company Limited . Base calling analysis of raw data was performed using the Oxford Nanopore GUPPY software (v0.3.0).G. elegans mitochondrial genome was assembled using a hybrid Illumina and Nanopore strategy. The mitochondrial genome assembly of G. elegans was performed using the default parameters (v1.7.5) of GetOrganelle software [G. elegans. The G. elegans mitochondrial genome’s circular chromosome was obtained after the contigs were merged by Bandage software [The software  on Illumsoftware , and thesoftware . The obtsoftware . The chlsoftware . The mitochondrial genome was annotated using Geseq software . The tRNhttps://webblast.ipk-gatersleben.de/misa/) (accessed on 6 July 2022) [https://tandem.bu.edu/trf/trf.unix.help.html) (accessed on 6 July 2022) [https://bibiserv.cebitec.uni-bielefeld.de/reputer/) (accessed on 6 July 2022) [MISA (ly 2022) , TRF (htly 2022) , and thely 2022)  were usely 2022) . Proteinly 2022) . https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) (accessed on 6 July 2022)  . The PCRN. attenuate (Mitochondrion: NC_036467.1) and H. niger (Mitochondrion: NC_026515.1) being set as outlier groups. The mitochondrial genomes of these nine species were downloaded from the NCBI database, the shared genes were extracted using PhyloSuite software [http://prep.unl.edu/) (accessed on 6 July 2022) [The mitochondrial genomes of eight species were selected for phylogenetic analysis, with software . The mulsoftware . The preG. elegans using a mixed strategy of Illumina and Nanopore. The G. elegans mitochondrial genome was two circles with 406,009 bp. A total of 51 genes were annotated in the mitochondrial genome of G. elegans, including 33 PCGs, 15 tRNA genes, and three rRNA genes. The G. elegans mitochondrial genome contained three forward repeats  and one pair of reverse repeats (360 bp) that mediate homologous recombination, resulting in the formation of one major conformation and five minor conformations. This study provides valuable information for a better understanding of the genetics of Gelsemium and other higher plants.We successfully assembled the mitochondrial genome of