Bacteroides, Escherichia, Clostridium, Lactobacillus, Fusobacterium, and Prevotella are dominant in piglets before weaning, then Prevotella and Aneriacter shift to be the predominant genera with Fusobacterium, Lactobacillus, and Miscellaneous as comparative minors in postweaned pigs. A number of 19 bacterial genera, including Bacteroides, Prevotella, and Lactobacillus can be found in more than 90% of pigs and three enterotypes can be identified in all pigs at different ages, suggesting there is a “core” microbiota in the gut of healthy pigs, which can be a potential target for nutrition or health regulation. The “core” members benefit the growth and gut health of the host. These findings help to define an “optimal” gut microbial profile for assessing, or improving, the performance and health status of pigs at different growth stages.Fully understanding the dynamic distribution of the gut microbiota in pigs is essential, as gut microorganisms play a fundamental role in physiological processes, immunity, and the metabolism of nutrients by the host. Here, we first summarize the characteristics and the dynamic shifts in the gut microbial community of pigs at different ages based on the results of 63 peer-review publications. Then a meta-analysis based on the sequences from 16 studies with accession numbers in the GenBank database is conducted to verify the characteristics of the gut microbiota in healthy pigs. A dynamic shift is confirmed in the gut microbiota of pigs at different ages and growth phases. In general, IMPORTANCE The ban on feed antibiotics by more and more countries, and the expected ban on ZnO in feed supplementation from 2022 in the EU, urge researchers and pig producers to search for new alternatives. One possible alternative is to use the so-called “next-generation probiotics (NGPs)” derived from gastrointestinal tract. In this paper, we reveal that a total of 19 “core” bacterial genera including Bacteroides, Prevotella, and Lactobacillus etc., can be found in more than 90% of healthy pigs across different ages. These identified genera may probably be the potential candidates of NGPs or the potential target of microflora regulation. Adding substrates preferred by these target microbes will help to increase the abundance of specific symbiotic species and benefit the gut health of pigs. Further research targeting these “core” microbes and the dynamic distribution of microbiota, as well as the related function is of great importance in swine production. Similar to humans, pigs have a complex and diverse community of microorganisms in their gastrointestinal tract (GIT), that plays a fundamental role in immunity, physiological processes, and the metabolism of nutrients. The diversity, composition, and function of gut microbial community are influenced by various factors including diet, age, stress, and the environment . This ca7–17–Here we first summarized the characteristics and the dynamic shifts of the bacterial community in the GIT of pigs at different growth phases based on the data reported in 63 published papers. A total of 57,875,211 16S rRNA gene sequences from untreated, healthy pigs from 16 different studies (from the 63 published papers) were downloaded from NCBI GenBank for a meta-analysis to verify characteristic microbial populations. Based upon this metadata, the contribution of the gut microbiota to growth phenotypes of the host were evaluated, in order to discuss functional microbes, and/or potential biomarkers in swine.Age is an important factor driving the maturation of the gut microbiota , 23. The14–Infancy is a critical period for microbial colonization, during which microbiota structure is unstable and susceptible to surrounding environmental conditions. Increasing evidence has demonstrated that the early colonization of “appropriate” microorganisms may determine the gut microbial composition and immunological maturation , 26, 27.31–Bacteroides, Escherichia, and Clostridium are the three most abundant genera. A marked shift from Bacteroides to Prevotella has been observed as piglets age/grow . In the modern pig industry, piglets start to receive commercial feed 3 to 5 days after birth. The shift from sow milk to solid feed helps shape the GIT microbiota of piglets. Microbes in the gut of neonates are adapted to use a wide range of both milk oligosaccharides and host-derived glycans  as their unique carbon source   , Prevotebacillus , and inchia coli , have behia coli , 47. Thehia coli . Such diWeaning is the most stressful time in the piglets’ life. During this period, piglets are switched from milk- to a solid-based diet –50. Accu–51–Prevotella, Roseburia, Blautia) in the GIT might offer piglets a stronger ability to digest glycan   , 56, afffections .Prevotella and Bacteroides. It is common that piglets do not eat feed in the immediate postweaning period due to weaning stress, and the abundance of Prevotella is temporarily decreased while Bacteroides is increased in abundance. For example, from day 21 (day of weaning) to day 24, the abundance of Prevotella (33.07%), Alloprevotella (12.81%), and Oscillibacter (5.52%) in the gut of piglets can decrease to 13.9%, 11.0%, and 4.8%, respectively. However, the abundance of Bacteroides (5.22%), Faecalibacterium (0.05%), and Roseburia (1.77%) can increase to 6.31%, 8.97%, and 3.51%, respectively (Prevotella can significantly increase from 12.93% (day 21) to 57.24% (day 28) (Prevotella (23.1%) is one of the three most abundant genera identified in the feces of weaning piglets (21 days old), and it becomes predominant (34.7%) in the feces of post-weaning pigs (28 days old), followed by Ruminococcus (5.2%) and unclassified bacteria belonging to the Lachnospiraceae (5.0%) (Prevotella and Lactobacillus is significantly higher in the gut of post-weaning piglets (28 days old) compared with weaned piglets (21 days old), accompanied by changes in the metabolism of carbohydrates and amino acids, which may be beneficial for the adaptation to the new diet post-weaning  . Prevoteevotella .07%, All-weaning . On the  10 days . This re 10 days .Campylobacter and the decrease of beneficial bacteria like Alloprevotella or Oscillibacter during the weaning transition period , which is a major problem in the swine industry and causes significant economic losses . The divn period . In the ecreased . Enterobecreased , 59. Somecreased . The enrecreased . Few stuecreased . Five meecreased .Prevotella spp. are the most abundant bacteria in the GIT of pigs between 28 to 91 days  sequences showed at least 97% similarity to sequences in the SILVA database. The donors range from newborn (day 0) to finishing (day 174), helping to draw a full picture of the dynamic distribution of the microbiota at different ages. Finally, a total of 12,595 bacterial operational taxonomic units (OTUs) were identified and assigned to 39 phyla and 509 genera. Differences in the α-diversity of microbial communities were observed among suckling, nursery, growing and finishing pigs, with finishing pigs harboring the most diverse microbiota . Principal coordinate analysis (PCoA) plots based on Bray-Curtis showed shifts in the microbial community among these four stages, especially before (lactation) and after weaning (nursery) phase . These rWe next tried to identify the “core” and “stage-associated” microbiome in the gut of healthy pigs. The definition of a “core microbiome” in the swine gut has been the focus of numerous publications , 68, whiBacteroides, Escherichia, and Lactobacillus are predominant in lactating pigs , while Prevotella, Lactobacillus, and Oscillispira are dominant in nursery pigs . In growing pigs, the three most abundant genera are Prevotella, Lactobacillus and Faecalibacterium , and the top 3 genera are Prevotella, Lactobacillus, and Streptococcus in finishing pigs . These dominant taxa may be the potential functional microbiota, not only for their relatively high abundance, but also for their higher frequency to contact with the intestinal mucosa of host.Although a “core” microbiota may not exist in swine according to a strict definition, here we still found several bacterial phyla and genera in more than 90% of the samples in all growth stages. Firmicutes and Bacteroidetes accounted for the majority of total sequences . A total of 19 genera were found in more than 90% of samples , which were defined as “core” bacteria here. Accordingly, Bacteroides, Escherichia, Lactobacillus, Fusobacterium, and Ruminococcus were identified as the lactation stage-associated genera. Blautia was identified as a nursery stage-associated genus. Growing stage-associated genera included Prevotella, Megasphaera, Treponema, and Faecalibacterium, while finishing stage-associated bacteria were characterized by Streptococcus, Clostridium, Prevotella, YRC22, and SMB53. It is worth noting that the abundance of bacteria belonging to the Proteobacteria (17.35%) showed higher relative abundance in suckling piglets compared with other growth phases . Ten genera, including Bacteroides, Escherichia. and Prevotella, were present across all stages  and Escherichia  were present in the gut of suckling piglets, they were markedly decreased in the subsequent growth stages . Bacteria belonging to the family Enterobacteriaceae, such as E. coli, often show over-growth in the case of dysbiosis , growing (27.79%), and finishing (15.25%) pigs , suggesting that Prevotella, a class of bacteria with powerful capacity to metabolize complex carbohydrates like hemicelluloses and xylans  and effect size (LEfSe), the “stage-associated core bacteria” were identified . In detail, ysbiosis  and may ysbiosis , 70. Preows milk . Notablyithelium . It can  T cells , 73. In d xylans , 41, 51,Bacteroides-Escherichia-Lactobacillus), E2 (Prevotella-Lactobacillus-Megasphaera) ,and E3 (Prevotella-Lactobacillus-Treponema), were identified from the collected sequences . Among them, E1 accounted for approximately 85% of the bacterial genera in the gut of suckling piglets , while E3 accounted for 88% in finishing pigs . For nursery and growing pigs, the division of enterotypes was not absolute. The proportion of E1, E2, and E3 in the gut of nursery piglets was 16%, 50%, and 34%, respectively . However, only two enterotypes, E2 (60%) and E3 (40%), were found in the gut of growing pigs . Bacteroides, Escherichia, and Lactobacillus were the three main genera of bacteria contributing to E1 . Megasphaera, Prevotella, and Lactobacillus were the other three main genera contributing to E2 , while Prevotella, Treponema, and Lactobacillus were the main genera contributing to E3 . These findings can help to understand the whole picture of how enterotypes change from suckling to finishing pigs, which can be one of the characteristics used to assess balanced or normal microbiota in healthy pigs at different growth stages.The term “enterotype” is now recognized as an important characteristic of the gut microbiome. A total of three enterotypes, E1  most strongly affected the composition of the swine feces microbiota (Table S7), and the result indicate that age is the most important factor affecting the gut microbiota of pigs. This aligns with our previous summary that the microbial community dynamically changes with age.in vitro models of piglet gut for the development and testing of new feed additives  influencing the pig gut microbiota, and focus on how commensal microorganisms impact the biochemical and metabolic process of host , 75. Difdditives . Here, wIn a recently published meta-analysis, the microbial load in the GIT and the dynamic shifts of the microbiota along with the GIT of pigs were discussed . A clearDue to the rapid development of sequencing techniques, data are available to link certain gut bacteria with swine phenotypes. The identification of specific bacterial biomarkers linked to growth performance in a supervised way will provide promising information to develop effective strategies for modulating the gut microbiota and improving growth performance of pigs , 80.Bacteroides (2.65% versus 4.54%), Anaerotruncus (0.01% versus 0.03%), and Anaerococcus (0.02% versus 0.01%) were significantly different in the gut of the heavier (16.70 to 22.75 kg) pigs compared with the lighter (8.09 to 11.89 kg) pigs  pigs . The bodkg) pigs .Prevotella-dominated enterotypes present lower growth rate during the lactation period, but higher body weight and average daily gain after weaning  and low FE (FCR = 2.85 ± 0.19), a remarkably high abundance of Prevotella sp. CAG:604 was only found in the gut of low-FE pigs, indicating the potential of Prevotella as a biomarker to distinguish the cecal microbiota of pigs with high and low FE (Butyrivibrio (42 days postweaning) and Prevotella (at weaning) are the two genera positively correlated with a low RFI value, which may be due to the introduction of cereal-based diet rich in complex carbohydrates post-weaning  is another important economic metric in addition to body weight and feed intake in swine production. Different compositional and functional characteristics of gut microbiota have also been reported to be associated with FE in pigs. Taking  FCR = 2. ± 0.19, d low FE . Of the -weaning . An OTU--weaning . Most ofin ileum , cecum , or as a biomarker to indicate the target growth performance or health status of host (a result).Bacteroides, Escherichia, Clostridium, Lactobacillus, Fusobacterium, and Prevotella, followed by age-associated shifts to a microbiota with Prevotella and Aneriacter as predominant genera and subsidiary genera, such as Fusobacterium and Lactobacillus. Postweaning diarrhea is a common issue in swine production which is traditionally controlled by ZnO or antibiotics in feed. However, the ban on feed antibiotics by more and more countries and the expected ban on ZnO in feed supplementation from 2022 in the EU urge researchers and pig producers to search for new alternatives. One possible alternative is to use novel probiotics, such as the so-called “next-generation probiotics (NGPs)” derived from GIT. We reveal that a total of 19 “core” bacterial genera dominated by Bacteroides, Prevotella, and Lactobacillus can be found in more than 90% of healthy pigs across different ages, and three enterotypes can also be identified. Without exception, both the composition of these “core” bacteria and enterotypes are gradually changed into an “adult-like” profile with age. These identified genera may probably be the potential candidates of NGPs or the potential target of microflora regulation. New evidence proves that some of these “core” microorganisms like Lactobacillus, Prevotella, Bacteroidetes, and Fusobacteria contribute to the growth phenotypes of pigs. Therefore, some consensus, such as the FCR of finisher pigs is much higher than piglets, or that piglets are prone to stress-induced diarrhea, may be partially due to their unique gut microbiota, in which the contribution of specific bacteria  cannot be ignored. Based on this, a precise nutritional strategy for pigs may have to be considered, along with the requirements of the host and the target microorganisms (such as the “core” microbiota we have screened). Adding substrates preferred by these target microbes will help to increase the abundance of specific symbiotic species and benefit the gut health of pigs. Although we have comprehensively summarized the dynamic shifts of gut microbiota in pigs, substantial work remains to investigate metabolites as the functional output of microorganisms to interact with the host phenotype. It is also possible to achieve the desired phenotype through effective regulation of constantly changing microbial communities or metabolites. Further research targeting these “core” microbes and the dynamic distribution of microbiota, as well as the related function is of great importance in swine production.The current work extends the understanding of the dynamic shift of gut microbes throughout each growth stage of pigs . It alsohttp://www.ebi.ac.uk/ena).The meta-analysis was carried out following the reference of Holman et al. . StudiesAll downloaded 16S rRNA gene sequences were processed using the QIIME software package (v. 1.9.1) . The maxP <0.05), and the effect size of each of these genera was estimated using linear discriminant analysis (10) of 4.0 was used as the cutoff for identifying differentially abundant genera. The between-sample (beta) diversity was assessed using the unweighted and weighted UniFrac distances and Bray-Curtis dissimilarities (post hoc pairwise comparison test (agricolae package)  was used to determine which genera were significantly enriched in each sample type. Genera that were relatively more abundant in a particular sample group were identified by LEfSe using the Kruskal-Wallis test  . All res


The first clinical signs patients present with are skeletal pain, most commonly involving ribs and vertebrae. (2) Methods: Retrospective analysis of 114 patients  receiving spinal surgery from March 2006 until April 2020. Neurological impairments and surgical instability were the criteria for intervention in this cohort. Analysis was based on demographic data, Spinal Instability Neoplastic Score (SINS), location of the lesion, spinal levels of tumor involvement, surgical treatment, histopathological workup, adjuvant therapy, functional outcome, and overall survival (OS). (3) Results: The following surgical procedures were performed: posterior stabilization only in 9 patients, posterior stabilization and decompression without vertebral body replacement in 56 patients, tumor debulking and decompression only in 8 patients, anterior approach in combined approach without vertebral body replacement and without biopsy and/or without kyphoplasty in 33 patients, 3 patients received biopsies only, and 5 patients received kyphoplasty only. The histopathology diagnoses were MM in 94 cases and SBP in 20 cases. Median OS was 72 months (53.4–90.6 months). Preoperative KPSS was 80% (range 40–100%), the postoperative KPSS was 80% (range 50–100%). (4) Conclusions: Surgery for patients with plasma cell neoplasia is beneficial in case of neurological impairment and spinal instability. Moreover, we were able to show that patients with MM and a low number of spinal levels to be supplied have a better prognosis as well as a younger age at the time of the surgical intervention. Plasma cell neoplasia is the second most common hematological malignancy following non-Hodgkin lymphoma ,2,3. It The two subtypes of plasma cell neoplasia are solitary bone plasmacytoma (SBP) and extramedullary plasmacytoma. SBP has a 3-year probability of progression to multiple myeloma (MM) by 10.1% . MM is slightly more common in men than in women, and is twice as common in African-Americans compared with Caucasians . In geneAdditionally, there are two other factors associated with aggressive disease: elevated serum lactate dehydrogenase and evidence of circulating plasma cells on routine peripheral smear examination. To combine both into tumor burden and disease biology, Palumbo et al. published a revised ISS in 2015 .The first clinical signs that patients present with are osteolytic bone lesions, fractures, bone pain, progressive anemia, hypercalcemia, renal insufficiency, recurrent infections, and/or bleeding ,20,21. IIn recent studies, the necessity of surgery in the case of spinal involvement due to plasma cell neoplasia has been discussed. They all reached the same conclusion: in case of structural instability and neurological impairment, surgery must be considered ,24,25. TA total of 114 patients with spinal lesions as the first clinical manifestation of plasma cell neoplasia and characterized by spinal instability or neurological impairment treated between March 2006 and April 2020 were reviewed retrospectively. A histopathological workup confirmed 94 patients with a diagnosis of multiple myeloma (MM) and 20 patients with solitary plasmacytoma of the bone (SPB) . The basNeurological impairments and/or surgical instability were the criteria for intervention in this cohort. Analysis was performed based on age, gender, location of the lesion, spinal levels of tumor involvement, surgical treatment, histopathological workup, functional outcome, and overall survival. Patients’ quality of life was assessed by using pre- and postoperative the Karnofsky Performance Status Scale (KPSS). OS was calculated from the initial diagnosis of plasma cell neoplasia until death or censored at the time of last follow-up. Furthermore, we calculated the overall survival from the time of surgery. The neurologic status of the patient was assessed pre- and postoperatively, according to the American Spinal Injury Association (ASIA) impairment scale. The ASIA impairment scale was rated on a scale from grade A–E, representing the patient’s motor and sensory function with grade A representing no motor and sensory function in the sacral segments S4–5 and grade E representing a normal motor and sensory function in all spinal segments. Statistical analysis was performed using STATA Version 13.1 . In the descriptive data analysis, we show non-normally distributed data as median and interquartile range (IR) and normally distributed variables as mean and standard deviation. Univariate OS distributions were compared using Kaplan–Meier estimates (Log-rank). Multivariate survival analysis was performed using a Cox proportional hazard regression model. In the univariate analysis, we included sex, age, KPSS at admission and day of discharge, SIN-Score, adjuvant radio- and chemotherapy, duration of hospitalization, ASA-Score, SIN-Score, and number of spinal levels supplied. In multivariate Cox regression analysis, the following parameters were included: sex, KPSS at admission, SIN-Score, and number of spinal levels supplied. Differences with a type one error probability of less than 0.05 were considered statistically significant.The study was approved by the local ethics committee (N°335-16S) of the Technical University Munich, School of Medicine. It was conducted in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments .This retrospective study analyzed 114 cases of spinal plasma cell neoplasia surgically managed in our tertiary care institute over a 14-year period (2006–2020) . Among tAt hospitalization, physical examinations showed neurological deficits or palsies resulting from spinal cord compression for 45 patients (39.48%) and pain in 97 patients (85.09%). The median OS was 72 months (53.4–90.6 months). A total of 36 patients died during follow-up. Median OS from the time of surgery was 58 months (32.2–83.7 months).Among all patients, 52 had one spine lesion, while 24 patients presented two lesions and 38 presented three or more spinal lesions.p = 0.000; CI: 95% 9.301319–482.6197) and shorter survival time .Subgroup analysis of patients older than 60 years independent of their diagnosis shows significantly longer hospitalization . In the case of SPB as diagnosis, no significance was shown.The following parameters were included in the Cox regression analysis: sex, KPSS at admission, SIN-Score, and number of spinal levels supplied. For patients with multiple myeloma, only the number of spinal levels supplied were significantly related to OS : 86 (range 58.6–113.4) vs. 62 (range 10.7–113.3) months. No significance was found for postoperative chemotherapy. In multivariate Cox regression analysis, postoperative radiotherapy was proven to be a significant protective factor for longer OS .If postoperative adjuvant therapy is divided according to histology, 18 patients with MM received radiotherapy alone, 27 patients received chemotherapy alone, and 27 patients received a combined adjuvant therapy. In case of SBP, nine patients received radiotherapy alone, two patients received chemotherapy alone and three patients received a combined adjuvant therapy . PatientT1 SE-weighted sagittal (A) and T1 FS-weighted axial  magnetic resonance images showed tumor suspected lesions at Th6 and Th9. Additionally, MRI showed pathological fractures at Th12 and L3. Postoperative CT-imaging (D) confirmed decompression via hemilaminectomy of Th6- and right Th9-vertebral body as well as vertebroplasty of Th9 and Th12. Histology confirmed the rare case of multiple myeloma with plasma cell leukemia.Multiple myeloma and SBP are common malignant primary tumors in vertebra and usually treated conservatively with chemotherapy and radiotherapy ,28. HoweThe analysis of this cohort represents the data of 20 SPB and 94 multiple myelomas. Of these, 77 patients had an initial manifestation of the disease in the spine with neurological deficits, unstable fractures, and pain. In 23 cases, the operation was urgent, and in 6 cases it was an emergency. However, apart from the need to provide surgical care for this cohort, out of these 77 initial diagnoses, 57 patients showed a systemic indication for treatment due to further manifestations in the entire skeleton and/or due to the results from the bone marrow biopsy. In the case of an emergency indication due to neurological deficits, laminectomy initially became established. Via this access, one can remove posterior elements of the spinal column, but less of a tumor, and this often fails to achieve immediate decompression . However, in order to produce less blood loss and smaller wounds, tumor debulking and decompression as already described by Qian et al. has proven effective. In our study, this approach was used in eight patients. According to this, faster access to adjuvant therapy is possible. However, this approach is only possible in the absence of spinal instability and can therefore not be considered a standard of care . Over the years, the operational possibilities have developed significantly . In combination with the imaging developments and the SIN-Score established by them, the approach via decompression of the spinal tumor and stabilization has been shown to be a great gain in treatment in the case of spinal instability, pain, and/or neurological deficit . HoweverIn our study, postoperative radiotherapy proved to be a significant factor for longer median OS. This high sensitivity to radiotherapy, has also been described in recent studies ,39,40,41Apart from surgical options, systemic therapies have improved survival. In the 1990s, a high-dose of melphalan followed by an autologous stem cell transplant (HDM-ASCT) was the standard of care for patients aged <65 years ,43, therAnother significant step was taken in 2016 and 2017 when second-generation proteasome inhibitors such as carfilzomib and ixazomib received approval as well as daratumumab and panobinostat. Other drugs such as pomalidomide, daratumumab, and panobinostat also received approval in this time frame [In summary, a multimodal concept should be implemented including surgical treatment and subsequent systemic therapy. This study offers a single-center experience only. A limitation of this study is its retrospective nature, as it introduces an unavoidable selection bias. Furthermore, due to the highly selected patient population, the number of patients included in this study is relatively small. Moreover, this study cannot reflect the impact of cytogenetic or molecular biological status on the prognosis. Therefore, most recent innovations in systemic chemotherapy are not reflected. Surgery for patients with plasma cell neoplasia is beneficial in the case of neurological impairment and spinal instability. Moreover, we were able to show that patients with MM and a low number of spinal levels to be supplied have a better prognosis as well as a younger age at the time of their operations.


Proganochelys quenstedtii represents the best-known stem turtle from the Late Triassic, with gross anatomical and internal descriptions of the shell, postcranial bones and skull based on several well-preserved specimens from Central European fossil locations. We here report on the first specimen of P. quenstedtii from the Late Triassic (Klettgau Formation) Frickberg near the town of Frick, Canton Aargau, Switzerland. Similar to other Late Triassic ‘Plateosaurus-bearing bonebeds’, Proganochelys is considered to be a rare faunal element in the Swiss locality of Frick as well. The specimen, which is largely complete but was found only partially articulated and mixed with large Plateosaurus bones, overall resembles the morphology of the classical specimens from Germany. Despite being disarticulated, most skull bones could be identified and micro-computed tomography (CT) scanning of the posterior skull region reveals new insights into the braincase and neurovascular anatomy, as well as the inner ear region. These include the presence of a fenestra perilymphatica, potentially elongated cochlear ducts, and intense vascularization of small tubercles on the posterior end of the skull roof, which we interpret as horn cores. Other aspects of the skull in the braincase region, such as the presence or absence of a supratemporal remain ambiguous due to the fusion of individual bones and thus lack of visible sutures . Based on the size of the shell and fusion of individual elements, the specimen is interpreted as a skeletally mature animal.The online version contains supplementary material available at 10.1186/s13358-022-00260-4. Proganochelys Baur, Proterochersis from the Late Triassic (Middle Norian) of Germany and Poland and Palaeochersis and Waluchelys  of China  of Germany  of the Gruhalde Member of the Klettgau Formation, which are Norian in age Fig. E. These Plateosaurus bones , a complete pelvis still in articulation and a fragmented postpelvic hypoischium. Due to the find situation Fig. D, the sines Fig. D. The skMechanical preparation of the fossil was done by one of us (BP), who also led the excavation. Missing parts of the carapace, especially on the left side, were mirrored and modelled for display purposes, based on preserved parts from the right side. The carapace has been found in visceral view and preparation was done from the inside first. Later the carapace was mounted on a bed of synthetic supporting material to prepare the outside of the carapace as well. Due to the extremely thin and fragile nature of the shell, it cannot be easily taken off or flipped over without risking breakage. The description of the internal morphology is, therefore, limited and based largely on photographs that have been taken during the process of preparation. See Tables The large posterior skull fragment of SMF 09-F2 was scanned using a NIKON XTH 225 ST CT Scanner housed at the Anthropological Department of the University of Zurich. The micro-computed tomography scan was taken with a voltage of 91 kV and a current of 345 µA, yielding a voxel size of 0.03758 mm, with no filter used. Reconstruction of the virtual model was performed using Mimics 23.0. CT scans and derivative 3D models are deposited on the online repository MorphoSource (see Declarations section).Proganochelys skull SMNS 16890 , and the asymmetric preservation in P. talampayensis raises at least some doubt for this species as well. For both species in question, alternative interpretations could potentially be supported, especially as the respective bone areas are slightly raised so that an interpretation of an osteoderm identity cannot be ruled out with certainty. The original proposition of a supratemporal for P. quenstedtii was likely influenced by outgroup comparisons of the time , although it should be noted that recent taxonomically comprehensive phylogenies do not actually support this taxon to be a stem turtle , the basis tuberculi basalis is unremarkable and not associated with a ridge , the hiatus acusticus remains unossified instead  in SMF 09-F2, we could not identify this structure, which apomorphically extends through the base of the processus interfenestralis in turtles Rieppel, , includiGaffney, . We inteGaffney, . The antGaffney, . The acoGaffney, . The facGaffney, , and opeGaffney, . SpecifiGaffney, C–F. ThisGaffney, D in alluGaffney, . The latGaffney,  are a miGaffney, . Our intGaffney,  of the fGaffney, .Proganochelys quenstedtii specimens  nerve foramina of SMF 09-F2 are also clearly identifiable canals, which traverse the basisphenoid anteroposteriorly at the dorsum sellae and exit the bone anteriorly halfway along the dorsoventrally deep pituitary fossa Figs. , 6, 7. TProganochelys quenstedtii  and 15 marginals with the 10th broken and overlapping the 9th approximately 3 cm . SMF 09-F2 thus appears to differ in the number of marginals compared to SMNS 16980 and MB 1910.45.2 which, according to Gaffney  were recovered Fig. .Fig. 13FProganochelys resembles already the morphology of other turtles  of turtles are known from extinct and extant terrestrial tortoises and extinct non-testudinoid taxa . A, Partially reassembled bones. B, Interpretative sketch and identification of the skull elements.Additional file 2:Fig. S2 Spatial comparison of the skull elements Proganochelys quenstedtii (SMF 09-F2) with newly generated 3D surface model of SMNS 16980  with interpretative drawings of scute sulci superimposed on the shell bones. A, Carapace in angled anterodorsolateral view. B, Right side of carapace in dorsolateral view. C, Right side of carapace in lateral view. Reconstructed parts of the carapace are delimited by grey-shaded area set off by a stippled white line and marked with a white r. The anterior aspect of the carapace is marked in each view by a black arrow. Abbreviations: c, cervical scute; m, marginal scute; pl, pleural scute; sc, supracaudal scute; sm, supramarginal scute; v, vertebral scute.Additional file 4:Fig. S4 Carapace of Proganochelys quenstedtii (SMF 09-F2) during preparation. Large white arrows indicate cranial direction. A, Internal/visceral view of carapace, with the dorsal side still resting on the plaster jacket. B, Close-up view of the anterior portion of the carapace with shallow grooves between costals being indicated by small white arrows.Additional file 5:Fig. S5 Right hind limb elements of Proganochelys quenstedtii (SMF 09-F2) during preparation. A, Matrix block with zeugopodial elements, limb osteoderms and few autopodial elements. B, Reverse side of matrix block showing most of the autopodium still in articulation.


Cordia dichotoma, a plant of medicinal importance native to the Jammu region of India, was selected for the isolation and characterization of culturable endophytic bacteria and evaluation of their antimicrobial activities. Standardized surface sterilization methods were employed to isolate thirty-three phenotypically distinguishable endophytic bacteria from the root, stem, and leaf parts of the plant. Shannon Wiener diversity index clearly divulged diverse endophytes in roots (0.85), stem (0.61), and leaf (0.54) tissues. Physio-biochemical features of the isolates differentiated the distinct variations in their carbohydrate utilization profile and NaCl tolerance. The endophytes produced an array of enzymes, namely, catalase, oxidase, amylase, cellulase, nitrate reductase, and lipase. The bacterial isolates belong to the genera Bacillus, Pseudomonas, Paenibacillus, Acidomonas, Streptococcus, Ralstonia, Micrococcus, Staphylococcus, and Alcalignes predominantly. However, the antibiotic susceptibility pattern indicated that the isolates were mostly sensitive to erythromycin and streptomycin, while they were resistant to rifampicin, amoxicillin, and bacitracin. Interestingly, majority of the bacterial endophytes of C. dichotoma showed antimicrobial activity against Bacillus subtilis followed by Klebsiella pneumoniae. The 16S rRNA sequence of Bacillus thuringiensis has been deposited in the NCBI GenBank database under accession number OM320575. The major compounds of the crude extract derived from endophytic B. thuringiensis OM320575, according to the metabolic profile examination by GC-MS, are dibutyl phthalate, eicosane, tetrapentacontane, heneicosane, and hexadecane, which possessed antibacterial activities. In conclusion, results indicated the potential of C. dichotoma to host a plethora of bacterial endophytes that produce therapeutic bioactive metabolites.Endophytic bacteria isolated from medicinal plants are crucial for the production of antimicrobial agents since they are capable of possessing bioactive compounds with diverse structures and activities.  Global health problems due to the evolution of defiance to accessible antibiotics by pathogenic fungi and bacteria, the inefficacy of present antifungal and antibacterial agents to various fungal and bacterial infections, and the emergence of life-threatening viruses require a critical need to search for novel and effective antimicrobial agents  that colonize inner healthy plant tissues without causing them any pathogenicity Wilson, . Nearly Bacterial endophytes are thought to have a symbiotic relationship with plants. In this mutualistic relationship, plants give shelter and nutrients to the endophytes , India, in sterile polythene bags and brought immediately to the laboratory and processed to reduce the risk of contamination. The specimen was deposited at the Department of Botany, University of Jammu, with accession number 16613.Healthy plant parts, i.e., root, stem, and leaf, were collected from three individual plants of The collected plant samples  were washed under running tap water to remove the dust and debris present on the surface, followed by washing in distilled water. They were carefully excised and exposed to four different reported surface sterilization methods. In Method I, samples were surface sterilized with 70% ethanol for 8 min  and oxidase reagent (1% tetramethyl-p-phenylenediamine dihydrochloride), respectively, on the smear of freshly grown endophytic bacterial cultures in nutrient agar plates. The presence of oxygen bubbles and purple color, respectively, was considered as positive.The endophytic bacterial strains were screened for the presence of several enzyme activities, such as catalase, oxidase, amylase, cellulase, and lipase. The monitoring was done by streaking the selected endophytic bacteria on culture agar media with the substrate . Endophytic bacterial strains were inoculated into the plates and incubated at 37°C for 6 days, and observations were recorded every 24 h.Staphylococcus aureus MTCC 737, Escherichia coli MTCC 1687, Bacillus subtilis MTCC 1789, Pseudomonas aeruginosa MTCC 1688, Klebsiella pneumonia MTCC 432, and Salmonella typhi MTCC 733. Each endophytic bacterial culture (10 ml) was centrifuged at 1,000 rpm for 10 min and the supernatant was used to determine antibacterial activity. Overnight grown cultures of the test organisms were spread by sterile cotton swabs onto the surface of the Muller Hinton agar plates. Wells (6 mm) were made and 100 μl of endophytic bacterial strain was added into it; an equal volume of sterile nutrient broth instead of bacterial endophyte was used as a negative control, and ampicillin was used as a positive control. All plates were wrapped with parafilm and incubated for 24 h at 37°C and observed for the zone of inhibition of pathogenic bacteria. Antibacterial activity was evaluated by measuring the diameter of the clear zone of inhibition  and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used to amplify the 16S rRNA gene. Total DNA (50–500 ng) was added to the PCR mix (30 μl), which contained 24 μl of DreamTaq PCR master mixture, 1 μl of 10 nm each primer, 2 μl of DNA, and 7.5 μl of 10X buffer in a total volume of 30 μl. This reaction was performed under the following conditions: one cycle of 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final extension of 10 min at 72°C. The amplified DNA products were separated and visualized under UV light using agarose gel electrophoresis. The amplicons were purified using the Genei PureTM quick PCR purification kit and quantified using a spectrophotometer at 260 nm. Biologia Research India sequenced the purified partial 16S rDNA amplicons. The sequences were assembled, edited, and aligned in MEGA11 before comparison with those in the GenBank database with the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the sequence homology with closely related organisms. In this study, the microorganisms with the highest level of identity (100%) were chosen as the closest match, and isolated bacteria were classified to the species level based on the information of the closest microbes.Bacterial isolates with high antibacterial activity were chosen for molecular identification using 16S rDNA sequence analysis. A loopful of freshly grown bacterial cells was dissolved in tubes with 500 μl of Tris-EDTA for DNA extraction. Then, 500 μl of phenol: chloroform: isoamyl alcohol (25:24:1) was added. This mixture was vortexed and centrifuged at 14,000 To analyze the various volatile bioactive constituents, an ethyl acetate extract of SMC212 was subjected to gas chromatography-mass spectrometry (GC-MS). The Indian Institute of Technology, Jammu, conducted the GC-MS analysis. SHIMADZU, QP2010 PLUS was used for GC-MS analysis. The injecting temperature was set to 250°C, the column temperature was set to 50°C, the pressure was set to 29.7 kPa, and the column flow rate was set to 0.72 ml/min. The sample's total running time was 40 min. The phytochemical compounds in ethyl acetate extract were identified based on retention time by matching MS with available standards using the Willey and NIST libraries. The constituents' names, molecular formulas, and molecular weights were determined.H/) was calculated as:Colonization rate was determined as the total number of plant segments colonized by bacteria divided by the total number of incubated samples. Isolation rate was calculated as the number of bacterial strains obtained from plant segments divided by the total number of incubated segments. The Shannon Weiner diversity index  were treated separately by a different amalgam of chemical disinfectants. Method I (70% ethanol) was not found productive individually as a high percentage of contamination was noticed along with the growth of endophytes. While in Methods II, III, and IV, plant samples were treated with different mixtures and duration of sodium hypochlorite, ethanol, and mercuric chloride to attain a sufficient result. In Method III, mercuric chloride was successful in removing the contamination, yet the survival percentage of endophytic bacteria declined. Our result shows that only Method IV  was found effective for surface sterilization of C. dichotoma plant tissues, with no contamination and a high percentage of survival rate. Reports are indicative of surface sterilization methods used in dye-yielding plants for the isolation of endophytic bacteria with 70% ethanol for 6–8 min and 0.1% mercuric chloride for 5–10 min showing promising results  of C. dichotoma of which 16, 9, and 8 isolates were obtained from root, stem, and leaf segments, respectively (C. dichotoma harbor diverse types of endophytic bacteria as compared with stem (0.61) and leaf (0.54) which are in line with the available reports for Chinese medicinal plants Berberis poiretii, Eucommia ulmoides, and Rhus potanini showing a high colonization frequency (47–63%) and isolation rates (0.7–0.9) of endophytic fungi , amylase (78%), cellulase (87%), nitrate reductase (39%), methyl red (30%), and indole (39%) production. The isolated endophytes were also screened for their ability to utilize carbohydrates, i.e., dextrose, fructose, sucrose, maltose, and lactose, in phenol red agar medium supplemented with 1% sugar. Dextrose was found to be the best-utilized carbon source by most of the bacterial isolates; bacterial isolates were moderate in fermenting fructose and sucrose, while very few isolates were fermenting maltose (6) and lactose (7).The bacterial endophytes of Bacillus, Pseudomonas, Paenibacillus, Acidomonas, Streptococcus, Ralstonia, Micrococcus, Staphylococcus, and Alcalignes. Similar endophytic bacteria have been reported from medicinal plants like Azadirachta indica, Gynura procumbens, Tephrosia apolline, Phyllanthus emblica, and Vitis vinifera  was 2.2. The evolutionary history was inferred by using the maximum likelihood method and the bootstrap consensus tree inferred from 1,000 to 3,000 iterations. The evolutionary relationship is represented as a dendrogram , which cB. thuringiensis OM320575 were analyzed using GC-MS which led to the identification of 38 different compounds. B. thuringiensis OM320575 produced bioactive chemicals with antimicrobial, antioxidant, and anticancer properties, according to GC-MS analyses.The results pertaining to GC-MS analysis  of the eC. dichotoma plant parts such as root, stem, and leaf collected from SMVDU, Katra, Jammu, for the isolation of endophytic bacteria. In India, a countable number of reports showed the diversity of endophytic bacteria and fungi in medicinal plants  sodium hypochlorite was found to be more effective than the low concentration of (1%) in removing plant surface microorganisms. Hence, sodium hypochlorite showed very low contamination because it is very effective as a disinfectant agent against many contaminated bacteria as previously reported  and dibutyl phthalate (C16H22O4) was discovered in larger percentages in the ethyl acetate fraction of Streptomyces strain . Antimicrobial estimation revealed that bacterial endophytes showed significant antibacterial activity against S. typhi, E. coli, P. aeruginosa, B. subtilis, S. aureus, and K pneumonia. The therapeutic properties of C. dichotoma may be a consequence of its endophytic microorganisms producing biologically active products. The bacterial strains were sensitive to antibiotic erythromycin and streptomycin, whereas most of them were resistant to rifampicin, amoxicillin, and bacitracin. Endophytes have been found to be abundant sources of novel natural chemicals with a wide range of biological functions and a high level of structural diversity. By synthesizing dibutyl phthalate; eicosane; tetrapentacontane; heneicosane; hexadecane; tetradecane; silane, trichlorooctadecyl; and 2,4-di-tert-butylphenol as bioactive chemicals, one potential endophyte isolated from C. dichotoma and identified as B. thuringiensis OM320575 by 16S rRNA demonstrated considerable antibacterial activity against pathogenic bacteria. Further exploration would provide us an insight into the potential use of isolated bacterial endophytes, and it will lead to the discovery of various high-value metabolites.This may be the first-ever report on endophytic bacteria isolated from The original contributions presented in the study are included in the article/MS conducted the experiments, recorded the observations, analyzed the results, and prepared the draft manuscript. SM contributed to designing the experiments, providing the facilities, analyzing the results, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflictof interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To explore the prognostic risk factors of ESD curative resection of gastrointestinal-neuroendocrine neoplasms (GI-NENs).  A total of 97 patients treated with ESD successfully in our hospital were selected, their surgical site, size, number of resection lesions, operation time, intraoperative complications (such as bleeding and perforation), and treatment status were recorded, and the number of hemostatic clamps used after the postoperative follow-up results and the independent risk factors for ESD complications were obtained through the comparison between the noncomplication group and the ESD complication group using regression analysis. P > 0.05). Postoperative bleeding was related to the operation time (P=0.017), but it was not an independent risk factor for postoperative bleeding . 59 cases were followed up by endoscopy after the operation, and recurrence or no new tumors were found.  A total of 97 patients with gastrointestinal neuroendocrine tumors were treated with ESD. 61 were males, 36 were females, the ratio of male to female was 1.7 : 1, onset age was 20–78 years old, and median onset age was 50 years old. In 81 cases, tumors were located in the stomach, 10 in the duodenum, and 6 in the rectum. A total of 103 lesions were detected by endoscopy, including 1 case with 2 sites in the stomach, 5 cases with 2 sites in the rectum, and the rest were single. The tumor diameter was 0.3 ∼ 2.5 cm, and the median diameter was 0.6 cm; there were 25 sites with a diameter less than 5 cm. There were 57 places with 10 mm, 16 places with 10–15 mm, and 5 places with >15 mm. All ESD operations were performed in one piece, with a total resection rate of 100%; 89.6% (60/67) of postoperative pathology showed negative basal, and 90.3% (56/62) showed negative resection margin, with a complete resection rate of 88.9% (48/54). ESD's operation time is 6 ∼ 66 min, and the median time is 18 min. During the operation, 5 cases had small amount of bleeding, 3 cases were perforated, 2 cases of delayed postoperative bleeding, 1 case of bleeding was caused by the patient's failure to follow the advice of the doctor to eat a large amount of solid food too early, and 1 case of delayed perforation . ESD operation that bled, age, gender, and perforation location, pathological grade, pathological classification, tumor diameter, tumor surface, operation time, number of titanium clips, origin, echo uniformity, and echo level were statistically insignificant ( ESD is an effective and safe treatment method for gastrointestinal neuroendocrine tumors with a diameter of 1-2 cm without invading the muscularis propria. The intraoperative complications seem to have little relationship with the patient; postoperative delayed bleeding is closely related to the ESD operation time but it is not an independent risk factor. Gastrointestinal-neuroendocrine neoplasms (GI-NENs) are a type of heterogeneous tumors originating from peptidergic neurons and neuroendocrine cells, which show obvious signs of slow growth from indolence and low-grade malignancy to high metastasis. A series of malignant biological behaviors  is distrAt present, the diagnosis of gastroenteric pancreatic-neuroendocrine neoplasm (GEP-NEN) mainly depends on clinical manifestations, tumor marker level, relevant imaging examinations (such as computerized tomography (CT) and magnetic resonance imaging (MRI) or digestive endoscopy), and “gold standard pathological examination.” According to the clinical manifestations of patients, gastroenteric pancreatic-neuroendocrine tumors (GEP-NET) can be divided into nonfunctional and functional tumors, most of which are nonfunctional tumors, which can be asymptomatic for many years. Most of them are accidentally found or manifested as compression symptoms caused by masses and signs of tumor metastasis. In this study, 154 patients with GEP-NEN showed typical carcinoid syndrome, accounting for only 1.3%, which is consistent with relevant literature reports at home and abroad [At present, ESD treatment is the main choice for GEP-NEN tumors with tumor diameters less than 2 cm, no lymph node metastasis, infiltration depth not to the muscular propria, and well-differentiated GEP-NEN tumors . HoweverThe main purpose of this paper is how ESD is used for treating patients in their particular age durations. For this purpose, ESD has different procedures and methods. Using ESD methods, patients were treated with some ratio from male to female. Tumors with ESD were diagnosed, and the diameter and different complications were identified. Patients of this disease have different conditions due to their ages, and young age patients can have a better treatment than the old.Participants were collected in our hospital from December 2019 to June 2021. Patients who successfully underwent ESD endoscopic resection and were pathologically diagnosed as gastrointestinal neuroendocrine tumors strictly implemented the inclusion and exclusion criteria. This study has been approved by the ethics committee of Xingtai People's Hospital and obtained the informed consent of patients. A total of 97 patients were enrolled, including 61 males and 36 females, aged 20–78 years old; the median age of onset was 50 years old.Age >18 years oldThe pathology department of our hospital diagnosed gastrointestinal neuroendocrine tumorTreat patients with ESDNeuroendocrine tumors in other systems except for the digestive tract The pathology department of our hospital has not diagnosed gastrointestinal neuroendocrine tumorThose who have ESD contraindicationsExclude other fatal diseasesOlympus UM-200 ultrasonic endoscopic system, UM-2R microprobe, OlympusQ260 or XQ260 electronic gastroscope, Olympus JF-240R electronic duodenoscope, Olympus JF-260R electronic duodenoscope, AGB high-frequency generator, HK knife, IT knife, injection needle, trap, hemostatic forceps, transparent cap, titanium clip, ventilator, and ECG monitoring were used.The lesion site and endoscopy results are recorded in detail according to the sex and age of the patient before surgery, and the blood biochemistry, coagulation function, electrocardiogram, chest X-ray, and lung function test are checked regularly. Possible complications such as substitution surgery, requirements and precautions for family members, intraoperative bleeding and perforation, late-onset bleeding, and late perforation should be informed to patients and families before surgery. Patients and their families can have a certain degree of understanding of the operation, eliminate their anxiety and fear, and sign the informed consent of the operation.Marking. The scope of the lesion was determined, and the spot electrocoagulation was marked on the outer edge of the lesion by about 5 mmSubmucosal Injection. Submucosal injection  should be performed at the mark until the lesion is uniformly elevatedIncision of the Mucosa. The lesion is incised at the lesion markerSubmucosal Dissection. After the incision of the mucosa around the lesion, the lesion was exfoliated to the whole lesionTreatment of Wounds. The small vessels exposed on the wound surface were stopped by thermal hemostatic forceps, and the wound surface was closed by titanium clipsSpecimen Processing. The diseased tissue was smoothed and fixed on a thin plate with a pin and fixed with 10% neutral formalinESD operation steps are as follows :Marking.Postoperative fasting for 2 to 3 days, routine fluid replacement, using antibiotics and hemostatic drugs if perforation or bleeding occurs, observing abdominal symptoms and signs, and monitoring exhaust and defecation are done in postoperative treatment. All resected lesions were immersed in a 10% formalin solution and sent for pathological examination to determine the nature of lesions, the cutting edge of specimens, and whether tumor cells were involved in the base.Lump-sum resection: the lesion was a lump-sum resection under endoscopyComplete resection: at the pathological level, both horizontal and vertical resection margins of the whole specimen were negativeCurative resection: complete resection with no or low risk of lymph node metastasisAll the enrolled patients were followed up with an endoscopic follow-up, which generally lasted from 1 to 3 months after ESD. The follow-up mainly included whether there were new lesions, whether the postoperative lesions were repaired, whether the levels were clear, and whether there were abnormal echo changes.n ≥ 40 and all theoretical frequencies ≥5, Pearson chi-square test was used. Fisher's exact test was used for data that did not meet the chi-square test conditions. Univariate analysis was first used for patients with ESD complications. After confounding factors were excluded, logistic multifactor analysis was used to obtain independent risk factors. P < 0.05 was considered statistically significant.The data were analyzed by SPSS18.0 statistical software, and the measurement data conforming to normal distribution were expressed as mean ± standard deviation; otherwise, the median was used as an expression, and the adoption rate or composition ratio of the series data was described. For qualitative data, when In this study, 97 patients with gastrointestinal-neuroendocrine tumors (GI-NET) were treated by ESD, including 61 males and 36 females, with a male to female ratio of 1.7 : 1. The onset age was 20–78 years, and the median onset age was 50 years.Of the 97 GEP-NEN patients, 81 were located in the stomach, 10 in the duodenum, and 6 in the rectum. A total of 103 lesions were detected by endoscopy, including 2 lesions in the stomach in 1 case, 2 lesions in the rectum in 5 cases, and the rest were single. The diameter of the tumor was 0.3–2.5 cm, and the median diameter was 0.6 cm. There were 25 lesions with a diameter of <5 mm, 57 with a diameter of 5–10 mm, 16 with a diameter of 10–15 mm, and 5 with a diameter of >15 mm.ESD was successfully performed on all patients to remove the lesions; among which 2 cases underwent additional surgery due to the involvement and infiltration of tumor cells in the base as indicated by postoperative pathology. The operation time was 6–66 min, with a median time of 18 min. During the operation, 5 cases had a small amount of bleeding, which was stopped by hot biopsy forceps or titanium clips. There were 3 cases of perforation, 2 of which were clamped by intraoperative titanium clip, and the other one was not treated. There were 2 cases of delayed bleeding after the operation and 1 case of bleeding caused by eating a large amount of solid food prematurely without following the doctor's advice. All patients were cured by internal conservative treatment. Delayed perforation occurred in 1 case, which was clipped with a titanium clip under endoscopy.P < 0.05 were included in the binary logistic regression analysis model, and their independent risk factors were obtained. According to the analysis, age, gender, location, pathological grade, pathological classification, tumor diameter, tumor surface, operation time, number of titanium clips, source, echo uniformity, and echo level were not risked factors in EDS surgery, but the number of lesions may cause perforation.In this study, a total of 5 patients had intraoperative bleeding, 3 patients had intestinal perforation, and rectal bleeding was more than other parts of the bleeding. Therefore, the intestine can be divided into upper gastrointestinal tract and gastrointestinal division. Similarly, the pathological stage can be divided into G1 stage and G1 stage. The high degree of echo is divided into higher than normal group and lower than normal group. The origin can be divided into mucosal layer and mucosa. Then, we performed single-factor analysis on the suspected risk factors of ESD complications  and intraoperative bleeding and perforation. After excluding confounding factors, factors with According to the analysis in Tables In this study, there were 2 cases of delayed postoperative bleeding and 1 case of delayed postoperative perforation. Considering that the incidence of postoperative perforation was very low <1%, this study focused on analyzing the risk factors of delayed postoperative bleeding. Suspicious risk factors for delayed bleeding were found in univariate analysis, including age, sex, location, pathological typing, tumor diameter, tumor surface, time of surgery, quantity, source, titanium forceps, echo uniformity, and echo height. And the operation time is related to postoperative bleeding. When included in logistic multifactor regression, it was found that it was not an independent risk factor for delayed postoperative bleeding. According to the analysis, age, gender, location, pathological grade, pathological classification, tumor diameter, tumor surface, operation time, number of titanium clips, source, echo uniformity, and echo level were not risk factors in EDS surgery  postoperative pathology suggested a negative base, 90.3% (56/62) suggested a negative margin, and the complete resection rate was 88.9% (48/54). Pathological results showed that 85 cases were G1, 4 G2, and 3 G3) were followed up by endoscopy. The endoscopic review showed that the resection sites of lesions were mostly recovered without new tumors or recurrence, and local mucosal thickening and clear hierarchical structure were most common.All the patients were followed up by endoscopy, of which 59 patients (52 Currently, there is no unified treatment specification for the treatment of neuroendocrine tumors –12, and Though some operation methods have reached a consensus, because of their low incidence, there is still considerable controversy in surgical methods and the lack of clinically credible and effective data to evaluate efficacy, so with the continuous development of endoscopic technology, compared with conventional surgery, endoscopic surgery has less trauma, fast recovery, and less cost, making more and more people choose endoscopic treatment. Since most gastrointestinal NENs tend to invade the submucosa , traditiBleeding and perforation are the most common complications of ESD treatment for GEP-NEN. Domestic reports reported that the incidence of intraoperative acute bleeding was 7.1% , and theA definitive diagnosis of GEP-NEN requires a pathological diagnosis. Compared with other similar studies at home and abroad , NEN hadGastrointestinal neuroendocrine tumors, as potential malignant tumors of the digestive system, have a low incidence rate, which can occur anywhere in the digestive tract and lack specific clinical manifestations. These factors have led to the difficulty of diagnosing the disease in the past. With the increasing incidence rate of the disease and the improvement of the level of diagnosis, GEP-NEN has gradually entered our field of vision. Surgical resection has been the preferred treatment for this disease. It has been popular. In recent years, with the development of endoscopic technology, ESD has been used as an effective and safe treatment for GEP-NEN. However, because of the low incidence rate of gastrointestinal neuroendocrine tumors, the sample size of this study is small, and patients need to choose bias. The research results of large samples are further confirmed.


Populus AAAP family, 83 PtrAAAPs were identified from Populus trichocarpa and grouped into 8 subfamilies. Subsequently, chromosomal distribution, genetic structure, cis-elements analysis, and expression pattern analysis of the AAAP family were performed and the potential gene AAAP21 regulating root development was screened by combining the results of RNA-Seq and QTL mapping. PsAAAP21 was proven as promoting root development by enhancing AR formation. Differentially expressed genes (DEGs) from RNA-seq results of overexpressing lines were enriched to multiple amino acid-related pathways, and the amino acid treatment to transgenic lines indicated that PsAAAP21 regulated amino acid transport, including tyrosine, methionine, and arginine. Analysis of the AAAP gene family provided a theoretical basis for uncovering the functions of AAAP genes. The identification of PsAAAP21 on root promotion and amino acid transport in Populus will help with breeding new woody plant species with strong rooting ability.The adventitious root (AR) is the basis for successful propagation by plant cuttings and tissue culture and is essential for maintaining the positive traits of a variety. Members of the amino acid/auxin permease (AAAP) gene family play indispensable roles in various plant metabolisms and have few studies on root growth and amino acid transport. In this study, with a systematic bioinformatics analysis of the  The root is an essential plant organ and plays roles in absorption, support, transport, and synthesis; good root development is of great significance to the growth and development of plants. Moreover, the research on plant root development has become increasingly in-depth and has become a research hotspot ,2,3,4. ANitrogen, an essential nutrient element for plant growth, is absorbed from the rhizosphere by the roots in the form of nitrates and ammonium salts and is stored in the form of amino acids and nitrogen-containing compounds . Amino aAtAAP1/NAT2 was the first identified plant amino acid transporter and was found in Arabidopsis thaliana in 1993 [AAAPs have been identified in several species, such as arabidopsis [ in 1993 . The AAA in 1993 . Aa_tran in 1993 , and acc in 1993 ,16. To dbidopsis , tea trebidopsis , etc.OsAAP4 increases rice tillering and grain yield as a result of enhancing the neutral amino acid concentrations of Val, Pro, Thr, and Leu. Exogenous Val or Pro significantly promotes the bud outgrowth and bud outgrowth overexpressing lines [OsAAP1 has similar functions in rice tillering and grain yield, the treatment with neutral amino acids can promote axillary bud outgrowth [AtAAP1 regulates root uptake and embryos loading neutral amino acids [The research on the function of AAAP genes is gradually deepening. OsAAP6 is the positive regulator of rice grain protein content (GPC) promotes amino acid uptake by the root system and influences amino acid distribution . Overexpng lines . OsAAP1 utgrowth . In arabno acids ,21  ranged from 0 to 117.13, with PtrAAAP15 and PtrAAAP41 being the smallest and largest, respectively. Exon ranged from 1 to 11. The subcellular localization of PtrAAAPs was also predicted, with all PtrAAAP proteins localized to the cytoplasm, membrane, nucleus, vacuole, plasma membrane, and extracellular Only one signal peptide was predicted in PtrAAAP35 for all PtrAAAP proteins  and ProT having the smallest number (3) (A phylogenetic tree of the mber (3) A.P. trichocarpa and arabidopsis. At the same time, the motifs and structural features of each protein were marked , methyl jasmonate (172), ethylene (179), and anaerobic induction (220). Three of these, abscisic acid, methyl jasmonate, and ethylene, belonged to hormone type, one belonged to stress type, and none belonged to growth and metabolism type  and to plot the expression heatmap. To further validate the correctness of the tissue-specific expression data, 14 AAAP genes were selected, except for ANT and ProT subfamilies, and two genes from each subfamily were selected to determine their expression patterns in Populus simonii ‘Tongliao1’, and the expression pattern results were generally consistent with those of Phytozome, meanwhile, all 14 genes were highly expressed in the roots  were found to be differentially expressed in the results of different individual root transcriptomes in the previous period. For these 39 genes, two expression patterns existed for higher and lower expression, with 15 and 24 genes, respectively, while the expression patterns of the 39 genes of the hybrid offspring and the parents essentially remained consistent [PtrAAAPs were searched in the results of QTL mapping for root and stem traits, and a total of three PtrAAAPs were found  and good rooting offspring (GR) was higher than that of P. simonii ‘Tongliao1’ (Ps) and bad rooting offspring (BR). Moreover, AAAP21 could respond to drought, but didn’t show significant reaction to salt, which indicated AAAP21 was the positive factor for root development and drought response  development. Traits of root fresh weight and number of AR, but not maximum root of length, showed differences. The inhibited expression of PsAAAP21 suppressed AR development, furthermore, this difference was caused by promoting the formation of AR   B. Moreovpathways C. The anAAAP21 expression and amino acid content was analyzed. The expression of AAAP21 was significantly correlated with tyrosine (Tyr) and arginine (Arg) content, especially with the opposite trend of Tyr content in phloem and xylem (−0.89 and 0.93)  on overexpression plants and WT plants than on inhibited expression plants, and this combined with the reduced inhibitory effect of 25 mM Arg on overexpression plants compared to WT and inhibited expression plants (In the RNA-seq results of overexpressed strains (OE#6), the enrichment results of KEGG with DEGs showed that many DEGs were enriched to amino acid related pathways, including arginine and proline metabolism, tyrosine metabolism, and cystine and methionine metabolism, which contained 9, 26 and 20 genes, respectively. Meanwhile, there were two expression modes of positive and negative correlation A–D. Combnd 0.93) . In orden plants .Camellia sinensis, Medicago truncatula, Phyllostachys edulis, and Liriodendron chinense [Populus, which important model and energy species and plays an important role in scientific research and production, is not comprehensive. [Populus and the potential role of AAAP proteins on plant root development, the first systematic genome-wide analysis was performed in the P. trichocarpa genome and genetic transformation of AAAP21 gene was performed to verify its regulation function on root development.Amino acid/auxin permease (AAAP), a family of proteins that perform amino acid transport functions in plants, has been identified in several plants, including chinense ,16,17,38hensive. ,24,25,39PtrAAAPs, and 83 PtrAAAP genes were identified from P. trichocarpa , used in this study was originally collected from a natural stand in Tongliao, Inner Mongolia Autonomous Region, and Populus deltoides ‘Danhong’ (Pd), a fast-growing and insect-resistant variety, were preserved in the nursery of Chinese Academy of Forestry. The 84K poplar  was now preserved in the experimental site and tissue culture room of the Chinese Academy of Forestry under 2500 lx and 25 °C.The http://pfam.xfam.org/, accessed on 19 February 2020) [Populus bHLH proteins were identified with HMMER 3.0   from P. trichocarpa (Pt) (v3), downloaded from Ensembl Plant . Meanwhile, the PtrAAAPs were confirmed on SMART [http://pfam.xfam.org/, accessed on 19 February 2020) [http://www.ncbi.nlm.nih.gov/cdd/, accessed on 19 February 2020). The PtrAAAPs characteristic and located information were identified with ProtParam  [http://linux1.softberry.com/, accessed on 19 February 2020) and SignalP 4.1  [Arabidopsis thaliana and Oryza sativa.After retrieving the Hidden Markov Model profiles of the Aa_trans domain (PF01490) from the Pfam database (ry 2020) , the Popon SMART , pfam(htry 2020)  and NCBIry 2020)  and TBtory 2020)  softwarery 2020) . TBtoolsry 2020)  was usedPtrAAAPs, extracted with Bio-Linux after being aligned by ClustalW with MEGA X , were divided into eight subfamilies according to their amino acid conservation. The Maximum likelihood estimate was used.Protein sequences of http://meme-suite.org/, accessed on 19 February 2020) [PtrAAAPs protein sequences, the length and number were set to 6–50 and 20, and other parameters were the default values. The structure information of PtrAAAPs was extracted and TBtools [PtrAAAPs structure map.MEME (ry 2020)  was used TBtools  was usedhttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 19 February 2020) of 2000 bp upstream sequences of the transcriptional start point for PtrAAAPs online website was used to identify cis-acting regulatory elements and then cis-acting regulatory elements were analyzed and classified after that. The PsAAAP21 protein sequence was submitted to the online website String  [PtrAAAPs on root growth. The P. trichocarpa was selected as reference.PlantCARE  (http://ry 2020)  to queryPtrAAAPs was downloaded from Phytozome . We analyzed the expression pattern of some genes from different subfamily in P. simonii ‘Tongliao1’ by qRT-PCR. RNAprep Pure Plant Kit  was used to extract the total RNA of roots, stems, and leaves of P. simonii ‘Tongliao1’ according to the manufacturer’s protocol, and then the total RNA was reverse transcribed into cDNA using a TIANScript II RT Kit . The Actin gene (Potri.001G309500) was used as the reference gene and the 2−ΔΔCT method was used to analyze. All the experiments were performed with three replicates, primer information in The expression pattern of PtrAAAP genes in the differentially expressed genes in the RNA-seq of root development (transcriptome data comes from previous research by the research group of badly rooted and good rooted hybrid offspring), and qRT-PCR was used to analyze the expression of differential genes in the roots, stems, and leaves of P. simonii ‘Tongliao1’.Analysis and selection of the PtrAAAP21 was selected out based on the result of QTL mapping and RNA-seq of root growth of hybrid of P. deltoides ‘Danhong’ and P. simonii ‘Tongliao1’. The CDS sequence fragments of the PtrAAAP21 gene was cloned from P. simonii ‘Tongliao1’ cDNA via specific primers, and the sequence was first cloned into the pDONR222 and then into pMDC32, utilizing BP and LR . The method of Agrobacterium-mediated genetic transformation was taken to transform 84K poplar. PsAAAP21 and LST8.1, SNF4, and MAF4, cloned from 84K poplar, were cloned into pGBDT7 and pGADT7-rec1, respectively.In the previous research of the research group, Transgenic 84K poplar lines  and wild-type 84K poplar (WT) were cultured in the tissue culture room of the Chinese Academy of Forestry, and the growth phenotype was determined after one month of growth.P. trichocarpa v4.0 was used as reference .Hybrid parents and offspring cutting seedlings with consistent growth were selected for treatment. The water content in the drought treatment group was controlled to be 60–70% of the maximum water holding capacity, the salt stress group was treated with 150 mM NaCl, and the control group was treated with water, and the water content of salt stress and control was 100%. After one month of growth, root tips were collected, frozen in liquid nitrogen and sent to Biomarker Technologies Co, Ltd.  for RNA-seq, three replicates per line. The 150 bp paired-end reads were generated on the Illumina NovaSeq 6000 platform and PsAAAP21 overexpression line #6 and wild type tissue culture were sampled, frozen in liquid nitrogen and sent to GENEDENOVO Biotechnology Co., Ltd.  for RNA-seq, three replicates per line, and each replicate was pooled from three plants. The 150 bp paired-end reads were generated on the Illumina NovaSeq 6000 platform, and P. trichocarpa v3.1 was used as reference .Roots from one-month seedlings of PsAAAP21, wild-type 84K poplar (WT) and transgenic plants  and a variety of amino acids were selected for verification, including tyrosine (Tyr), methionine (Met), and arginine (Arg), their concentrations were 2 mM, 4 mM, and 25 mM, respectively, and the amino acid solution was added to sterilized 1/2 MS medium after filtration sterilization. The culture conditions were 25 °C and 2500 lx with three replicates at least per line.In order to verify the amino acid transport function of PtrAAAPs in Populus and PsAAAP21 was identified to integrate adventitious roots development and amino acid transport. Consequently, 83 PtrAAAPs were identified and characterized in Populus, and systematic bioinformatics analysis was performed and the possible regulatory role of PtrAAAPs was predicted. Combining RNA-seq and QTL mapping results, PsAAAP21 was screened and cloned from P. simonii ‘Tongliao1’. The phenotype of transgenic plants showed that PsAAAP21 promoted adventitious root development by regulating auxin. The results of the RNA-seq and amino acid content determination showed that PsAAAP21 was related to amino acid transport, which was proved by amino acid treatment experiment. This study helps us to understand the AAAP gene family of Populus and uncover the theoretical basis for improving rooting and growth traits. Finally, we could preserve excellent traits of woody plants by using AAAP gene family and increase plant nitrogen utilization by the AAAPs function on amino acid transport.In this study, HMMER search was used to identy


Malignant mesothelioma (MMe) is a rare and fatal cancer with a poor prognosis. Our study aimed to compare the overall survival (OS) of MMe patients across various sites and develop a prognostic model to provide a foundation for individualized management of MMe patients.From the Surveillance, Epidemiology, and End Results (SEER) database, 1,772 individuals with malignant mesothelioma (MMe) were identified. The X-tile software was used to identify the optimal cut-off point for continuous variables. The Kaplan–Meier method was employed to compare the survival of MMe across different sites. The Cox proportional hazards model was applied to identify the independent risk factors of overall survival (OS) and a nomogram was constructed.In the survival analysis, MMe originating from the reproductive organs and hollow organs showed a relatively better prognosis than those originating from soft tissue, solid organs, and pleura. Age, gender, location, histological type, grade of differentiation, extent of disease, lymph node status, lymph node ratio (LNR), and chemotherapy were all found to be independent risk variables for the prognosis of MMe patients (P<0.05) in a multivariate Cox analysis and were included in the construction of nomogram. In the training and testing sets, the C-index of the nomogram was 0.701 and 0.665, respectively, and the area under the ROC curve (AUROC) of the 1-, 3-, and 5-year overall survival rate was 0.749, 0.797, 0.833 and 0.730, 0.800, 0.832, respectively. The calibration curve shows that the nomogram is well-calibrated.This is the first research to examine the prognosis of MMe patients based on the location. However, previous studies often focused on malignant pleural mesothelioma or malignant peritoneal mesothelioma with high incidence. Furthermore, a nomograph with good prediction efficiency was established according to the variables that influence patient survival outcomes, which provides us with a reference for clinical decision-making. Malignant mesothelioma (MMe) is an uncommon and aggressive cancer  mostly aPrevious studies mostly focused on malignant mesothelioma originating in the pleura or peritoneum; and age, gender, histology, differentiation, lymph node status, chemotherapy, and other characteristics have been demonstrated to be independent prognostic factors –11.It is worth mentioning that the overall concordance between biopsies and surgical resection specimens is 80.6% . ClinicaThe prediction model of MMe has been reported in several population-based studies. Some studies have established a prognosis model of malignant pleural mesothelioma after surgical treatment or systemic chemotherapy , 15, andThe SEER 18 registries (2000–2018), which encompass roughly 28% of the population of the United States, were used to compile the data. ICD-O-3/WHO 2008 morphology codes 9050–9053 were used to identify patients with mesothelioma. And tumor behavior records that were malignant were included in the analysis. After initial screening, candidates were enrolled in the study.The criteria of inclusion were as follows: (a) patients diagnosed with malignant mesothelioma according to ICD-O-3/WHO 2008 morphology codes; (b) patients diagnosed between 2004 and 2015; (c) Patients who have MMe as their first primary malignancy. The exclusion criteria were as follows: (a) Patients whose location was not otherwise specified (NOS); (b) Patients who did not have surgery and were just subjected to a biopsy or whose surgical status was unknown; (c) Patients without survival information; (d) The number of lymph nodes examined or the number of positive lymph nodes is unknown. Accordingly, a total of 1772 patients were included in our study. After strict inclusion and exclusion, the research object of this article is the primary malignant mesothelioma diagnosed pathologically from 2004 to 2015, which avoids the impact of secondary malignant tumors on the results. Moreover, the pathological information of lymph nodes in all subjects is complete, which can be further used to analyze the relationship between them and the prognosis of patients with malignant mesothelioma.The data from the SEER database is free and publicly available; no institutional review board permission was required. The relevant clinical data were extracted, including the year of diagnosis, age, gender, marital status, race, location, histologic type, grade, extent of disease, TMN stage, T classification, lymph node status, regional nodes examined, regional nodes positive, distant metastasis, treatment , survival time and vital status.The variables of age, number of lymph nodes examined and number of positive lymph nodes were extracted from the Seer database. The lymph node ratio (LNR) is the ratio of positive lymph nodes to examined lymph nodes. The optimal cut-off value was determined by X-tile software and the variables were subdivided into 3 groups to facilitate further statistical analysis. The specific group information was as follows: the age of the patients , number of nodules dissected , number of positive nodules  and LNR . Following the screening, the locations were sorted into the following seven groups: (a) Pleura; (b) Peritoneum, retroperitoneum; (c) Male genital organs, including testis and other male genital organs; (d) Female genital organs, including ovary and fallopian tube, round ligaments and uterine adnexa; (e) Soft tissues; (f) Solid organs, including lung, heart or mediastinum, thymus, liver, and spleen;(g) Hollow organs, including colon, small intestine, and stomach. The Seer surgery code was used to gather surgical information, which was divided into three types of procedures: palliative, radical, and not otherwise specified (NOS). Regional lymph nodes dissection was grouped into Yes and No/Unknown according to the records from the SEER database. Chemotherapy was considered to have been administered if the chemotherapy record stated Yes. According to the radiotherapy records in the Seer database, the remaining patients were considered to have received radiotherapy, excluding those who refused radiotherapy or whose radiotherapy status was unclear.The optimal cut-off points of the continuous variables, including age, the number of lymph nodes dissected, the number of positive lymph nodes, and LNR were identified by the X-tile program, which utilized the minimum P-value method to find them. The specific group information is displayed in For statistical analyses, we utilized R software version 4.1.2. The nomogram was created and drawn using the regplot package. A two-sided P value of less than 0.05 was considered statistically significant.A total of 1,772 patients from the National Cancer Institute’s SEER program were enrolled in this research. The patients diagnosed between 2007 and 2015 were assigned to the validation group and those diagnosed between 2004 and 2006 to the training group. The demographic, pathology and treatment information for the training  and testing cohorts (n = 440) are displayed in The majority of patients in the primary cohort were married (66.6%), white (91.1%), aged <75 (78.3%), male (71.0%), and had MMe originating from the pleura (74.6%). Epithelioid histology was seen in 836 individuals (47.2%), biphasic histology in 203 patients (11.5%), and fibrous histology in 146 patients (8.2%), but the degree of differentiation in the majority of MMe patients was unknown (84.9%).There were 245 patients with stage I (13.8%), 217 with stage II (12.2%), 430 with stage III (24.3%), 504 with stage IV (28.4%), and 376 with an unknown stage (21.2%). Distribution of the T stage was 310 (17.5%), 369 (20.8%), 398 (22.5%), 323 (18.2%) and 372 (21.0%) for T1, T2, T3, T4 and unknown, respectively. 393 (22.2%) patients had lymph node metastasis, 242 (13.7%) with distant metastasis.Most patients (72.6%) underwent palliative surgery, and 410 patients (23.1%) underwent radical surgery. Most patients (68.5%) did not undergo lymph node dissection. Only 305 patients (17.2%) were treated with radiotherapy. Chemotherapy was performed on 1,037 patients (58.5%).The MMe death rate reached 100% during the follow-up period in the two cohorts. In the whole group, the median OS was 16 months (95% CI 14.811–17.189 months). MMe originating from soft tissue, solid organs and pleura had a poor prognosis, whereas MMe originating in reproductive organs and hollow organs showed a relatively better prognosis. The specific median survival times and their 95% CIs for different anatomical sites are shown in Several demographic and tumor characteristics were significantly associated with OS in the univariate Cox regression model Table 3,The independent risk variables identified through the multivariate analysis were used to create a nomogram Figure 4As shown in Both in the training set  Our study is a retrospective analysis based on the Seer database, and selection bias is unavoidable. (2) The SEER database lacks detailed clinical data such as surgical methods, specific radiotherapy, and chemotherapy regimens, which may imply other unknown variables. (3) With advances in surgical –67 and tA large prospective trial with strict design needs to be carried out to verify our results. Information not recorded in the Seer database can be included in the trial, such as specific treatment plans, molecular pathology or blood indicators. Besides, external validation is needed to demonstrate its performance in clinical practice.In conclusion, this study is based on the Seer database, including large sample size, clear pathological diagnosis, and complete follow-up. Our study found that there were differences in prognosis for malignant mesothelioma at different anatomical sites. Further, age, sex, histological type, location, differentiation, the extent of disease, N status, ratio, and chemotherapy were identified as independent factors affecting the overall survival (OS) of MMe patients. Based on these factors, we built a nomogram that can be used to assess the prognosis of individual MMe patients at different sites. Prognostic model established in our study provides a reference for clinical decision-making and helps medical workers to individualize the care of patients with malignant mesothelioma.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SS, YL and YW conceived and designed the experiments. SS, LS, KQ and TY collected and analyzed data. SS, YW and XJ wrote this manuscript. All authors contributed to the article and approved the submitted version.Funding from Outstanding Doctoral Project Funding Program of Air Force Military Medical University (2021D08) and Shandong Province Medical and Health Science and Technology Development Plan Project (2018WS383).www.home-for-researchers.com) for language enhancement of this paper.The authors are grateful for the invaluable support and useful discussions with other members of the Department of Thoracic Surgery, and thank Home for Researchers (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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