Eucommia ulmoides, also known as the industrially and medicinally important hardy rubber tree, is the sole species of Eucommiaceae. Nevertheless, its dioecious property hinders sex recognition by traditional morphological observation at very early developmental stages, thus inhibiting breeding and economic cropping. In this study, double-digest restriction site-associated DNA sequencing (ddRAD-seq) was applied to screen sex-linked molecular markers for sex identification and investigation of the sex determination system in 20 male and female E. ulmoides individual plants, respectively. In consequence, five candidate male-specific loci but no female-specific loci were predicated among the 183,752 male and 147,122 female catalogue loci by bioinformatics analysis. Subsequent PCR (polymerase chain reaction) amplification and Sanger sequencing examinations were performed on another 24 individuals, 12 for each sex, from a separate population. One ideal sex-linked locus, MSL4, was identified among the five putative male-specific loci that were found using ddRAD data. MSL4 is 479 bp in length and highly conserved in all the male individuals, suggesting its feature of being stable and repeatable. Our results also indicated that the sex of E. ulmoides is likely determined genetically. In short, this study provides a consistent and reproducible ddRAD marker (MSL4) that is able to discriminate male from female seedlings in E. ulmoides, which will be valuable for rapid breeding practice and better commercial production of this economically important tree. Eucommia ulmoides Oliver, belonging to the monotypic family Eucommiaceae [E. ulmoides is therefore to potentially supersede the commonly known para rubber tree (Hevea brasiliensis in Euphorbiaceae) that is restricted in the tropical zones [E. ulmoides is rather difficult for its dioecious sexual system and long life cycle (up to 8-10 years), which limits the efficiency of breeding for improving the gutta (Eu-rubber) yield or other agronomic traits [mmiaceae , is an emmiaceae . It is ammiaceae . In recemmiaceae –6. Becaual zones , 8. Howec traits .Actinidia chinensis [Asparagus officinalis [Carica papaya [Silene latifolia [Populus trichocarpa [Rumex acetosa, evolve an XO sex determination system, in which the sex of one individual is determined by the X-to-autosome ratio [E. ulmoides has a diploid genotype with a basic composition of 2n = 34 chromosomes and circa 1.2 Gb of genome size [E. ulmoides has a genetic sex determination or heteromorphic sex chromosome [E. ulmoides during the vegetative growth phase via karyotype [Dioecy represents the sex separation in distinct individuals, which resembles the majority of mammals that include individuals of males and females . Only abhinensis , Asparagicinalis , Carica a papaya , and Silatifolia . In contchocarpa . Some otme ratio . Until nme ratio . The dioome size . Neverthromosome , 22, leaaryotype .Actinidia spp.), Y-encoded genes SyGI and FrBy act independently as the suppressor of feminization (SuF) and the maintenance of male (M) [Diospyros spp.), a Y-encoded pseudogene OGI is sufficient for the expression of androecium and repression of gynoecium through encoding a small RNA and targeting its autosomal counterpart gene MeGI [SOFF and aspTDF, have been discovered in garden asparagus  for the expression of maleness and repression of female development [C. papaya [P. trichocarpa [S. latifolia [E. ulmoides based on transcriptomic analysis [E. ulmoides is still in the dark to date.Moreover, with the advent of genomics, sex-determining genes are emerging in several plants . For insmale (M) . In persene MeGI . Two Y-eelopment . Nonreco. papaya , P. tricchocarpa , and S. atifolia . Recentlanalysis . NeverthE. ulmoides [E. ulmoides [Eucommia orchard [E. ulmoides long before phenomorphological features appear to satisfy the optimal sex ratio for gutta production.Sexual dimorphisms for instance morphological and physiological differences between the genders have been reported in dioecious plants , 33. Theulmoides . The fruulmoides . An opti orchard . It is tHippophae rhamnoides) [Excoecaria agallocha [S. latifolia [MiddRAD) technique [E. ulmoides during the vegetative growth phase.Ideal sex-linked molecular markers can assist to select off the low-value male seedlings and to keep pistillate trees at the juvenile stage, consequently to enlarge economic benefits of the orchard, as that applied in the kiwifruit  and sea mnoides) . Restricmnoides) , 37. Congallocha , kiwifrugallocha , and S. atifolia , in thisE. ulmoides cultivated on the campus of Northwest A&F University, Yangling, Shaanxi, China  were sampled for ddRAD marker development. Their sexes were identified by flower traits during the flowering season (April in 2017), when discrimination of the staminate and pistillate genotypes was decided  in April 2018 as a separate population to validate the sex-specific markers. The distance between these two sampling points is about 650 km.A total of 20 female and 20 male trees from a population of  decided . For eacv) agarose gel and NanoPhotometer® spectrophotometer . We constructed a ddRADseq library for the 20 male individuals and 20 female individuals following the MiddRAD protocol [AvaII+MspI enzyme pair was used to digest the purified genomic DNA and ligated the barcoded adaptors. Then, the DNA samples were pooled and fragments with 500-600 bp in length were selected for PCR amplification. The enriched DNA was purified and measured by gel and Qubit 2.0 Fluorometer . Finally, the library was sequenced on the Illumina HiSeq 2500 platform at Novogene, Beijing, China, to generate paired-end (PE) reads with 150 nucleotides in length.Genomic DNA was isolated from the leaf samples using the CTAB method . The quaprotocol . In brieRaw reads were demultiplexed using the process_radtags algorithm in Stacks software version 1.24 , 43. Aveustacks module in Stacks was used firstly to merge short-read sequences into tags/loci at the set of –m 10, -M 3, in which m is the minimum number of identical reads required to create a stack and M is the maximum distance (in nucleotides) allowed between stacks [cstacks program (−n 3) for all these individuals, in which n is the maximum number of mismatches allowed between the loci in the catalog. Finally, the output of sstacks (matches.tsv) from each sample was used to identify putative sex-specific loci by the R script [N-1 individuals of one sex  while absent in all individuals of the other sex was defined as putative sex-specific markers [The n stacks . Then, aR script . The ddR markers .Since each member of the PE reads was treated as an independent locus in the above analysis, we further investigated if there were cases where two of the predicted sex-specific ddRAD loci represented two members of a PE read pair. In addition, clean reads of the opposite sex were mapped to each of the chosen loci by the Linux grep command to rule out the possibility of any exact matches in sequence to the selected locus. These two steps can narrow down the number of candidate markers .μL for PCR amplification reaction: 12.5 μL PCR mix , 2.0 μL random decamer primers (25 μmol·μL−1), 0.5 μL genomic DNA (100 ng·μL−1), and 10.0 μL ddH2O. The following PCR profile was used: an initial denaturation at 94°C for 3 min followed by 30 cycles of denaturation (30 s at 94°C), annealing (30 s at 59°C) and extension (1 min at 72°C), and a final extension 72°C for 5 min. The PCR amplification products were finally examined on a 1.0% agarose gel. Each PCR experiment was repeated twice to validate both the reliability and stability.We subsequently conducted PCR to validate these putative sex-specific ddRAD loci based on the additional 24 individuals (12 for each sex) collected from one separate population in Henan. The forward and reverse primers were designed with Primer3  and anchin silico predicted sex-specific ddRAD loci by blastn with e value < 10−5 [https://www.ncbi.nlm.nih.gov/) to investigate their potential functional information. Furthermore, we run the local blastn by setting the marker MSL4 as query and Eucommia genome scaffolds (https://bigd.big.ac.cn/gwh/Assembly/13/show) [In addition, for the verified ddRAD marker that was only amplified via PCR in males but not in females , we sequenced the band by Sanger sequencing on ABI 3730X at Sangon Biotech, Shanghai, China. We used the MSL4-F primer Table  as the se < 10−5  to confi13/show)  as databE. ulmoides, with each sex having 20 individuals, were sequenced according to the ddRAD-seq protocol. About 2-3 Gb data were produced for each individual, reaching a total of 180,645,228 paired-end (PE) reads . After demultiplexing and quality filtering, we got a total of 178,740,749 reads . The number of reads in the 40 samples varied from 2,186,096 to 6,742,116 , we preliminarily detected 11 candidate male-linked ddRAD loci that were present in 19/20 males and none in the 20 females. Based on further checking, if these loci represented a pair of PE reads and had no exact match of female clean reads, we finally identified five loci as putative male-specific ddRAD markers, which were denoted as MSL1, MSL2, MSL3, MSL4, and MSL5 . Luckily, we detected a high similarity between the positions of 25,047-25,525 in GWHAAAL00024598#OriSeqID=scaffold571_obj#Len=331914  and MSL4. The detailed results are as follows: Score = 835 bits (452), Expect = 0.0, Identities = 470/479 (98%), Gaps = 0/479 (0%), and Strand=Plus/Plus. We furthermore ascertained the annotation information of this scaffold  and found that MSL4 is probably non-protein-coding sequences located between CDS17  and CDS18  of a putative gene with unknown function.We further sequenced the male-specific band (MSL4) from 12 male individuals by Sanger sequencing. Sequences of MSL4 were highly conserved, with no sequence variations, in the 12 staminate genotypes . The conFragaria virginiana) and garden asparagus , which are difficult to be cytologically detectable for the lack of visible crossovers or of pairing [E. ulmoides are missing and gender discrimination by karyotype is undependable [Actinidia, Asparagus, Diospyros, and so on [E. ulmoides is controversial [E. ulmoides based on the high-throughput sequencing technology.The evolution of dioecy has long been an intriguing issue since Darwin's time . However pairing , 51. Untpendable , 22. Morpendable , 24. Up nd so on , 52. Genoversial . Sex-speHumulus lupulus [A. officinalis, 760 primers were used for developing a SCAR marker associated with the M  locus [E. ulmoides, it is even more difficult to screen sex-linked markers via conventional methods  since experimental crosses and linkage map constructions are often impractical. For instance, in P. vera [H. rhamnoides [E. ulmoides explored a pair of primers (EST-Eu059) out of 140 pairs of EST-SSR primers as sex-linked marker that displayed an extra band in male plants [E. ulmoides but with far less labor and cost. We obtained a high density of ddRAD tags for 20 males  and 20 females  of E. ulmoides (Molecular markers such as SSR (simple sequence repeat), AFLP (amplified fragment length polymorphism), SCARs (sequence-characterized amplified regions) and RAPD (random amplification of polymorphic DNA) have been commonly used in exploring sex-specific markers of dioecious plants , 35. How lupulus ; in A. og) locus . Especia P. vera , only onamnoides . A previe plants . In contulmoides  across tulmoides .E. ulmoides , this study provides a convenient and cost-effective method to screen sex-specific markers for gender discrimination. In addition to present study, the ddRAD-PCR technique has been proven solid in identifying sex-specific markers in the dioecious mangrove tree, E. agallocha [More importantly, ddRAD-seq technique in present study directly used multiple adult males and females in a natural population to screen sex-specific markers, which is largely different from previous studies exploring sex-linked markers based on experimental crosses and genetic segregation , 40, 58.ulmoides . Based oulmoides  and 3, wulmoides . It is t species , 56. Givgallocha  as well.E. ulmoides is substantially important in the breeding practice [E. ulmoides has to experience 8-10 years of juvenility period before flowering and fruiting [E. ulmoides long before maturity when morphological characteristics of flowers are invisible. Additionally, this marker identified in the Shaanxi population of E. ulmoides also worked in the Henan population, which is suggesting that MSL4 probably is a universal marker for all populations of E. ulmoides across their national or global distributions. E. ulmoides has drawn much attention for hardy rubber (gutta) production during recent years [E. ulmoides with the molecular marker MSL4 to assure optimal sex ratio in E. ulmoides orchard, which will largely enhance their economic value.Gender identification of the young seedlings of practice , 60. As fruiting ; therefont years , 61. Prent years  and the nt years . Therefohttps://www.ncbi.nlm.nih.gov/), we luckily found a homologous sequence of MSL4 in the Eucommia genome database  with nucleotide identities being 98% . It is noteworthy that the individual E. ulmoides plant used for genome sequencing was also a male tree [E. ulmoides in the future.Exploring sex-determining regions in nonmodel plant species may help us to understand the evolution of recombination suppression within young sex chromosomes , 62. We ale tree , which eE. ulmoides is probably determined genetically. Noteworthily, we only detected male-specific ddRAD markers but no female-specific loci, similar to a recent study that only found a male-specific SSR marker (EST-Eu059) in E. ulmoides [E. ulmoides likely has an XX/XY sex determination system. But, we should keep it in mind that additional sex-linked markers need to be explored to dissect the genetic architecture of sex determination in E. ulmoides in the future.Sex-specific markers have also been used to infer sex determination mechanisms in several species, e.g., kiwifruit , 39 and ulmoides . The 479E. ulmoides, based on which we then identified five candidate male-specific loci while no female-specific loci. One of the five male-specific loci (MSL4) was further validated by PCR amplification and Sanger sequencing in a geographically distant population. As a result, the male-specific ddRAD marker is a reproducible, quick, and precise tool for discriminating males from female seedlings in E. ulmoides. The results also indicated that E. ulmoides probably has a genetic sex determination system. Collectively, reliable gender identification of E. ulmoides at the juvenile stage with the male-specific marker is not only valuable for marker-assisted breeding program but also helpful to ascertain optimal sex ratio in E. ulmoides orchard to maximum increase gutta (Eu-rubber) yield. The combination method of ddRAD and PCR also shed light on identifying sex-specific markers in other dioecious tree species.In this study, we performed ddRAD-seq to screen putative sex-linked markers in the hardy rubber tree,


In Europe, pulmonary histoplasmosis is rarely diagnosed except in travelers. We report a probable autochthonous case of severe chronic pulmonary histoplasmosis in an immunocompetent man in Switzerland without travel history outside of Europe. Diagnosis was achieved by histopathology, fungal culture, and serology, but the source of the infection remains speculative.  A 48-year-old man in Switzerland sought treatment for a 1-year history of progressive dyspnea, cough, 20-kg weight loss, and increased sweating; he was receiving oxygen therapy. Results of previous consultations had been inconclusive. An HIV screening test was negative. Medical history included hyperreflexia, depression, and chronic hepatitis B. The man had stopped cocaine inhalation and heroin consumption 20 years earlier but continued smoking cigarettes and cannabis. Regular medications included omeprazole and trimipramine. Except for a short trip to Greece and Italy many years before, the patient reported no foreign travel. In the absence of travel history to an endemic area, histoplasmosis was not initially considered at the time this patient sought treatment. A prolonged diagnostic process and delayed treatment initiation had meanwhile resulted in significant deterioration of health, including need for oxygen therapy, and loss of ability to work. Meanwhile, the patient was cachectic and had clubbing on his fingers and toes. Spirometry revealed nearly normal dynamic lung volumes. Forced expiratory volume was 3 L (75%) and forced vital capacity 4.1 L (83%), but diffusion capacity was severely impaired; diffusing capacity for carbon monoxide was 20%. A 6-minute walking test was limited to 400 m (59% predicted), initial oxygen saturation dropping from 90% to 78%. A chest computed tomography (CT) scan showed a diffuse reticulonodular pattern with predominantly upper lung opacifications and bronchiectases indicating fibrotic lung disease . A wedge biopsy showed predominantly upper-lobe fibrosis and multiple, confluent, necrotizing granulomas harboring yeasts, establishing the diagnosis of pulmonary histoplasmosis  Figure. https://www.immy.com) was positive for antibodies in plasma, but an antigen immunoassay for Histoplasma in urine (IMMY) was negative; a beta-1,3-D glucan test  was highly positive . At prolonged incubation , a fungal culture on BD Difco dehydrated culture media Sabouraud brain heart infusion agar base (with chloramphenicol and cycloheximide) (https://www.bd.com) showed flat, floccose to powdery, whitish growth. We found microscopically large, tuberculated macroconidia (7–12 µM) and small round microconidia on short, lateral pegs consistent with Histoplasma capsulatum. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry  results confirmed the diagnosis. Molecular identification was done using an in-house panfungal PCR assay with consecutive sequence analysis. We used the internal transcribed spacer region as target and internal transcribe sequences 1 and 2 for amplification primers (https://www.microsynth.ch) performed DNA sequencing. Sequences produced alignments of H. capsulatum in BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and CBS  databases. A qualitative immunodiffusion test (IMMY, Some radiologic features were unusual. There was no cavity formation (Pneumocystis jirovecii pneumonia prophylaxis. At 3-month follow-up, the patient had improved considerably. Repeated spirometry was nearly normal, showing persistent impairment of diffusion capacity. Follow-up chest CT scan (Underlying lung disease likely predisposed this patient for severe disease. However, his clinical response to treatment was remarkable. We initiated antifungal treatment with liposomal amphotericin B and oral prednisolone. After a few days, the patient improved substantially, and oxygen supplementation was stopped. At 10 days, therapy was switched to oral itraconazole. Steroid treatment was continued at a tapered dosage over 3 months, with trimethoprim/sulfamethoxazole used as H. capsulatum var. capsulatum is less common in that region (The source of infection for this patient remains speculative. However, possible risk exposures were guano from flying bats in the garden (H. capsulatum in Switzerland. Although diagnoses of autochthonous histoplasmosis have been rare, and few autochthonous cases have been described (In addition to previous findings of histoplasmosis in badgers (Additional information on an autochthonous case of pulmonary histoplasmosis in Switzerland.


Alcaligenes faecalis is usually causes opportunistic infections in humans. Alcaligenes faecalis infection is often difficult to treat due to its increased resistance to several antibiotics. The results from a clinical study of patients with Alcaligenes faecalis infection may help improve patients’ clinical care.Alcaligenes faecalis infection from January 2014 to December 2019. The medical records of all patients were reviewed for demographic information, clinical symptoms and signs, comorbidities, use of intravenous antibiotics within the past three months, bacterial culture, antibiotics sensitivity test, and clinical outcomes.We conducted a retrospective analysis of all patients presenting with Alcaligenes faecalis infection were seen during the study period, including 25 cases of cystitis, nine cases of diabetic foot infection, eight cases of pneumonia, seven cases of acute pyelonephritis, three cases of bacteremia, and nine cases of infection at specific sites. Thirty-seven patients (60.7%) had a history of receiving intravenous antibiotics within three months of the diagnosis. Fifty-one (83.6%) cases were mixed with other bacterial infections. Extensively drug-resistant infections have been reported since 2018. The best sensitivity rate to Alcaligenes faecalis was 66.7% for three antibiotics  in 2019. Two antibiotics (ciprofloxacin and piperacillin/tazobactam) sensitivity rates to A. faecalis were less than 50%.Sixty-one cases of Alcaligenes faecalis infection sites, in order, are the bloodstream, urinary tract, skin and soft tissue, and middle ear. The susceptibility rate of Alcaligenes faecalis to commonly used antibiotics is decreasing. Extensively drug-resistant Alcaligenes faecalis infections have emerged.The most frequent The online version contains supplementary material available at 10.1186/s12879-020-05557-8. Alcaligenes faecalis  is a Gram-negative, obligate aerobic, oxidase-positive, catalase-positive, and nonfermenting bacterium. It is commonly found in soil, water, and in hospital settings, such as in respirators, hemodialysis systems, and intravenous solutions [A. faecalis has been associated with endocarditis, bacteremia, meningitis, endophthalmitis, skin and soft tissue infections, urinary tract infections, otitis media, peritonitis, and pneumonia [A. faecalis infection is often difficult to treat due to its increased resistance to several antibiotics, such as anti-pseudomonas penicillin, cephalosporins, carbapenems, aminoglycosides, and quinolones [A. faecalis has not been well established in the literature. We report the results of a clinical study of patients with A. faecalis infection in this article. This study aims to emphasize the emergence of extensively drug-resistant A. faecalis and to provide local susceptibility data for the same.olutions , 2. It iolutions –6. A. faneumonia , 2, 7–28inolones , 24, 26.A. faecalis infection who were admitted to Dalin Tzu Chi Hospital from January 2014 to December 2019. Patients were diagnosed with A. faecalis infection when their clinical symptoms and signs indicated infection.We conducted a retrospective analysis of all patients presenting with A. faecalis infections , patient demographics, clinical symptoms and signs, details of hospital course, comorbidities, prior intravenous antibiotic use within 90 days, bacterial cultures, antibiotics sensitivity tests, and clinical outcomes.The data were obtained from the hospital’s clinical information system, microbiology laboratory report system, and medical chart review. We obtained details on all A. faecalis, including piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, cefotaxime, ceftriaxone, ampicillin-sulbactam, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, levofloxacin in our microbiology laboratory which was reference from clinical and laboratory standards institute (30th edition). Our microbiology laboratory susceptibility test report for A. faecalis included piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, and ampicillin-sulbactam. If the susceptibility test showed resistant to all antibiotics, tigecycline susceptibility in A. faecalis would be done by the disc diffusion method.Antibiotic susceptibility was tested using the VITEK® II system with VITEK® II Gram Negative Susceptibility cards  with Clinical & Laboratory Standards Institute interpretive criteria M100–25. One Gram-negative (GN) identification card and another VITEK II AST-N322 card  were placed in the neighboring slots, along with the transfer tube and the corresponding suspension tube . The comAlcaligenes faecalis with no language restrictions.We also searched PubMed and google scholar for topics related to The definition of MDR/XDR cites the literature of Maggiorakos et al. Our study was a retrospective analysis of medical records. The six-year study represented the lowest risk to the research subjects, and all information was made anonymous before being made available for research. The project was approved by the Buddhist Dalin Tzu Chi General Hospital Research Ethics Committee (Approved IRB No. B10802024), which exempted the study from the requirement for informed consent.p-value < 0.05 was considered statistically significant.Continuous variables were expressed as mean ± standard deviation or median (range), whereas categorical variables were expressed as frequencies and percentages. The trend in change of antimicrobial susceptibility analyzed by linear-by-linear association in chi-square test. All statistical analyses were conducted using the statistical package SPSS for Windows . A A. faecalis infection were identified during the study, including 25 cases of cystitis, nine cases of diabetic foot ulcer with infection, eight cases of pneumonia, seven cases of acute pyelonephritis (APN), three cases of bacteremia, and nine cases of infection at specific sites. There were nine hospital-acquired infections cases, including three cases of diabetic foot ulcer with infection, two cases of pneumonia, one case of pleural empyema, one case of peritonitis, one case of surgical wound infection, and one case of leg burn wound infection. Thirty-one community-onset infection cases (31/52 = 59.6%) had a history of previous hospitalisation within 90 days. Thirty-seven patients (60.7%) had a history of intravenous antibiotic use within the past 90 days. The most commonly used antibiotics were ceftazidime (11 cases) and levofloxacin (ten cases). There were only two cases of receipt of antibiotics during the same hospitalisation but before the onset of A. faecalis infection, including one case of prolonged mechanical ventilation with pneumonia and one case of peritonitis. The length of stay before A. faecalis isolation of the two cases were 190 days and 22 days, respectively. Fifty-one (83.6%) cases were mixed with other bacterial infections. The most common mixed infection pathogens were Enterococcus species (nine cases), Proteus vulgaris (nine cases), and Pseudomonas aeruginosa (eight cases).Sixty-one cases of A. faecalis cystitis were seen during the study period, which accounts for 41% of A. faecalis infections in this report. There were ten females and 15 males in our study. The mean age was 76.9 years (range: 25 to 98 years). Sixteen cases (64%) were catheter related cystitis. One patient had no comorbidity, while 24 patients had comorbidities such as diabetes mellitus, hypertension, dementia, cerebrovascular accident and chronic kidney disease . All seven patients had a risk of obstructive uropathy . Four patients had a history of intravenous antibiotic exposure within the past 90 days. Six patient urine cultures displayed polymicrobial infection. The most common mixed infectious pathogen was Providencia rettgeri, which affected three patients. The antibiotic sensitivity test showed no presence of XDR A. faecalis. All patients received adequate IV antibiotic therapy, and all were discharged from the hospital in excellent condition.Seven patients  with A. faecalis infection. The mean age was 57.2 years (range: 41 to 85 years). All patients had other comorbidities. All patients’ foot ulcer lesions were chronic (range: 14 days to 18 months). Two patients had no history of prior intravenous antibiotic use within 90 days. All patients’ wound cultures displayed polymicrobial infection. The antibiotics sensitivity test showed the presence of XDR A. faecalis infection beginning in 2019. All patients required surgical intervention. The wounds did not heal in three patients.Six male patients and three female patients had diabetic foot ulcers with A. faecalis pneumonia were seen during the study period. The mean age was 70.0 years (range: 51–83 years). All eight patients were at risk of pneumonia . One patient had no history of prior intravenous antibiotic use within 90 days. Six patient sputum cultures displayed polymicrobial infection. The antibiotics sensitivity test showed the presence of XDR A. faecalis beginning in 2018. Two patients died from A. faecalis pneumonia. Six patients received adequate intravenous antibiotics therapy and were discharged in excellent condition.Eight cases  of Enterococcus faecalis, and another patient had a mixed infection with Morganella morganii). Antibiotic sensitivity tests showed no presence of XDR A. faecalis. All patients received adequate antibiotics therapy and were discharged in excellent condition. Nine specific sites of A. faecalis infection cases are shown in Table A. faecalis to commonly used antibiotics (except ciprofloxacin and cefepime) is decreasing year by year.Three patients developed bacteremia during the study period. These three patients included a 78-year-old male, a bedridden stroke survivor; an 81-year-old male, a bedridden dementia; and an 81-year-old female with cholangiocarcinoma and dementia. The two male patients had a history of intravenous antibiotic exposure within the past 90 days. Two patients’ blood cultures displayed polymicrobial infection , and UTI. In our series, the most frequent cases were, in order of occurrence, UTI, SSTI, and pneumonia. The cases reported in the literature and our cases indicate that the most frequent A. faecalis infection sites, in order, are the bloodstream, urinary tract, skin and soft tissue (diabetic foot ulcer accounts for 56.5% of skin and soft tissue infections), and middle ear.According to the literature, there were 130 sporadically reported cases of on Table  [1, 2, 7cteremia . A. faecn Angola . The useA. faecalis cases were mixed with other bacterial infections [A. faecalis otitis media cases were mixed with other bacterial infections [A. faecalis peritonitis and concluded that it was important to view A. faecalis as a pathogen rather than a contaminant [A. faecalis. Therefore, rather than a contaminant, A. faecalis should be regarded as a pathogen, because global cases of life-threatening infections caused by A. faecalis are emerging [A. faecalis strain isolated from two outpatients and three inpatients, including four wounds cultures and one urine culture [A. faecalis specimens such as urine, pus, blood, and body fluids [A. faecalis infections cases exist but have not been reported in the literature. Our series of A. faecalis infection cases were about 10 cases every year, which was only a small fraction of the infectious diseases in our hospital. We concur with Junejo and believe A. faecalis to be an infectious pathogen rather than contaminant. In our series, the majority of cultures were mixed with other well-established pathogens. However, one should consider that A. faecalis may be a contaminant in some cases, particularly for those who were cured despite lack of active treatment.In Tena’s report, two out of five skin and soft tissue fections . In Filifections . Kahvecitaminant . Junejo taminant .. Al-Zakemerging . In 2017 culture . Howevery fluids . We beliA. faecalis strains were resistant to amoxicillin, ticarcillin, and gentamicin [A. faecalis infection. In 2000, Pereira reported that a strain of A. faecalis resistant to expanded-spectrum beta-lactamase cephalosporins was isolated from the urine of an inpatient [A. faecalis with ESBL in a patient with a concurrent urinary tract infection [A. faecalis pneumonia were reported by Agarwal and Junejo [A. faecalis bacteremia who was treated with double-dose tigecycline had a successful treatment outcome [A. faecalis were published in 2020. Al-Zakhari reported a fatal case of cavitary pneumonia caused by pandrug-resistant A. faecalis. The patient died despite the aggressive antibiotic treatment (linezolid and polymyxin B) [A. faecalis hospital acquired urinary tract infection patient; the patient died in hospital [Alcaligenes faecalis from human clinical samples [In 1997, Bizet first reported that nfection . In 2017d Junejo , 23. In  outcome . Three amyxin B) . Majewskhospital . Ngbede  samples .A. faecalis exhibited sensitivity only to imipenem and meropenem in a pneumonia patient in our hospital. In May 2018, a strain of XDR A. faecalis susceptible only to tigecycline was isolated from a pneumonia patient. There were four cases with XDR A. faecalis infection in our series, including two cases of pneumonia and two cases of diabetic foot infection.In March 2015, the strain A. faecalis from 2014 to 2019. Piperacillin/tazobactam was effective in significantly decreasing the susceptibility rate of A. faecalis since 2016. Emerging resistant strains of A. faecalis to imipenem and meropenem have been found since 2018. A high resistance rate of many antibiotics was also found in 2019. The best sensitivity rate to A. faecalis was 66.7% for three antibiotics . In two antibiotics (ciprofloxacin and piperacillin/tazobactam) sensitivity rates to A. faecalis were less than 50%.In view of individual antibiotics, ciprofloxacin revealed a very low susceptibility rate of A. faecalis infection patient according to the results of his or her antibiotics sensitivity test. If the A. faecalis organism is an ESBL strain, carbapenem is an appropriate antibiotic. Four articles mentioned that A. faecalis is susceptible to colistin [A. faecalis susceptibility to colistin in this study. If the A. faecalis is an XDR strain, we recommend that tigecycline is effective to XDR A. faecalis.Based on our prior experience, we selected an appropriate antibiotic for a susceptible colistin , 22, 23.A. faecalis infection, there were seven treatment failure cases, including two cases of pneumonia, two cases of cystitis complicated with sepsis, and three cases of diabetic foot infection. The overall treatment failure rate was 11.5%. A. faecalis is a low virulence bacterium. With adequate intravenous antibiotic therapy, patients with A. faecalis infection will typically experience a positive treatment outcome.Among our reported cases of Four cases were cured with non-covering antibiotics, including two cases of diabetic foot infection, one case of pleural empyema, and one case of surgical wound infection. The four cases had received appropriate wound care, adequate abscess drainage, and surgical intervention which may be crucial for curing of infections.A. faecalis infection was a small case series and therefore can provide only minimal clinical experience. Additional case series reports of A. faecalis infection will add to the knowledge of how to treat A. faecalis infection. Misidentification of Acinetobacter baumannii as Alcaligenes faecalis by VITEK II system was reported in the literature [A. faecalis, matrix-assisted laser desorption ionization time of flight mass spectrometry and 16S rRNA sequencing can be performed. An accurate distinction between Acinetobacter baumannii and A. faecalis has substantial clinical significance.Our clinical study of terature . Matrix-terature . HoweverA. faecalis exhibited decreasing sensitivity rate to commonly used antibiotics in 2019. Extensively drug-resistant Alcaligenes faecalis infections have emerged recently. We select an antibiotic for patients susceptible to A. faecalis infection based on the results of the antibiotics sensitivity test. With adequate intravenous antibiotic therapy, patients with A. faecalis infection will typically experience a positive treatment outcome.Additional file 1.


Fusarium oxysporum f. sp. lini. It is a challenging pathogen presenting a constant threat to flax production industry worldwide. Previously, we reported chromosome-level assemblies of 5 highly pathogenic F. oxysporum f. sp. lini strains. We sought to characterize the genomic architecture of the fungus and outline evolutionary mechanisms shaping the pathogen genome. Here, we reveal the complex multi-compartmentalized genome organization and uncover its diverse evolutionary dynamics, which boosts genetic diversity and facilitates host adaptation. In addition, our results suggest that host of functions implicated in the life cycle of mobile genetic elements are main contributors to dissimilarity between proteomes of different Fusaria. Finally, our experiments demonstrate that mobile genetics elements are expressed in planta upon infection, alluding to their role in pathogenicity. On the whole, these results pave the way for further in-depth studies of evolutionary forces shaping the host–pathogen interaction. Fusarium wilt of flax is an aggressive disease caused by the soil-borne fungal pathogen  Fusarium oxysporum species complex (FOSC) is a group of soil-borne pathogens causing economically important blights, crown or root rots, and vascular wilts. Although the fungus attacks a wide range of hosts, individual strains exhibit surprising host selectivity towards one species. Strains with the same host range are classified into one forma specialis (f. sp.) [(f. sp.) .F. oxysporum ff. spp. genome architecture, revealing remarkable similarities in its organization and sparking an on-going debate on genomic composition and evolvability. The current, prevalent hypothesis suggests a dual compartmentalization of FOSC genomes, dividing them into core and accessory ) parts that differ in gene density, composition, and evolutionary dynamics. The core compartment is evolutionary stable both between and within FOSC strains, whereas the variable part is linage-specific and is allegedly solely responsible for host adaptation. The variable compartment features sparse gene density, high transposon abundance, specific repertoire of protein-coding genes. Additionally, in contrast to the core, it carries a large number of variants and exhibits rapid evolutionary dynamics [Genomics have fueled the studies of dynamics ,3,4. These observations regarding genome architecture gave momentum to the concept of a “two-speed genome”, as a driver of adaptive evolution ,6,7. A hF.oxysporim f. sp. cepae and F.oxysporim f. sp. lycopersici (Fol) have clearly demonstrated that three chromosomes attributed to the core compartment of the genome, are more divergent, express a greater number of genes encoding secreted proteins and cell wall degrading enzymes, i.e., genes associated with cell defense and virulence [Over a past decade, however, the two-speed genome model faced criticism as over-simplistic and loosely-defined . Studiesirulence ,14. ThesAvr) [Avrs are secreted in xylem (SIX) proteins encompassing 14 distinct families of small cysteine-rich secreted molecules [Plant cell wall being the main frontline of host defense against pathogen is attacked by a wide range of pathogen-secreted enzymes , namely pectinases, cellulases, and hemicellulases . The hosAvr) ,17. Perholecules . The comolecules ,18,19. F. oxysporum f. sp. lini is a cause of a wilt disease in Linum usitatissimum (flax), limiting the crop cultivation in almost all flax and linseed growing countries. Recently, we reported genome assemblies of five highly pathogenic Fusarium oxysporum f. sp. lini (FOLINI) strains: the reference hybrid assembly of monoisolate 39 (MI39) sequenced with SMRT technology and Illumina-based assemblies of strains F329, F324, F282, F287 [82, F287 . SynteniFusarium oxysporum f. sp. lini genome architecture and presents its comparison with genomes of other FOSC strains, thus putting the flax pathogen into the global context of fungal plant diseases.The study provides a detailed annotation of F. oxysporum f. sp. lycopersici. The synteny-based partitioning is further supported by statistically significant differences in density of protein coding genes, single nucleotide polymorphisms (SNPs), and other genomic features . Previously, MI39 whole genome assembly chromosomes were assigned to core and variable  compartments of the size of 47.55 and 21.77 Mb, correspondingly . Chromosfeatures . Thus, ifeatures . The parfeatures c, regardp-values 0.002 and 0.02, correspondingly), as shown in In contrast to the core part, the lineage-specific chromosomes are characterized by low abundance of genes responsible for pathogenicity and virulence of the fungus, e.g., effectors and CAZYmes  (F. oxysporum f. sp. lycopesici (Foli) chromosomes 11, 12, and 13, which are distinguished from the rest of Fol4287 core chromosomes for different evolutionary properties  [Interestingly, a noticeable difference in gene density is observed within core compartment between chromosomes 3, 4, and 9 and others constituents of the core part (× 10−28) . The afoy, etc.) . An in-depth analysis carried out using various quantitative features related to genome stability and variability hinted at the possibility of non-binary partitioning of FOLINI genome. Clustering of density values computed over SNPs and repeats in 4Kb windows, dN/dS values associated with 2326 protein-coding genes, as well as local genome integrity score a revealsF. oxysporum f. sp. cubense, F. oxysporum f. sp. meloni, F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. pisi, F. oxysporum f. sp. vasinfectum, F. oxysporum f. sp. cotton, F. oxysporum f. sp. raphani, FO47 strain, F. oxysporum f. sp. radicis, F. oxysporum f. sp. conglutinans, Fusarium verticilloides) and MI39 proteome. Domain enrichment analysis revealed a group of 35 PFAMs significantly over-represented in LS compartment of MI39. Unsurprisingly, the core part is quite homogeneous in terms of domain repertoire, whereas the LS compartment shows noticeable differences between various formae specialis . Both proteomes are characterized with relatively high frequency of domains associated with activity of transposable elements apparently absent in other f. spp.A comprehensive genome-wide annotation of FOLINI was performed using the PFAM database, a collection of protein families based on functional domains. PFAM domain frequencies were computed for proteomes of 11 Fusarium species obtained from Ensembl Fungi  terms (FDR < 0.001). The results encompassing terms such as nucleic acid binding, DNA integration, DNA binding, DNA helicase activity, DNA binding transcription factor activity presented in . lini are clustered together with another strain FOLIN of the same formae specialis and with strain hdv-247 which belongs to f. sp. pisi.To determine whether the 5 sequenced strains are monophyletic, we inferred RPB2-TUB2-EF1α maximum likelihood phylogeny . The 5 slini at all has polyphyletic or monophyletic origin. We tested F. oxysporum f. sp. lini strains collected from different locations and constructed a phylogenetic tree based on alignment of EF1α sequences  domains were obvious candidates for exploratory analyses. From the list of proteins containing the aforementioned domains 10 candidates were selected at random and their expression profiles were assessed in planta. In a like manner, 5 candidates were chosen from the list of proteins containing DDE and endonuclease domains. SIX genes identified in FOLINI were profiled as infection markers, whereas E2F1 and CYC were used as reference controls. All 11 genes tested have shown clear expression in planta, suggesting their role in the infection process  is referred to as a conserved “core” part of the genome. The so-called “variable”, dynamic compartment is just the opposite of the core and includes chromosomes 12–15. This part of the genome is highly syntenic with LS chromosomes of Fol  and featcubense is located on chromosomes 1 and 5. Conversely, all copies of SIX genes belong to the dynamic genome compartment (chromosomes 12 and 15). Undoubtedly, in-depth analysis involving other omics data modalities is essential to refine the proposed genome compartmentalization scheme to decipher the evolutionary model of fungal pathogenicity.Pathogen- and virulence-specific genes are not solely bound to the variable genome, hinting at the existence of complex genetic and epigenetic mechanisms in charge of pathogenicity and host adaptation. For instance, two homologs of cerato-plantanin gene FocCP1 , responsIn contrast to housekeeping genes, members of SIX1 and SIX13 gene families demonstrate host-related clustering as revealed by phylogenetic analyses , thus beF. oxysporum f. spp. protein repertoire reveals the dominating role of the variable compartment in diversification of the formae specialis. Remarkably, the main contributors to proteome divergence are PFAM domains associated with DNA-binding and mobilization of transposable elements, possibly suggestive of active transcription and ongoing genome reshuffling in variable compartment .The nucleotide sequences assembly, de novo prediction, and annotation of coding regions of monoisolate 39 and 4 strains  have been described in our previous publication . De novoProtein sequences predicted by BUSCO  as descrFo f. sp. lini, 12 genomes from Ensembl Fungi  were extracted from BLAST databases using blastdbcmd script. Sequences of the same genes of Fusarium graminearum and F. solani were downloaded from NCBI and used as outgroup. Each of 3 gene datasets were aligned using ClustalX [For the phylogenetic analysis of housekeeping genes, we used publicly available sequences of the genes on NCBI: RPB2 (GB: KX434985.1), EF1α (GB: KC889026.1), and TUB2 (GB: KP964895.1) as queries for BLASTN search. Databases were created using makeblastdb script for our published genomes of bl Fungi , Fo f. sClustalX  and thenClustalX . MaximumClustalX  with dislini available on NCBI and one strain XJ37 belonging to f. sp. cubense  as per manufacturer’s instructions. After quantification using NanoDrop 8000, the first strand of cDNA was synthesized with MMLV-RT commercial kit . We used Oligo(dT)17-primer for first strand cDNA synthesis, as per the manufacturer’s instructions. The PCR was carried out using the Veriti 9000 instrument (Applied Biosystems) in technical triplicate using the indicated primer pairs see . Agarose


In this study, comparative genomic analyses were conducted to mine homologs of canonical D. melanogaster and C. elegans immune pathway genes from eight tardigrades  and four non-arthropod ecdysozoans  in order to provide insights into the tardigrade antimicrobial system. No homologs of the intracellular components of the Toll pathway were detected in any of the tardigrades examined. Likewise, no homologs of most of the Imd pathway genes were detected in any of the tardigrades or any of the other non-arthropod ecdysozoans. Both the JNK and Tir-1 pathways, on the other hand, were found to be conserved across ecdysozoans. Interestingly, tardigrades had no detectable homologs of NF-κB, the major activator of antimicrobial response gene expression. Instead, tardigrades appear to possess NF-κB distantly related NFAT homologs. Overall, our results show that tardigrades have a unique gene pathway repertoire that differs from that of other ecdysozoans. Our study also provides a framework for future studies on tardigrade immune responses.Tardigrades are microscopic animals known to withstand unfavorable abiotic conditions. These animals are also constantly exposed to biotic stresses, including parasites and internal microbiomes. However, the tardigrade immune mechanisms against these biotic stresses are largely uncharacterized. Due to the contentious phylogenetic position of tardigrades, it is not intuitive whether they possess an immune system more similar to that of arthropods ( These fungi release spores which adhere to the tardigrade cuticle, bore through it, and release their contents internally  microbiomes , which is also composed of tyrosine kinases, is involved in the C. elegans immune response . For this, we used eight tardigrade species that represent four orders and seven families based on the new taxonomy proposed by Echiniscoides cf. sigismundi (Echiniscoidea: Echiniscoididae) and Echiniscus testudo (Echiniscoidea: Echiniscidae), one apotardigrade: Milnesium tardigradum (Apochela: Milnesiidae), and five eutardigrade: Hypsibius exemplaris , Mesobiotus philippinicus (Macrobiotoidea: Macrobiotidae), Paramacrobiotus richtersi (Macrobiotoidea: Macrobiotidae), and Richtersius cf. coronifer (Macrobiotoidea: Richtersiidae). To further understand the diversity of immune responses within Ecdysozoa, we also identified homologs in assembled genomes and transcriptomes of species from three other ecdysozoan phyla. In order to provide a more comprehensive view of Panarthropoda, we included two onychophorans, one from each of the two extant families: Epiperipatus sp. (Peripatidae) and Opithopatus kwazululandi (Peripatopsidae). We also included the nematomorph Paragordius varius to represent the other phylum in the clade Nematoida. Lastly, we included the priapulan Priapulus caudatus as a representative of Scalidophora and used it as a control to check the stringency of our homolog detection method. To identify homologs, we used a step-by-step approach of combining sequence similarity searches, detection of conserved domains, and phylogenetic reconstruction. This provided us with a list of putative homologs that not only share sequence similarity with D. melanogaster and C. elegans immune pathway genes but also contain domains that are considered functionally important in the immune response. This study constitutes the first comprehensive search of antimicrobial immune genes in Tardigrada, Nematomorpha, and Priapulida, thus enabling us to understand the diversity of the ecdysozoan immune responses.In this study, we used genomic and transcriptomic sequence data from both of these model organisms and identified homologs of Mesobiotus philippinicus and Echiniscus testudo). The transcriptome of M. philippinicus was obtained from a single tardigrade sample using a modified method described in E. testudo was assembled using 20 individually-sequenced tardigrade samples (see File S1 for detailed methods of sample collection and library preparation of the tardigrade samples). We also obtained two transcriptome raw read files (https://www.ncbi.nlm.nih.gov/sra/): Echiniscoides cf. sigismundi (SRX421163), Richtersius cf. coronifer (SRX4213802); two transcriptome assemblies from NCBI TSA database (https://www.ncbi.nlm.nih.gov/genbank/tsa/): Milnesium tardigradum (GFGZ00000000.1), Paramacrobiotus richtersi (GFGY00000000.1); and sequences from two proteomes (http://ensembl.tardigrades.org/index.html): Hypsibius exemplaris (Hypsibius dujardini nHD.3.1.5.proteins), and Ramazzottius varieornatus (Rv101.proteins). For the onychophoran dataset, we used two novel onychophoran transcriptome assemblies of Epiperipatus sp. and Opisthopatus kwazululandi (see File S1 for detailed methods of sample collection and library preparation of the onychophoran samples). Lastly, raw transcriptome reads of one nematomorph (Paragordius varius NCBI SRA: ERX1879698) and the proteome sequences of one priapulan (Priapulus caudatus NCBI Genome: Priapulus_caudatus-5.0.1) were obtained from online databases.For the tardigrade dataset, we used two novel transcriptome assemblies . Trimmed reads were aligned against small and large ribosomal RNAs (rRNAs), and tardigrade mitochondrial sequences  obtained from the SILVA non-redundant database release 128 . For the onychophoran transcriptome reads, the same steps were used, but were aligned against rRNA and Panarthropoda mitochondrial sequences obtained from the SILVA and MetAMIGA databases, respectively. The same alignment step was also done after assembling the onychophoran transcriptomes.The quality of the raw tardigrade and nematomorph transcriptome reads were first checked using Fastqc v0.11.5 and random sequencing errors were corrected using the k-mer based method Rcorrector v1.0.2 . After rease 128 . FinallyA BUSCO v3.0.2 . For theD. melanogaster and nematode orthologs of the C. elegans immune genes (Table S2) were first obtained from OrthoDB v9  in a BLASTp search against the first set of candidate genes as database. Hits with an e-value less than 10−6 were retained and formed the second set of candidate genes. This set was then used as query in an online BLASTp search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the D. melanogaster or C. elegans protein database. The third set of candidate genes were composed of genes that showed the specific D. melanogaster or C. elegans immune gene as the top hit and had a bit score greater than 80, a percent identity greater than 20%, and e-value less than 10−6 (based on https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to check whether they contained the domains of the D. melanogaster or C. elegans immune genes with the corresponding domain identifiers (Table S4). This was also done to quantify the gene homolog numbers if there were multiple candidate genes from the third set. Different genes were considered as different paralogs if they shared the same protein domains , and they were counted as one gene if their protein domains were parts of a whole gene .Protein sequences of arthropod orthologs of thoDB v9  and usedbased on . FinallyCaenorhabditis elegans genes were identified as homologs of D. melanogaster immune genes if they were the corresponding top hits in the homology section of Flybase and Wormbase. The same method was used for identifying D. melanogaster homologs of C. elegans immune genes.Since the Trinity assembler can predict and provide different putative isoform sequences of a single gene, a gene can be represented by multiple sequences. Thus, the longest isoform was used as the representative sequence of a given gene. http://www.lrrfinder.com/, http://www.cbs.dtu.dk/services/TMHMM/, http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1, LRR domains of the Toll homologs were determined using the LRRfinder . Alignments were not trimmed since some of the sequences were incomplete. The phylogenetic trees were built using the best-fit model obtained under the Akaike Information Criterion in ProtTest v3.4  availablM. philippinicus (PRJNA509138), E. testudo (SAMN10601501-SAMN10601521), Epiperipatus sp. (SRP173483), and Opisthopatus kwazululandi (SRP173472). All Trinity transcriptome assemblies were deposited in Harvard Dataverse (https://doi.org/10.7910/DVN/CFNUGF). Supplemental material available at figshare: https://doi.org/10.25387/g3.9864353.The raw transcriptome reads were deposited in NCBI SRA: D. melanogaster Toll pathway (D. melanogaster and C. elegans), on the other hand, have multiple cysteine cluster TLRs (mccTLRs), which possess two or more CF motifs and another cysteine cluster at the N-terminal end of LRR   . Cnidari; LRRNF)  and was ; LRRNF) .E. cf. sigismundi, H. exemplaris, M. tardigradum, P. richtesi, R. cf. coronifer, and R. varieornatus have homologs containing all three domains. E. testudo and M. philippinicus have homologs for the LRR and TIR domains (Table S6) but appear to have no transcripts coding for the transmembrane domain in the dataset . Phylogenetic analysis clustered the nematomorph, priapulan, and O. kwazululandi TLR homologs with other mccTLRs  and peptidoglycan recognition proteins (PGRPs) recognize and bind to these pathogens. GNBP3 is involved in fungal recognition (PGRP-SA/SD and GNBP1/3 homologs in any samples (data not shown). This suggests that the Spaetzle-mediated Toll pathway activation during pathogen infection could have originated only within hexapods since the molecules involved in Spaetzle cleavage were present only within this group.To further explore this hypothesis, we looked at the presence of upstream genes in the D. melanogaster, two types of NF-κB pathways, the Toll and Imd pathways, are involved in the regulation of antimicrobial responses (NF-κB proteins are a superfamily of transcription factors which contain a highly conserved Rel-homology domain (RHD) in their N-terminal sequences required for DNA binding, dimerization, and nuclear localization. Pathways that lead to the nuclear translocation of these proteins are called NF-κB pathways . In D. mesponses .D. melanogaster Toll pathway are present across Arthropoda. Gene mining results  were present in all tardigrades, except for M. tardigradum that had no detectable Tir-1 and Nsy-1 homologs  has been shown to be involved in multiple stress responses , such as the JNK and Tir-1 pathways, could have been selectively retained. Experimental studies can also be performed to determine if these tyrosine kinase pathways are involved in the tardigrade immune response and if tardigrades possess novel immune mechanisms and antimicrobial peptides. Overall, this study provides a framework for future studies to elucidate how immune systems function in these extremely resilient organisms.The tardigrade species we examined showed similar patterns in terms of the absence and presence of immune-related


Salvia daiguii, was assembled in this study. The genome is 151,434 bp in length and contained 134 encoded genes in total, including 88 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The result of phylogenetic analysis based on 17 chloroplast genomes revealed that S. daiguii is clustered with Salvia miltiorrhiza in Lamiaceae.The complete chloroplast genome of Zhangjiajie sage,  Salvia daiguii Y. K. Wei & Y. B. Huang, is a newly described species in Salvia subgenus Glutinaria  according to IUCN guidelines and suggest that immediate measures should be taken to ensure the long-term survival of this species in its original habitat ). DNA was extracted from its leaf using DNA Plantzol Reagent . The raw data were generated using the Illumina platform . In total, about 3.8 G high-quality clean reads (150 bp PE read length) were obtained with adaptors trimmed. Following Liu et al.  has a length of 151,434 bp with a typical circle structure and a GC content of 38%. It consists of a large single-copy region , a small single-copy region , and two inverted repeat regions . In total, there were 134 genes in S. daiguii, including 88 protein-coding genes, eight rRNA genes, and 37 tRNA genes. Among them, six protein-coding genes , seven tRNA genes  and all four rRNA genes  have two copies. Nine of these protein-coding genes  have one intron each and two (ycf3 and clpP) of them have two introns.The complete chloroplast genome of S. daiguii (S. daiguii is closely related to S. miltiorrhiza.Seventeen species with available chloroplast genomes in Lamiaceae were selected to study the phylogenetic placement of  daiguii . The seq daiguii . We drew daiguii  with 500


In recent years, a variety of imaging techniques operating at nanoscale resolution have been reported. These techniques have the potential to enrich our understanding of bacterial species relevant to human health, such as antibiotic-resistant pathogens. However, owing to the novelty of these techniques, their use is still confined to addressing very particular applications, and their availability is limited owing to associated costs and required expertise. Among these, scattering-type scanning near field optical microscopy (s-SNOM) has been demonstrated as a powerful tool for exploring important optical properties at nanoscale resolution, depending only on the size of a sharp tip. Despite its huge potential to resolve aspects that cannot be tackled otherwise, the penetration of s-SNOM into the life sciences is still proceeding at a slow pace for the aforementioned reasons.In this work we introduce SSNOMBACTER, a set of s-SNOM images collected on 15 bacterial species. These come accompanied by registered Atomic Force Microscopy images, which are useful for placing nanoscale optical information in a relevant topographic context.The proposed dataset aims to augment the popularity of s-SNOM and for accelerating its penetration in life sciences. Furthermore, we consider this dataset to be useful for the development and benchmarking of image analysis tools dedicated to s-SNOM imaging, which are scarce, despite the high need. In this latter context we discuss a series of image processing and analysis applications where SSNOMBACTER could be of help. Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) _[FOV_number] [imaging_mode_abbreviation].Achromobacter xylosoxidans ATCC 27061_1 M2A.gsf” indicates that the respective *.gsf file corresponds to the second harmonic of the topography error image, collected on the first FOV for A. xylosoxidans strain ATCC 27061. In each FOV folder we provide a *.txt file that presents all the acquisition parameters used for the respective measurement . In Fig. S. aureus ATCC 25923.For instance, the filename “Each file of SSNOMBACTER  can be dThe SSNOMBACTER dataset consists of 4,400 images collected with s-SNOM and AFM modalities on 15 bacterial species. The dimensions of this dataset can be further expanded by processing the available images to extract other types of data representations . A diffBy employing digital restoration methods, an image whose quality is affected by noise, artifacts, or improper acquisition conditions is processed to obtain a better estimate of the original object. The aforementioned causes are technique or equipment dependent; thus they greatly differ between imaging modalities that rely on optical and scanning probe principles. In the case of the latter, probe damage, mismatch between probe and sample geometry, scanner drift, vibrations, surface contamination, and others impede an unbiased visualization of the imaged sample , 76. FurThe proposed dataset can be used to develop novel image restoration methods oriented towards s-SNOM and AFM imaging and benchmark existing ones. We envision 2 potential scenarios for such efforts. In the first, the images available in our set can be restored  or depicting complementary information (amplitude and phase), can be processed on the basis of a methodology that relies on the oscillating point dipole model, in order to extract a nanoscale map of precise values of the dielectric function, and intrinsic optical properties  , 60 Fig. In a foImage segmentation is the process of partitioning an image into sets of pixels known as segments to allow for easier and meaningful representation . In bioiPrevious studies have shown that morphological features such as shape, cell size and size distribution, cell wall thickness, and many others can be useful in distinguishing between various bacterial species or between different types of the same species . MeasuriImage acquisition of bacteria can be performed at different scales to allow for visualization and examination of details at different scales, such as imaging a group of bacteria at low magnification versus imaging a single bacterium at high magnification. These different imaging scales will of course cover different levels of detail, which can be merged to allow a more comprehensive view (and understanding) of the specimen. Furthermore, when high-magnification image acquisition of particular cells or features is performed in an unsupervised manner, identifying them in a group of many similar ones imaged at low magnification  is timeFurthermore, similar to the popular computer vision application of panorama creation , 102, imWe introduce SSNOMBACTER, a collection of 4,400 images collected with s-SNOM and AFM modalities on 15 bacterial species, including harmless species regarded as model organisms as well as pathogens included in the ESKAPE group. By publishing this carefully crafted collection, our interest is 3-fold: (i) we wish to increase the awareness of relevant stakeholders in the life sciences field of this valuable imaging technique, s-SNOM; (ii) we wish to draw the attention of more groups active in the field of s-SNOM towards its huge potential for enabling novel high-impact studies and applications in microbiology; and (iii) we wish to offer to the computer vision community the means to interact with s-SNOM outputs, leading to the advent of novel s-SNOM–oriented methods for automated image analysis. With respect to the latter, we carried out a detailed discussion on relevant use-cases. In the future we plan to extend this collection of images to cover additional pathogens, imaged with multiple laser wavelenghts. We hope that our effort will inspire similar ones, originating from other groups, leading to wider availability of datasets collected on bacterial species with emerging imaging modalities that could enhance our current understanding of prokaryotic organisms.All data supporting this work are openly available in the OSF Platform .Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species; FOV: field of view; .gsf: Gwyddion Simple Field file format; nano-FTIR: nanoscale Fourier transform infrared spectroscopy; RI: refractive index; s-SNOM: scattering-type scanning near-field optical microscopy; SRM: super-resolution microscopy; .tiff: tagged image file format.AFM: atomic force microscopy; ESKAPE: The authors declare that they have no competing interests.The work of S.G.S., D.E.T., and R.H. was supported by UEFISCDI grant PN-III-P1–1.1-TE-2016–2147 (CORIMAG). The use of the Neaspec NeaSNOM Microscope was possible due to European Regional Development Fund through Competitiveness Operational Program 2014–2020, Priority axis 1, Project No. P_36_611, MySMIS code 107066,—INOVABIOMED. The work of D.U. was realized during his STSM visit to Rome Tre University supported by the COST Action CA15124 NEUBIAS. The work of M.L. was supported by the COST Action CA17121 COMULIS.M.L., P.V., and G.C. designed this dataset in terms of bacterial species to be imaged. S.G.S., G.C., and G.A.S. designed the experiment in terms of imaging configurations. M.L. and A.M.H. prepared the samples, under the guidance of P.V., G.A.S., G.C., and S.G.S., whereas M.L. and D.E.T. collected the images. L.N. and R.H. verified the dataset for potential inconsistencies and helped in its organization. D.U. and S.G.S. identified use-cases of the dataset for developing s-SNOM–oriented computer vision applications. All authors wrote and reviewed the manuscript.giaa129_GIGA-D-20-00201_Original_SubmissionClick here for additional data file.giaa129_GIGA-D-20-00201_Revision_1Click here for additional data file.giaa129_GIGA-D-20-00201_Revision_2Click here for additional data file.giaa129_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giaa129_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giaa129_Reviewer_1_Report_Original_SubmissionChris Armit -- 7/16/2020 ReviewedClick here for additional data file.giaa129_Reviewer_2_Report_Original_SubmissionJoachim Heberle -- 7/27/2020 ReviewedClick here for additional data file.giaa129_Reviewer_2_Report_Revision_1Joachim Heberle -- 10/24/2020 ReviewedClick here for additional data file.


Fibigia clypeata (L.) Medik. is a poorly studied plant species belonging to the Brassicaceae family, and usually used as cress in the salads. The current investigation aimed at assessing the antioxidant potential and inhibitory activity of ethyl acetate, methanol, and aqueous extracts of F. clypeata against key enzymes targeted in the management of type II diabetes (α-amylase and α-glucosidase), Alzheimer’s disease (acetylcholinesterase and butyrylcholinesterase), and skin hyperpigmentation (tyrosinase). Cytotoxicity of the extracts was also determined using normal VERO and cancer FaDu and SCC-25 cell lines. Besides, LC-MS was employed to investigate the detailed phytochemical profiles of the extracts. The methanol extract showed potent enzyme inhibitory activity  and antioxidant potential . Interestingly, caffeic acid-O-hexoside derivative, caffeyl alcohol O-glucopyranoside, and ferulic acid derivative were identified in all extracts. F. clypeata extracts showed no cytotoxicity towards VERO cell line and a weak cytotoxic potential against FaDu and SCC-25 cell lines. Interesting scientific evidence gathered from the present study support further investigation on F. clypeata in the view of designing and developing a novel therapeutic agent for the management of Alzheimer’s disease, type II diabetes, skin hyperpigmentation problems, as well as cancer. Ethnopharmacological literature compiled by our ancestors has indicated the potential of plants in the treatment of several health complications affecting mankind. The therapeutic properties of plants have been ascribed to biologically active chemical compounds called secondary metabolites. Alkaloids, terpenoids, and phenolic compounds are the different classes of secondary metabolites, which naturally occur in plants and have been found to exhibit biological effects, such as anti-inflammatory, anti-cancer, anti-diabetic, and antioxidant properties, amongst others ,2,3,4. BF. eriocarpa (DC.) Boiss. is used against the common cold - at m/z 193, was identified as ferulic acid. Moreover, several sesquiterpenes  were also tentatively characterized  and methanol (1.83 mg GALAE/g for AChE and 1.08 mg GALAE/g for BChE) extracts [F. clypeata were stronger than F. eriocarpa. In the literature survey, several researchers reported the significant cholinesterase inhibitory abilities for other solvents  rather than water [The methanolic extract (4.87 mg GALAE/g) was most active against acetylcholinesterase. Both the ethyl acetate (3.54 mg GALAE/g) and methanol (3.52 mg GALAE/g) extracts showed comparable inhibition against butyrylcholinesterase. In our previous study, the best cholinesterase inhibition abilities of extracts . Based oan water ,34,35. TF. clypeata showed potent inhibition against tyrosinase. The inhibition of tyrosinase is crucial in the management of skin hyperpigmentation conditions, such as melasma and freckles. Inhibiting tyrosinase is directly related to the reduced production of the dark brown melanin. Moreover, the increased public interest towards naturally derived products, including cosmetic and dermatological products, has fueled the need for natural tyrosinase inhibitors. Another species of the Fibigia genus, namely F. eriocarpa, was previously reported to inhibit tyrosinase [F. eriocarpa were not active on tyrosinase. This fact could be explained with the differences of chemical profiles in these extracts and the complex interactions of phytochemicals. However, methanolic extracts exhibited stronger actions on tyrosinase in earlier studies conducted by some researchers [Likewise, the methanolic and ethyl acetate extracts of rosinase . In contearchers ,37.F. clypeata exhibited potent α-glucosidase inhibitory action. It was noted that, in general, the water extract of F. clypeata exhibited the lowest enzymatic inhibition. The higher activity of the ethyl acetate and methanol extracts might be related to the synergistic action of the different bioactive compounds present in these extracts. Anti-diabetic properties of the ethyl acetate and methanol extracts of F. clypeata were stronger than those of F. eriocarpa  in our previous paper. Additionally, it is worth mentioning the level of the different bioactive compounds might have also affected enzyme activity. The alkaloidal amine, sinapine, tentatively characterized from the ethyl acetate extract of F. clypeata was previously reported to inhibit acetylcholinesterase isolated from rat cerebral homogenate with an IC50 of 3.66 µmol/L [p < 0.01), tyrosinase , α-glucosidase , and BChE  inhibition values  and methanol (24.68 mmol ACAE/g) extracts of 6 µmol/L . Regardin values .F. clypeata extracts. As presented in F. clypeata showed potent radical scavenging properties.The role of oxidative stress in the onset and/or progression of human ailments supports the systematic antioxidant evaluation of studied plant extracts. Antioxidants can act by different mechanisms, namely, hydrogen atom transfer, single electron transfer, or transition metal chelation . In thisFibigia or Brassicaceae species. For example, the antioxidant properties in F. eriocarpa extracts can be ranked methanol > ethyl acetate > water. However, the antioxidant abilities of the water extract of F. clypeata were stronger than that of F. eriocarpa. [F. clypeata methanolic extract might be responsible for the observed activity. Sinapic acid was reported to exhibit radical scavenging potential [F. clypeata showed the highest activity. These findings suggest that bioactive compounds present in the methanolic extract exhibited mostly radical scavenging and reducing potential, while compounds present in the aqueous extract were potential metal chelators. Regarding the potential correlation between TPC and TFC values, we found strong correlation coefficients between TPC and some antioxidant assays, such as DPPH , CUPRAC , FRAP , and ABTS  values. In addition, total flavonoids were inversely correlated to chelating activity  and strongly correlated to DPPH values   and FRAP (104.85 mg TE/g) assays. Obtained results could be compared with earlier studies on other iocarpa.  Besides,iocarpa. ,41,42. Aotential . Caffeicotential . In term < 0.01) .Fibigia clypeata extracts, their cytotoxicity was measured on two cancer cell lines belonging to the group of head and neck squamous cell carcinomas (HNSCC), i.e., squamous cell carcinoma of the pharynx (FaDu) and squamous cell carcinoma of the tongue (SCC-25) and compared with the results obtained for the normal VERO cell line. The results of cytotoxicity evaluation are presented in To assess the anti-cancer potential of In the case of VERO cells, no cytotoxicity was observed for methanolic and aqueous extracts. The ethyl acetate extract showed a cytotoxic effect in concentrations above 125 µg/mL, but it was impossible to calculate the CC50 value because even in the highest tested concentration of 1000 µg/mL the viability of VERO cells was above 50% .p < 0.001). In the case of methanolic extract tested on FaDu and SCC-25, the CC50 values were similar, but a significant difference was found for the CC10 values (p < 0.001)  .The criteria of classification of plant extract cytotoxicity according to the protocols suggested by the National Cancer Institute (NCI)  and seleF. clypeata extracts assessed on the THP1 human monocyte cell line by colorimetric determination of cell viability using the oxidation–reduction indicator Alamar Blue. The aqueous and dichloromethane extracts showed CC50 of 124.9 and 123.6 µg/mL, whereas in the case of methanol extract, it was above 250 µg/mL.According to this criterion, all tested extracts can be classified as non-cytotoxic towards the VERO cell line. In the case of cancer cell lines, ethyl acetate and methanolic extracts tested on FaDu and methanolic and aqueous extracts tested on SCC-25 can be classified as weakly cytotoxic. However, the selective activity towards cancer cell lines encourages further investigations focused on the isolation of bioactive constituents and testing their anti-cancer potential. Di Giorgio et al.  studied Fibigia clypeata in the management of Alzheimer’s disease, type II diabetes, and skin hyperpigmentation problems. Cytotoxicity studies also supported the action of F. clypeata against cancerous cell lines, thereby advocating further investigation geared towards assessing the anti-cancer properties of F. clypeata. Higher enzyme inhibition and in vitro antioxidant capabilities were noted for the methanolic extract, showing that the choice of solvent greatly affected the extraction of bioactive compounds from the plant material. Therefore, considering the potential health-implication given by this plant-food for human nutrition, both in vivo and bioavailability studies appear to be strongly required to further support the preliminary data collected in the present study.The present work presents interesting scientific data on the potential of


Cuscuta, commonly known as dodder, is a genus of family convolvolaceace. Approximately 170 species of Cuscuta are extensively distributed in temperate and subtropical areas of the world. Species of this genus are widely used as essential constituents in functional foods and traditional medicinal systems. Various parts of many members of Cuscuta have been found efficacious against a variety of diseases. Phytochemical investigations have confirmed presence of biologically active moieties such as flavonoids, alkaloids, lignans, saponines, phenolics, tannins, and fatty acids. Pharmacological studies and traditional uses of these plants have proved that they are effective antibacterial, antioxidant, antiostioporotic, hepatoprotective, anti-inflammatory, antitumor, antipyretic, antihypertensive, analgesic, anti hair fall, and antisteriogenic agents.  Plant-based medicines are an integral part of virtually all cultures since immemorial times. The journey of information from prehistoric texts to various indigenous folklores and modern preparations have witnessed the presence of bioactive moieties with therapeutic potential in these herbs -4. The iEver growing consumption of natural products by local masses has forcefully motivated the scientists to acquire systematic, elaborated, and practical knowledge about their constituents by using advanced technologies . Herbal Cuscuta L. commonly known as dodder is one of the essential herbal constituents of pharma foods and curative tonics that are frequently prescribed to nourish various body parts. It is used to enhance the nutritional value of porridge and alcoholic beverages . These stem and leaf parasites depend entirely on their host plant, thus reducing the growth and yield of the host. They mostly infect many broadleaf crops, ornamentals plants, weeds, and a few monocot crops. Some of the species are strictly host-specific while others thrive on diverse hosts -N-[(4-methoxyphenyl)ethyl]propenamide and 7’--N-[(4-butylphenyl)ethyl]propenamide -β-D-glucopyranoside), cuscutoside B (2′-hydroxyl asarinin 2′-O-β-xylopyranosyl-(1→6)-β-glucopyranoside), cuscutoside C (2′-hydroxyl asarinin 2′-O-β-D-glucopyranoside), cuscutoside D (2′-hydroxyl asarinin2′-O-β-D-apiofuranosyl-(1→2)-[β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranoside) and neo-sesamin  were analyzed for quantity of phenolics and flavonoids content. Their antioxidant capacity was measured by using a variety of assays including reducing power, DPPH scavenging activity, percent inhibition of linoleic acid peroxidation and δ-tocopherol. It was observed that there was a strong correlation between amount of total phenolics and antioxidant capacity  and lipid contents were saponified to separate unsaponifiable materials and fatty acids. The extract was fractionated by using various solvents, and antioxidant activity of all extracts  was appraised by DPPH free radical assay. The methanol extract was found most potent , alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin have confirmed hepatic damage after CCl4 administration. C. epithymum prevented the toxic effect in both anticipatory and curative models, which may be due to the presence of various bioactive moieties, including phenolics, flavonoids, and alkaloids  cells  cells . C. chin glucose -248. In  glucose . A Chine effects . Recentlas genes . It is cAnti-inflammatory C. reflexa and its ethyl acetate fraction showed remarkable anti-inflammatory activity in in vitro and in vivo tests. Inflammation was induced by various chemicals like histamine and lipopolysaccharide. It was observed that extracts inhibited inflammatory responses that can be related to the presence of flavonoids, phenols, and polyphenols in this plant  is a widely spread infectious agent causing a huge loss in food crops with visible symptoms and leave shedding. Aqueous decoction and infusion extract of C. pedicellata were evaluated for antibacterial activity against diverse pathovars of XC using in vitro well diffusion method. Inhibition zone diameter was observed from 1.0 to 5.0 cm   . As the empferol  and querin vitro -269. Theiability . C. campestris also has anticancer agents  to a noticeably high level by affecting Th1 and Th2 cell functions  inhibition at a dose of 400 mg/ml. Four caffeoylquinic acid derivatives were isolated from the active fraction having inhibitory effects on ACE activity. Presence of these metabolites, at least in part is responsible for the antihypertensive activity extract  bacterial strains. The extract revealed noteworthy antimutagenic activity against 4-nitro-o-phenylenediamine and sodium azide for S. typhimurium strains  disorders comprise 12 % of deaths worldwide and are still a hugely challenging endeavor for health care systems. Plenty of rnatives .C. campestris affects the CNS action and decreases motor activity of mice sited on a rotarod. Various tests applied indicated the CNS-depressant activity of the extract, which probably seems due to an anesthetizing effect  prepared by a pharmacist were found effective for major depression patients. In a study period of eight weeks depression was measured before and after by Beck Depression Inventory and Hamilton Depression Inventory  and 79 % (aqueous) as compared to the standard drug  after six hours of treatment  involved in seizure activity was noticeably increased in the brains of mice (C. reflexa significantly reduced convulsions by delaying onset and duration of seizures in an albino mice model. A dose of 400 mg/kg showed maximum delay in pentylenetetrazole induced convulsions ( seizure . Methano of mice . Ethanolvulsions .C. pedicellata is widely used for management of obesity. Ethanolic extract of C. pedicellata has significantly reduced the bodyweight along with serum lipid profile in high-fat diet-fed rats (fed rats . Recentlfed rats .Cytotoxicity, insecticidal, antiarthritic, and wound healing activity C. reflexa, parasitizing Nerium oleander, exhibited promising cytotoxic activity (C. europaea demonstrated the cytotoxic effects of LLP and LLP on C127 and B-16 cells (C. reflexa protects against arthritis and nephrotoxicity. A dose at 600 mg/kg considerably reduced paw edema and joint swelling up to 71.22 % (C. reflexa stem at 200 mg/kg and 400 mg/kg were able to heal wounds in a rat model (The ethanolic extract activity . Lectin-16 cells . Various16 cells . C. refl 71.22 % . Aqueousat model .Cuscuta genus is a rich and diverse source of many valuable chemical components. It is loaded with flavonoids, alkaloids, lignans, polysaccharides, steroids, volatile oils, and resin glycosides. Medicinal importance of its various species is part of prehistoric texts. Traditionally it is considered a miracle genus equipped with broad spectrum of remedial values. Decoctions, extracts, paste, powder, juice, and infusions of different parts of the plants are important herbal prescriptions in traditional medicinal systems.C. reflexa, C. chinensis, C. pedicellata, C. approximate, C. monogyna, C. campestris, and C. mitraeformis have shown impressive antioxidant activity. C. chinensis, C. australis, C. reflexa, and C. epithymum are significantly hepatoprotective in nature. Some species of Cuscuta including C. reflexa, C. chinensis, C. campestris, C. japonica, and C. kotschyana have been reported potentially antitumor against various cancer cell lines. Moreover crude extracts and compounds from the various parts possessed antibacterial, antiosteoporotic, anti-inflammatory, antihypertensive, analgesic, anti hair fall, analgesic, and antiestrogenic properties. A lot of experimentation has been employed to verify its phytotherapy as claimed by traditional healers and local inhabitants. C. reflexa and C. chinensis out of nearly 170, while the rest of the members are partially or fully undiscovered in terms of phytochemistry and pharmacology. Most of the efforts are limited to in vitro and in vivo animal models or cell line level. Very few clinical studies are reported in humans. Although a good deal of secondary metabolites with multitudinous pharmacological attributes have been isolated, identified, and characterized but most of the pharmacological investigations are extract-based. Further studies must be conducted to clarify the mechanism and to figure out the active principle behind the activity to use these compounds as leads and template in development of new drugs. Rich and unrivaled medicinal history demands verification with modern scientific methodologies. Only a few of the species are thoroughly investigated up till now, especially


Triboliumcastaneum is a known pest of various grains and stored-products such as wheat flours; however, T.castaneum feeds on and infests soybean and soy products. For more than 60 years, soy flour has been suggested to be unstable food for Tribolium spp. because it causes larval development failure. However, it remains unknown whether soy flour affects adult beetles. The objective of the present study was to examine the effects of soy flour and its related isoflavones against T. castaneum using an artificial dietary intake assay. Beetles were fed gypsum (a non-digestible compound) mixed with either water (control) or soy flour. Significantly fewer beetles survived after being fed the soy flour treatment. Although the soy isoflavone genistein, a defensive agent and secondary metabolite, decreased the T. castaneum adult survival, it required a long time to have a lethal effect. Therefore, the cytotoxic effects of soy flour, i.e., the rapid biological responses following isoflavone addition, were also examined using a cultured cell line derived from T. castaneum. Both genistin and genistein significantly affected the survival of the cultured cells, although genistein had a stronger lethal effect. This study demonstrated the toxicity of genistein found in soybean against T. castaneum cultured cells within 24 h period. Genistein may be used as an oral toxin biopesticide against T. castaneum.The red flour beetle  Tribolium castaneum (Herbst) is a serious stored-grain pest that attacks various foods, such as wheat flour, dried fruits, nuts, pasta, cereal germs, and processed foods . Dead cells were counted using colorimetry based on the LDH (lactate dehydrogenase) assay (Dojindo). Absorbance was measured using a microplate reader at 450 nm and 495 nm. The experimental procedures were conducted according to the protocols of the manufacturers, with a minor adjustment of the enzyme reaction temperature, i.e., 25 °C was used . The sigT. castaneum adult beetles recognized and digested gypsum-containing soy flour.The dietary intake was estimated by measuring gypsum excreta based on the TribUTE assay . The amoT. castaneum markedly decreased from 6 to 8 days. Soy flour did not extend the lifespan of T. castaneum. These results also indicated that the T. castaneum beetles were affected by certain toxins in soy flour rather than by the nutritional contents.The number of alive beetles were counted daily . SignifiT. castaneum beetles by TribUTE assay . Therefore, it was difficult to identify the effects by isoflavones, including genistein, using the TribUTE assay. The present study assessed the effect of isoflavones, such as daidzin, daidzein, glycitin, glycitein, genistin, and genistein, on TE assay . DaidzinTE assay . GenisteTribolium adults.The Tc81 cells were exposed to media containing each isoflavone for 24 h . DMSO wa50) of genistein was 12.7 ± 0.9 µM for 24 h.The viability of Tc81 cells exposed to isoflavones was examined using WST-8 at 24 h after exposure. Significant Tc81 viability was observed in the presence of daidzin, daidzein, glycitin, and glycitein. However, compared to the DMSO control level, viability decreased when cells were exposed to genistin and genistein in the culture medium A. Both gTribolium spp. because the larval growth rates of T. castaneum and T. confusum fed only soy flour were significantly delayed compared to those fed whole wheat flour supplemented with yeast [T. castaneum remains unclear. Despite the willingness of T. castaneum adults and larvae to consume soy flour (T. confusum larvae compared with whole wheat flour (T. castaneum. Rather, the T. castaneum beetles seemed to be affected by certain toxins in soy flour. The present study examined the insecticidal effects of flavonoids that occur naturally in plants as candidate toxic insecticides against T. castaneum. Many flavone and isoflavone derivatives have been identified in the soybean plant [Diabrotica virgifera virgifera and Spodoptera litura [T. castaneum beetles. Although the TribUTE assay was able to show the effects against T. castaneum adults (For over 60 years, studies have shown that soy flour is an unstable food for th yeast ,6. Howevoy flour , the nutat flour . Soy floan plant . Plants an plant . In insean plant . Some fla litura ,31. Soy m adults , it requT. castaneum cultured cell line Tc81 (Acorus calamus, was evaluated using the Spodoptera frugiperda cell line Sf9 [T. castaneum beetles in the present study, I decided to assess the lethal effects of these compounds using insect cultured cells. The Tc81-containing vesicles shrunk under genistin and genistein 24 h after exposure. It was unclear whether the genistein cytotoxicity directly affected the Tc81 cells or indirectly affected the cells via the vesicle disruptions. The viability and lethality of Tc81 cells were then examined using the WST-8 colorimetric assay and the LDH assay (T. castaneum. Glycoside derivatives of daidzein and genistein and acetate derivatives have been shown to have toxic effects on the larvae of mosquito, e.g., the LC50 value of genistein for mosquito larvae was 40 µM [T. castaneum adults. The most effective toxic component in soya will become a biopesticide candidate. The cultured Tc81 cells are available to conduct chemical screening.The present study also examined the cytotoxic effect of isoflavones using the ine Tc81 . Insect line Sf9 . Owing tDH assay . Actuallas 40 µM . The finas 40 µM . These tA. pisum [T. castaneum also expresses an estrogen-like receptor, however, the function of such receptors remains unclear. Given that genistein had a lethal effect on Tc81 cells in the present study, these insect cultured cells may become a useful tool for studying certain mechanisms, e.g., regarding these estrogen-like receptors. Collectively, the findings of the present study and those of previous studies indicate that genistein may have multiple functions, and that these functions may be species-dependent.Several studies have shown that genistein, a secondary plant metabolite, can have deterrent or lethal effects on polyphagous insects ,22,35. FA. pisum . GenisteA. pisum ,39,40,41A. pisum . T. castT. castaneum and its cultured cells. The present findings that genistein has a cytotoxic effect on Tc81 cultured cells will help to elucidate the mechanisms underlying soy-stored insect interactions. Secondary metabolites such as phenols, saponins, and flavonoids are synthesized by plants after perceiving herbivore attacks. These natural chemicals in plants show various properties of repellents, feeding deterrents, growth retardants, and toxicants against insects, and thus they may have commercial applications as botanical insecticides for crop protection [T. castaneum. My findings indicated that genistein may act as an oral toxin and biopesticide against T. castaneum. Further studies are required to assess the interactions of other stored-grain pests and soy compounds.The present study demonstrated the toxicity of isoflavone genistein found in soybean against otection . Isoflav


Aquilegia barnebyi, belonging to the genus Aquilegia (Ranunculaceae), is a member of basal eudicot species. In this study, we obtained the complete chloroplast (cp) genome of A. barnebyi. The genome size is 161,954 bp with a GC content of 38.98%. A total of 113 unique genes including 79 protein-coding genes, 30 tRNA genes, four rRNA genes were annotated. The large single-copy region and small single-copy region contains 91,250 bp and 17,359 bp, respectively. The inverted repeat regions are 26,671 bp in length. The phyologenetic analysis indicated that A. barnebyi had a close relationship with A. coerulea. And four species in genus Aquilegia formed a monophyletic group with high support value. The availability of A. barnebyi cp genomic resources will greatly helpful for taxonomy, phylogeny and conservation genetic studies of basal eudicot specie. Aquilegia genus (Ranunculaceae) belongs to basal eudicot angiosperms which consists of approximately 75 species occurring in North America (22 spp), Asia (23 spp), and Europe (21 spp)  Nold . These spp) Nold . The invof A. barnebyi were collected from rifle falls of northwest Colorado, USA , and the speciman was deposited at the herbarium of department of ecology, evolution and marine biology, university of California (speciman code Aquilegia_BA). The chloroplast reads were filtered from whole genome Illumina sequencing data of A. barnebyi which was downloaded from NCBI Short Read Archive (SRR7965809)   divided by two single-copy regions . The overall GC content of the cp genome was 38.98%. There were a total of 113 unique genes, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes, in A. barnebyi cp genome. Among these genes, nine protein-coding genes and six tRNA genes contained a single intron, and two protein-coding genes possessed two introns. The gene rps12 found to be trans-spliced; with the 5′-end exon located in the LSC region and two copies of 3′-end exon and intron in the IR regions. Moreover, the rpl32, infA and clpP were identified as pseudogenes because of the partial duplication.The complete cp genome of A. barnebyi within the genus Aquilegia and the family Ranunculaceae, we downloaded the complete cp genome of nine species in Ranunculaceae including three species in genus Aquilegia. The sequences were aligned using MAFFT v7.307 (Katoh and Standley Ranunculus occidentalis as outgroup. All nodes in the complete plastome tree were strongly supported. The phylogenetic tree showed that four species in genus Aquilegia formed a monophyletic group with high support value. And A. barnebyi was closely related to A. coerulea and far away from A. rockii (A. barnebyi cp genome will provide useful information for phyogenetic and evolutionary studies in the genus Aquilegia and Ranunculaceae.To understand the phylogenetic position of . rockii . This pu


Lilium dauricum Ker Gawler is a wild lily species that belongs to section Sinomartagon and is one of the ancestors of the Asiatic hybrid lilies. Unique traits such as disease resistance and early flowering make L. dauricum a desirable resource for interspecific hybridization. However, in Korea, the natural resources of L. dauricum are being exhausted by excessive exploitation and require urgent conservation. In this study, the complete chloroplast genome of L. dauricum was generated using Illumina paired-end sequencing technology, and its structure was compared with that of other Lilium species. The chloroplast genome was 152,063 bp long, with a typical quadripartite structure including a pair of inverted repeat regions  separated by a large  and small  single-copy (SC) region. The genome encodes 131 different genes, including 85 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. A phylogenetic analysis strongly supported the relationship of L. dauricum with other members of Sinomartagon and Martagon lilies. Lilium dauricum Ker Gawler (genus Lilium) is a perennial bulb-producing flowering plant known for its high degree of resistance to Fusarium oxysporum var lilii, and exhibits early flowering, making it a valuable genitor for both interspecific and intersectional hybridizationhybridisation  under accession number 2015-10 high-throughput sequencing was performed using an Illumina HiSeq 4000 platform. The raw reads were quality trimmed by Trimmomatic  genome sequence of L. dauricum, the complete cp genome of 15 species in the family Liliaceae, including 11 representative species from the genus Lilium and two species each from Fritillaria and Cardiocrinum, and that of Smilax china as an outgroup were selected. A neighbour-joining (NJ) tree was constructed with Mega 6.0 using 1000 bootstrap replicates , Martagon (L. hansonii and L. tsingtauense) and Leucolirion (L. brownii and L. longiflorum). Another group comprised Sinomartagon (L. fargesii) and Leucolirion (L. henryi and L. leucanthum)  and Martagon (L. hansonii and L. tsingtauense) formed a monophyletic clade with a high bootstrap value, indicating a close relationship among these species.To ascertain the phylogenetic status of canthum) . Fritill