Persistent orthostatic hypotension (OH) is a lesser-known complication of lung transplantation (LTx). In this retrospective case series, we describe the clinical manifestations, complications, and treatment of persistent OH in 13 LTx recipients.  We identified LTx recipients who underwent transplantation between March 1, 2018, and March 31, 2020, with persistent symptomatic OH and retrospectively queried the records for clinical information. 2, respectively. Six (46%) patients were deceased at the time of chart abstraction with a median (IQR) posttransplant survival of 12.6 months ; the 7 remaining living patients were a median of 19.6 months  posttransplant. Signs and symptoms of OH developed a median of 60  days after transplant. Patients were treated with pharmacological agents and underwent extensive physical therapy. Most patients required inpatient rehabilitation , and patients commonly developed comorbid conditions including weight loss, renal insufficiency with eGFR <50 , gastroparesis , and tachycardia-bradycardia syndrome . Falls were common . The incidence of OH in LTx recipients at our center during the study period was 5.6% (13/234).  Thirteen patients were included in the analysis, 9 (69%) had underlying pulmonary fibrosis, and 12 (92%) were male. The median age, height, and body mass index at LTx were 68 years, 70 inches, and 27 kg/m Persistent OH is a lesser-known complication of LTx that impacts posttransplant rehabilitation and may lead to comorbidities and shortened survival. In addition, most LTx recipients with OH at our center were tall, thin men with underlying pulmonary fibrosis, which may offer an opportunity to instate pretransplant OH screening of at-risk patients. Lung transplantation (LTx) is a lifesaving procedure for patients with end-stage lung disease; however, it is associated with several complications that impact both quality of life and longevity. Persistent orthostatic hypotension (OH) is an unusual complication of LTx with significant clinical implications. OH is defined as a reduction of systolic blood pressure (SBP) of at least 20 mmHg or diastolic blood pressure (DBP) of at least 10 mmHg within 3 minutes of standing and/or an increase in heart rate ≥20 beats/minute but not exceeding the upper limit of normal [OH may also be part of a clinical syndrome of autonomic dysfunction (AD), which is characterized by cardiac, gastrointestinal, and genitourinary disturbances. AD may present as arrhythmias , ileus, Ogilvie syndrome, gastroparesis, urinary complaints , and bowel complaints (diarrhea and constipation) .Physical therapy and rehabilitation are key components of LTx recovery . Despiten = 9), 3 of these patients were symptomatic, and another 3 had supine hypertension [Persistent OH, alone or in conjunction with AD, can lead to significant comorbidities after LTx and can also impair posttransplant rehabilitation. In a pilot sample of 30 consecutive stable LTx recipients from the University of Zurich, the prevalence of undiagnosed OH was 30% . Patients who underwent LTx between March 1, 2018, and March 31, 2020, at Norton Thoracic Institute, Phoenix, Arizona, with persistent symptomatic OH any time after LTx were included. OH was defined as a reduction of SBP of at least 20 mmHg or a reduction of DBP of at least 10 mmHg within 3 minutes of standing and/or an increase in heart rate ≥20 beats/minute but not exceeding the upper limit of normal [Induction therapy included a high-dose corticosteroid (methylprednisolone) before perfusion of each lung allograft and antilymphocyte therapy with basiliximab, rituximab, or antithymocyte globulin (ATG). At our center, the vast majority of patients are induced with basiliximab, with the exception of highly sensitized patients and patients undergoing retransplant who developed allograft failure as a result of recurrent or persistent acute cellular or antibody-mediated rejection. Highly sensitized patients typically receive rituximab or ATG at the time of transplant; whereas, patients undergoing retransplant commonly receive ATG. The maintenance immunosuppressive regime was uniform across the study period and consisted of a corticosteroid (prednisone), an antiproliferative agent (mycophenolate mofetil or mycophenolic acid), and a calcineurin inhibitor ((CNI), tacrolimus or cyclosporine). A selective T cell costimulation blocker (belatacept) was used to reduce CNI-induced nephrotoxicity in patients with progressive and advanced chronic kidney disease.Descriptive statistics were used. Median and interquartile range (IQR) were reported for continuous variables. Frequencies and percentages were reported for categorical variables. The Kaplan–Meier method and log-rank test were used for comparison of survival estimates between included subjects and a control group of remaining subjects who underwent LTx during the study period. Analyses were performed with Stata Statistical Software, Release 13 .During the study period, 234 patients  with a median (IQR) age of 67.2 years  underwent LTx at our center.n = 12) were male. Median height and body mass index (BMI) at LTx were 70  inches and 26.6  kg/m2, respectively; however, 2 of the patients had a BMI <18.5 kg/m2. Most patients had idiopathic pulmonary fibrosis , and the remaining patients had a either chronic obstructive pulmonary disease  or combined pulmonary fibrosis and emphysema  (Thirteen patients with post-LTx OH were included in this series. The incidence of OH in LTx recipients at our center during the study period was 5.6% (13/234). The median age of the study cohort at the time of LTx was 68  years, and 92.3%  .n = 11) of patients and grade IV was observed in 15.4% (n = 2). Median oxygen requirement at rest was 3  liters per minute, and 15.4% (n = 2) of patients required continuous positive airway pressure for sleep apnea. The median pre-LTx forced expiratory volume in one second (FEV1) was 45% , and the median forced vital capacity (FVC) was 49% ; the lowest FEV1 was 16% of predicted, and the lowest FVC was 18% of predicted. Pre-LTx hypercapnia with nonobstructive pathology was seen in 30.8% (n = 4) of patients. None of the patients had valvular heart disease.All patients were functionally impaired prior to LTx; New York Heart Association Grade III functional class was observed in 84.6% (n = 8), only 1 patient carried a pre-LTx diagnosis of OH and was treated with midodrine. Pre-LTx arrhythmias were common  with atrial fibrillation (AFib) in 23.1% (n = 3) of patients and left bundle branch block in 7.7% (n = 1). Pre-LTx diabetes was present in 30.8% (n = 4) of patients; 23.1% (n = 3) had peripheral neuropathy, and 1 patient was insulin-dependent. Gastroparesis was present in 15.4% (n = 2) of patients, one of whom was diabetic. Osteopenia or osteoporosis was present in 30.8% (n = 4) of patients, and 15.4% (n = 2) had chronic kidney disease (stage III).Although systemic hypertension was common . Cardiopulmonary bypass was used in 1 patient and extracorporeal membrane oxygenation in 2 patients. Concomitant left atrial appendage clipping was performed in 15.4% (n = 2) of patients. A thoracic epidural catheter was placed before induction of anesthesia uneventfully in 76.9% (n = 10) of patients. Intraoperative arrhythmias were seen in 38.5% (n = 5) of patients: AFib needing cardioversion in 23.1% (n = 3), supraventricular tachycardia in 7.7% (n = 1), and ventricular tachycardia in 7.7% (n = 1). Intraoperative right ventricular function and pulmonary vein velocity were normal in all patients. Median blood loss was 1  liter, and blood products were transfused in 46.2% (n = 6) of patients.Most patients underwent bilateral LTx  developed grade 3 primary graft dysfunction (PGD), and the median time to extubation of these patients was 6 days ; 1 of these patients had a tracheostomy for prolonged mechanical ventilation. The median time to extubation was 4 days  in the entire cohort.Most patients developed cardiac arrhythmia in the immediate postoperative period. Nine patients (69.2%) developed AFib with rapid ventricular response within the first postoperative week, which was managed with antiarrhythmic agents and cardioversion. A dual-chamber pacemaker was placed in 1 patient 2 weeks after LTx, and radiofrequency ablation was performed in 1 patient 5 months after LTx.n = 13) required a >1-week stay in the ICU; the median duration of hospitalization was 40 days , and 76.9% (n = 10) of patients were discharged to an inpatient acute rehabilitation facility. Readmission within the first year of LTx was common; 84.6% (n = 11) had ≥1 readmission within the first year, with a median number of 2 readmissions. The median duration of readmission hospital stay was 18 days , and 3 patients required readmission to an inpatient acute rehab facility.Protracted post-LTx hospitalization was common. All patients (n = 11), limited ambulation , falls , and muscle weakness . Patients also commonly developed comorbid conditions including weight loss (median decrease in BMI compared to pre-LTx BMI was 2.7 kg/m2) and renal insufficiency with eGFR <50% . Fractures sustained during mechanical fall episodes occurred in 46.2% (n = 6) of patients, and 38.5% (n = 5) required orthopedic surgery. Deconditioning and failure to thrive were present in all patients (n = 13) with decubitus ulcers in 15.4% (n = 2). Clinical depression was treated in 30.8% (n = 4) of patients, and 1 patient had generalized anxiety related to fear of syncope.OH was typically diagnosed early in the post-LTx course with 12 patients (92.3%) diagnosed within 3 months of transplant. The median decrease in SBP and DBP from supine to standing position was 56  and 25  mmHg, respectively. Patients commonly reported dizziness  also had additional symptoms of AD that could not be attributed to OH alone. The presentation of AD included urinary complaints , bowel dysregulation , gastroparesis , ileus , Ogilvie syndrome with cecal dilation >10 cm , labile blood glucose levels requiring frequent insulin dose adjustments , tachycardia-bradycardia syndrome , sweating paroxysms with new-onset peripheral neuropathy , and dry mouth with oral mucosal fragility   .Testosterone levels were measured in 8 male patients, 7 of whom had hypogonadism with a median level of total testosterone, bioavailable testosterone, and free testosterone of 151 ng/dL, 59 ng/dL, and 25 pg/mL, respectively. Six (46%) of the patients were deceased at the time of chart abstraction with a median posttransplant survival of 12.6 months ; 7 living patients were a median of 19.6 months  posttransplant. Follow-up was available for a median of 17.7  months, and median survival was 17.4  months.n = 13), fludrocortisone , and droxidopa , fluid retention, and hyponatremia, with the latter two particularly common among patients treated with fludrocortisone. In an attempt to reduce tacrolimus-induced OH, 1 patient was transitioned to a cyclosporine-based regimen and 1 patient was treated with belatacept, which allowed us to target lower tacrolimus troughs. Compression socks, TED hose, and abdominal binders were used in all patients. A port for outpatient administration of intravenous fluids on a biweekly basis was placed in 1 patient. Despite ≥2 treatment strategies, orthostatic signs were challenging to treat in 53.8% (n = 7) of patients. Four patients were on triple therapy ; none of these 4 patients were alive at the end of the study period. Of those who were alive (n = 7), OH was self-limiting in 1 patient and recalcitrant to therapy in 6 patients who still require some form of treatment.Pharmacologic and mechanical agents were used to treat OH. Commonly used medications included midodrine  had additional symptoms of AD with resultant significant morbidity and mortality. The etiology of OH and AD after LTx remains unclear; however, several hypotheses have been proposed. One hypothesis focuses on vagus nerve injury. The right and left vagus nerves contribute to the cardiac, pulmonary, and esophageal plexuses and enter the diaphragm through the esophageal hiatus as the posterior and anterior vagal trunks. In light of its course through the thoracic cavity, it may be subject to mechanical, ischemic, and hypothermic injury at the time of transplant, and the manifestations of the injury may vary depending on which branch was affected.We identified 13 LTx recipients at our center with OH; 100% (n = 1) developed OH and AD 17 months after surgery. Notably, the patient who developed OH and AD >6 months after transplant was >65 years old. OH is a well-described complication of aging and senescence [α-1 adrenergic vasoconstrictor response to sympathetic stimuli, and parasympathetic activity may be altered. In addition, renal salt and water conservation, comorbidity burden, and impaired thirst response to dehydration may be associated with poor homeostasis to orthostatic stress [Although intraoperative vagus nerve injury may explain OH and AD in the early post-LTx course, additional factors need to be considered since 7.7% , diarrhea, labile blood glucose levels, tachycardia-bradycardia syndrome, gastroparesis, ileus, Ogilvie syndrome with cecal dilation >10 cm, sweating paroxysms with new-onset peripheral neuropathy, and dry mouth with oral mucosal fragility . These sPersistent OH can be treatment refractory despite the use of both pharmacological and mechanical therapies. Our standard protocol is to rule out adrenal insufficiency and use intravenous fluids, mechanical strategies , midodrine, fludrocortisone, and droxidopa on a case-to-case basis, along with outpatient physiotherapy. Escalating doses of medications and polypharmacy may lead to untoward side effects including supine hypertension, edema, and electrolyte disturbances. Mechanical devices such as TED hose may be difficult to put on, particularly by weak patients, and abdominal binders may restrict breathing and food intake. The complications of AD are also difficult to treat. For example, gastroparesis often required multiple interventions, including endoscopy with pyloric dilation and botulinum toxin injection, and patients are often left malnourished, nauseous, and feeding tube dependent.Our report has several limitations. First, it is a descriptive, single-center case series with low levels of evidence. Nevertheless, to the best of our knowledge, this is the first series in the LTx literature describing OH and AD as possible complications of LTx. Second, we have likely underestimated the incidence of post-LTx OH and AD as patients are not routinely screened for these conditions, and therefore, minimally symptomatic patients were not identified. Third, since the clinical presentation of AD is so variable, it is difficult to differentiate between symptoms attributable to AD versus those attributable to other etiologies. However, despite these challenges, the syndrome of OH and AD is worthy of formal description in the literature as well as ongoing study as it has a profound impact on post-LTx morbidity and mortality. In fact, as a result of these observations after LTx, our center has incorporated a formal assessment of orthostasis as part of the standard pre-LTx evaluation. However, further large studies are needed to better define this syndrome and its impact on the clinical outcomes of lung transplant recipients.OH with concurrent AD is a newly described complication of LTx that leads to significant comorbidities, impairs post-LTx rehabilitation, and may be associated with shortened post-LTx survival. The majority of LTx recipients with OH at our center were tall, thin men with underlying pulmonary fibrosis, which may offer an opportunity to instate pretransplant OH screening of at-risk patients. Further studies are needed to identify risk factors for OH and AD as well as to identify the etiology and pathophysiology of this syndrome.


Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical single-nucleotide-polymorphism (SNP) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cutoff threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis with human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly available for application by users, including those with little bioinformatics knowledge.Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for  Bacillus anthracis is a Gram-positive, rod-shaped, spore-forming bacterium and the etiological agent of anthrax in wildlife, livestock, and humans worldwide (B. anthracis produces spores that remain viable in the soil and can be dispersed by wind and vectors (B. anthracis has two toxin-carrying plasmids (pXO1 and pXO2) that can also be present in other Bacillus species leading to atypical anthrax-causing strains  compared to the Ames Ancestor strain (NC_007530.2)  and multilocus variable-number tandem repeat (VNTR) analysis (MLVA). Canonical SNP typing employs a selection of representative branch-specific SNPs for phylogenetic branches, which can be used to define key phylogenetic positions for descendant strains  . Standarstrains 79. Based strains 7–10, B. alyzing 8 , 15 (9),lyzing 8 , 25 13)B. anthralyzing 8  VNTR locathogens . While cathogens , internaathogens . In factathogens . TherefoB. anthracis strains were used for scheme setup, application, and validation . First, we selected 57 B. anthracis genomes (referred to as query genomes) available at RefSeq (April 2019) and used for the cgMLST scheme setup.A total of 753 B. anthracis into major clades and subclades as previously defined and accepted by anthrax laboratories around the globe .Then, for scheme application and validation, we used three data sets. Data Set 1 was aimed at (i) investigating the overall cgMLST scheme typeability  and (ii) investigating the backward compatibility of the new scheme to standard canonical SNP grouping to classify he globe . This dahe globe , 20–22 aB. anthracis strains isolated from heroin users in four European countries  includes (i) eight epidemiologically related strains from the three most recent anthrax outbreaks in Germany in 2009, 2012, and 2014, as well as (ii) 35 epidemiologically unrelated strains from 35 different outbreaks in Italy with one strain per outbreak (Table S1). Strains of this data set were sequenced in this study, except three German strains previously sequenced using the Ion Torrent or Pacific Bioscience platform –25. Data–ountries  (Table SB. anthracis strains (Data Set 2) were sequenced using Illumina MiSeq . DNA was extracted using the Genomic-tip 100/G and genomic DNA buffer kit  for the German strains. For the 35 strains from Italy, the DNAeasy blood and tissue kit  was used. Paired-end sequencing libraries were prepared using the Nextera XT DNA Library Preparation kit  with an average sequencing depth of between 40× and 104× for the strains. Genome assembly was performed for sequence data produced in this study or downloaded from the NCBI using shovill v.1.0.4 (option -trim [https://github.com/tseemann/shovill]) for paired-end Illumina data or SPAdes v.3.12.0 (-careful option) ( option)  for sing option) .B. anthracis using canonical SNPs and Bayesian analysis to select representative genomes of all phylogenetic groups. For that, we used 172 genomes from the RefSeq database (Table S1). Briefly, we estimated the pairwise average genomic nucleotide identity between all genomes using pyani v.0.2.9 (module ANIm)  .For scheme setup, we first investigated the genetic population structure of le ANIm)  and Fastle ANIm) . We thenle ANIm)  to buildle ANIm)  using Pyle ANIm)  and canole ANIm)  and Van NC_007530.2) was used as a reference. The following criteria were applied to exclude reference genes that (i) have a length of less than 50 bp (“minimum length filter”), (ii) lack a start and/or a stop codon (“start codon filter” and “stop codon filter”), (iii) have paralogues with identity of >90% and overlap of >100 bp (“homologous gene filter”), and (iv) are overlapping large genes (“gene overlap filter”). Retained genes from the reference strain were then searched through BLAST v.2.2.12 against the 57 query genomes. Genes lacking a single start or stop codon in 80% of the query genomes as well as genes that match plasmid sequences by BLAST analysis (NC_007322.2 and NC_007323.3) were excluded. BLAST thresholds include 90% identity and 100% coverage, with parameters involving a word size of 11, mismatch penalty of 1, match reward of 1, gap open costs of 5, and gap extension costs of 2.The cgMLST Target Definer tool v.1.5 within Ridom SeqSphere+ v.7.1.0  was usedN50 of less than 40 kb from the evaluation data sets. For all investigated genomes, cgMLST allelic profiles  were compared pairwise (untypeable genes were ignored), and the resultant calculated pairwise distances were used to generate a neighbor-joining (NJ) tree and a minimum-spanning tree (MST) using SeqSphere+. Genes were regarded untypeable if they were not assigned an allele number because of no BLAST match or due to the incidence of internal stop codons or ambiguities in the gene.The application of the cgMLST was performed using Ridom SeqSphere+ v.7.1.0 . This inBacillus cereus database (https://pubmlst.org/) together with gene-allele libraries of the B. anthracis genomes (https://rest.pubmlst.org)  and pXO2  in the database as previously described (https://gitlab.com/bfr_bioinformatics/chewieSnake]) was used with default options, including 0.6 as the minimum BLAST score ratio for locus similarity (–bsr 0.6) and 20% length boundaries for allele definition (–st 0.2). GrapeTree (The developed cgMLST scheme was incorporated into the PubMLST  genomes . In addilst.org) . We haveescribed . Finallyescribed , a commaescribed  applicatrapeTree  was usedrapeTree .We used Parsnp v.1.2  to identPRJNA656733. Additional sequence data analyzed in this work are available in the NCBI SRA database and listed in Table S1.Raw sequencing data generated in this study are available under NCBI Bioproject accession no. n = 220) or repetition (n = 26) or because the genes had internal stop codons (n = 265)  in any of the 57 query genomes or carrying an internal stop codon (n = 2) in >80% of the query genomes were discarded. Following these criteria, we determined 3,803 core genes were suitable for cgMLST typing comprising 58.2%  of the reference genome . These genes have an average GC content of 36.02% and average length of 801.3 bp .Of the 5,357 genes with CDS in the chromosome of the Ames Ancestor strain, 511 genes were excluded due to gene overlapping (n = 265) . AdditioB. anthracis genomes to evaluate the applicability of the cgMLST scheme (see Data Set 1 in Table S1). Twelve genomes (out of 596) did not belong to the highly clonal B. anthracis lineage and had less than 90% of the cgMLST targets. The pairwise ANI values for these strains were 93 to 98% compared to the B. anthracis reference genome (Table S1). For the remaining 584 strains (out of 596) of classical B. anthracis lineage, the 3,803 cgMLST targets were identified on average with 99.1%  per genome, with more than 99% of the cgMLST genes being conserved and typed in a total of 382 genomes (∼65.4% of the data set). Decayed genes  represented the majority of untypeable genes  (Table S1). The 584 genomes were clustered into 473 cgMLST profiles , resulting in a Simpson’s index of discrimination of 0.998 . This was slightly less than the number of unique SNP profiles (n = 508) identified for the 584 genomes based on 15,081 whole-genome SNPs, with a Simpson's index of diversity of 0.999 . Furthermore, there was a high topological concordance between cluster analysis inferred from cgMLST and the maximum likelihood tree based on whole-genome SNPs . These results emphasize that the developed scheme provides enough discrimination between strains and is highly concordant to whole-genome SNPs.We used a set of 596 publicly available B. anthracis using the cgMLST profiles clearly defines the three different A, B, and C clades of B. anthracis (n = 481), followed by clade B (n = 87 [14.8%]), and then clade C (n = 7 [1.9%]). Clade A splits into six subclades, with subclade “Ancient A” being basal to all other subclades and the subclades “V770,” “Sterne/Ames,” and “Australia 94” having evolved from a single branch (A.Br.004). Only one strain could not be assigned to any of the defined subclades of clade A. The subclade “TransEurAsia”  is the most abundant, comprising ∼32% of the data set (n = 188), with a very wide geographic distribution in Europe (n = 88), North America (n = 72), Asia (n = 23), South America (n = 3), and Africa (n = 2). Although trees based on cgMLST and SNPs resulted in the same subclade definition, there were some differences in the branching points between both trees that led to switches in the positions of a few groups of the TEA subclade.As presented in nthracis . Clade AB. anthracis . We also identified specific SNPs within the cgMLST targets that are specifically present in each of the B. anthracis (sub)clades but simultaneously absent in all other genomes . These group-specific SNPs are positioned within the identified cgMLST targets and can serve as further targets for fast identification of new strains (Table S4).The average allele distance within and between each of the identified (sub)clades and the average allele distance of each (sub)clade to the closest sample in the other groups show that cgMLST can discriminate between the established subgroups of n = 8) and strains that have no epidemiological relationships (n = 35). For the epidemiologically linked strains, we investigated sequence data of the three most recent anthrax outbreaks that occurred in the cattle population in Germany since 2009 . The 2009 outbreak was distant from the other outbreaks and caused by a clade B strain. Using cgMLST, a maximum of five allelic variations could be identified between isolates from a single outbreak , Apulia (n = 12), Sicily (n = 5), Sardinia (n = 2), Tuscany (n = 2), Veneto (n = 2), Lazio (n = 3), Campania (n = 2), Calabria (n = 1), Lombardia (n = 1), and Umbria (n = 1). The strains were recovered from cattle (n = 21), sheep (n = 8), goats (n = 4), a horse (n = 1), and a human (n = 1) (Table S1). The cgMLST analysis confirmed classical canonical SNP typing in which one strain belong to clade B (B.Br.004) and one strain of subclade Ancient A (A.Br.034). The remaining 33 strains belong to the TEA (A.Br.008) group (28 strains of A.Br.011/009 and 5 strains of A.Br.008/011 [Pasteur]). MLVA typing classified 31 strains as singletons. Two MLVA genotypes, MLVA31-8 and MLVA31-27, comprised two strains each. However, MLVA31-8 was found to contain strains that differed in 58 alleles, as determined using cgMLST. MLVA31-27 contained strains that differed by six alleles, as determined using cgMLST . The strains were isolated from human cases with injectional anthrax, with in some cases several isolates being recovered from a single patient  available in the database. In addition, the database includes a hierarchy of cluster definitions for the B. anthracis cgMLST profiles at thresholds of 200, 100, 50, 25, 10, and 5 different alleles, using the single-linkage method. This is in order to explore potential local and regional transmission chains of B. anthracis strains in future investigations. Along with cgMLST targets, 1,263 accessory genes are hosted at PubMLST that can be combined with the cgMLST profiles to perform a whole-genome-based MLST (wgMLST) to further improve interstrain resolution if required.In order to facilitate cooperation in the control of lst.org/ . New strhttps://doi.org/10.6084/m9.figshare.13220735). All cgMLST profiles defined using SeqSphere for the 584 B. anthracis strains in the evaluation data set have been correctly identified using chewBBACA. However, chewBBACA split some cgMLST profiles into several profiles, leading to a slightly larger number of profiles being described. A total of 499 cgMLST profiles were defined using chewBBACA, compared to 473 profiles identified using SeqSphere , resulting in a Simpson’s index of diversity of 0.999 . Using cgMLST results from chewBBACA as the primary typing method, the adjusted Wallace value was 0.995 , while using SeqSphere as the primary typing method, the adjusted Wallace value was 0.678 .Besides PubMLST, we applied the freely available tool chewBBACA  to the dhttps://pubmlst.org/. However, the method suffers poor resolution for B. anthracis, which is a highly monomorphic and evolutionarily stable pathogen. The high virulence potential of this pathogen, its worldwide and unknown dispersal of spore-contaminated fields (“champs maudit”) in the environment but also its potential use as an agent for bioweapons motivate in-depth genotyping for routine surveillance and outbreak investigation. As such, whole-genome SNP analysis has been applied by many laboratories for detailed subtyping of B. anthracis  technologies generate sequence data for bacterial pathogens at high throughput, at affordable costs, and with high speed. Due to the advantages it offers, the use of NGS in clinical laboratories has become essential for outbreak investigations and surveillance. However, challenges associated with NGS include bioinformatics analysis and standardized analytic workflows and nomenclature for pathogens and clades. In the pregenomic era, MLST was regarded as the “gold standard” for typing of many pathogens thanks to the unified allele nomenclature and centralized gene allele library that can be accessed via public web services: e.g., nthracis , 15. Howre tools  togetherre tools  may faciB. anthracis cgMLST scheme involved the analysis of an extensive collection of B. anthracis genomes with very different geographical origins to determine the species population structure. The explicit definition of B. anthracis clades and subclades using cgMLST was characterized by a high degree of congruence to the well-established and widely used canonical SNP nomenclature. The results were also very consistent with the methods for SNP typing of the whole genome , corresponding to a low number of alleles  in all genomes studied. Nevertheless, shifts in the phylogenetic positions of a few groups within the TEA subclade (A.Br.008) were observed in the cgMLST-based phylogenetic tree. This can be attributed to the abundance of simultaneous descendant lineages and the frequent occurrence of extremely short phylogenetic branches in the TEA subclade  carried the anthrax plasmid genes of pXO2 (Table S1). These results on one hand indicate that the developed cgMLST system can reliably distinguish the classical B. anthracis lineage  without the need for a prior estimation of ANI for the strains or phenotypic identification as shown with cgMLST . On theBrucella melitensis and Francisella tularensis, where 81 and 65% of the genes with coding sequences are used, respectively  and reference-based SNP approaches, which can provide slightly higher resolution than cgMLST . Howeverectively , 47.B. anthracis. This scheme reliably identifies B. anthracis strains down to the strain level and can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis.In conclusion, we present a cgMLST scheme that provides a high-resolution strain genotyping for the highly monomorphic bacterium


Haemophilus and Porphyromonas for RHG; Bacilli for HAP and Prevotella for TI. In conclusion, RHG, the biotic substratum, is able to support a highly viable and diverse microbiome. In contrast, the viability and diversity of the biofilms on the abiotic substrata were influenced by the substrata type, pH of the environment and the richness of the growth media. These results suggest that the host  plays a vital role in the oral ecology.The microbial composition of a specific oral niche could be influenced by initial bacterial adherence, nutrient and physiological property of the local surface. To investigate the influence of nutrient and surface properties on microbial composition, saliva-derived biofilms were grown in agar on three substrata: Reconstructed Human Gingiva (RHG), a hydroxyapatite (HAP) surface, and a titanium (TI) surface. Agar was mixed with either Brain Heart Infusion (BHI) or Thompson (TP) medium. After 1, 3, or 5 days, biofilm viability (by colony forming units) and microbiome profiles (by 16 S rDNA amplicon sequencing) were determined. On RHG, biofilm viability and composition were similar between BHI and TP. However, on the abiotic substrata, biofilm properties greatly depended on the type of medium and substratum. In BHI, the viability of HAP-biofilm first decreased and then increased, whereas that of TI-biofilm decreased in time until a 6-log reduction. In TP, either no or a 2-log reduction in viability was observed for HAP- or TI-biofilms respectively. Furthermore, different bacterial genera (or higher level) were differentially abundant in the biofilms on 3 substrata:  Niche-specific structure maintains the ecological stability of the microbiome community and ensures a healthy balance between host and microbes. The loss of this structure can eventually lead to diseases6. Hence, identification of major factors influencing the niche-specificity is critical for maintaining a healthy microbial ecology.The human body is colonized by millions of microbes. These microbes form diverse and dynamic microbial communities distributed over various body habitats, including the skin, gut and oral cavity. High-throughput sequencing technologies revealed that the microbial community of each niche has its own distinct biodiversity features7. These sites include natural substrata, such as hard (teeth) and soft (mucosa) tissues, and therapeutic substrata, such as dental implants and dentures, where various microbes can colonize and flourish. Oral microbiomes also show niche-specific structures: separate sites within the oral cavity, whilst being bathed in the same saliva, harbor considerably different microbial communities9. For example, the keratinized gingiva is mainly occupied by Gemella haemolysans and Streptococcus mitis group species, whereas the supragingival plaque on tooth surfaces is dominated by Rothia, Streptococcus and Corynebacterium10. Thus, Welch et al. proposed a “Site-Specialist Hypothesis”: an oral microbe can actively colonize its preferred site, grow and divide there. When outside its preferred site, it will be in much lower abundance and display altered metabolism, gene expression and spatial organization.The oral cavity is a complex habitat, comprising of diverse sites characterized by different anatomic structures and physiochemical factorsVeillonella and Neisseria in the  peri-implant microbiome11. However, an in vitro study investigated the effect of different abiotic substrata (glass versus hydroxyapatite discs) and two types of growth media on the composition of saliva-derived microcosm biofilms and revealed that the growth media, not the substrata, mainly affected the structure of the microbiome12. Therefore, the influence of nutrients, substrata and their interactions on the niche-specific microbiome structure warrants further investigation.According to this hypothesis, the ability of a microbe to adhere to a surface, directly or indirectly, determines its site-specificity and thereby the local microbial community. Bacterial adherence is only the first step in biofilm development. Other factors during biofilm formation, e.g. nutrients supplied by saliva or local substrata, also affect the niche-specific microbiome structure. It was demonstrated that titanium released from dental implants led to the reduction of alpha-diversity and enrichment of 13), were examined. BHI is a protein-rich medium. Multi-species oral biofilms have been successfully cultured in BHI, supplemented with menadione and hemin15. TP is a complex medium containing proteose peptone, mucins and serum, which supports nutrient requirements of subgingival biofilms17. Three substrata were used to support the growth of biofilms: reconstructed human gingiva , hydroxyapatite discs (HAP) and titanium discs (TI), representing 3 oral sites respectively: gingiva, tooth and dental implant. Previously, RHG has shown similar reactions to oral multi-species biofilms as native gingiva, such as the induction of defensive cytokines and antimicrobial peptides19. RHG represents a biotic substratum, whereas HAP and TI are abiotic substrata.Our aim was to investigate the influence of nutrient and oral substrata on the community structure of a saliva-derived microcosm biofilm grown for 1, 3 and 5 days. Two biofilm growth media, BHI (brain heart infusion broth) and TP (Thompson medium10CFU/biofilm at inoculation (dotted line) to 8.5 ± 0.5 log10CFU/biofilm after 1 day and remained at this level until day 5. However, the viable counts of those on HAP decreased to 6.4 ± 0.1 log10CFU/biofilm after 1 day but increased to 8.4 ± 0.1 log10CFU/biofilm on day 3 and 5. The viable counts of those on titanium (TI), decreased over time and was below detection limit (2.5 log10CFU/biofilm) from day 3. In contrast, with TP, biofilm viability was much less affected by the type of substratum. On RHG and HAP, biofilm viable counts reached approximately 8.4 log10CFU/biofilm after 1 day and remained at this level until day 5. On TI, biofilm viable counts significantly decreased over time. However, the reduction was within 1.9 log10CFU/biofilm, much less than when biofilms were grown in BHI. The unexposed RHG showed a differentiated and stratified epithelium on a fibroblast-populated collagen hydrogel. Application of the agar, with or without microcosm biofilm, moderately disrupted the epithelium in localized places, irrespective of the growth media .In order to understand the growth media- and substrata-dependent shifts in biofilm compositions over time, principal component analysis (PCA) and LEfSe were performed. The PCA plot identified four major clusters Fig. . HAP- anHaemophilus and Porphyromonas; HAP-biofilms had a higher abundance of Bacilli and TI biofilms had a higher abundance of Prevotella  and substratum type influenced the microbiome structure. When the biofilms grew on the surface of the biotic substratum RHG, the microbial profiles were independent of the type of growth media. In contrast, when the biofilms grew on abiotic substrata, HAP and TI, the microbial profiles were considerably affected by the type of growth media. Moreover, different bacterial genera (or higher level) were differentially abundant in the biofilms on various substrata: Haemophilus and Porphyromonas for RHG; Bacilli for HAP and Prevotella for TI.Niche-specific community structures are important for the stability of microbial communities in the oral cavity and hence critical in maintaining healthy homeostasis12. However, our results indicate an interactive effect of growth media and substratum type on the microbiome structure. When the medium was TP, our results were in line with Li et al.12, where the biofilm viability and composition were not affected by the substratum type. TP medium was developed to mimic the nutrients available in the subgingival environment13. It is a complex medium comparable to the biofilm media used in Li’s study, which might explain the similar findings. In contrast, when using BHI medium, the biofilms were affected by the types of abiotic substrata: viability of HAP-biofilm first reduced and then recovered, but viability of TI-biofilm continuously decreased resulting in different species becoming predominant. To understand the observed decreasing viability in TI-biofilm and reduced Shannon index in HAP-biofilm when grown in BHI, we measured the pH of all biofilms and found that the biofilm pH was neutral except the pH of TI-biofilm grown in BHI, which was around 5.5 throughout the entire experiment. Hence, it is possible that the low buffer capacity of BHI was not sufficient to neutralize the acid produced by the biofilms on the surfaces of TI, resulting in low viability of TI-biofilm. In the case of HAP-biofilm, the initial pH-drop possibly led to the dissolution of HAP which subsequently prevented the pH from dropping further. As a result, the initial pH drop led to reduced biofilm viability followed by a neutral pH favoring the growth of specific bacterial species, e.g., Staphylococcus. Taken together, our data indicated that the microbial composition was shaped by a combination of multiple factors, including nutrient availability, environmental pH and substratum type.A previous study reported that the growth media , not the substrata (glass versus HAP), affected the microbiome structure of in vitro biofilms23. Although BHI is a nutrient-rich medium, it misses substances, like haemin and vitamin K, which are critical for the growth of black-pigmented bacterial species, e.g., Porphyromonas gingivalis24. BHI supported a highly diverse bacterial community in vitro only when supplemented with mucins, haemin and vitamin K21. In another study, even the supplemented BHI was reported to cause low viability and low species richness in biofilms22. Therefore, our results indicate that living RHG tissues are able to provide key nutrient components for the growth of various bacteria and supported a highly diverse microbiome. Furthermore, the neutral pH of RHG-biofilm grown in BHI indicated that RHG might either neutralize the pH directly or reduce the acid production indirectly by limiting the growth of acid producing bacteria. Histological analysis of RHG revealed a moderate disruption in RHG epithelium both in agar and microcosm + agar groups compared to unexposed RHG which was probably caused by exposing to the high temperature of unsolidified agar (around 50 °C) during inoculation. We are currently exploring an improved inoculation method in order to maintain an optimal epithelium after application of agar. Nevertheless, our data do convincingly demonstrate the sufficient nutrient support of RHG to the microcosm biofilms over time.Here we investigated the composition of oral multi-species biofilms grown on the surface of a biological tissue. Surprisingly, BHI, without any additional supplements, did not lead to any loss in viability or low species richness in RHG-biofilm, which is in contrast to HAP-biofilm and TI-biofilm. Microbial viability and composition in BHI were the same as those in TP. The components in TP, such as mucins and proteose peptone, were believed to be crucial for supporting the cultivation of fastidious bacteria and increasing the proportion of anaerobesNeisseria and strictly anaerobic Porphyromonas through our network analysis. Notably, all biofilms were grown in 7.5% CO2 with ambient air in this study. Surprisingly, the strictly-anaerobic bacteria, Porphyromonas, Fusobacterium and Prevotella, were detected in our microcosm biofilms. Lambooij et al.25 showed that Candida albicans was able to induce the growth of strictly-anaerobic oral bacteria in air through mitochondrial activity. In order to illustrate the interaction between aerobic and anaerobic bacteria, we performed an additional experiment, where the strictly-anaerobic P. gingivalis was cultured with or without aerobic Neisseria perflava. The results clearly showed that the full growth of P. gingivalis in 7.5% CO2 could be achieved only when N. perflava was present  could reduce the composition similarity between biofilms and inocula and recommended 5% serum in biofilm growth medium. Our model showed that high similarity could be achieved even without serum addition to the growth medium.To exclude the effect of microbial site-specific adhesion, we grew microcosm biofilms by mixing the saliva inoculum with 0.7% agarose. This gel-entrapped biofilm model allows microbes to form a dense and structured biofilm within the gel matrix and mimics wound or lung biofilms in vivo19 showed that the biofilm growth condition  seemed to have greater influence on biofilm compositions than the source of saliva inoculum . However, another study found that the in vitro biofilm compositions varied among different saliva donors17. Further studies are needed to understand the donor effect on in vitro microcosm biofilms.In this study, all microcosm biofilms were grown from the saliva collected from one donor. A previous studyIn conclusion, this in vitro study demonstrated that the type of media and substrata interactively influenced viability and composition of the microcosm biofilms. RHG, the biotic substratum, was able to support a highly viable and diverse microbiome, even in nutrient poor growth media which lacks some essential growth elements. In contrast, the viability and diversity of the biofilms on the abiotic substrata were influenced by the substrata type, pH of the environment and the richness of the growth media. These results suggest that the host  plays a vital role in the oral ecology.28. In detail, fibroblasts (8 × 105 cells/ml) were mixed with a collagen hydrogel and transferred to transwell inserts of 0.4 μM pores  in a 6-well tissue culture plate. After 24 h, keratinocytes were seeded on top of the fibroblast-collagen hydrogel at a density of 5 × 105 cells/well. After 3 days submerged, RHGs were then lifted to the air-liquid interface to induce epithelial differentiation and cultured for another 10 days in the RHG differentiation medium. The RHG differentiation medium contains DMEM/Ham’s F12 , supplemented with 1% Fetal Clone III , 1% penicillin-streptomycin (Gibco), 0.1 μM insulin , 2 μM hydrocortisone (Sigma-Aldrich), 1 μM isoproterenol (Sigma-Aldrich), 10 μM carnitine (Sigma-Aldrich), 10 mM l-serine (Sigma-Aldrich), 0.4 mM l-ascorbic acid (Sigma-Aldrich) and 2 ng/mL epidermal growth factor (Sigma-Aldrich). The medium was changed every 3 days, and the RHGs were grown at 37 °C, 7.5% CO2. One day before the addition of microcosm biofilms, the RHG was refreshed with the same medium but without penicillin-streptomycin and hydrocortisone. During microcosm biofilm exposure, RHG, cultured in 6-well plates, were refreshed once daily with the RHG differentiation medium without penicillin-streptomycin and hydrocortisone.RHG was constructed using immortalized human gingival keratinocyte  and fibroblast  cell linesStandardized HAP discs  and TI discs  without any surface treatment were sterilized by autoclaving before use.Unstimulated saliva was collected from a single healthy donor who was systemically healthy, had no periodontal disease or active caries, and had not taken any antibiotics for at least 3 months. The study was approved by the Medical Ethical Committee of the University Medical Center, Amsterdam UMC (document number 2011/236) with signed and informed consent from the donor. On the day of saliva collection, the donor was asked not to brush the teeth for 24 h and not to drink or eat for at least 2 h prior to sampling. The collected saliva was mixed with 60% glycerol (ratio of 1:1) and stored at −80 °C before use.29 containing biofilm growth medium, BHI or TP, on 3 substrata. The Brain heart infusion broth (BHI) was prepared following the instruction of the manufactory (BD Difco). The Thompson medium (TP) contains 0.25% pig gastric mucin, 0.07% Bacto proteose peptone (BD Biosciences), 0.1 mol/L K2HPO4, 0.3% trypticase peptone , 0.5% yeast extract, 0.25% KCl, 10 μg/ml hemin, 1 μg/ml menadione, 0.05% cysteine hydrochloride, 1 mmol/L lysine, 1 mmol/L glycine, 1 mmol/L urea and 5 mmol/L arginine13. In contrast to original TP, the TP in this study does not contain fetal horse serum.The microcosm biofilms were grown in 0.7% agar (BD Biosciences)7 CFU of total viable cells per sample and inoculated onto the surface of each substratum . The reduced volume on air-exposed RHG was used to prevent contamination of culture medium from the overflow of the inoculum during co-culture. For RHG, a control of 50 μL BHI-agar or TP-agar without saliva inoculum was included as well as an unexposed RHG. At inoculation, the sterile HAP and TI discs were placed in 24-well plates (one disc/well). The 6-well plates containing RHG with biofilms and the 24-well plate containing HAP or TI discs with biofilms were incubated at 37 °C, 7.5% CO2.To form microcosm biofilms, a saliva stock was diluted in a freshly-prepared mixture of BHI or TP with 0.7% agar to reach 10On day 1, 3 and 5 after inoculation, biofilm samples were harvested for viable cell counts and microbial composition analysis. The RHGs were fixed in 4% formaldehyde and processed for paraffin embedment. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E).All experiments were repeated 3 times and duplicate samples was included in each experiment.Each biofilm sample was collected using a microbrush into 2 mL cysteine peptone water (CPW). The biofilms were dispersed by 10 s vortexing, followed by 1 min sonication on ice 1 s (with 1 s pulse) at an amplitude of 40 W . Each sample was then serially diluted and plated onto trypticase soy agar containing 5% sheep blood, 5 μg/mL hemin and 1 μg/mL menadione, and further incubated anaerobically at 37 °C for 7 days before the colony forming units (CFUs) were counted. The remaining sonicated samples were centrifuged and stored at −80 °C for sequencing.31. After purification, paired-end sequencing of the amplicons (251-bp length) was conducted on the Illumina MiSeq platform at the UMC Cancer Center Amsterdam  using the MiSeq reagent kit V3 . The obtained paired-end reads were quality-filtered, merged and clustered into operational taxonomic units (OTUs) at 97% similarity. The most abundant sequence of each OTU was assigned a taxonomy using the ribosomal database project (RDP) classifier (min. confidence 0.8) and the SILVA rRNA database (v132)32. The OTU table was subsampled at a depth of 7900 reads per sample.Genomic DNA of the biofilm pellets was extracted and the V4 region of 16 S rDNA was amplified according to established protocolst test. Differences were considered statistically significant if p < 0.05.Viable counts were log10 transformed and analyzed for the changes in time for each substratum using one-way ANOVA in SPSS (version 25). The viable counts of day 1 were also compared to those of the inoculum using Student’s 33: namely Shannon diversity index, principal component analysis (PCA) and one-way permutational multivariate analysis of variance (PERMANOVA). The OTU table was log2-transformed before the latter two analyses.Several analyses on the OTU table were conducted in PAST 34 and network analysis were conducted on the OTUs with a relative abundance higher than 0.01%. To perform a LEfSe analysis, the relative abundance data were first split by biofilm growth media into two groups, BHI and TP. Next, LEfSe was carried out for each medium separately, using the type of substrata as class with default settings, except that the threshold on the logarithmic LDA score for discriminative features was set to 4.0. The interaction between bacterial species in the biofilms was inferred using Sparse Inverse Covariance Estimation for Ecological Association and Statistical Inference 20. The parameters of SPIEC-EASI are: “MB” neighborhood selection procedure, lambda minimum ratio of 1 × 10−2, nlambda of 20 and 50. The SPIEC-EASI calculated weighted network adjacency network was generated in R (v4.0.3) and imported into Cytoscape (v3.8.2). A community clustering based on Girvan-Newman fast greedy algorithm in clusterMaker plugin was used to identify the clusters inside the network. The identified clusters were visualized in Cytoscape using different color labels.Linear discriminant analysis effect size (LEfSe)Further information on research design is available in the Reporting SummarySupplementary Information


Myrtales is a species rich branch of Rosidae, with many species having important economic, medicinal, and ornamental value. At present, although there are reports on the chloroplast structure of Myrtales, a comprehensive analysis of the chloroplast structure of Myrtales is lacking. Phylogenetic and divergence time estimates of Myrtales are mostly constructed by using chloroplast gene fragments, and the support for relationships is low. A more reliable method to reconstruct the species divergence time and phylogenetic relationships is by using whole chloroplast genomes. In this study, we comprehensively analyzed the structural characteristics of Myrtales chloroplasts, compared variation hotspots, and reconstructed the species differentiation time of Myrtales with four fossils and one secondary calibration point.A total of 92 chloroplast sequences of Myrtales, representing six families, 16 subfamilies and 78 genera, were obtained including nine newly sequenced chloroplasts by whole genome sequencing. Structural analyses showed that the chloroplasts range in size between 152,214–171,315 bp and exhibit a typical four part structure. The IR region is between 23,901–36,747 bp, with the large single copy region spanning 83,691–91,249 bp and the small single copy region spanning 11,150–19,703 bp. In total, 123–133 genes are present in the chloroplasts including 77–81 protein coding genes, four rRNA genes and 30–31 tRNA genes.The GC content was 36.9–38.9%, with the average GC content being 37%. The GC content in the LSC, SSC and IR regions was 34.7–37.3%, 30.6–36.8% and 39.7–43.5%, respectively. By analyzing nucleotide polymorphism of the chloroplast, we propose 21 hypervariable regions as potential DNA barcode regions for Myrtales. Phylogenetic analyses showed that Myrtales and its corresponding families are monophyletic, with Combretaceae and the clade of Onagraceae + Lythraceae  being sister groups. The results of molecular dating showed that the crown of Myrtales was most likely to be 104.90 Ma (95% HPD = 87.88–114.18 Ma), and differentiated from the Geraniales around 111.59 Ma (95% HPD = 95.50–118.62 Ma).The chloroplast genome structure of Myrtales is similar to other angiosperms and has a typical four part structure. Due to the expansion and contraction of the IR region, the chloroplast genome sizes in this group are slightly different. The variation of noncoding regions of the chloroplast genome is larger than those of coding regions. Phylogenetic analysis showed that Combretaceae and Onagraceae + Lythraceae were well supported as sister groups. Molecular dating indicates that the Myrtales crown most likely originated during the Albian age of the Lower Cretaceous. These chloroplast genomes contribute to the study of genetic diversity and species evolution of Myrtales, while providing useful information for taxonomic and phylogenetic studies of Myrtales.The online version contains supplementary material available at 10.1186/s12870-021-02985-9. The Myrtales belong to the Rosidae, which is one of the most speciose groups in the Rosanae clade of angiosperms , 2. AccoWith the rapid development of second-generation sequencing technology, the cost of sequencing has made phylogenomic approaches feasible on large scales, ushering in a new exploration of plant identification and classification. Complete plastome sequences have become powerful tools to answer questions about plant evolution from inferred phylogenies –18. The Heimia myrtifolia, an important medicinal plant with a variety of pharmacological alkaloids in the Lythraceae. Later, combined with 22 samples of other species in the Lythraceae, the chloroplast genome structure was comprehensively analyzed and compared with that of other species in Myrtales. The chloroplast genomes of 22 species of Lythraceae ranged from 152,049 bp to 160,769 bp, and included 10 variation hot spots that were selected as potential molecular markers  Fig. . MelastoThe results of the BEAST analysis of species divergence time in Myrtales are shown in Fig. Pelargonium hortorum, Cryptomeria fortunei, Geranium, Pisum sativum, Vicia faba, and Erodium in which the size of the IR is increased, decreased or even completely lost [Previous studies have shown that the size of chloroplast genomes in angiosperms are between 120 and 180 kb, and the size of IR region is 20–30 kb . The sizely lost –34. In aely lost  though cely lost , 37.Panax notoginseng, the variation of the IR region of Myrtales is smaller than that of the SC region [rbcL failed to resolve the phylogenetic position of the order. Our analyses showed that the nucleotide diversity of rbcL is relatively low compared to other loci (PI < 0.05)  Fig. , which hCompared with previous studies based on a few chloroplast genome fragments, our results based on the major lineages of Myrtales  showed a highly resolved phylogenetic relationship of Myrtales by using whole chloroplast genomes , 6, 28. Biogeography estimates generally suggested that the Myrtales originated in Gondwana , 51, 52 In this study, we analyzed and compared the structural characteristics of chloroplast genomes of Myrtales, and inferred the phylogenetic divergence time of Myrtales. The chloroplast genomes of Myrtales has a typical four part structure, including 77–81 protein coding genes, 29–31 tRNA genes and four rRNA genes, with a total length of 152,214–171,315 bp. We found 21 mutation hotspots, which can be used as potential DNA barcodes in the future phylogenetic study of Myrtales. Phylogenetic relationships (Ml / BI) based on whole chloroplast genome and multiple datasets showed that Myrtales and its families were monophyletic, as well as Combretaceae and Onagraceae + Lythraceae strongly supported as a clade, . Reconstructing the divergence time of Myrtales shows that the crown of Myrtales is 104.90 Ma (95% HPD = 87.88–114.18 Ma), and it differentiated from Geraniales around 111.59 MA (95% HPD = 95.50–118.62 MA) in the Albian of the early Cretaceous. The species divergence of Myrtales ranged from 60 to 90 Ma. These chloroplast genomes contribute to the study of genetic diversity and species evolution of Myrtales, while providing useful information for taxonomic and phylogenetic studies of Myrtales. In the future, we will expand genomic sampling, including nuclear genomes, to comprehensively compare and discuss the phylogeny and evolution of Myrtales species.Combretum kraussii Hochst., Eucalyptus grandis W. Mill ex Maiden, Melaleuca leucadendra Linn., Combretum littoreum (Engl.) Exell, Syzygium forrestii Merr. et Perry, S. cumini (Linn.) Skeels and Tibouchina semidecandra Cogn. were collected from the Ruili Botanical Garden . Combretum malabaricum Linn. and Terminalia catappa Linn. were collected from Hainan University . The sampling of nine newly sequenced species was approved by Ruili Botanical Garden  and Hainan University (Hainan province of China) and met local policy requirements. Table Viviania marifolia, NC_023259.1; Pelargonium tetragonum, NC_031205.1; Pelargonium quercifolium, NC_031203.1) were downloaded from NCBI with detailed information presented in Table Leaf material from nine species, representing seven genera and three families in Myrtales, was collected and stored in silica gel. rbcL gene of Arabidopsis thaliana from NCBI (accession number: U91966) as a seed and assembled chloroplast genomes for each species using the clean reads with NOVOPlasty [We used a modified cetyltrimethyl ammonium bromide (CTAB) method to extract high quality DNA from dried leaves . QualityVOPlasty . The lonVOPlasty . QualityVOPlasty  to verifVOPlasty .https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) [Plastome sequences were initially annotated using Geneious R11.0.4 , then further annotated with Dual Organellar GenoMe Annotator (DOGMA)  to modifaw.html)  Fig. 3)https://cgenome.lbl.gov/vista/index.shtml [Vochysia acuminate (NC_043811) as a reference. To reveal highly variable regions for future species identification studies and to evaluate different plastome regions that may show different evolutionary patterns, we sequentially extracted both coding regions and noncoding regions (including intergenic spacers and introns) after alignment with MAFFT v7 [Plastome comparisons across 24 Myrtales species  were performed in Shuffle-LAGAN mode on the mVISTA program , usingMAFFT v7  using thMAFFT v7 . The IR MAFFT v7 .Viviania marifolia (NC_023259), Pelargonium tetragonum (NC_031205), and Pelargonium quercifolium (NC_031203) as outgroups based on a previous study [Phylogenetic analyses were conducted on 95 species, using olia NC_03259, Pelus study  and manuus study . Maximumus study  with 100us study . Bayesiaus study . Two indus study  and the us study  were useMyrtaceidites lisamae (83.5 Ma) fossil from Gabon, Africa during the Santonian [Eucalyptus frenguelliana (51.69 Ma) dated to the early Eocene from Laguna del Hunco in Chubut Province, Argentina [Lythrum elkensis of Lythrum/Peplis from the Late Cretaceous  in Wyoming, USA [Sonneratioxylon preapetalum Awasthi [The complete 92 plastome dataset of Myrtales was analyzed using the GTR + GAMMA+I model selected by MrModelTest  in BEASTantonian , 77, 78 rgentina , 80. We  Awasthi , the cla Awasthi  and the  Awasthi  was used Awasthi  was usedAdditional file 1: Figures S1–S6. are phylogenetic relationships inferred by Maximum Likelihood and Bayesian inference based on: coding genes; noncoding loci; the LSC (the Large Single-Copy); the SSC ; NO-IRa data set (data set composition is described in the methods) and IRb (Inverted Repeat region). Support values are maximum likelihood bootstrap support/Bayesian posterior probability. The families of Myrtales are indicated by different colors. For each figure, the inset shows the same tree as a phylogram (except for some inconsistencies in the phylogenetic relationships of IR dataset construction). The support value on the branch is bootstrap value/Bayesian posterior probability: “*” means 100% /1.0 support value, and “-” means bootstrap value/Bayesian posterior probability is less than 60 / 0.7. The families of Myrtales are represented by different colors. The small picture in the upper left corner is the ML phylogenetic tree (showing branch length).Additional file 2: Figure S7. Optimal phylogenetic tree resulting from analyses of 92 complete chloroplast genomes of Myrtales and 20 outgroups using Maximum Likelihood (ML). Support values are maximum likelihood bootstrap support posterior probability. The families of Myrtales are indicated by different colors. . The support value on the branch is bootstrap value, “*” means 100% support value, and “-” means bootstrap value is less than 60. The families of Myrtales are represented by different colors. The small picture in the upper left corner is the ML phylogenetic tree (showing branch length).Additional file 3: Table S1. Eta, Pi value, H, Hd, PICs, the length and aligned length of 188 Myrtales homologous loci across.Additional file 4: Table S2. Species information and chloroplast genomes GenBank accession number of Outgroups in this study.


Sclerotinia sclerotiorum, affects Brassica crops. Brassica crops produce a broad array of compounds, such as glucosinolates, which contribute to the defense against pathogens. From their hydrolysis, several products arise that have antimicrobial activity (GHPs) whose toxicity is structure dependent. S. sclerotiorum may overcome the toxic effect of moderate GHP concentrations after prolonged exposure to their action. Our objective was to identify the molecular mechanism underlying S. sclerotiorum response to long exposure to two chemically diverse GHPs: aliphatic GHP allyl-isothiocyanate (AITC) and indole GHP indol-3-carbinol (I3C). We found that the transcriptomic response is dependent on the type of GHP and on their initial target, involving cell membranes in the case of AITC or DNA in the case of I3C. Response mechanisms include the reorganization of chromatin, mediated by histone chaperones hip4 and cia1, ribosome synthesis controlled by the kinase-phosphatase pair aps1-ppn1, catabolism of proteins, ergosterol synthesis, and induction of detoxification systems. These mechanisms probably help S. sclerotiorum to grow and survive in an environment where GHPs are constantly produced by Brassica plants upon glucosinolate breakdown.White mold disease, caused by the necrotrophic fungus IMPORTANCEBrassica species, including important vegetable crops, such as cabbage, cauliflower, or broccoli, or oil crops, such as rapeseed, produce specific chemical compounds useful to protect them against pests and pathogens. One of the most destructive Brassica diseases in temperate areas around the world is Sclerotinia stem rot, caused by the fungus Sclerotinia sclerotiorum. This is a generalist pathogen that causes disease over more than 400 plant species, being a serious threat to economically important crops worldwide, including potato, bean, soybean, and sunflower, among many others. Understanding the mechanisms utilized by pathogens to overcome specific plant defensive compounds can be useful to increase plant resistance. Our study demonstrated that Sclerotinia shows different adaptation mechanisms, including detoxification systems, to grow and survive when plant protective compounds are present. Sclerotinia sclerotiorum is a necrotrophic fungal pathogen that attacks at least 408 species of plants from 278 genera, belonging to 75 families , aromatic , and indolic (derived from tryptophan)  . Myrosinptophan) . Dependiathogens , 5. Amonathogens .S. sclerotiorum in planta  with GO-Tool2. We describe the most important features found under the biological process (BP) category .P ≤ 0.05 and −1 ≤ log2 fold change ≥ 1), from which 181 were induced and 31 were repressed. The most significant induced GO terms were related to mitosis and cell division . Other categories were related to the organonitrogen compound metabolic process (GO:1901564) and the small-molecule metabolic process (GO:0044281). Otherwise, there is a large set of significantly enriched terms associated with the positive regulation of the proteasomal protein catabolic process (GO:1901800 and GO:0032436). The first six most significantly repressed GO terms are related to toxin biosynthetic pathways . Several terms were related to apoptosis and cell death . Other significant terms included processes related to mitochondrial transmembrane transport (GO:1990542 and GO:0045041), mitochondrial DNA repair (GO:0043504), and maturation of LSU-rRNA (GO:0000463 and GO:0000470).Transcript analysis resulted in 212 significantly enriched GO terms , threonine (GO:0009088), and sulfur amino acids (GO:0000097). The ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway (GO:0043162) and glyoxylate metabolic process (GO:0046487) were also significant. A large part of significant GO repressed terms were related to translation and ribosome biogenesis and assembly . Among the most significant GO downregulated terms, we could find several terms related to stress and stimulus response . Other significantly repressed terms were involved in the small-molecule metabolic process and nucleobase-containing small-molecule metabolic process (GO:0044281 and GO:0055086) and toxin biosynthetic process (GO:0009403).Expression of 296 GOs was higher in mycelia treated with AITC than in mycelia treated with I3C. Among them, we can find terms related to cell division, such as positive regulation of metaphase/anaphase transition of the cell cycle (GO:1902101), positive regulation of cell cycle phase transition (GO:1901989), or actin polymerization or depolymerization (GO:0008154). Other terms are related to catabolic processes, such as positive regulation of the proteasomal protein catabolic process (GO:1901800) and positive regulation of proteolysis (GO:0045862). Translation (GO:0006412), ribonucleoprotein complex assembly (GO:0022618), endoplasmic reticulum (ER)-nucleus signaling pathway (GO:0006984), cellular response to sterol depletion (GO:0071501), and fungus-type cell wall biogenesis (GO:0009272) were also upregulated by AITC. The expression of four GO terms was higher in the treatment with I3C compared to AITC: mitochondrial DNA repair (GO:0043504), cellular response to light stimulus (GO:0071482), mitochondrial DNA metabolic process (GO:0032042), and response to light stimulus (GO:0009416).mad2, klp9, mug190, ish1, hsp9, and SPAC32A11) is shared with the network of the AITC versus control comparison. In both comparisons, mad2 and klp9 are induced and mug190, ish1, hsp9, and SPAC32A11 are repressed.There was a higher number of DEGs in response to AITC than in response to I3C compared to control samples. Thirty genes were commonly induced by both treatments, and 98 were repressed  and B. Ihip4, hhf1, mst1, and ngg1, are induced by AITC, whereas DEGs rhp6, ubc7, rev1, and rhp14 are induced by I3C. Both branches are connected by DEG cia1, which is induced by I3C and repressed by AITC.Seventy-four DEGs are interacting in the AITC versus I3C comparison . crz1 is a target of the calcineurin protein that is related to resistance to antifungal molecules and homeostasis of cations in Saccharomyces cerevisiae  pathway, and both are downregulated by both GHPs. The expression of ish1 increases after S. pombe exposure to osmotic stress or glucose deprivation  is induced by AITC. Histone chaperones modulate nucleosome assembly and, therefore, the structure of chromatin (hip4 and cia1 in S. pombe (homologous to hpc2 and asf1 of S. cerevisiae) are chaperones acting on histones H3 and H4. hip4 is a component of the HIR complex. Deletion of hip4 provokes cycle delay  play a central role in signaling and reprogramming cell metabolism through their phosphorylation/dephosphorylation. Asp1 is an inositol kinase that participates in the synthesis of the diphosphate group of PP-InsP5 and (PP)2-InsP4 , and preubc11 was induced by AITC, whereas ubc7 was induced by I3C.Both treatments induced protein catabolism. AITC induced the ubiquitin-proteasomal system, whereas I3C induced the multivesicular (MVB) body sorting pathway. The ubiquitin-proteasome system has an essential role in cell growth and differentiation, survival, stress tolerance, and adaptation to environmental changes , 35. ThiS. sclerotiorum cells, whereas I3C did not affect it. The sterol biosynthetic process was induced in fungi treated with AITC compared to I3C. Sterols play an important structural role in regulating membrane fluidity and permeability  was repressed after AITC treatment, probably because, when samples were taken, oxidative stress caused by AITC was reduced after several days of adaptation.As a detoxification mechanism, glutathione rotiorum  and A. bsicicola , 43 afteith AITC . Exposurith AITC , 10. Con to AITC . HoweverS. sclerotiorum after the invasion of B. napus plants  plates and incubated at 24°C in the dark for 4 days. Fresh colonies were obtained through routine growth of mycelium-agar plugs from the margin of the fungal colony on PDA and incubated at 24°C for 72 h. GHPs were purchased from Sigma-Aldrich, including allyl-ITC (AITC) (36682) and indole-3-carbinol (I3C) (I7256-5G). Fungal growth in 2× YPG medium was monitored spectrophotometrically for 4 days at 25°C with the addition of each GHP in concentrations ranging from 0 to 250 μM. A sublethal concentration of 100 μM for both compounds then was selected to be used in the transcriptomic analysis.We collected Fungi were grown in PDA medium supplemented with each GHP, which was added to the medium before solidification to reach a concentration of 100 μM. Petri dishes were coated with one layer of cellophane membrane . Agar plugs of 2.5-mm diameter containing fresh fungal colonies then were transferred to petri dishes and incubated at 24°C in darkness. After 5 days, mycelium mass was harvested from cellophane membranes and homogenized in liquid nitrogen. The resulting material was immediately used to isolate total RNAs. Two replicates per treatment and control sample were carried out.In another experiment, the diameter of fungal mycelium growing radially from the center was measured for 8 days, until it reached the end of petri dishes.RNA was extracted with a Spectrum plant total RNA kit  according to the manufacturer’s protocol. In order to remove any traces of genomic DNA, the RNA was treated with DNase by following the manufacturer’s instructions. Massive analysis of 3′-cDNA end (MACE) libraries was generated by using GenXPro’s MACE kit  as described previously . Brieflyhttps://github.com/marcelm/cutadapt/]) and poly(A) tails were clipped by an in-house python script. Reads were aligned to reference sequences using bowtie2. On this genome, since the functional annotation was poor, a cluster_bed step was performed. Reads that successfully map the genomic regions with no annotation (feature) were clustered. Clusters (consensus sequences) were annotated via BLASTX to the UniProt database (Swiss-Prot and TrEMBL). Reads that did not match the genome were taken into a de novo assembly step and annotated via BLASTX to the UniProt database (Swiss-Prot and TrEMBL). The assembly was completed with Trinity RNA-Seq (https://github.com/trinityrnaseq/trinityrnaseq/wiki). As for the functional annotation, UniProt (http://www.uniprot.org/) and NCBI fungal sequences were employed.NextSeq-derived sequence reads were processed with GenXPro’s online in-house MACE analysis pipeline. Briefly, libraries were sorted according to their respective index, and all duplicate reads detected by the TrueQuant technology were removed from raw data sets. Additionally, quality filtering  analysis with all the annotated transcripts was done for each comparison (GHP versus control and AITC versus I3C) with GO-Tool2. The likelihood of a specific GO term was calculated by using Fisher’s exact test.2 fold change (FC) ≥ 1 were considered to be differentially expressed genes (DEGs) between treatments. The web tool STRING (v. 10.5) was used to study the interconnections between DEGs. In addition to well-supported protein–protein interactions experimentally observed, this resource includes indirect and predicted interactions on top  of ≤0.05 and −1 ≤ logs on top . Howevert  =  0 to t  = 210  min using a Promega fluorescence measurement system (GloMax) at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Fluorescence values were corrected by the absorbance of samples measured at 600 nm. Measurements were carried out in triplicate.The SYTOX green uptake assay was carried out by following reference GSE144344.Data supporting the findings of this study are openly available in the Gene Expression Omnibus repository under reference number


Cervical cancer (CC) is the second most common cancer and the leading cause of cancer mortality in women. Numerous studies have found that the development of CC was associated with multiple genes. However, the mechanisms on gene level are enigmatic, hindering the understanding of its functional roles. This study sought to identify prognostic biomarkers of CC, and explore their biological functions. Here we conducted an integrated analysis to screen potential vital genes. Candidate genes were further tested by experiments in clinical specimens and cancer cell line. Then, molecular modeling was used to predict the three-dimensional structure of candidate genes’ proteins, and the interaction pattern was analyzed by docking simulation technique. Among the potential genes identified, we found that TAF1A and ZBTB41 were highly correlated. Furthermore, there was a definite interaction between the proteins of TAF1A and ZBTB41, which was affected by the activity of the p53 signaling pathway. In conclusion, our findings identified TAF1A and ZBTB41 could serve as biomarkers of CC. We confirmed their biological function and deciphered their interaction for the first time, which may be helpful for developing further researches. Cervical cancer (CC) is the second most common type of cancer affecting women . It is aThe current advances in screening and treatment methods have improved the prevention and management of CC; however, most patients are diagnosed late, thus complicating their management. The main treatment methods for CC are radical surgery and local radiotherapy, supplemented by chemotherapy, which often cause various complications, and severely affect the patients’ quality of life . TherefoResearch has implicated the HPV infection as an independent risk factor for CC . The HPVThe development of large-scale genomic technologies has promoted studies involving tumor biomarkers. The Weighted Gene Co-Expression Network Analysis (WGCNA) is among the most powerful genomic techniques used in research . It applLikewise, a more detailed in silico analysis with ab initio modeling serves as an essential and effective auxiliary method for simulating the macromolecular structure of proteins with unconfirmed crystal structures. This method has been applied in most proteins to generate new structures from sequences, crucial for protein model design and protein folding. Additionally, this technique has been widely used to analyse protein structures and intermolecular interaction patterns of various proteins , 13.This study focused on the association between gene sets and the common phenotypes of CC. We identified two candidate genes (TAF1A and ZBTB41) by combining a set of complex molecular analyses. Subsequent experiments showed that the TAF1A and ZBTB41 gene expression in the tumour and normal tissues were significantly different. Moreover, the results revealed that these genes were involved in the proliferation and migration of tumour cells. Co-immunoprecipitation results suggested that TAF1A and ZBTB41 form a complex, and their binding ability was affected by the activity of the p53 signaling pathway. Subsequently, we predicted the protein structures of TAF1A and ZBTB41. Molecular docking results showed that TAF1A and ZBTB41 interacted through hydrophobic, van der Waals, hydrogen, and electrostatic bonds. This finding may have significant prognostic value for CC and unveil the potential mechanism of the interaction between TAF1A and ZBTB41 for the first time.2 = 0.918) was determined as the soft threshold to obtain a scale-free network  pathway and WikiPathways (WP) pathway analysis. According to the KEGG pathway analysis, genes were mainly involved in the p53 signaling pathway and valine, leucine, and isoleucine degradation. The enrichment pathway ID was marked in the radar chart, where 1-P represented the radius of each enrichment. The radius was proportional to the enrichment significance  curve analyses were performed to determine the diagnostic efficiency of the high and low pathological grades of cancer. Two genes in the yellow module (TAF1A and ZBTB41) were negatively associated with patients’ overall survival (OS) based on multiple comprehensive analysis data Fig. A, B.Fig.The Geneset enrichment analysis (GSEA) was performed to obtain further validation of the functional enrichment of TAF1A and ZBTB41. Based on the cut-off criteria of false discovery rate (FDR) < 0.05, we found that “the p53 signaling pathway” was enriched significantly in both TAF1A and ZBTB41 highly expressed samples, compared to the low-expression samples Fig. . TherefoP < 0.05, **P < 0.01, Fig. Furthermore, the TAF1A and ZBTB41 were significantly overexpressed in 304 high-grade tumour samples retrieved from the TCGA database (*P < 0.001) and the other 32 types of cancer  data from the Human Protein Atlas (HPA) database demonstrated significantly higher protein levels in tumour tissues than in normal tissues . Additionally, the expression levels of the two genes demonstrated a strong correlation with CC . More convincingly, the qRT-PCR clinical tissue samples showed a significant upregulation of TAF1A (P < 0.01) and ZBTB41 (P < 0.05) in tumours compared to the normal healthy tissues  revealed that TAF1A and ZBTB41 knockdown significantly inhibits cell proliferation Fig. . AdditioAs TAF1A and ZBTB41 were significantly co-expressed, we sought to determine whether TAF1A interacts with ZBTB41, and to explore the influence of the activity of the p53 signaling pathway on their interaction ability. We used the PFTα as an inhibitor of the p53 signaling pathway based on the published literature , 15.Immunofluorescence results showed no significant intracellular overlap between TAF1A (red fluorescence) and ZBTB41 (green fluorescence) in the PFTα-treated experimental group Fig. . HoweverP < 0.01). Their interaction was significantly suppressed by PFTα, which was likely attributable to the malignancy of cancer cells influenced by p53 signaling pathway.A Co-immunoprecipitation (Co-IP) assay using endogenous proteins extracted from the vehicle- or PFTα-treated HeLa cells showed that the ZBTB41 and TAF1A were coimmunoprecipitatedFig. C, D, demThe I-TASSER server modeling showed TAF1A protein as a globular protein, and its secondary structure mainly consisted of a spiral and random curl Fig. . After eTo further explore their structure-activity relationship, docking analysis demonstrated that TAF1A docked with high affinity to the BTB domain of ZBTB41 Fig. E, F. We Cervical cancer (CC) is a heterogeneous disease accompanied by a high clinical recurrence and metastasis rate . The proSimilar studies have shown altered gene expression profiles in gynecological tumors, including CC compared with the normal tissues , 20. AmoPreviously, WGCNA has been widely used in microarray data analysis for gene annotation, prediction of potential gene function, discovery of new biomarkers for diseases, identification and validation of chemotherapy drugs –23. We cThe TATA-Box Binding Protein Associated Factor, RNA Polymerase I Subunit A (TAF1A) is a protein-coding gene component of the SL1/TIF-IB transcription factor complex playing involved in the assembly of the RNA polymerase I preinitiation complex. It can recruit RNA polymerase I to ribosomal DNA promoters, which is the essential for ribosomal biogenesis . A previOn the other hand, Zinc Finger and BTB Domain Containing 41 (ZBTB41) is an important paralog of the Zinc Finger Protein 407 (ZNF407). The BTB domain of the ZBTB41 is highly conserved and mediates protein–protein interactions. The BTB is currently known to play an important role in transcriptional regulation, chromatin remodeling, protein degradation, and cytoskeletal regulation. It is also closely associated with functions of mammalian growth and development, including lymphocyte development, axonal orientation, gonadal morphogenesis, among others . The prop53, a transcriptional factor, has been generally viewed as an effective anticancer target. It is mutated in over 50% of the human tumours and is related to rapid tumour progression and resistance to anticancer therapy . PifithrMoreover, the PFTα inhibits the heat shock proteins, glucocorticoid receptor signal transduction, and the NF-κB activity, and blocks the induction of some receptors, such as the androgen receptor and CD95 –40. ThesWestern blot analyses demonstrated a dose-dependent inhibitory effect of the PFTα on the p53 protein HeLa cells  database. This dataset included 28 CC cases of different stages and pathological grades. After all data filtering and background noise correction, the “affy” package from Bioconductor (http://www.bioconductor.org) was used to normalize and summarize. The top 25% most variant genes were then selected for subsequent analysis.The gene expression profiles of CC were extracted from the Metabolic gEne RApid Visualizer  based on dissimilarity measure . Minimump < 0.05 was selected as the significant module to determine highly correlated genes with CC phenotypes. The selected gene module was then annotated, integrated, and analysed using g:Profile (https://biit.cs.ut.ee/gprofiler/gconvert.cgi) at default settings. The enrichment comprised five categories: molecular function, cellular component, biological process, KEGG pathway and WP. The results were categorised according to the cut-off criterion of the adjusted P < 0.05 and demonstrated in a radar chart. A value of 1-P represented the radii of each group. The full names corresponding to the pathway ID are presented in the supplementary Table In this study, the module with the highest absolute correlation coefficient and Considering the interaction of genes within the modules, we used the co-expression analysis method and a circos plot to illustrate the genes’ interaction within the three most significant modules.https://cancergenome.nih.gov/) database and normalized using the edgeR package. Each candidate gene identified in the selected module was assessed using survival analysis and receiver operating characteristic (ROC) analysis. Samples with missing information were excluded. Correlation analyses were performed across the CC samples and the other 32 types of cancer from TCGA database . The GSEA was performed using GSEA software (http://www.broadinstitute.org/gsea). Other analyses were conducted using custom-written code in R software.The CC samples in the TCGA dataset were divided into high-expression and low-expression groups according to the median expression values of each selected gene. To verify the pathway enrichment of the candidate genes in the TCGA data set, Geneset enrichment analysis (GSEA) was performed using data from the KEGG database, and compared with the previous results. FDR < 0.05 was set as the cut-off criteria. Subsequently, we measured the expressions of hub genes by the immunohistochemistry using the Human Protein Atlas  at 4 °C overnight, and then incubated with a secondary antibody at 37 °C for 40 min. The target proteins were visualized using 3,3’-diaminobenzidine as the colour substrate.We measured the expression of hub genes in clinical specimens by the immunohistochemistry. The paraffin-embedded CC tissue and the corresponding paracancerous tissue samples were deparaffinised in xylene and hydrated in graded ethanol concentrations in descending order. The endogenous peroxidase was removed with 3% HQuantitative real-time PCR (qRT-PCR) experiments were performed with the SYBR green-based detection system. Total RNA was extracted using an RNA extraction kit . cDNA synthesis was carried out with PrimeScript RT Master Mix , and the amplified products were detected by using SYBR pre-mix EX Taq . The qRT-PCR was performed on a 7500 real-time PCR system  with the primer shown in Supplementary Table The human cervical carcinoma cell line (HeLa) was purchased from the ATCC . The siTAF1A and siZBTB41 synthesized by RiboBio Co.  were selected for transient knockdown experiments, which were determined by qRT-PCR and western blot . After 4 days, the cells were fixed with 4% paraformaldehyde for 15 min, and then stained with 1% crystal violet. A wound scratch healing assay was used for determining the migration of cells. Briefly, the cultured cells were allowed to reach confluence on six-well culture plates, and a wound was created by scraping the middle of the cell monolayer across the entire diameter of the well with a P10 pipette tip. Floating cells were washed twice with Phosphate-Buffered Saline (PBS). Experiments were carried out without any proliferation inhibitor. Photographic images were captured using an inverted microscope to monitor the number of colonies (>50 cells) and the wound closure, and then analysed with ImageJ software . All assays were conducted more than two times.Cell proliferation was measured by colony formation experiment. Different groups of cells were inoculated into a six-well plate at a density of .The PFTα, an inhibitor of the p53 signaling pathway, was purchased from Sigma–Aldrich (USA). Briefly, 2 × 102 cell culture dishes (2 × 106 cells) for 24 h, and then treated with PFTα 10 μM for 24 h. Dimethylsulfoxide (DMSO) was used as the control reagent. Subsequently, the medium was removed, and cells were washed thrice with PBS.HeLa cells were seeded in 10 cmThe RIPA lysate containing protein inhibitors was added, and the dishes were then placed on ice to lyse cells for 60 minutes. The lysates were collected into the EP tubes and centrifuged at 4 °C to remove the precipitation and retain the supernatant. The target antibody was added, and the setup was incubated at 4 °C for 2 h. After that, protein A/G-beads were added and incubated overnight at 4 °C. After the immunoprecipitation reaction, the tubes were centrifuged to precipitate the beads bound to the Ag–Ab complex. The collected protein A/G-beads were cleaned, SDS buffer was added, and boiled for 5–10 min. Western blotting was then performed to detect the proteins. Simultaneously, the study sample and IgG antibody served as the positive and negative controls, respectively. The primary and secondary antibodies against TAF1A, ZBTB41, and IgG were obtained from Invitrogen .Protein concentration was measured using BCA Protein Assay reagent . Samples were separated on an SDS-polyacrylamide gel and blotted to poly-vinylidene difluoride (PVDF) membranes. Signal detection was performed with ECL western blotting detection reagent . Antibodies used were anti-TAF1A , anti-ZBTB41 , anti–GAPDH , β-actin , and anti–p53 .http://zhanglab.ccmb.med.umich.edu/I-TASSER/) online database [http://www.uniprot.org/). During the modeling process, the Procheck server (https://servicesn.mbi.ucla.edu/PROCHECK/) was used to established the optimal model with low energy based on relevant theories. After model construction, the Ramachandran plot of candidate protein structures was drawn to evaluate the quality of the prediction model.For ab initio modeling, 3D structure predictions were carried out by the I-TASSER server  and GraphPad Prism software version 7.0 . Image analysis was performed using Image J (NIH). The schematic representation of this process is illustrated in Fig. The Pearson’s correlation test and logistical regression analyses were performed to investigate the correlation between the candidate genes. The log-rank test was used to compare the survival curves. Two-tailed Student’s supplementary tablesSupplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5supplementary figure legends