Calendula officinalis, Celosia argentea, Daucus carota, Helichrysum bracteatum, Matthiola incana, and a Zinnia elegans-Zinnia hybrida mixture. At least four species were in bloom from early June through late September. The flowers attracted diverse pollinator groups including Diptera, Hymenoptera, Coleoptera, and Lepidoptera. Bees most often visited Ca. officinalis, H. bracteatum, and Celosia spicata whereas flies most often visited D. carota. Bombus were the most oft-collected bees from the flowers and were found on all six cut flower species. Wasp abundance varied little across the cut flowers, but wasp community composition was distinct. The highest diversity of wasp families was collected from the Zinnia mixture (seven families) in contrast to less diverse collections from Ce. spicata (two families). The most abundant wasp families collected were Crabronidae and Sphecidae. Our experiment documented that ornamental cut flower species attract pollinator insects into high tunnel environments. All cut flower species tested were visited by multiple types of beneficial insects. Planting a mixture of specialty cut flowers can support insect diversity while also diversifying on-farm agricultural products through sale of cut flower stems.Various strategies incorporate floral resources into agricultural landscapes to support beneficial insects. Specialty cut flower production offers a rarely explored approach to offer floral resources while yielding a marketable product for growers. We characterized insect visitation to six species of specialty cut flowers. Due to Wyoming’s growing conditions, the flowers were grown in high tunnels, thus offering insight into insect abundance in this unique semi-controlled environment. The flower species tested were  Floral resources in managed landscapes support beneficial insects that contribute to pollination, pest control, and overall biodiversity . EffectiDiverse wildflower plantings on farmland is an oft-researched practice , but groSpecialty cut flower production offers another potential strategy to simultaneously enhance pollinator activity and provide economic benefits to growers. The specialty cut flower market—flowers other than roses, carnations, and mums—has grown in the U.S. via cut flowers produced for local markets . SpecialAlthough many cut flowers are produced in open-field settings, they also can be produced in greenhouses and high tunnels . Cut floThe experiment was conducted at the University of Wyoming Laramie Research and Extension Center in Laramie, WY . The property, approximately 3.5 ha, consists of greenhouses, small research plots, and the student farm. Two high tunnels were used: one oriented east-west and one oriented north-south . The high tunnels were 12 × 16 ft. with an arch roof style, covered in a double layer of six-mil uninflated polyethylene plastic . TunnelsCalendula officinalis L. ‘Princess Golden’ , stock Matthiola incana (L.) W.T. Aiton ‘Lucinda Mix’ , strawflower Helichrysum bracteatum (Vent.) Andrews ‘Double Mix’ , ornamental carrot Daucus carota L. ‘Dara’ , cockscomb Celosia argentea ‘Celway Mix’ , Zinnia hybrida ‘Profusion Yellow’, and Zinnia elegans Jacq. ‘Peppermint Stick’  . Flowers were selected to span a range of plant families and morphology types used in floral arrangements , other native bees (Hymenoptera: Anthophila), honey bee (Apis mellifera L. [Hymenoptera: Apidae]), wasps, butterflies and moths (Order Lepidoptera), flies (Order Diptera), and beetles (Order Coleoptera).Timed observations recorded the number of unique plant-pollinator interactions during a set amount of time for each flower species. Observations occurred between 10 am and 2 pm approximately every two weeks from 7 June 2020 until 23 September 2020. Observations were made for 15 min in each species plot. A season-long total of 360 min of observation time was spent on each flower species. We used a two-minute transition period between plots to minimize disturbance. During the 15 min observation period, an individual insect was counted a single time even when visiting multiple flowers to determine the number of visiting insects per site. We observed open flowers for insects landing on reproductive flower parts, not stems and leaves. Observations were recorded as bumble bee , using square-root transformation if needed to meet assumptions. Means separations were conducted with Tukey tests. Bi-partite networks were created with the package ipartite  in R-proipartite  to visuaipartite .n = 831), followed by bees , wasps , beetles , and butterflies and moths . Bombus spp. were 67% of the total number of observed bees, other native bees comprised 32%, and A. mellifera were 1% of the total. Observations of Diptera differed between flower species , with the highest visitation to D. carota, more than twice as often as the other flower species , with the highest visitation occurring on Ca. officinalis, H. bracteatum, and Celosia spicata, more often than M. incana and D. carota , with the highest visitation again on Ca. officinalis, followed by H. bracteatum. Significantly fewer observations of non-Bombus native bees occurred on Ce. spicata, D. carota, M. incana, and the mixture of Z. elegans and Z. hybrida (Zinnia mixture was visited by wasps more often than M. incana; otherwise, wasp observations were similar across the flowers.Overall, we observed flies visiting the specialty cut flowers most often (45%,  species . Bumble . carota . For nat hybrida . The ZinBombus were collected from all flower species (Ca. officinalis and H. bracteatum.For Diptera, 42% of collected flies were identified as Syrphidae. The remainder of the flies were nonsyrphids, found on all of the flower species . We coll species . In contZinnia mixture, in comparison to Ce. spicata, from which we only collected two families (Ten unique wasp families were collected; these families span a diversity of lifestyles ranging from parasitic to predatory . The mosfamilies .The number of flowers in bloom varied throughout the season with peak number of blooms from mid-August through late September for all flower species .Ornamentals used as floral resources can be as attractive or more attractive than native flowers and can provide continual flowering for pollinators . In our Aculeate wasps, including crabronids, sphecids, and vespids, also have the potential to act as dual service providers through their often generalist predatory behavior and may be ‘backup’ pollinators in certain environments . ParasitH. bracteatum and Ca. officinalis, hosted all bee taxa identified here; species-level identification could reveal more specific relationships. Ca. officinalis offers a high reward in both pollen and nectar based on resource per flower (Zinnia mixture. Z. elegans also was used in a high tunnel experiment to test methods to improve biological control, and lady beetles and lacewings foraged on Z. elegans (Zinnia cultivar can affect the attractiveness to different pollinator taxa suggesting that they vary in nutritional reward, floral advertisement, or accessibility (Two flower species, r flower . The mos elegans . Zinnia sibility .Matthiola, which does not regrow after cutting (Many species can be grown for cut flower use. Cut flowers must be carefully handled at every stage of production and postharvest, from seeding, planting, harvesting, grading, marketing, selling, and accounting. When we grew these flowers for cut flower stems in past experiments, we harvested multiple times each week , in cont cutting . Future  cutting .toac051_suppl_Supplementary_MaterialClick here for additional data file.


Vibrio harveyi belongs to the family Vibrionaceae of class Gammaproteobacteria. Around 12 Vibrio species can cause gastroenteritis  in humans. A large number of bacterial particles can be found in the infected cells, which may cause death. Despite these devastating complications, there is still no cure or vaccine for the bacteria. As a result, we used an immunoinformatics approach to develop a multi-epitope vaccine against the most pathogenic hemolysin gene of V. harveyi. The immunodominant T- and B-cell epitopes were identified using the hemolysin protein. We developed a vaccine employing three possible epitopes: cytotoxic T-lymphocytes, helper T-lymphocytes, and linear B-lymphocyte epitopes, after thorough testing. The vaccine was developed to be antigenic, immunogenic, and non-allergenic, as well as have a better solubility. Molecular dynamics simulation revealed significant structural stiffness and binding stability. In addition, the immunological simulation generated by computers revealed that the vaccination might elicit immune reactions Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher codon adaptation index value, which was then included in the cloning vector pET2+ (a). Altogether, our experiment implies that the proposed peptide vaccine might be a good option for vibriosis prophylaxis. Vibrio species and their antibiotic resistance is, however, poorly understood. The virulence components of Vibrio spp. that have been associated to animal and human illnesses are often not accessible or common in the environment [Vibrio has a highly plastic genome, there is a substantial likelihood that pathogenic and ambient Vibrio will share genes for virulence. As a result, there has been an increase in the number of pathogenic Vibrio strains in the aquatic environment [Vibrio harveyi is one of the most serious infections affecting farmed fishes, and Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio campbellii have also been found in numerous tropical nations [V. harveyi infection include anemia, necrosis of the intestine, ascetic fluid, petechial hemorrhages, tail erosion, infection of the eye, mucous secretions, and frequent mortality in fish [Vibrio spp. over the last few decades [V. harveyi is highly pathogenic to salmonids, sea bass, and tilapia, and generates an extracellular product with a high titer of hemolytic activity against fish erythrocytes. V. harveyi has a single chromosome with a length of 6,374,398 base pairs [V. harveyi causing disease in fish, hemolysin is well-known as a virulence factor linked to both fish and human diseases [in-silico design of multi-epitope vaccines against pathogens. Epitope-based vaccines have been successfully created in the postgenomic period to stimulate responsiveness against some of the worst human viruses, including influenza, nipah, chikunguniya, zika, ebola, Middle East respiratory syndrome coronavirus, rota, and others [in-silico technique in fish had not been developed due to a lack of understanding of the differences between major histocompatibility complexes (MHC class I and II) and human leukocyte antigen (HLA) [in-silico techniques [in-silico technique was effective in predicting epitopes and multiepitopes with significant responsiveness against Streptococcus agalactiae, Edwardsiella tarda, and Flavobacterium columnarie, three harmful bacteria that induce streptococcosis, edwardsiellosis, and columnaris in fish, separately [V. harveyi infection.The disease, called vibriosis, affects both farmed and marine fish across the globe. The pathogenicity of ironment . Becauseironment . Vibrio  nations -6. Among in fish . Moreove decades . V. harvdiseases . The hemdiseases . Cytotoxdiseases  and CD4+diseases  by epitodiseases . Immunizdiseases . The prod others -20. Preven (HLA) ,22 but rchniques -25. Bothchniques ,26. Lateparately -29. Expeparately ,31. As aArchitectural flow chart is being given in https://www.ncbi.nlm.nih.gov/) database to find accessible Vibrio harveyi proteomes for antigen selection. Hemolysin is a crucial protein that allows viruses to penetrate the host cell wall, making it a suitable target antigen for V. harveyi vaccine development [V. harveyi for multi-epitope vaccine design because of its direct role in pathogenesis. After the hemolysin was isolated, the chosen amino acid sequences of the bacteria were obtained as FASTA files (GenBank: ACF32997.1). VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/) server was used to assess the protective antigens of hemolysin [http://scratch.proteomics.ics.uci.edu/) server [We used the NCBI  server . Subsequhttp://www.cbs.dtu.dk/services/NetCTL/) to predict CTLs epitope [http://tools.iedb.org/immunogenicity/) [http://crdd.osdd.net/raghava/toxinpred/) [https://ddg-pharmfac.net/OP/) [CTLs are basic kinds of immune responsive cells that have the ability to directly destroy other infectious cells . They im epitope . It inte epitope , followenicity/) , ToxinPrinpred/) , and Allnet/OP/)  online shttp://tools.iedb.org/mhcii/. With a percentile rank of 5%, the HTL epitopes were chosen using the CONSENSUS technique [http://crdd.osdd.net/raghava/ifnepitope/) [http://crdd.osdd.net/raghava/il4pred/) [http://crdd.osdd.net/raghava/IL-10pred/) [In response to external antigens, helper T-lymphocytes (HTLs) detect and activate B lymphocyte and CTL and causing the infectious pathogen to be destroyed . The HTLechnique . The IEDechnique . This topitope/) , IL4predl4pred/) , and IL110pred/)  servers,http://www.thegleelab.org/iBCE-EL/) to identify the linear B-lymphocyte (LBL) epitopes using default settings [To promote humoral or antibody-mediated immunity, B-cell epitopes are required. B-cells are made up of amino acid groups that bind with secreted antibodies and stimulate the immune system to fight infections . As a cosettings . It is asettings . This sehttps://bioserv.rpbs.univ-parisdiderot.fr/services/PEP-FOLD3/) server was used to simulate the chosen CTL and HTL epitopes. For the procedure, the sOPEP sorting scheme with 200 simulations was employed [https://www.rcsb.org/) [PEP-FOLD v3.0 (employed . HLA-B*3sb.org/)  followedsb.org/) . To compThe vaccine was created by combining the chosen CTL, HTL, and LBL epitopes with a suitable adjuvant and linking them with the proper linkers ,53. Becahttps://web.expasy.org/protparam/, was used to predict the vaccine's physicochemical properties to comprehend the vaccine's essential essence [The physiochemistry of a protein describes its fundamental characteristics. The ProtParam server, which can be found at  essence . We also essence , MHC-I i essence , AllerTo essence , and SOL essence  servers.https://npsa-prabi.ibcp.fr/NPSA/npsa_seccons.html [The two-dimensional (2D) structural features such as alpha-helix, beta-turn, and random coils of the construct were identified by SOPMA (Self-Optimized Prediction Method with Alignment) server at ons.html  and PSIPons.html  with defons.html . To furthttp://raptorx.uchicago.edu/) [http://galaxy.seoklab.org/refine) online web-based server. The CASP10 refining approach was used to operate this webserver [https://prosa.services.came.sbg.ac.at/prosa.php) tool [The constructed vaccine was submitted into the RaptorX server (go.edu/) . Using ago.edu/) . The C-sebserver . The RMSebserver . The Ramebserver ; and Z-shp) tool .https://cluspro.bu.edu/, to submit the refined vaccine model as a ligand and the TLR4 protein as an immunological receptor for molecular docking [The binding interactions between modeled proteins and receptor molecules can be revealed through molecular docking experiments. For this, we used the ClusPro v2.0 server, which can be found at  docking . The TLR docking . DiscoveThe complex structure of the selected candidate compounds was evaluated using 50 ns molecular dynamic simulations (MDS) to evaluate their binding stability to the desired protein to the active site cavity of the protein . The MDShttp://www.cbs.dtu.dk/services/C-ImmSim-10.1/), the entire construct was uploaded for assessment of the vaccine's potential immunological response [Using the C-IMMSIM v10.1 server (response . As prevresponse . Three ihttp://jcat.de/). We employed the commonly used E. coli K12 as the host in this study, and the entire procedure was carried out while avoiding the following three criteria: Sites of restriction enzyme cleavage, binding sites of prokaryotic ribosomes, and rho-independent transcription termination. The codon adaptation index (CAI) value and guanine–cytosine (GC) concentration of the modified sequence were used to evaluate it [in-silico cloning of the adapted nucleotide sequence into the pET28a (+) expression vector was performed using the modified nucleotide sequence. SnapGene v4.2 software was used to carry out the entire in-silico cloning procedure [Codon optimization is required for the expression of a foreign gene in a host organism . As a reluate it . Lastly,rocedure .V. harveyi proteomes featured hemolysin protein. We chose a hemolysin protein with the highest antigenic score of 0.4070 (VaxiJen) and 0.617 (ANTIGENpro) from all examined proteins based on antigenicity. The chosen hemolysin had a length of 418 amino acids and a GenBank accession number of ACF32997.1. For subsequent investigation, the main sequence of the chosen protein was employed.The retrieved To design a rational vaccine, accurate predictions of CTL epitopes are crucial. Furthermore, they can minimize the amount of experimental effort needed to identify epitopes. From the chosen hemolysin protein, a total of 52 CTL epitopes with a length of nine amino acids were predicted by using NetCTL v1.2 server. 22 CTL epitopes were shown to be antigenic, immunogenic, non-toxic, and non-allergenic. We chose the top three CTL epitopes for the final vaccine design based on the antigenicity score due to the large number of possible epitopes . C-scoreInitially, the IEDB server was used to identify 358 HTL epitopes, each with a length of 15 amino acids. Only 14 HTL epitopes were able to trigger the three kinds of cytokines tested, including IFN-γ, IL-4, and IL-10. Similarly, based on the antigenic score, we examined the top three HTL epitopes for incorporation into the final vaccine design .To develop epitope-based vaccines, produce antibodies, and prevent and diagnose diseases, B-cell epitopes must be identified. In this study, a preliminary investigation found 10 LBL epitopes, each of which is 12 amino acids long. Later with further evaluation, two epitopes were found as antigenic, non-toxic non-allergenic .The docking approach was utilized to confirm the efficiency of chosen epitopes in binding their HLA alleles. The vaccine was created utilizing eight epitopes from three distinct classes that had previously been chosen . As illustrated in 1214H1914N310O379S4, the instability index of 20.25, the aliphatic index of 82.05, and the grand average of hydropathicity of ‒0.237 were also present. The construct's physicochemical properties and immunological efficacy were also assessed. For example, the construct's antigenicity was 0.7017, whereas its immunogenicity was 1.59238. Furthermore, the vaccine was non-allergenic and soluble, with a score of 0.891723 out of 1 , as advised by the server, out of the five. With GDT-HA score 0.8176, RMSD value 0.519, MolProbity 2.993, Clash score 25.7, and Poor rotamers score 0.8, the vaccine (model 1) exhibited 87.7% residues in the favorable area in the Ramachandran plot after refinement. The ProSA-web servers were used to further evaluate the refined 3D vaccine model. The vaccine's Ramachandran plot showed 78.5% residues in the favorable zone, 18.7% in approved regions, and 0.5% residues in prohibited regions before refining. The Ramachandran plot of the refined vaccine model showed 87.7% residues in the favorable region and 10.5% in allowed regions, while 0.5% residues in disallowed regions . SimilarTo predict their binding affinity and interactions, the vaccine (ligand) and TLR4 (receptor) were docked. As a result, the ClusPro v2.0 server produced ten docked complexes in various positions. We chose the complex with the lowest energy score and the binding posture with functional interactions from among them. As a result, model 1 met the inclination criterion. As a result, it was chosen as the best vaccine–TLR4 complex, with a –937.6 energy score. Binding interactions and residues implicated in active site residues were investigated in the chosen complex. A total of eight hydrogen bonds were found in the interaction surface. There were eight classical hydrogen bonds among the hydrogen bonds. The interacting residues in the CHB from the vaccine were Lys39, Lys20, Ser45, Asn47, His62, Arg67, and Asn44. Moreover, associated TLR4 active site residues are shown in We calculated the RMSD for the vaccine complex and the vaccine. The vaccine complex had an average RMSD of 4.76Å, which indicated structural stability during the interaction. 3) than the Th1 type immune reaction (The simulated immune response in reaction . Expandereaction  and 11G.E. coli K12 on the JCat service. The nucleotide sequences created by the peptide vaccine construct (254 amino acid residues) totaled 761 lengths  vector. Using the SnapGene program, the optimized vaccine design was cloned into the pET28a (+) cloning vector  value and aliphatic index, on the other hand, indicated that the vaccine was hydrophilic and thermostable, respectively. According to the prediction of physicochemical properties and scores on all parameters, there is a high probability for this vaccine to be a valid candidate against hemolysin protein of V. harveyi. The detected models were revised and the best model (based on the lowest energy score) was chosen after the 3D structure prediction (based on C-score). We observed a reasonable number of Z-score (–6.01) and superior features of most favored, acceptable, and prohibited areas for the Ramachandran plot in the validation test of 3D structure. It was suggested by the lowest energy score of 937.6 for a molecular docking between the peptide vaccine and virus glycoprotein binding convenient receptor of TLR4 that the vaccine could have infection-inhibiting activity and might interact tightly with TLR4 receptor. The molecular dynamics simulation is a potentially useful tool for understanding how proteins function and how their structure is derived. Anatomical movement can be simulated by protein dynamic simulations as a function of time. We have performed dynamic simulations of the vaccine candidate for 50 ns, and analyzed the results using the RMSD and RMSF scores. When comparing distinct atomic conformations of a molecular system, the RMSD value is employed. A significant flexibility and departure of vaccine candidates from receptor structure was determined using the RMSD value, whereas the displacement of our particular vaccine candidate's atoms from receptor structure was determined using RMSF of the complex structure. The calculated average RMSD and RMSF value was 4.77 Å and 4.0 Å, respectively. The fluctuation was not observed to be larger in the vaccine section, but it smoothed out after 5 ns, suggesting that the modeled vaccine and receptor are stable. Lastly, we examined the optimal target clearance and cell density parameters for the best immunologic response against the pathogen by constructing an immune simulation. As a result of the upgraded vaccine doses, the immune system created memory B-cells  and T cells. The vaccination efficiently imitated a humoral immune response to increased immunoglobulin production in this way. In order to optimize the multi-epitope vaccine production, the MD simulation was done to evaluate stability of the vaccine candidate with the receptor, in which codon optimization was done for stability of the construct vaccine within the host. Eventually, the codon was adjusted, and in-silico cloning of the intended vaccine candidate into the E. coli K12 expression host pET28a (+) vector was successful.The current diabolical emergence of industry , which iindustry . Acquireindustry . As a re species . As vibr species ,23. The  species . The adj species . A total species . A solubV. harveyi hemolysin protein, which were finally stitched into a multi-epitope mRNA vaccine. The newly developed vaccine possesses the immunodominant qualities that are sought. Significantly, it was capable of binding to the immunological receptor TLR4 and induce a substantial immune response in regard to V. harveyi infection. Based on our findings, we believe that developing a vaccine against the etiological agent of the V. harveyi outbreak in fish should begin with the vaccine candidate. In addition, the possible epitopes discovered in this study can be employed in future research. Nevertheless, more testing is needed to show that our designed vaccine is an effective preventive against V. harveyi infection in fish species.A range of computational techniques were used in this work to find possible T- and B-cell epitopes in


Association of insurance type with colorectal surgical complications, textbook outcomes (TO), and cost in a safety-net hospital (SNH).SNHs have higher surgical complications and costs compared to low-burden hospitals. How does presentation acuity and insurance type influence colorectal surgical outcomes?Retrospective cohort study using single-site National Surgical Quality Improvement Program (2013–2019) with cost data and risk-adjusted by frailty, preoperative serious acute conditions (PASC), case status and open versus laparoscopic to evaluate 30-day reoperations, any complication, Clavien-Dindo IV (CDIV) complications, TO, and hospitalization variable costs.P = 0.009) versus Private. Medicaid/Uninsured  patients were more likely to undergo urgent/emergent surgeries compared to Private. Medicare patients had increased odds of any and CDIV complications while Medicaid/Uninsured had increased odds of any complication, emergency department or observations stays, and readmissions versus Private. Medicare  and Medicaid/Uninsured  patients had lower odds of achieving TO versus Private. Variable cost %change increased in Medicaid/Uninsured patients to 13.94% (P = 0.005) versus Private but was similar after adjusting for case status. Urgent/emergent cases  and any complication  increased %change hospitalization costs.Cases  with patient mean age 55.2 years (SD = 13.4) and 53.1% male. Adjusting for frailty, open abdomen, and urgent/emergent cases, Medicaid/Uninsured patients had higher odds of presenting with PASC  assessing the association of insurance type  with complications and costs adjusted for frailty, open versus laparoscopic approach, and case status. Markedly increased rates of urgent/emergent surgeries in Medicaid/uninsured patients drive the increased odds of complications and costs compared to Private patients. Additionally, the impact of frailty on patient outcomes is not captured using standard Hierarchical Condition Category (HCC) risk adjustment.4 High Social Vulnerability Index scores in colorectal surgery patients were associated with higher risk of postoperative complications and index hospitalization expenditures,2 but are not included in risk adjustment. Safety-net hospitals (SNH) serve higher proportions of low socioeconomic status (SES) patients with higher severity of illness scores, higher rates of emergency surgeries, and longer hospital length of stay (LOS).5 Hospital Readmission Reduction Program and other pay for performance (P4P) programs unintentionally contribute to widening disparities in healthcare and outcomes, penalizing SNH, and further limiting resources to treat vulnerable populations.8Frailty10 Insurance status/type is a common proxy used for patient SES.13 Moreover, dual-eligibility for Medicare and Medicaid is indicative of poverty and plays a fundamental role in predicting surgical outcomes,14 along with urgent/emergent surgeries,15 frailty,18 and open compared to laparoscopic surgeries.12 Uninsured or Medicaid patients experience increased rates of emergency procedures, complications, and mortality.20 Additionally, the current NSQIP risk calculator underestimates the risk of complications for emergency surgeries21 and does not provide risk prediction for urgent cases.15 Failure to account for factors beyond clinicians’ control  in value-based medicine when assessing outcomes of high-burden facilities may continue to widen disparities in access, care, and outcomes in vulnerable populations.18High-burden, SNH have higher postoperative complications and costs compared to low-burden hospitals.23 Social risk factors may not consistently affect outcomes; therefore, including multiple outcomes of interest may more comprehensively assess the effects and costs of social risk factors, especially in underinsured/uninsured patients.Textbook outcomes (TO) is a composite metric that has been increasingly used to assess surgical outcomes.We assessed the association of Private, Medicare, and Medicaid/Uninsured  health insurance type with complications and cost after colorectal surgeries in an SNH with a large range of SES patients. We hypothesize that after adjusting for frailty and open versus laparoscopic procedure type, increased presentation acuity, measured by presenting with acute serious conditions and urgent or emergent cases in patients with Medicaid/Uninsured insurance type will be associated with higher complications and index hospitalization costs compared to patients with Private insurance.24 and used local, identified data on all patients undergoing colorectal procedures present in the 2013-2019 American College of Surgeons National Surgical Quality Improvement Program (NSQIP) at a single facility, serving as an academic medical center and SNH. NSQIP registry was used for cohort identification. NSQIP provides standardized definitions of preoperative risk factors and complications.25 The Institutional Review Board of the University of Texas Health San Antonio approved this study.This retrospective cohort study followed STROBE Reporting Guidelines26 using preoperative variables from NSQIP as previously described.16 The RAI has been validated using both Veterans Affairs Quality Improvement Program and NSQIP datasets26 and exhibits collinearity with the Charlson Comorbidity Index.1 RAI has been used for risk adjustment for medical comorbidities in multiple studies.28 RAI scores were grouped into robust (≤20), normal (21–29), frail (30–39), and very frail (≥40).Frailty was measured using the recalibrated Risk Analysis Index (RAI)http://links.lww.com/AOSO/A177 lists the NSQIP variable names).16 Case status was determined from NSQIP variables with urgent cases being defined as neither elective nor emergency, as determined by ”no” responses to the ELECTSURG and EMERGNCY variables.15 Procedures were categorized as open or laparoscopic surgeries using NSQIP principal Current Procedural Terminology Codes .Patients presenting with preoperative acute serious conditions (PASC) were defined using 6 NSQIP present at the time of surgery (PATOS) variables and NSQIP variables defining acute renal failure  within 2 weeks before surgery, as previously described  complications using the NSQIP variables of postoperative septic shock, postoperative dialysis, pulmonary embolus, myocardial infarction, cardiac arrest, prolonged ventilation, reintubation, or stroke as previously reported.16 Unplanned reoperations were defined using the NSQIP variable REOPERATION1 as present or absent. Any complication was defined using the CDIV and reoperation NSQIP variables plus an additional 11 NSQIP variables defining postoperative complications.Clavien-Dindo classifies complications based on their treatments.TOs were defined as surgeries with the absence of 30-day CDIV complications, unplanned reoperations, 30-day mortality after the date of surgery, and 30-day after the date of discharge from the index hospitalization readmissions and emergency department or observations stays (EDOS).Mortality was defined as death within 30 days of the index colorectal surgical procedure. Dates of death were obtained from ACS NSQIP and cross referenced from our data warehouse using the electronic health records of the local health system as well as the Social Security Death Master File.http://links.lww.com/AOSO/A177). “Other” included encounters billed to the Veterans Administration, Department of Corrections, or self-pay with >1% of charges collected and were excluded (n = 13).The identified, local NSQIP data were merged with electronic health records and managerial accounting data to determine insurance type and the variable cost of the index hospitalization. Insurance type was categorized based upon billing data for the encounter supplemented by EHR data and defined as (1) Private including TRICARE and workers compensation; (2) Medicare; and (3) Medicaid/Uninsured including Medicaid, dual enrollment in Medicare/Medicaid, charity care, self-pay with ≤1% of charges paid, or county indigent care programs  perineal and transsacral only procedures; (2) missing or inaccurate cost variables; and (3) “Other” insurance type.Clinical outcomes of interest were 30-day unplanned reoperations, any complication, severe/life-threatening CDIV complications, readmissions, EDOS, TO composite variable, and variable costs for the index surgery hospitalization adjusted for RAI, case status, open versus laparoscopic procedure, and insurance type.33 Percent change/relative difference was calculated using the exponential function; %change = (eestimated coefficients − 1) × 100. Analyses were performed using R version 4.1.0 (2021-05-18).Categorical data were summarized using count and percentage and continuous data using mean and standard deviation (SD). Chi-square tests and F-tests were used to test for difference between groups for categorical and continuous variable. Logistic regression analyses were performed for (1) PASC and case status adjusting for RAI, open abdominal procedures, and insurance types; (2) open abdominal procedures adjusting for RAI, insurance type, PASC and case status; (3) complications, EDOS, readmissions and TO adjusting for RAI, open abdominal procedures, insurance type, and case status; and (4) TO subgroup analyses for elective and urgent/emergent cases. Natural logarithms were used to normalize the skewed LOS and variable costs for the index hospitalization, which reduces the impact of extreme values, as previously described.Our cohort consisted of 1,078 cases of inpatient procedures at a major urban SNH  and Medicaid/Uninsured patients  compared to Private. Patients presenting with PASC had higher odds of undergoing urgent/emergent surgeries . All 3 insurance types had similar odds of undergoing open abdominal surgeries after adjusting for frailty, PASC, and urgent/emergent procedures.Rates of patients presenting with PASC 13.8%) were highest in Medicaid/Uninsured (15.8%) and Medicare (15.5%) patients versus Private  was similar between insurance types, except for sepsis which was lowest in Medicare patients. Rates of urgent/emergent cases were highest in the Medicare (100.0%) and Medicaid/Uninsured (94.9%) patients presenting with PASC.PASC variable distribution . The distribution of CDIV complications  was similar between insurance types except myocardial infarction was highest in Medicare patients (25.0%) and Private (23.1%) compared to Medicaid/Uninsured .Rates of CDIV complications were 10.5% overall and were higher in Medicare and Medicaid/Uninsured patients , while Medicaid/Uninsured patients had higher odds of any complication  versus Private.Medicare patients had higher odds of any complication  compared to elective cases.Urgent/emergent surgeries greatly increased 30-day mortality odds  or urgent/emergent  cases demonstrated that Medicare patients had higher aOR for CDIV complications for elective cases and any complication for the urgent/emergent subgroup compared to Private insurance patients. Medicaid/Uninsured patients had higher aOR only for any complication in the urgent/emergent subgroup compared to the Private insurance group.Subgroup analysis including only elective . Subgroup analysis including only elective  or urgent/emergent  cases showed EDOS had a higher aOR for Medicaid/Uninsured versus Private insurance group in the elective and urgent/emergent subgroups.Medicaid/Uninsured patients had higher odds of EDOS occurring  patients had decreased odds of achieving TO compared to Private. Subgroup analyses of elective and urgent/emergent case status also demonstrated that Medicare and Medicaid/Uninsured patients exhibited decreased odds of TO compared to Private. Rates of TO for Private, Medicare and Medicaid/Uninsured patients were 79.4%, 67.4%, and 62.0% for elective cases (P < 0.001) and 72.7%, 46.2%, and 51.2% for urgent/emergent cases (P = 0.001), respectively.Both Medicare  patients had longer %change LOS compared to Private. However, after adjusting for urgent/emergent case status, LOS of Medicaid/Uninsured patients were similar to Private. Urgent/emergent cases resulted in a 70.42% change in LOS compared to elective cases (P < 0.001).Medicaid/Uninsured  had higher %changes in variable costs compared to Private (Table P < 0.001). Adjusting for any complication or CDIV complications increased the %change by 78.34% and 153.07%, respectively . For the TO patient subgroup, Medicaid/Uninsured patients had 13.48% higher variable costs versus Private and 9.58% higher variable costs after adjusting for urgent/emergent cases .Subgroup analysis for patients without any postoperative complications showed that Medicaid/Uninsured patients had 8.84% higher variable costs compared to Private insurance patients but had similar costs after adjusting for urgent/emergent cases  in the Medicaid/Uninsured group (Table Medicare and Medicaid/Uninsured patients had higher odds of 30-day complications with decreased odds of achieving TO compared to the Private group. Contributing to the worse outcomes in Medicaid/Uninsured patients were the increased odds of presenting with PASC (aOR = 2.02) and undergoing urgent/emergent surgeries aOR = 1.80) versus Private. Presenting with PASC was associated with 94.6% rate and aOR = 26.65 of undergoing an urgent/emergent surgery. Consistent with our data, uninsured patients were 3.54 times more likely to undergo emergent colorectal surgeries..80 versu36 reoperations,36 costs,37 and lower odds of achieving TO compared to laparoscopic surgeries. Medicaid/Uninsured patients displayed similar odds of undergoing open procedures as Private after adjusting for PASC and urgent/emergent cases  and readmissions (aOR = 1.69). Emergency departments often serve as the primary healthcare source for low-SES patients with limited access to care.42 Our data demonstrate the utility of this approach. While all component variables had increased aOR for Medicare and Medicaid/Uninsured patients compared to the Private insurance group  to the previously reported complication rate (32.8%) of privately insured cohorts treated in low-burden hospitals.The Medicaid/Uninsured group was associated with increased adjusted odds of longer index hospitalization LOS and higher variable costs, but both were similar to Private after adjusting for urgent/emergent cases. Major factors impacting the %change in variable costs Table  were urg19 Healthcare providers are increasingly being held accountable for quality, outcomes, and cost of care.53 Decreasing rates of nonelective surgeries is a potential target for policy change. Medicaid expansion was associated with a 1.8-percentage point increase in the probability of an early, uncomplicated presentation for several surgical conditions compared to states that did not expand Medicaid.54 Improving access to health care to decrease the incidence of PASC and urgent/emergent operations may be a better approach to improving surgical outcomes and reducing costs than P4P programs. A Cochrane review55 concluded that hospital P4P programs have an uncertain impact and effects on patient outcomes were at most small on quality of care, equity, or resource use. Further studies should assess the impact of improved healthcare access for vulnerable patients on reducing urgent/emergent surgeries, complications, and costs. Numerous studies indicate that SNH and academic medical centers are disproportionately penalized in current value-based medicine programs.18 Integration of socioeconomic risk factors into evaluation of high- and low-burden hospitals could improve the distribution/allocation of resources, improving resources to SNH and mitigating disparities potentially propagated by P4P programs.Our findings suggest insurance type plays a significant role in outcomes and costs after colorectal surgery. Worse outcomes in Medicaid/Uninsured, low-SES patients were driven by increased presentation acuity, measured by increased odds of presenting with PASC and undergoing urgent/emergent surgeries, driving the increased complications and costs, consistent with a previous publication.This study is a retrospective review and cannot establish causal relationships. NSQIP provides a representative sample of surgeries but does not include all procedures performed at our institution. High complication and mortality rates may occur in cases performed for palliation, rather than for the purpose of extending life, but these data do not clearly define procedures that were performed specifically for palliation. While we included frailty, laparoscopic procedures and case status variables, multiple other variables could have been included.47 and costs of SNH serving a higher proportion of low-SES patients. Improving access to health care could provide a more significant impact on patient outcomes and decrease index hospitalization costs, by decreasing the incidence of urgent/emergent colorectal surgeries, particularly in low-SES, vulnerable patients.Medicaid/Uninsured insurance type was associated with decreased odds of achieving TO and increased odds of presenting with PASC and urgent/emergent cases, driving higher odds of complications, and index hospitalization costs. This suggests that factors beyond the surgeons’ control, such as increased presentation acuity and insurance type, impact surgical outcomes. Socioeconomic factors profoundly affect patient outcomes, which can account for the higher complication ratesJ.C.T. and M.A.J. are co-first authors and contributed equally to the contents of this study. P.K.S., J.K., M.A.J., and L.S.M. had full access to the data and take responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: P.K.S., S.S., M.A.J., J.K., B.B.B., and P.D. Acquisition, analysis, or interpretation of data: All authors. Drafting of the manuscript: J.C.T., M.A.J., and P.K.S. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: M.A.J., J.K., C.-P.W., and P.D. Obtained funding: P.K.S., B.B.B., S.S., P.D., and C.-P.W. Administrative, technical, or material support: All authors. Supervision: P.K.S.


Vanilla planifolia, within its cultivated origin, Mexico. High genomic heterozygosity levels within the globally cultivated ‘Daphna’ genome have raised questions on the possibility of a hybrid origin and analogous genomic signatures of vanilla cultivated within its origin. This study investigated these questions by assessing whether the genomic structure of Mexican V. planifolia reflected domestication events. Whole genome re-sequencing was used to compare genome complexity between 15 cultivated accessions from different regions and gene pools. Results showed high levels of heterozygosity, ranging from 2.48% to 2.85%, in all but one accession, which exhibited a low level (0.403%). Chromosome-level comparative analyses revealed genomic variability among samples, but no signals of chromosome rearrangements. These findings support the hypotheses that cultivated vanilla resulted from hybridization and that multiple domestication events have shaped cultivated vanilla leading to the formation of landraces. High cultural diversity within this region further supports the occurrence of multiple domestication processes. These results may help to improve breeding and conservation efforts aiming to preserve the genetic diversity of this beloved spice threatened by climate change.Although vanilla is one of the most valuable spices, there is a lack of understanding of the genomic variability of the main vanilla producing species,  Vanilla planifolia Andrews is a tropical vine of the family Orchidaceae, which produces vanilla, one of the most widely known and valuable spices worldwide [Vanilla planifolia is self-compatible, but incapable of self-fertilization without natively co-occurring pollinators [V. planifolia’s cultivated center of origin may offer a novel gene pool to increase the genetic diversity within the species and ensure crop sustainability under future climate scenarios. Analyzing the genomic structure of regionally cultivated vanilla in Mexico will offer important insight into this crop’s genetic resources and a better understanding of the processes leading to its domestication.orldwide . With a orldwide ,2. By coorldwide . Vanillalinators . Outsidelinators . Inhibitlinators ,6,7,8. Olinators  and funglinators , the raplinators . GeneticVanilla species in Mexico [V. planifolia.Ancient and contemporary cultural groups have shaped vanilla in its center of cultivation for centuries. Historical records indicate that vanilla was used as a flavoring and medicinal beverage by multiple cultures in Mesoamerica, including the Totonacs, the Mayans and the Aztecs ,12. Aften Mexico , which eV. planifolia. Single nucleotide polymorphism (SNP) analyses have revealed variation, clustering vanilla accessions into three main groups (types 1–3), with accessions cultivated in Mexico clustering into only two [V. planifolia in Mexico, haplotype variation, inferred using ITS, was revealed, uncovering ten different haplotypes [V. planifolia, where cultivated vanilla exhibited substantially higher levels [Recent advances in genomic sequencing technology and the publication of a reference genome ,20 have only two . Furtherplotypes . At the r levels .V. planifolia to be 2.32% [V. planifolia cultivated outside of its native range raise the questions of what evolutionary processes contributed to this genomic structure and whether vanilla cultivated in its origin exhibits the same genomic signals. It has been hypothesized that these high levels of heterozygosity within cultivated vanilla were attributed to the accumulation of somatic point mutations brought about by clonal propagation [Manihot esculenta Crantz [For examining an organism’s genetic diversity and evolutionary history, genome-wide patterns of heterozygosity offer a valuable metric. Using GenomeScope, a recently developed software designed to assess the relative abundance of homozygous and heterozygous sequences within k-mer frequency distributions ,23, recebe 2.32%  and 2.47be 2.32% , therefopagation , as showa Crantz . The extV. planifolia and six species: V. pompona Schiede [V. aphylla Blume [V. odorata C. Presl, V. ×tahitensis J.W. Moore [V. phaeantha Rchb.f. [V. palmarum Salzm. ex Lindl [V. planifolia [Hybridization has previously been suspected as a contributing agent to phylogenetic incongruences between nuclear and plastid signals  and chro Schiede , V. aphyla Blume , V. odorW. Moore , V. phae Rchb.f. , and V. ex Lindl . Owing tanifolia  and coulV. planifolia in its origin will help shed new light on the domestication processes and genetic resources of this beloved spice threatened by climate change.Within this study, we aimed to explore the evolutionary mechanisms underpinning the high levels of genome-wide heterozygosity in vanilla and shed light onto the evolutionary processes that have affected this crop in its cultivated center of origin, Mexico. Due to the phenotypic and genetic variation observed in Mexico and the long histories of regional cultivation by different ethnic groups, we hypothesized that the gene pool of cultivated vanilla in Mexico has been influenced by multiple domestication events. On top of that, due to the extent of genome-wide heterozygosity levels found within globally cultivated vanilla, we hypothesized that cultivated vanilla stems from a hybrid origin. To assess if the genome structure of regionally cultivated haplotypes reflects domestication processes, we compared the genome structure of regionally cultivated vanilla against the available reference ‘Daphna’ genome , evaluatThe data and reproducible workflow  associated with this study are available on GitHub , and a cV. planifolia, a predominantly vegetatively propagated crop, were comparatively high [The query for studies that have used GenomeScope to infer genomic heterozygosity resulted in 455 publications deposited on PubMed, of these 142 pertained to plants . For allely high ,20  were detected among all samples. Pruning for linkage disequilibrium (LD) at thresholds 0.2 and 0.8 reduced the number of filtered SNPs to 9297 and 419,885, respectively. Independently of LD thresholds, results from the principal components analysis (PCA) remained similar . With a V. planifolia samples exhibited high genome-wide heterozygosity levels, except one, MEX67, which exhibited a notably low level (0.403%), (Artemisia tridentata Nutt. (Asteraceae) and, based on the abundance of AAB (26%) and AABB (14%) k-mer ratios along with the dominant AB (49%) k-mer pairs [V. planifolia, however, results from Smudgeplot confirmed the diploid status of all accessions by uncovering almost exclusive AB k-mer pairs  were observed in Litchi chinensis Sonn. and were attributed to the hybridization of two distinct haplotypes [V. planifolia, candidate parental species may include other less cultivated species like V. pompona or V. odorata. One sample, MEX67, did not exhibit the genomic signal of hybridization as the others did , . Similarer pairs , were attructure  and contand AABB % k-mer rtructure , howeverplotypes . In addihers did B, and thV. planifolia leading to their similar, but variable, levels of heterozygosity. These results indicate that multiple haplotypes exist within the AB sub-genomes identified through Smudgeplot analyses  of methods within this study are available on GitHub . AdditioTo obtain a reference of plant genomic heterozygosity levels inferred using GenomeScope ,23, a liVanilla planifolia plants within the origin of vanilla cultivation in Mexico in October of 2019 from the northernmost region around Papantla, Veracruz to the southernmost region around Chinantla, Oaxaca (V. planifolia ‘Daphna’ genome (BioProject ID: PRJNA633886), downloaded from the National Center for Biotechnology (NCBI) website, was used as a reference in this study.Samples were collected from 15 , Oaxaca . Sampleshttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/ accessed on 5 May 2020) and all reads were cleaned and trimmed using Trimmomatic [Genomic DNA was extracted from all lyophilized leaf samples using the Qiagen DNeasy Plant Mini Kit  following the manufacturer protocol. DNA yield was quantified using a Qubit fluorometer . Extracted genomic DNA with concentrations greater than 20 ng/µL was sent to GENEWIZ, Inc.  for library preparation and sequencing of 150 bp paired-end reads using an Illumina HiSeq platform aiming for a sequencing depth of 50X to allow for sufficient coverage on the reference ‘Daphna’ genome  [mmomatic  with minhttps://github.com/KamilSJaron/smudgeplot accessed on 2 April 2022) using k-mer coverage (kcov) values inferred from GenomeScope, where L = (kcov/2) − 5.Genomic sequences were characterized  by k-mer frequency analyses (k = 21) using Jellyfish  and GenoV. planifolia ‘Daphna’ genome using Bowtie 2 [https://github.com/tpoorten/dotPlotly accessed on 1 June 2022). For visualization of genomic variability among samples, a heat map was produced in R using ‘gplots’ [Chromosome-scale genomes were reconstructed by mapping cleaned, trimmed reads to the reference Bowtie 2 . VariantBowtie 2 . ChromosBowtie 2  to assesBowtie 2 , chromosBowtie 2  and chro‘gplots’  to show Reconstructed genomes were analyzed using BCFtools  to call


To explore the active compounds of the Chinese medicine prescriptions of Bushen Hugu Decoction (BHD) and demonstrate its mechanisms against malignant tumor bone metastasis (BM) through network pharmacology and molecular docking analysis. The main components and targets of BHD were retrieved from the TCMSP database, and the targets were normalized by UniProt. The Herbs-Components-Targets network of BHD was established by Cytoscape. The main BM targets were obtained from GeneCards, TTD, DrugBank, and OMIM. STRING and Cytoscape were used to construct a PPI network and obtain hub genes. DAVID and Metascape were used for GO and KEGG enrichment analyses. According to the network topology parameters, the top 4 components were selected for molecular docking verification with the core targets. p < 0.05), and 164 signaling pathways (p < 0.05) were found in KEGG, including PI3K-Akt signaling pathway, proteoglycans in cancer, prostate cancer, MAPK signaling pathway, and IL-17 signaling pathway. Molecular docking analysis showed that the active components of BHD, quercetin, luteolin, kaempferol, and aureusidin have good binding activity to the core targets.  Compound–target network of BHD mainly contained 51 compounds and 259 corresponding targets including 107 BHD-BM targets. PPI interaction network and subnetworks identified ten hub genes. GO enrichment analysis found 1970 terms ( The potential molecular target and signaling pathways were found for BHD major active components. It provides guidance for the future mechanism research of the BHD in malignant tumor bone metastasis. This study also established the foundation for the new strategy for the pharmacology study of Chinese medicine. Cancer incidence increased worldwide. Cancer, especially malignant tumor, ranks as the disease with the highest fatality rate after cardiovascular disease globally . Most maΒ ligand (RANKL). The RANK/RANKL/OPG axis is critical for the regulation of bone formation and resorption. Inhibition of RANKL may serve as an ideal therapeutic strategy for excessive bone resorption [The standard cares for bone metastases in cancer patients are bisphosphonates and denosumab. Bisphosphonates, the inhibitors of osteoclast activity, can inhibit tumor growth by blocking prenylation. However, clinical studies showed minimal efficiency on tumors due to limited uptake at tumoral sites . Denosumsorption . Howeversorption . Standarsorption , 15.Chinese medicine is a traditional treatment method original to China. A variety of Chinese medicine prescriptions, herbs, and extracts have been confirmed with the potential to treat malignant tumors by experimental studies and clinical trials . Combiniα signaling pathway [Cistanches Herba extract phenylethanol glycosides increase the sensitivity of liver cancer patients to oxaliplatin by inhibiting HIF-1 pathway .Cuscutae Semen, Dipsaci Radix, Drynariae Rhizoma, and Eucommiae Cortex can inhibit bone destruction through the immune system and inflammation-related pathways –24. In thttps://old.tcmsp-e.com/index.php) was used to authenticate chemical composition of various herbs in BHD. TCMSP consists of 499 Chinese herbs registered in the Chinese Pharmacopoeia, with 29,384 ingredients, 3,311 targets, and 837 associated diseases [https://www.uniprot.org). “Herbs-components-targets” network of BHD was constructed based on network topology parameters (NTP) through Cytoscape 3.7.2. The larger the node, the more targets associated with it, and the more likely it will be a key ingredient in BHD.Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , OMIM database (http://www.omim.org), TTD database (http://bidd.nus.edu.sg/group/cjttd), and DrugBank database (https://www.Drugbank.ca) using “Bone Metastasis”, “Bone Metastases”, “Osseous Metastasis”, “Bone Metastasis of Malignant Tumor” and “Metastatic Carcinoma of Bone” as keywords. The targets were supplemented through literature search. Use Venn script in R language to get the intersection of the targets of BHD components and BM. BHD-BM genes were uploaded to STRING11.0 (https://string-db.org) to build a PPI interaction network. Initial screening of the BHD-BM gene by setting the degree score. The data were corrected by setting the biological species to 'Homo sapiens'. The PPI interaction network of BHD-BM genes was visualized with Cytoscape using 0.9 as the highest confidence level. Topological analysis of BHD-BM genes by Cytohubba in Cytoscape to obtain the hub genes [The bone metastases-related targets were identified in the GeneCards database (ub genes .https://david.ncifcrf.gov/) online analysis tool and Metascape platform (http://metascape.org/gp/index.html). A p < 0.05 was used to screen BHD-BM-related biological process (BP), molecular function (MF), and cellular component (CC). Then, the top ten results were chosen for visualization. Similarly, a p < 0.05 was used to screen KEGG pathway analysis. BHD compounds, BHD-BM genes, and top 20 model pathways were used in KEGG enrichment analysis. The NTP  was analyzed by the built-in tool of Cytoscape to build the BHD compound, BM target, and pathway network.GO and KEGG enrichment analyses of BHD-BM genes were performed using DAVID (https://pubchem.ncbi.nlm.nih.gov/) [http://www.RCSB.org/) [Molecular docking is a well-established computer-based structural method for structurally docking small molecules to target proteins and assessing their binding affinity . It is wih.gov/) . And savSB.org/) . Use AutSB.org/) .As shown in Database searching found 8,691 nonduplicated BM-related genes. By setting the degree score greater than the median three times, 1086 targets were finally obtained. Combined with the target screened for BHD active compounds in TCMSP dataset, 107 genes associated with both BHD and BM were considered to build the BHD-BM PPI interaction network . PPI netGO enrichment analysis found 1,970 terms. Multiple targets of BHD were closely related to BM. The major biological process enriched for BHD-BM has a response to an inorganic substance, positive regulation of cell death, response to cytokine, positive regulation of cell migration, and regulation of apoptotic signaling pathway. The molecular function related to BHD-BM has kinase binding, protein kinase binding, ubiquitin-like protein ligase binding, DNA-binding transcription factor binding, and cytokine receptor binding. BHD-BM-related cellular components have membrane raft, transcription regulator complex, vesicle lumen, serine/threonine protein kinase complex, and extracellular matrix . KEGG paAccording to the NTP analysis, the core compounds  A3 quer PTGS2, HBone is one of the common sites of cancer metastasis. The unique structure and the dynamic metabolism pattern in bone make bone metastasis a different mode of malignant tumor metastasis. The mechanism of BM is still not very clear yet.β1, PDGFs, etc. [The “seed and soil” theory for tumor metastasis proposed by Paget in 1889 believed that the spread of tumor cells is determined by the interaction between cancer cells (seed) and host organs (soil) . After mFs, etc. . TME incFs, etc. . Bone maFs, etc. . The croThe interaction between TME and BME involves multiple pathways as a comprehensive network. However, during the drug development process, it is very common to identify or design pharmacologically effective agents specifically targeting a single molecule or site. Drugs that act on a single molecular target often show unsatisfactory therapeutic effects or have large side effects when used to treat complex diseases in clinical. Therefore, drug development has gradually shifted from a “single target and single drug” model to a “network target multicomponent therapy” model , 41. MulIn this study, a total of 51 active components and 259 targets for BHD and 1086 BM targets were obtained. Combination of these two dataset, 107 BHD-BM intersection targets, was obtained finally. PPI interaction network analysis obtained the hub targets. GO and KEGG enrichment analyses showed that multiple targets of active components of BHD were closely related to BM. Membrane raft, transcription regulator complex, vesicle lumen, serine/threonine protein kinase complex, and extracellular matrix are the top cellular components found in GO and KEGG enrichment analyses. These cellular components are closely related to the formation of extracellular vesicles (EVs). EVs include exosomes, activation- or apoptosis-induced macrovesicles, and apoptotic bodies, which are key players in intercellular communication . EVs are+ helper T cells [KEGG enrichment analysis of BHD-BM showed that BHD compounds can affect BM through various pathways including regulation of apoptosis, immunity, inflammatory response, and treatment of primary tumors. A3 (quercetin), A2 (kaempferol), DR13 (aureusidin), DR9 (luteolin), A1 (beta-sitosterol), EC13 (syringetin), DR10 , and B2 (stigmasterol), the top 8 active components of BHD, were obtained after the bioinformatic analysis. Their combination to core targets was verified by molecular docking, which confirmed the multitarget and multichannel therapeutic effect of BHD on BM. PI3K-Akt signaling pathway was identified as one of the top signaling pathways in BHD-BM network. PI3K-Akt could participate in multiple cellular events to promote tumor initiation, progression, and metastasis. PI3K/Akt significantly increases bone metastases and osteolytic bone lesions in prostate cancer by mediating the stabilization of histone methyltransferase WHSC1 . TLRs ar T cells . Th17 ce T cells . By bloc T cells . All thiIn this study, we built the network between BHD compounds and BM target and screened and verified the in vivo combination of BHD components and their target employing network pharmacology and molecular docking analysis. Network pharmacology showed that quercetin and kaempferol were the top 2 core compounds of BHD.Quercetin and kaempferol, as natural bioflavonoids, are widely found in a large number of natural herbs and have broad-spectrum antitumor effects , 53.Animal and cellular experiments have shown that quercetin and kaempferol could downregulate AKT1, PTGS2, and VEGFA expressions –77 and uThis study established the foundation of the new strategy for the pharmacology study of Chinese medicine. This study shows that the main active components of BHD can participate in the regulation of tumor and bone microenvironment through extracellular vesicles and affect the occurrence and development of malignant tumor bone metastases through various mechanisms such as immunity, apoptosis, and inflammation. It provides a basis for the study of the molecular mechanism of BHD and provides a new option for the treatment of bone metastases.


Vibrio vulnificus necrotizing soft tissue infection (NSTI) is associated with higher mortality compared with non-Vibrio NSTI. This study’s objective was to compare outcomes including in-hospital mortality and prognosis between patients with V. vulnificus NSTI and those with non-Vibrio NSTI.It remains unclear whether V. vulnificus infection. We performed multiple imputation using chained equations followed by multivariable regression analyses fitted with generalized estimating equations to account for clustering within matched pairs. All-cause in-hospital mortality and length of stay during hospitalization were compared for NSTI patients with and without V. vulnificus.A retrospective 1:2 matched-pair cohort study of hospitalized patients with NSTI diagnosed by surgical finding was conducted in two tertiary hospitals in southern Taiwan between January 2015 and January 2020. In-hospital outcomes  were compared between patients with and without V. vulnificus NSTI group and 90 in non-Vibrio group. The V. vulnificus NSTI patients had higher mortality and longer hospital stays. Multivariable logistic regression analysis revealed that V. vulnificus NSTI was significantly associated with higher in-hospital mortality compared with non-Vibrio NSTI .A total of 135 patients were included, 45 in Vibrio vulnificus NSTI was associated with higher in-hospital mortality and longer hospital stay which may increase health care costs, suggesting that preventing V. vulnificus infection is essential. Vibrio vulnificus is a life-threatening condition with rapid progression and high mortality. It usually occurs through the injuries sustained when handling seafood, wound exposure to seawater, and ingestion of contaminated under-cooked seafood. Immunocompromised patients are also at higher risk of developing this disease  = 12.7), and 66.4% of the patients were male. Table V. vulnificus NSTI and non-Vibrio NSTI after 1:2 matching. The V. vulnificus NSTI group tended to have hypotension, bacteremia, history of seawater or seafood contact, comorbidity with liver disease, and higher serum lactate. All-cause in-hospital mortality was 13.3% in the V. vulnificus NSTI, whereas it was 7.8% in the non-Vibrio NSTI . Higher mortality was significantly associated with higher serum lactate, higher LRINEC score, and with liver disease.A total of 135 patients were included, 45 in Vibrio vulnificus is a gram-negative marine bacterium that is usually present in warm coastal waters. The main clinical manifestations of V. vulnificus infections in humans are gastrointestinal illnesses, primary septicemia, and wound infections. The clinical course can progress rapidly by releasing hemolysins and proteases and result in hemorrhagic bullae and severe skin necrosis [V. vulnificus include wound infections while handling seafood, exposure of a preexisting wound to seawater, and ingestion of contaminated under-cooked seafood [necrosis , 12, 13. seafood .V. vulnificus NSTI than those in non-Vibrio group. In our study, in-hospital mortality among patients with V. vulnificus NSTI was 13.3%. Previous studies have reported vulnificus NSTI mortality around 10–13% [Aeromonas spp. revealed high mortality rates, even through aggressive surgical debridement and antibacterial therapies. They all raised concern for increasing risk of monomicrobial infection and gram-negative pathogen. As comparison, the non-Vibrio group are including gram-positive, negative, and culture no growth patients, and possibly monomicrobial or polymicrobial. Mainly pathogens included Aeromonas spp., Pseudomonas, Escherichia coli, Klebsiella pneumoniae, Staphylococcus, and Streptococcus spp. Using two tertiary hospital database in Southern Taiwan, our study showed that mortality was higher in patients with d 10–13% , 15, whid 10–13%  found thd 10–13%  revealedV. vulnificus NSTI, there are 17 patients with positive blood and wound culture (37.8%), 12 patients with positive blood culture (26.7%), and 16 patients with positive wound culture (35.5%). In non-Vibrio group, there are 19 patients with bacteremia (21.1%). V. vulnificus group had higher incidence of bacteremia than non-Vibrio group (64.4% vs 21.1%). Tsai et.al conducted a study about NSTI patients with V. vulnificus and methicillin-resistant Staphylococcus aureus (MRSA). The incidence of bacteremia was 65% vs 18.75%; V. vulnificus group was also higher than MRSA group [V. vulnificus are more likely to develop blood stream infections [V. vulnificus group. Vibrio vulnificus can produce Vibrio vulnificus hemolysin/cytolysin (VVH), multifunctional autoprocessing RTX toxin, Vibrio vulnificus serine protease (VvsA), and Vibrio vulnificus protease (VVP). Both VVH and RTX toxin contribute to bacterial invasion to the blood stream and play a role in the development of hypotensive septic shock. The RTX toxin may promote the colonization at the primary infection site and cause severe tissue damage by releasing degrading cytotoxins to induce the inflammatory responses, that Vibrio vulnificus can then spread to the blood stream to result in systemic manifestations. Both VVP and VvsA are produced in the interstitial tissues of limbs and cause serious collagenolytic, hemorrhagic, or edematous skin damage in the extremities [V. vulnificus may explain a relatively higher rate of blood steam infection than other pathogens. However, the comparison of Vibrio vulnificus bacteremia between NSTI and food-borne infection settings needs further studies.In culture 2.7%, and remities –23. The Aeromonas species than for those with V. vulnificus [V. vulnificus and Klebsiella pneumoniae [V. vulnificus and MRSA [V. vulnificus versus non-Vibrio NSTI. In the present study, we clearly demonstrated that mortality was higher in patients with V. vulnificus than in patients with non-Vibrio NSTI. Besides, longer hospital stay may lead to higher hospitalization cost in V. vulnificus NSTI.Studies have shown conflicting results on the difference in mortality between patients with NSTI in subgroups. One study had shown higher mortality for NSTI patients with lnificus . One stueumoniae . Anotherand MRSA . But fewV. vulnificus infections and mortality. There were studies considered liver cirrhosis as risk factor with increased mortality among NSTI patients [V. vulnificus infection. Because NSTI and its rapidly progressive infection remain associated with high mortality, the LRINEC score, developed by Wong et al. [V. vulnificus [V. vulnificus NSTI but display a significant role with prognosis, associated with higher mortality in V. vulnificus NSTI patients.Our study showed that patients with hepatic dysfunction had significant associations with patients , 26–29. patients , 31. Theg et al.  based onlnificus . HoweverThis study still has some limitations. First, this study lacks external validation. The validity of the result still requires other studies to confirm. Second, the retrospective design of this study had its inherent limitation in data collection, such as unverified comorbidities. Third, the sample size is relatively small due to the rarity of NSTI. Fourth, the degree of liver disease in each group is not clearly stated.V. vulnificus NSTI than for those with non-Vibrio NSTI in this matched-pair cohort study. Preventing V. vulnificus infection is essential. The validity of the result still needs to be confirmed by further studies.In summary, adjusted in-hospital mortality was significantly higher for patients with


Staphylea bumalda DC, belonging to family Staphyleaceae, is a woody understory tree that is both edible and medicinal and produces oil with high economic value. This study reports the first complete chloroplast genome sequence of S. bumalda. The complete chloroplast genome sequence of S. bumalda is 160,319 bp in length with an overall GC content of 32.79%, which is composed of a large single-copy region , a small single-copy region , and two inverted repeat regions . A total of 130 genes were predicted in this genome, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic analysis based on 14 complete chloroplast sequences from related species revealed that S. bumalda is a sister to S. holocarpa. Staphylea bumalda (Candolle 1825) is a shrub species in family Staphyleaceae mainly distributed throughout China, Japan, and Korea. Also called pearl flower, its floral buds contain a high content of crude protein, crude fat, and essential amino acids and are consumed as a vegetable in the mountainous regions of China for their unique flavor  of Henan Province, China. The voucher specimen is deposited in the National Center for Forestry and Grassland Genetic Resources . Total genomic DNA was extracted according to a modified CTAB protocol (Doyle and Doyle http://soap.genomics.org.cn/soapdenovo.html) short sequence assembly software  software. The genome was annotated using the CpGAVAS2 pipeline . The new annotated chloroplast genome sequence was deposited in GenBank (Accession No. CRA005805).The fresh leaves of S. bumalda was 160,319 bp in length, consisting of a small single-copy region , a large single-copy region , and two inverted repeat regions , with the overall GC content of 32.79%. The chloroplast genome encoded a total of 130 genes, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Twenty-seven introns were distributed across twenty-one genes contained. The gene content of the S. bumalda cp genome was nearly identical to those of related taxa. However, two genes, LhbA and InfA were lost in S. bumalda compared with other species.The chloroplast genome sequence of Sambucus species, one Bischofia species, and one Acer lucidum species as outgroup. Genome sequences were downloaded from the GenBank database and were aligned using MAFFT v7.0 (Katoh and Standley S. bumalda was sister to S. holocarpa. The topology shows Tapiscia sinesis is distantly related from S. blualda and other Staphylea. This supports previous research describing the unique characters of Tapiscia that warrant its treatment as a separate family (Ding and Yu To explore the phylogenetic relationships within tree species of related families and genera, a maximum likelihood phylogeny analysis was performed using IQ-TREE 2.1.3 with 1000 bootstrap replicates (Nguyen et al.


Arabidopsis thaliana using immunolocalizations with commercially available antibodies. We detected the presence of AGO proteins associated with posttranscriptional gene silencing  in the cytoplasm and the nucleus, while AGOs associated with transcriptional gene silencing (AGO4 and 9) localized exclusively in the nucleus. These results indicate that the localization of different AGOs correlates with their predicted roles at the transcriptional and posttranscriptional levels and provide an overview of their timing and potential role during meiosis.Meiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have been proposed to carry a specific activity during meiosis, but the pathways involved during this process remain unclear. Here, we explored the subcellular localization of different ARGONAUTE (AGO) proteins, the main effectors of RNA silencing, during male meiosis in The online version contains supplementary material available at 10.1007/s00497-021-00434-z. Arabidopsis, the miRNA pathway is crucial for the development of male gametes , at 24–25 °C and 45% relative humidity.https://www.mirbase.org/) was used for miRNA alignments  were normalized data were extracted for both GSE10229 (http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=195) and GSE13000 (http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=46). RNA sequencing data were downloaded, adapter trimmed, and filtered by length and quality using Trim Galore! . For gene expression analysis, paired reads were aligned to the Arabidopsis TAIR10 genome using bowtie2  provide an overview of the subcellular localization, timing, and potential role of different RNA silencing pathways during meiosis. Furthermore, our work complements previous analysis that analyzed RNA silencing activity in meiocytes and opens the door for future molecular analysis of the specific role of AGO proteins during specific meiosis stages, which are technically challenging to purify for their analysis through high-throughput sequencing techniques at the moment.C.O and G.M. design the experiments and wrote the manuscript. C.O. performed the experiments and analyzed the data. G.M. analyzed the bioinformatic data.Supplementary file1 (XLSX 38 KB)Supplementary file2 (PDF 239 KB)Supplementary file3 (PDF 1634 KB)Supplementary file4 (PDF 78 KB)Below is the link to the electronic supplementary material.