Single-cell RNA sequencing (scRNA-seq) is a new technology that can be used to explore molecular changes in complex cell clusters at the single-cell level. Single-cell spatial transcriptomic technology complements the cell-space location information lost during single-cell sequencing. Coronary artery disease is an important cardiovascular disease with high mortality rates. Many studies have explored the physiological development and pathological changes in coronary arteries from the perspective of single cells using single-cell spatial transcriptomic technology. This article reviews the molecular mechanisms underlying coronary artery development and diseases as revealed by scRNA-seq combined with spatial transcriptomic technology. Based on these mechanisms, we discuss the possible new treatments for coronary diseases. Coronary artery disease may lead to insufficient blood and oxygen supply to the heart, resulting in myocardial necrosis, which can be fatal in severe cases . Accordiin situ hybridization or paired cell sequencing or complex algorithms with scRNA-seq data , complex computational algorithms, and cell pairing, is based on the principle that some components of spatial information are compared with scRNA-seq data to obtain the spatial information of a single cell  is a powerful tool that can generate and validate many predictions that need to be confirmed experimentally –6. Howevseq data , 10. Thegle cell , 11, 12.hnology.  These tehnology. . FurtherIn this study, we explored the findings of scRNA-seq and spatial transcriptomic techniques in a multicellular coronary microenvironment. We integrated recent results from single-cell sequencing and spatial transcriptomic studies of coronary developmental stages and diseased coronary arteries to provide the latest insights into the cellular origins and molecular mechanisms of coronary artery disease development and coronary lesions. We found that cells such as venous sinuses are the major contributors to coronary development during the developmental stages. Furthermore, epicardial cells play key roles in promoting the development of coronary arteries. Focusing on the composition of the pathological microenvironment in coronary atherosclerotic lesions, we discovered a much-debated collateral circulation and its formation. Various types of cardiac cells participate in a coronary-capable collateral circulation through different mechanisms. Based on the mechanism targets and gene expression discovered using single-cell spatial transcriptomics technology, we propose possible molecular targets and methods to achieve precision medicine for coronary artery disease, with the hope of providing prospective future precision medicine treatment ideas.1.1.Determining the mechanisms underlying coronary arterial origin has always been a striving direction for researchers , 21. WitFrom the earliest “aortic sprouting origin” theory to the belief that there was a transient structure “precordial” in the embryo, coronary arterial origin has been studied for decades. Using single-cell sequencing technology, we identified the venous sinus origin of the coronary arteries. The formation of coronary vasculature is a complicated and unintuitive process involving multiple cellular sources. Advances in single-cell sequencing technologies have allowed us to analyze the different origins of coronary arteries at the single-cell level. Studies have shown that the venous sinus, endocardium, and cardiac neural crest are the main sites of coronary artery development. In addition, epicardial cells express vascular chemokines that promote coronary artery development in a paracrine manner .Slc45a4, marked the pre-arterial population at an early stage  gene promotes the proliferation of subendocardial endothelial cells to generate endocardium-derived coronary arteries ..15).in situ hybridization was used to display the spatial information of the different cell populations. They identified three populations of epicardial cells with distinct spatial preferences. Only clusters of epicardial cells expressing Sfrp2, Dkk3, and Pcsk6 were uniformly dispersed in infarct border areas. This confirmed that epicardial cells undergo multi-layered divisions under ischemia and hypoxia, which might be related to the different roles of different epicardial cell clusters in myocardial infarction. Deciphering the spatial distribution of the epicardium helps explain disease development and provides clues for treatment. Studies have shown that adult epicardial cells exposed to chronic hypoxia have developmental characteristics similar to the endothelial-mesenchymal transformation at the embryonic stage. They can differentiate into various cell types and promote coronary angiogenesis in a paracrine manner (in situ hybridization were combined (In a single-cell spatial transcriptomic study, Hesse et al. simulated myocardial infarction by ligating the left anterior descending artery in mice . All care manner –68. To ecombined . The relcombined . Genes ecombined . Previoucombined . Mesothecombined . Epicard3.Based on the current efforts in single-cell research on coronary artery development and disease, we propose the following new ideas for the treatment of coronary artery disease: 1. Targeting vascular chemokines 2. Induction of mature cardiac cells to reverse differentiation and proliferate to form new blood vessels 3. Induction of myocardial regeneration.Human induced pluripotent stem cell therapy has shown great potential in experiments and has even been clinically applied . Single-β4 plays an important role in promoting coronary vascular development. In order to explore a new method for the treatment of myocardial infarction, Chen et al. used an engineered exosome to continuously and slowly release thymosin β4 in the myocardial infarction area of model mice (β4 exosomes could significantly increase the level of neovascularization and restore blood supply after myocardial infarction (As an emerging molecular technology, spatial transcriptomics plays an important role in treatment of coronary artery disease. The coronary sinus, an adult derivative of the embryonic sinus vein, possesses vascularization ability similar to that of the sinus vein. Furthermore, adult coronary veins and arteries have the same origin during development . Based odel mice . It was farction . Echocarfarction . A recenfarction , 75. The4.Single-cell transcriptome sequencing is a revolutionary technology that has been successfully applied to different organs and tissues of an organism from an unprecedented perspective. Direct spatial transcriptome sequencing compensates for the loss of spatial information in single-cell sequencing and is not only used to verify the results of single-cell sequencing but also makes it possible to study the complex changes of coronary artery lesions in space. The biology of coronary artery disease involves multiple cell types and complex cell-to-cell interactions, and scRNA-seq allows for the detailed study of these multicellular microenvironments. scRNA-seq revealed important molecular mechanisms underlying the origin of coronary artery development and disease progression. Venous sinus cells must differentiate into pre-arterial cells before participating in coronary artery formation. Prdm6 promotes differentiation of cardiac neural crest cells into pericytes through epithelial-mesenchymal transformation by up regulating the expression level of WNT1. Next, pericytes differentiate into coronary smooth muscle cells under the activation of the NOTCH3 signaling pathway to participate in coronary artery formation. Furthermore, endocardial cells are also involved in coronary artery formation during embryonic development. However, current research on coronary artery disease is still identifying changes in gene expression levels in various cells at different stages, and research on various molecular mechanisms is still immature. Therefore, there is insufficient evidence regarding the accuracy of each molecular mechanism in disease progression. In the future, extensive spatial transcriptomic studies should be conducted to compensate for the lack of current molecular mechanistic studies . Based o


It is well established that the neonatal immune system is different from the adult immune system. A major task of the neonatal immune system is to bridge the achievement of tolerance towards harmless antigens and commensal bacteria while providing protection against pathogens. This is highly important because neonates are immunologically challenged directly after birth by a rigorous change from a semi-allogeneic sterile environment into a world rich with microbes. A so called disease tolerogenic state is typical for neonates and is anticipated to prevent immunopathological damage potentially at the cost of uncontrolled pathogen proliferation. As a consequence, neonates are more susceptible than adults to life-threatening infections. At the basis of a well-functioning immune response, both for adults and neonates, innate immune cells such as monocytes and monocyte-derived macrophages play an essential role. A well-responsive monocyte will alter its cellular metabolism to subsequently induce certain immune effector function, a process which is called immunometabolism. Immunometabolism has received extensive attention in the last decade; however, it has not been broadly studied in neonates. This review focuses on carbohydrate metabolism in monocytes and macrophages in neonates. We will exhibit pathways involving glycolysis, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation and their role in shaping neonates’ immune systems to a favorable tolerogenic state. More insight into these pathways will elucidate potential treatments targets in life-threatening conditions including neonatal sepsis or expose potential targets which can be used to induce tolerance in conditions where tolerance is harmfully impaired such as in autoimmune diseases. Neonates (those <28 days of life) are born into a world rich in microbes as opposed to the semi-allogeneic sterile intra-uterine environment where fetal development takes place. This immediately results in a challenge to the neonate´s immune system as tolerance towards harmless antigens and commensal bacteria needs to be developed while protection against invading pathogens is also vital. The need to develop tolerance against commensal bacteria results in a higher susceptibility to infections, which has long been attributed to an immature innate immune system and lack of memory mechanisms of the adaptive immune system. However, numerous recent studies suggest that neonatal immunity is highly evolved, regulated and life stage appropriate. Not immaturity but a highly regulated tolerogenic status distinguishes the neonatal innate immune system from that of adults , Enterobacter cloacae (Gram-negative), Klebsiella pneumoniae (Gram-negative) and Escherichia coli (Gram-negative) . Th. ThCandiMALT1 signalosome genes were found to be reduced in monocytes from neonates born preterm  showed a diminished expression of the costimulatory molecules CD80 and CD86 compared to adults (42 days) . In lineGLUT1 was found to be reduced likely impairing the influx of glucose. Secondly, phosphofructokinase M (PFKM) mRNA was reduced, which is an enzyme needed to convert fructose 6-phosphate (F6P) into glyceraldehyde 3-phophate (GAP). As well, mRNA levels of mitochondrial pyruvate carrier 1 (MPC1) and 2 (MPC2), forming a heterodimer (MPC1/MPC2), were diminished. This heterodimer is essential to translocate pyruvate from the cytosol into the mitochondria. Moreover, the expression of the enzymes pyruvate carboxylase (PC) and malate dehydrogenase 2 (MDH2) were downregulated impairing the processing of pyruvate to oxaloacetate and malate to oxaloacetate, respectively. These molecular differences are in line with the absence of glycolysis induction upon immune stimulation observed in mononuclear phagocytic cells from neonates.In addition, on a genetic or epigenetic level, several metabolic regulators of glycolytic metabolism and the TCA cycle were found to be altered in CBMOs compared to PBMOs  Figure . First, Regarding neonatal sepsis, a large screening approach, using genome-wide transcriptional analysis, was conducted to gain insight into immune-metabolic differences between healthy human neonates and neonates with bacterial infection . In neonEscherichia coli-mediated sepsis [Another metabolic regulator with a divergent expression between neonates and adults is the mammalian target of rapamycin (mTOR), a key regulator of the metabolic switch towards anaerobic glycolysis during immune activation . mTOR trd sepsis . Since mMycobacterium tuberculosis (Mtb) stimulation, the causative agent of tuberculosis [Importantly, one study observed a rapid increase in glycolysis in cord blood macrophages (similar to adult macrophages) in response to LPS or rculosis . It is pIn pursuit of an altered glucose metabolism, in neonatal monocytes and macrophages, the following question arises: which environmental factors dictate a divergent macrophage metabolism in neonates? Or are they the result of genetically or epigenetically encoded developmental programs or a combination of both? The observed differences between neonatal and adult phagocytic cells can be partly explained by a concept called trained immunity . This coAs well, the counterpart of trained immunity, known as ‘innate immune tolerance’, may likely be involved in functional reprogramming of neonatal monocytes and macrophages. Hereby, immune cells are unable to activate gene transcription and its connected pro-inflammatory pathways upon re-stimulation by microbial particles . As an eS. aureus or LPS induced infection with a strongly enhanced mortality [Potential candidates, recently found to be involved in reprogramming neonatal phagocytic cells, are the S100 calcium binding proteins S100A8 and S100A9 and their extracellular heterodimer complex form S100A8/S100A9. These antimicrobial proteins were found in high amounts in human breast milk, fecal samples and serum from newborns ,64. S100ortality ,65. Thisortality . Of inteortality ,65. As aNext to S100 proteins, breast milk contains a great many of other biologically active components such as immunoglobulins, chemokines/cytokines, oligosaccharides, microRNAs, hormones, immune cells and microorganism all priming the perinatal immune system . Of inteFurthermore, the detection of pathogens by innate immune cells dictates immune-metabolic pathways including macrophage metabolism and its associated polarization. Mechanistically, pathogen-associated molecular patterns (PAMPs), such as LPS, activate microbial sensors  including C-type lectin receptors, nucleotide-binding oligomerization domain (NOD)-like receptors and TLRs. Of interest, signaling via PPRs lead to intracellular metabolic changes ,68. For IL-1β, IL6, TNF and CCR2, was associated with a matching gene expression [Differently programmed neonatal mononuclear phagocytic cells may also be the result of epigenetic changes and transcriptional remodeling. Epigenetic mechanisms can activate or silence gene transcription by modulating chromatin structure and stability without affecting the DNA sequence itself. Epigenetic modifications include DNA methylation, histone modifications and microRNA (miRNA) expression. These modifications are known to regulate immune cell differentiation and function in adults but have sparsely been studied in neonates. In neonatal monocytes a unique histone modification landscape was observed . During pression .Additionally, in human monocytes from neonatal cord and adult blood, a difference in miRNA-146 was observed. MiRNAs are small, single-stranded, non-coding RNA molecules (21 to 23 nucleotides) and are involved in RNA silencing and post-transcriptional regulation of gene expression. More specifically, miRNA-146 was found to be a regulator of Toll-like receptor 4 (TLR4) through a negative feedback loop mechanism . MiRNA-1Escherichia coli and Listeria monocytogenes. Hence, transgenerational immune modifications could be beneficial to survive dangerous pathological conditions and may limit innate immune responsiveness during bacterial colonialization in early fetal life.Interestingly, trained immunity and effective protection against infection can be inherited from parents . A recenIn conclusion, an altered glucose metabolism and its related immune modifications, observed in neonates, are the result of environmental factors  and epigenetic changes that may partly be inherited from parents.Emerging evidence show significant differences between neonatal and adult phagocytic cells regarding immunometabolism. One recurring difference is that neonatal monocytes and macrophages exhibit lower rates of aerobic glycolysis due to epigenetic alternations connected to a life stage compatible microenvironment. The myth that the neonatal immune and metabolic system is an underdeveloped version of the adult system does not match the published findings. Currently, it is hypothesized that the observed dissimilarities, between newborns and adults, are attributed to the fact that both groups have different health benefiting priorities considering their developmental state. The adult immune system is primed to remove the invading pathogens at the cost of tissue injury (disease resistance). The fetal immune system is more tolerant towards invading microbes (disease tolerance) as allowance is needed for commensal bacteria to colonize the gut. Of note, when microbial colonialization starts is not precisely known. Novel findings indicate that prior to birth, already certain bacterial and fungal strains are present in the womb determining immune cell fate and functions of the neonate ,80,81. HThe critical balance, which seems to exist between disease resistance and disease tolerance, shifts between different life stages. In addition, disturbance of this balance may lead to fatal consequences, such as uncontrolled microbial infections. In a healthy state, the neonate is better off in a disease-tolerant state as this allows for antigen sensitization and colonialization by harmless pathogens in the first days and weeks after birth. However, under inflammatory conditions, such as sepsis, a more disease-resistant state is desirable. This potentially explains a higher incidence of infection in preterm and term newborns. Interestingly, septic neonates can bear a much higher number of bacteria in blood compared to septic adults ,83,84. OOf interest, adult patients with severe sepsis that result in immunoparalysis show a phenotype with similarities to the neonatal immune metabolic phenotype. During immunoparalysis, leukocytes show broad metabolic defects including lower rates of aerobic glycolysis . NotablyBefore applying immunomodulatory interventions in newborns suffering from sepsis as described above, a deeper understanding of neonatal immunometabolism at cellular and molecular level is needed. One major limiting factor here is the amount of suitable research material. Most research is performed using cord blood as this is commonly the major reliant source of immune cells from newborns. The umbilical cord is a waste product and the use of this source for immune cells does not harm the baby or the mother. Herewith, it is important to keep in mind that knowledge obtained by using cord blood cannot be translated one-to-one into postnatal immunity as immunological alterations, driven by microbial interactions, occur rapidly after birth . To overMycobacterium tuberculosis [In addition, knowledge obtained using cord blood cannot always be translated to immune cells residing at different immune sites. A tissue/organ-specific immune pathway is, for example, observed in the peritoneal cavity of neonatal mice. In mouse peritoneal neonatal macrophages, an altered cytokine and chemokine profile was observed whereby the pro-inflammatory factors MIP-1α, MCP-1 and IL-6 were upregulated compared to peritoneal macrophages from adult mice . The uprrculosis . Hereby,rculosis ,94. One rculosis .Although time is needed to develop immunometabolic modulatory interventions to treat neonatal sepsis, knowledge on immune-metabolic pathways are starting to be used as a diagnostic tool to detect and predict severity of infections . In relaA greater understanding is required regarding the link between metabolism and immunity in neonates. Not only our knowledge regarding the link between carbohydrate metabolism and immunity needs to be expanded, but also other metabolic pathways, such as lipid metabolism , nucleotide metabolism and amino acid metabolism, are of particular interest and sparsely studied in neonatal immunity. This knowledge may be used to fine-tune the balance between disease resistance and tolerance benefiting neonates suffering from infections and other immune-related pathologies.


Similarly, no significant differences could be observed with regard to major morbidity  and mortality rates  within the first 90 days following surgery. Propensity-score matching analyses revealed, however, a higher occurrence of post-pancreatectomy hemorrhage after pancreatogastrostomy (p = 0.015). Conclusion: Similar CR-POPF rates suggest no crucial role of the applied reconstruction technique. Increased incidence of intraluminal post-pancreatectomy hemorrhages following pancreatogastrostomy demands awareness for meticulous hemostasis.Background: Pancreatoduodenectomy is still hampered by significant morbidity. So far, there is no universally accepted technique aimed at minimizing postoperative complications. Herein, we compare three different reconstruction techniques. Methods: This is a retrospective study of a prospectively maintained database including 283 patients operated between January 2010 and December 2020. Three reconstruction techniques were compared: (1) the Neuhaus-style telescope pancreatojejunostomy, (2) the pancreatogastrostomy, and (3) the modified Blumgart-style, duct-to-mucosa pancreatojejunostomy. The primary endpoint consisted in determining the rates of clinically relevant postoperative pancreatic fistulas (CR-POPF); the secondary endpoints included 90 days morbidity and mortality rates. A propensity score matching analysis was used. Results: Rates of CR-POPF did not differ significantly between the groups , neither in the unmatched nor in the matched analysis ( Pancreatoduodenectomy (PD) is still hampered by significant morbidity varying between 20% and 50% ,2. The mAimed at minimizing the occurrence of CR-POPF, numerous techniques to restore the continuity between the pancreatic stump and the gastro-enteric tract following PD have been proposed. However, so far, no universally accepted reconstruction procedure has shown a clear superiority. Recent prospective randomized controlled trials (RCTs) as well as retrospective analyses comparing different reconstruction techniques resulted in discordant conclusions and lacked in identifying a technique able to clearly decrease the occurrence of CR-POPF ,15,16,17Not only the use of different anastomosis techniques but also the application of several mitigation strategies has been suggested in the past years to reduce the rates of CR-POPF. While the efficacy of sealants or of an omental roll-up in preventing postoperative morbidity is still a matter of debate ,19,20, tOver the last ten years, three different reconstruction techniques following PD have been performed at our department. Rather than deciding on the basis of specific organ features such as the consistency of the pancreatic parenchyma or the size of the main pancreatic duct, the different techniques represent three different historical phases of the department. Two dunking or telescope techniques and one duct-to-mucosa technique were used. First, the one-layered telescope pancreatojejunostomy using inverted mattress sutures according to Neuhaus  was perfThis study aimed to review postoperative outcomes focusing on the three different reconstruction techniques performed at our institution with regard to the rate of CR-POPF as well as the overall complication rate. To obviate shortcomings of the retrospective character, a propensity score matching model, based on an inverse probability of treatment weights (IPTWs)  was applA retrospective analysis of prospectively collected and auditable medical records from all patients who underwent a PD between January 2010 and December 2020 at the Department of Visceral, Transplant and Thoracic Surgery, Innsbruck, Austria, was performed. Reporting is consistent with the STROBE guidelines  for obseData collected included baseline demographics and clinical characteristics, fistula risk score (FRS) , intraopPatients were divided into three groups according to the reconstruction technique used: (1) one-layered telescope pancreatojejunostomy using inverted mattress sutures according to Neuhaus tsPJN,  26], , post-pancreatectomy hemorrhage (PPH), and biliary fistula, as well as the rates of major complications (Clavien–Dindo ≥ 3a) and mortality within the first 90 days after surgery .All PDs were performed by experienced surgeons. Excluding the years 2011, 2014, and 2015, more than 20 PDs per year were regularly performed at our institution with up to three surgeons operating the yearly occurring caseload. They were mostly performed according to the Traverso–Longmire procedure , and onlThe tsPJN was performed as originally described . In brieConcerning the pancreatic drain, the external end is guided through the intestinal wall 20 cm distally from the pancreatic anastomosis using a small metal trocar. Following fixation using the Witzel tunnel technique, the external end is finally guided through the abdominal wall with the metal trocar before abdominal closure.The PG was performed as previously published . FollowiThe dtmPJB was slightly modified compared to the original publication . BrieflyThe last step of the procedure consists in passing the U-sutures through the seromuscular layer of the jejunum before tying them. Hereby, the cut pancreatic surface is entirely covered by the jejunal loop . In the Mitigation strategies such as perioperative somatostatin or positioning of an omental roll-up around the pancreatic anastomosis were performed depending on the surgeons’ preferences. In all patients, prophylactic antibiotics and low molecular weight heparin were administered. The amylase and lipase levels in drain fluid were measured daily. Levels of these enzymes were considered pathological if more than threefold higher than the serum levels on or after postoperative day 3 . In the The externally draining pancreatic stents (in tsPJN and dtmPJB) were removed 6 weeks following PD.p-values less than 0.05 were considered statistically significant. Statistical analysis was performed using R 2016 statistical software  . ContinuTo overcome biases due to observed confounders among preoperative variables, we performed a matched propensity score (PS) analysis to assess treatment effects. A logistic regression model including all the covariates from Following recommendations in the current literature ,41, weigCase matching with IPTW allows the inclusion of all study subjects in the matched analysis and is applied to estimate the Average Treatment Effect (ATE), hence, overcoming the bias of a small observational study with many confounders.Balance measurements were conducted for each group against the other two groups. The overall assessment of the discrimination was evaluated using the z-difference, with larger values indicating a better discrimination . PropensThis study included 282 patients undergoing PD. In total, 116 patients underwent a tsPJN, 75 a PG, and 91 a dtmPJB. One patient was excluded from the general statistical analysis because of an incomplete data set. Regarding the demographic data, the unmatched analysis revealed some statistically significant differences between the three groups such as pre-existing diabetes, alcohol consumption, neoadjuvant chemotherapy, ASA classification, and preoperative biliary drainage (ERCP and/or PTCD) as well as endoscopic stenting. However, following the PS analysis with IPTW estimation, there were no more statistically significant differences between the analyzed groups. Similarly, mitigation strategies did not differ significantly between the three reconstruction techniques. With regard to the different items in the FRS, only the intraoperative blood loss showed a statistical significance with a higher number of patients losing more than 400 mL of blood in the PG group. The low number of patients with portal/mesenteric reconstruction and those where sealants were applied did not allow any further analysis .Chronic infections including any stable bacterial/fungal colonization of the skin (decubitus) or the mucosa (candidiasis) and jaundice and cholangitis were excluded. Previous malignancies included any liquid or solid tumor in the past patient history (before pancreatic surgery). Low-risk pathologies according to the FRS were pancreatic ductal adenocarcinoma and chronic pancreatitis. High-risk pathologies according to the FRS were ampullary cancer, duodenal carcinoma, cholangiocarcinoma of the distal bile duct, neuroendocrine neoplasms, any cystic lesions, benign entities, and distant metastases to the pancreas (specific diagnosis are listed in the p = 0.993). The PS confirmed the missing significant differences in CR-POPF rates between the three groups (p = 0.901). The postoperative complication rates are indicated in p = 0.607; matched p = 0.253) or DGE . Of note, PG showed numerically higher DGE rates .Similarly, no significant differences could be detected for the occurrence of biliary fistulas (unmatched p = 0.556), PS matching revealed, that PG was associated with significantly higher PPH of any grade compared to both PJs (p = 0.015). The difference was still there when comparing each single PPH grade between the three different techniques; however, the higher occurrence in the PG group did not reach statistical significance (p = 0.064).While there was no statistical significance in the unweighted analysis . Equally, both analyses showed no difference concerning 90 days mortality between the three different techniques , even if restricted to cases with POPF type C .Neither of the three techniques revealed a significant reduction in severe complications (Clavien–Dindo ≥ 3a) both in the unmatched and matched analyses (p < 0.001).The median follow-up time for our patients was 30 months (range 0–134) and differed significantly between the three study groups  or an externally draining (tsPJN and dtmPJB) pancreatic stent. This resulted in similar rates of CR-POPF for all techniques, supporting data showing no difference between the two stenting strategies .Even regarding the occurrence of biliary fistulas, there was not any significant difference in the three analyzed patient groups. This is in line with other studies that did not find any relation between the pancreatic anastomosis technique and early biliary complications more in general  or betweReported rates of DGE following PD range from 5 to 59% . Even thAnother particularity observed in the PS analysis was the higher occurrence of any grade of PPH following PG reconstruction. Of note, despite the comparable rates of PV/SMV resection and therapeutic anticoagulation in all groups, the intraoperative bleeding rates of the PG group were significantly higher. These results are concordant with the RECOPANC study  and alsoConcerning major surgical morbidity rates, a recent PS matching analysis comparing three different PJs showed that the Blumgart-anastomosis could reduce them . SimilarOn the contrary, the RECOPANC study showed no significant correlation between any type of pancreatic anastomosis and surgical complications other than PPH . SimilarSeveral studies highlight the importance of center volume when discussing mortality and major morbidities as well as the failure to rescue rates following pancreatic resections. A nationwide comparison between German and Dutch audits highlights a significant center volume–outcome relationship concerning in-hospital mortality for very low- and low-volume centers (defined as ≤20 procedures per year) . Similarp = 0.239). Still, the low number of patients presenting with postoperative diabetes precludes any apodictic conclusions.There are several limitations of this study, including incomplete documentation of the pancreatic texture as well as the relatively small number of patients per year over a relatively long “recruiting” time. The statistically significant difference in follow-up times among the different study groups reflects the different time periods when different anastomosis techniques were performed. Regarding postoperative records within the first 90 days, we could only define the presence of postoperative diabetes (type IIIc) according to the need for anti-diabetic drugs and by observing the mean fasting glycemia of the whole cohort. Unfortunately, records concerning HbA1c could only be retrieved for 50 patients. This sub-cohort of patients with no diabetes at the time of diagnosis and developing type IIIc diabetes after PD showed no differences in relation to the anastomosis technique  using techniques using an IPTW-based, PS-matched analysis. The presented data suggest that the applied reconstruction technique following pancreatoduodenectomy does not necessarily represent a crucial factor with regard to CR-POPF occurrence. These results are in line with those of a recent RCT and thus confirm the validity and reliability of PS-matched analysis on retrospective databases. The increased incidence of intraluminal postoperative bleedings following PG in these patients demands special awareness concerning intraoperative hemostasis but also during the early postoperative period.


Background and study aimsIn patients with familial adenomatouspolyposis (FAP), endoscopic resection of duodenal adenomas is commonly performed to preventcancer and prevent or defer duodenal surgery. However, based on studies using differentresection techniques, adverse events (AEs) of polypectomy in the duodenum can be significant.We hypothesized that cold snare polypectomy (CSP) is a safe technique for duodenal adenomas inFAP and evaluated its outcomes in our centers.Patients and methodsWe performed a prospective internationalcohort study including FAP patients who underwent CSP for one or more superficialnon-ampullary duodenal adenomas of any size between 2020 and 2022. At that time, thistechnique was common practice in our centers for superficial duodenal adenomas. The primaryoutcome was the occurrence of intraprocedural and post-procedural AEs.ResultsIn total, 133 CSPs were performed in 39 patients with FAP(1–18 per session). Median adenoma size was 10 mm (interquartile range 8–15 mm), ranging from5 to 40 mm; 27 adenomas were ≥20 mm (20%). Of the 133 polypectomies, 109 (82%) were performedafter submucosal injection. Sixty-one adenomas (46%) were resected en bloc and 72 (54%)piecemeal. Macroscopic radical resection was achieved for 129 polypectomies (97%). Deep muralinjury type II occurred in three polyps (2%) with no delayed perforation after prophylacticclipping. There were no clinically significant bleeds, perforations or other post-proceduralAEs. Histopathology showed low-grade dysplasia in all 133 adenomas.ConclusionsCSP for (multiple) superficial non-ampullary duodenaladenomas in FAP seems feasible and safe. Long-term prospective research is needed to evaluatewhether protocolized duodenal polypectomies prevent cancer and surgery. Familial adenomatous polyposis (FAP) is an inherited disorder resulting in the developmentof numerous colorectal adenomas, requiring colectomy at a young age to prevent colorectalcancerConventionally, extensive duodenal polyposis is considered a criterion for prophylacticduodenectomy to prevent malignant transformation; however, treatment burden for duodenectomyis high with significant morbidity (Clavien-Dindo III/IV 16%-53%) and mortality (0%-2%) risksSeveral studies shown that endoscopic treatment of duodenal adenomas resulted indownstaging of duodenal polyposis graded by Spigelman stageIn the large bowel, current guidelines advice cold snare polypectomy (CSP) fornon-pedunculated colorectal polyps < 10 mm and accumulating evidence suggests that CSPmight also be effective and safe in 10- to 19-mm and even larger colorectal polypsCompared to the colon, the complication risk of polypectomy in the duodenum is higher,presumably due to the thinner wall and increased vascularity. In two retrospective studies inFAP that included different resection techniques, the risk of perforation after endoscopicpolypectomy in the duodenum was 2% to 3% and the risk of delayed bleeding 13% to 20%We aimed to evaluate the safety of CSP for superficial non-ampullary duodenal adenomas ina consecutive series of patients with FAP in our expert centers.APCgene and/or having more than 100colorectal adenomas and a family history of FAP.From 2020 on, centers that participate in the European FAP Consortium have collectedfindings of endoscopic surveillance of patients with FAP in a prospectively maintaineddatabase. One of the aims of this prospective database is to study the safety and efficacyof endoscopic resection techniques for duodenal adenomas. For this study, we evaluated allCSPs for non-ampullary duodenal adenomas performed between 2020 and 2022 during one or moresessions in patients with FAP. A diagnosis of FAP was defined as having a constitutionalpathogenic variant of theThe study was approved by the institutional review boards of all participatinghospitals. All included patients gave informed consent prior to data collection. The studywas registered at ClinicalTrials.gov (NCT04677998).An en bloc CSP followed by a piecemeal CSP in the same session in a patient withextensive duodenal polyposis.Video 12insufflation was used.In the centers of the European FAP Consortium, the aim of duodenal polypectomy, apartfrom cancer prevention, is to prevent endoscopically unmanageable disease in the future,thereby obviating the need for duodenal surgery. The indications for performing polypectomyof non-ampullary duodenal adenomas are: adenomas ≥10 mm and adenomas ≥5 mm when in totalmore than 20 duodenal adenomas are present. All superficial adenomas are resected using CSP,while more protruding lesions are resected using HSP. Duodenal polypectomies were performedunder sedation (propofol or midazolam plus fentanyl) by endoscopists with expertise in FAPon dedicated endoscopy lists in tertiary referral centers. Patients on vitamin K antagonistsor direct-acting anticoagulants temporarily discontinued these drugs. Single-agentantiplatelet use was continued or discontinued depending on local protocols. Gastroscopes,duodenoscopes or pediatric colonoscopes were used for the included procedures.COThe procedure included assessment of the duodenum and stomach after which the adenomaswith an indication for polypectomy were removed during one or multiple procedures, dependingon the number and complexity of the lesions. The location, morphology, and size of theadenoma was assessed by the endoscopist prior to polypectomy, with or without narrow bandimaging at the discretion of the endoscopists. Whether the submucosa was injected to liftthe lesion, with or without adrenaline, was also left to the discretion of the endoscopist.Submucosal lifting and CSP was referred to as cold endoscopic mucosal resection (EMR) insome previous studiesPatients were evaluated 2 to 4 weeks after the procedure at the outpatient clinic or viaa telephone consultation, to ensure that possible AEs were identified, discussed, andevaluated. Follow-up endoscopy was scheduled after 3 to 6 months when macroscopic resectionwas incomplete, high-grade dysplasia was present in the resected adenoma and/or when therewere other lesions in the duodenum and/or stomach that required intervention. Otherwise,follow-up endoscopy was scheduled after 1 year. During follow-up endoscopy, the polypectomyscar(s) were assessed to detect recurrences.The primary outcome was the occurrence of procedure-related AEs occurring within 30 daysafter CSP. AEs were evaluated using the validated Adverse Events in GI EndoscopyclassificationDescriptive statistics were used for this study. Continuous variables are presented asmeans with standard deviation for normally-distributed variables and as medians withinterquartile ranges (IQRs) for skewed-distributed variables. Categorical variables arepresented as numbers and percentages. All analyses were performed using SPSS 26 .Thirty-nine consecutive FAP patients from three centers underwent CSP for a total of 133superficial non-ampullary duodenal adenomas. During the same time period, 25 duodenaladenomas were resected with HSP. One patient underwent combined CSP and HSP of a largeduodenal adenoma which, on histopathology, appeared to be an adenocarcinoma.Patient characteristics are presented inIn terms of intraprocedural AEs, no bleeding or perforation occurred. Three CSPs (2%) inthree different patients resulted in full exposure of the muscularis propria, classified asdeep mural injury type II (n=3). All three cases of muscularis propria exposure occurredafter submucosal lifting and piecemeal resection for adenomas measuring 25, 20, and 45 mm,with the use of a dedicated cold snare. In two lesions there was a suspicion of submucosalscarring, which might have been caused by previous biopsies. All three resection sites wereprophylactically closed with hemoclips to prevent delayed perforation, which did not occur. All thAfter eight of 42 polypectomy sessions (19%), the patients were admitted for one or twonights due to either the resection of large adenomas (n=3) or a high number of resectedadenomas (n=5). These admissions were planned and, therefore, not counted as AEs. Also, inthe three cases of deep mural injury, the admission was had been planned prior to theprocedure because of the number or size of the lesion(s). No delayed complications wereobserved either during the admission period nor the 4-week follow-up. Histology showed atubular adenoma in 90% of lesions and tubulovillous adenoma in 10%. All lesions containedlow-grade dysplasia; no high-grade dysplasia or cancer was detected.In this multicenter study, we investigated the safety of CSP for the removal ofsuperficial non-ampullary duodenal adenomas in FAP. We demonstrated that, in experiencedhands, CSP is a safe technique, regardless of the size or number of adenomas resected duringthe same procedure.Our series confirms the safety of CSP for duodenal adenomas because no bleeding orperforation was observed.Two studies retrospectively compared CSP to HSP, showing that AEs occurred less frequentlyafter CSPIn terms of efficacy, evaluating adenoma recurrences after duodenal polypectomy might beof importance. In this study, we did not report on recurrences after CSP. FAP patientsundergoing duodenal polypectomy usually have numerous adenomas and sometimes multiple adenomasare removed during the same procedure. We believe that this clinical setting does not lenditself to studying adenoma recurrence, because this is clinically less relevant, given thatpatients usually have multiple duodenal adenomas and should undergo regular surveillanceendoscopies anyway. This is in contrast to the sporadic setting, in which usually one polyp isremoved in an otherwise normal duodenum and surveillance might be terminated after completeremoval. Two studies in the sporadic setting comparing CSP to HSP did not find a difference inrecurrence rateWe demonstrated the safety of CSP in FAP patients, but not its efficacy in preventingcancer and surgery. Long-term prospective data from a large FAP cohort are needed to evaluatewhether protocolized removal of duodenal adenomas is an effective strategy and, morespecifically, which adenomas should be removed. As a European consortium, we hope to providesuch data in future years.


Anastrepha fraterculus and A. obliqua, which show evidence of gene flow despite clear evolutionary divergence. We find that their transcriptional patterns are a mosaic between those typically observed within and between allopatric species. Transcripts showing transgressive expression in hybrids or cis-regulatory divergence between species are associated with greater sequence divergence. This may reflect pleiotropic constraints that make them resistant to gene flow or they may be more likely to experience divergent selection. Although these more divergent gene classes are likely to be important contributors to species differences, they are relatively rare. Instead, most differentially regulated transcripts, including those linked to reproduction, show high degrees of dominance in hybrids and trans-regulated divergence between species, suggesting widespread genetic compatibility that potentially allowed for introgression. These findings provide insights into how postzygotic isolating mechanisms might evolve in the presence of gene flow: regions showing cis-regulatory divergence or transgressive expression contribute to reproductive isolation, whereas regions with dominant expression and trans-regulatory divergence allow for introgression. These patterns create a genomic mosaic of transcriptional regulation that is tied to sequence divergence.The extent to which hybridization disrupts a gene's pattern of expression likely governs its propensity for introgression, whereas its extent of molecular divergence can itself underlie such disruption. Together, these phenomena shape the landscape of sequence and transcriptional divergence across the genome as species diverge. To understand this process, we characterize gene expression inheritance, regulatory divergence, and molecular divergence in the reproductive transcriptomes of species linked by gene flow: the fruit flies  Divergence in gene expression regulation between species can impose a barrier to hybridization. Nonetheless, many species pairs experience substantial gene flow, often appearing as a mosaic of highly and lowly divergent genomic regions. To understand gene expression regulation and molecular divergence in such systems, we characterize transcription and its link to sequence divergence in hybrids of species connected by gene flow. We find that transcriptional patterns are a mixture of those typically observed within and between allopatric species. Although most transcripts exhibit regulatory patterns unlikely to be disruptive in hybrids, which presumably allows for introgression, those that exhibit disruptive patterns show greater molecular divergence. These findings provide important insights into how transcriptional regulation may shape gene flow.Although species were once considered to be well-defined evolutionarily independent lineages, there is increasing evidence that there is a continuum of reproductive isolation and, hence, genetic exchange . As a recis-regulatory differences  between genomes with low divergence, such as intraspecific crosses  and regulatory divergence . We then relate these patterns to the degree of sequence divergence and gene flow to gain insights into how postzygotic isolating mechanisms might evolve in the presence of gene flow, while buffering against complete reproductive breakdown to facilitate introgression.To understand the relationship between regulation and inheritance of gene expression in hybrids and patterns of sequence divergence between species, we use a pair of neotropical fruit fly species, ene flow . These s America . Despite America . Detecte America . Indeed,se males , are notse males . Becausese males . We compA. fraterculus and A. obliqua and sampled male and female reproductive tissues from the two parental species and their hybrids. By sequencing transcriptomes from these samples, we characterized gene expression inheritance and regulatory divergence, and the degree of molecular divergence at these transcripts. We refer to the reciprocal hybrids as OF and FO . Because the ♀ A. fraterculus × ♂ A. obliqua cross only produces female progeny  between the species. All reads were then mapped back to this reference using SNP-tolerant alignment as implemented in genomic short-read nucleotide alignment program (GSNAP) , which relies on the identification of the parental origin of reads sequenced in hybrid samples. To account for potential species-mapping bias to the reference, we generated a “common diploid  (GSNAP)  to ensurA. fraterculus and A. obliqua derived references) in the common diploid Anastrepha genome. The same number of reads was simulated for each polymorphic site and then mapped back to the reference; transcripts in all samples followed an expression ratio of 1  of 0.05. We found that the power to detect differential gene expression is maximized and approximates the canonical 80% when using a threshold of log2FC > 1.25, which is a standard threshold used in other ASE studies , indicating that higher coverage does not lead to biased result of gene expression.2FC between species following an FDR correction of 0.05  exhibited ED between species, whereas 1,177 ED transcripts (11%) were detected in males  (based on log2FC) following additive, dominant , transgressive (overdominant and underdominant), or as conserved when no significant differential expression was detected . The number of detected transcripts was greater for females than males across categories (fig. 2c). The majority of transcripts show dominance (more often of A. fraterculus over A. obliqua), followed by the transgressive categories, with overdominance being significantly more common than underdominance (fig. 2c). The direction of the cross  did not significantly affect the number of transcripts detected under these categories.We used a generalized linear model (GLM) analysis to investigate differences in the distribution of the remaining transcripts across categories between male and female reproductive tissues  online. fig. 2cis and trans-regulatory effects for each transcript based on significant log2FC estimates of differential gene expression (b), following cis-only, trans-only, cis + trans, cis × trans, and compensatory . The remaining transcripts were classified under five regulatory categories, and their distribution across profiles was compared using a GLM analysis , with the majority of transcripts showing trans-only effects and interactions between cis and trans (cis × trans and compensatory), whereas only a few transcripts showed evidence for cis-only or cis + trans-regulatory effects (fig. 2d). The interaction effect indicates that the total number of transcripts or their distribution across categories is different between males and females (fig. 2d). The number of trans-only transcripts was greater in female than in male reproductive tissues. On the other hand, the number of transcripts with evidence for cis and trans interactions (cis × trans and compensatory) was higher for male transcriptomes (fig. 2d). The direction of the cross  did not significantly affect the number of transcripts detected under these categories . Because this could bias estimates of regulatory divergence in males, we investigated the potential for X linkage to influence our results by using ASE patterns in hybrid males to identify transcripts showing expression consistent with X linkage. For this, we identified transcripts with significant allelic imbalance due to the complete absence of reads from the A. fraterculus allele in hybrid males, reflecting the fact that X-linked transcripts in males are inherited from A. obliqua . We found 49 transcripts consistent with this pattern, distributed in four categories of regulatory divergence: cis-only (3 transcripts), cis*trans (10 transcripts), compensatory (21 transcripts), and ambiguous (15 transcripts). Although we cannot determine what proportion of these 49 transcripts are, in fact, on the X chromosome, the fact that this small proportion  represents the upper limit to their potential total contribution indicates that they are unlikely to introduce meaningful biases in our analysis. Because these transcripts cannot be unambiguously assigned to the X chromosome and represent such a tiny percentage of transcripts with regulatory divergence, they have not been removed from our analyses. However, their hemizygous expression could potentially introduce bias if it were to lead to a large proportion of these transcripts being incorrectly classified as dominant-obliq . For example, such a bias could potentially explain the higher proportional representation of the dominant-obliq category within the set of transcripts that show nonconserved expression inheritance in males compared with females (c). However, we find that only 5 of the 189 (2.6%) transcripts classified as dominant-obliq show an expression profile consistent with X linkage in males, which is the same number that shows dominant-frat inheritance, indicating that no such bias exists in our analysis. Thus, it appears that the presence of unidentified X-linked transcripts is unlikely to be an important source of bias in our findings.In the absence of a reference genome for  females c. Howevea and fig. 3b and additive transcripts, followed by dominant and, finally transgressive transcripts (over and underdominant) (fig. 3a). These results may, in part, simply reflect the classification process for gene expression inheritance because the hybrid expression is more likely to fall somewhere in between the expression values of the parental species when the two are very different. Similarly, in cases where the difference between the parental species is small, any expression difference in hybrids is more likely to place their expression outside the range of the parental species. On the other hand, ED was substantially higher for transcripts exhibiting cis + trans regulatory divergence, whereas the lowest ED was detected for transcripts in the compensatory category and no substantial differences across the remaining patterns (fig. 3b). The direction of the cross  did not significantly affect the level of ED detected under these categories  across inheritance modes of expression . The cis index tended to be higher for transgressive transcripts (over and underdominant), followed by dominant transcripts (often higher in dominant-obliq than dominant-frat), and finally additive transcripts (c). The direction of the cross  did not significantly affect the cis index detected across categories of expression inheritance (We compared the pression  online. ractions c. The cinscripts c. The dieritance  online.a). The molecular divergence estimates for these three indices differed between male and female reproductive transcriptomes, with males exhibiting significantly higher values when compared with those of females. Similarly, molecular divergence estimates were consistently higher for transcripts with significant AI, but no significant differences between males and females were found (b).We estimated molecular divergence based on the rate of synonymous (Ks) and nonsynonymous substitutions (Ka) and their ratio (Ka/Ks) for the entire dataset in male and female reproductive transcriptomes. Then, we compared molecular divergence in transcripts showing significant ED between species and allelic imbalance in hybrids (AI), with nonsignificant transcripts . The level of molecular divergence obtained from Ka, Ks, and Ka/Ks estimates followed similar patterns, being significantly higher for transcripts with significant ED between species when compared with no-ED transcripts a. The more found b.conserved transcripts, and additive transcripts show the highest levels of molecular divergence (a). Likewise, dominant and transgressive transcripts showed significantly higher Ks divergence estimates, whereas Ks estimates from additive transcripts are not significantly different from conserved transcripts (a). All categories have higher Ka/Ks ratios when compared with conserved transcripts, although male additive transcripts and female transgressive transcripts values are not significant, perhaps due to a large variance. Interestingly, transgressive transcripts show the highest molecular divergence for Ks values between males and females (a).When comparing molecular divergence across categories of expression inheritance, we found that Ka and Ks estimates followed slightly different patterns. All categories of inheritance show significantly higher Ka divergence when compared with vergence a. Likewinscripts a. All ca females a.b). In this case, all regulatory divergence categories show significantly higher molecular divergence when compared with conserved transcripts. The only exception is that female cis-only transcripts are not significantly different from conserved transcripts, whereas male cis-only transcripts exhibit the highest molecular divergence (b), even higher than that of transgressive transcripts (b). These results suggest that transcriptional differences in expression and regulation between Anastrepha species correlate with patterns of molecular divergence.Estimates of molecular divergence are also different for categories of regulatory divergence, showing consistent patterns across Ka, Ks, and Ka/Ks ratios b. In thivergence b, even hnscripts b. These A. fraterculus and A. obliqua, we performed gene ontology (GO) enrichment analysis (trans-only divergence or dominant-frat expression in hybrids between these Anastrepha species (a). Although the rest of the categories did not show significant gene enrichments in female transcriptomes, these two sets of transcripts were strongly associated with important functions previously linked to the female reproductive tract of numerous insect species (b). These transcriptional changes are enriched for the following three groups of functional pathways (b): 1) epithelial modifications, 2) protease/protease inhibitors, and 3) immune/defense response. Similarly, male transcriptomes were significantly enriched for at least three functional pathways that have been previously associated with male reproductive tissues.To analyze the functional pathways associated with gene expression inheritance and regulatory divergence between analysis . We foun species a. Althou species b. These pathways b: 1) epiA. fraterculus and A. obliqua), we have been able to characterize regulatory divergence and expression inheritance and link these to sequence divergence. Expression inheritance characterizes the relationship between the pattern of gene expression in hybrids and that of the parental species, whereas regulatory divergence uses the patterns of ASE in hybrids to characterize the expression regulation of the gene copies coming from the two parental species. We find that transcriptional patterns in hybrids show a mosaic between those typically observed within and between allopatric species. A large proportion of the differentially expressed transcripts between the species show an interaction between cis and trans regulatory divergence, which is frequently seen in interspecific hybrids , followed by transgressive (over- or underdominant) and additive modes of inheritance (2–6%). Transgressive transcripts were not as common as described in other interspecific hybrids (transgressive transcripts (3–13%) is over three times lower than that observed in interspecific Drosophila hybrids (35–69%)  is consistent with the observation that lab generated hybrids are viable, fertile, and show no visible abnormalities aside from the inviability of males in one of the interspecific crosses, which follows Haldane's rule   and even(35–69%) . In cont changes . Transgrpression , and hene's rule . Those tDrosophila . The mechanisms underlying this pattern, or how widespread it is across species, are still unclear. The most accepted hypothesis suggests that divergence at cis elements evolves more slowly because it results from accumulated changes at individual genes that are independent of the background genome . In contrast, a single episode of mutation and evolution at a trans regulator  can potentially cascade across multiple target genes . Therefore, the overall contribution of trans regulation to species divergence likely depends on the nature and extent of pleiotropic interactions between trans regulators and their target genes.Although the number of studies evaluating ASE in hybrids between species is limited, comparisons across osophila  and planosophila  have shovergence . This sud genome   account for 67% and 34% of transcripts for males and females, respectively. These transcripts show opposing effects on gene expression, with a large proportion of transcripts showing compensatory regulation. This pattern, along with the high proportion of transcripts with conserved expression (over 83%), strongly indicates the presence of evolutionary constraints on gene expression  relative to females in the same cross . It is important to recognize, however, that this scenario does not represent a bias in the estimation of gene expression inheritance in males in this cross. Rather, the imbalance of X-linked regulatory factors in the heterogametic hybrid simply reflects the biological basis of differential gene expression regulation in males and females, which is expected in any system with XY sex determination. The presence of Haldane's rule in only one direction of the interspecific cross, however, does impose a biological constraint on the experimental design that means that the estimated patterns of expression inheritance and transcriptional regulation in males cannot be compared with the opposite direction of the cross. Therefore, the causes and consequences of gene expression inheritance and regulatory divergence in males should be interpreted with the potential influence of X-linked trans-regulatory factors in mind.Because the cross between transgressive expression and those showing cis-only regulatory divergence exhibit the greatest molecular divergence. More interestingly, these groups of transcripts also show the highest difference in patterns of molecular evolution between sexes, with males showing higher evolutionary rates than females. These two groups of transcripts represent outliers in the distribution of evolutionary rates across the whole genome. Transgressive female transcripts exhibit the highest level of divergence at synonymous sites, whereas male cis-regulated transcripts had the highest Ka/Ks divergence. Additional genomic features may correlate with sequence divergence, such as gene length and absolute expression values  suggests that synonymous sites are not saturated, and therefore our estimations are likely reflecting the influence of genetic drift and/or introgression , indicating that these transcripts are not experiencing recurrent divergent positive selection. The rates of molecular evolution in cis-regulated female transcripts are not significantly different from those of conserved transcripts . On the other hand, male transcripts exhibited an elevated Ka/Ks in both transgressive and cis-regulated transcripts, with the later showing the highest Ka/Ks estimates across the genome, which indicates a more relevant role of positive or relaxed selection in males . Our estimations of Icis  further support this hypothesis, because transgressive transcripts also had the highest level of cis regulation. This is consistent with previous research and models of hybrid incompatibility , trans-only regulation , and dominant inheritance in hybrids. This is consistent for species that diverged recently and still experience gene flow, because their transcriptional incompatibilities are weak enough to allow hybrids to survive and reproduce in order to account for the presence of introgression between the species  and Southeastern  regions of Brazil, respectively. Field collected flies were identified using wing, ovipositor, and other morphological markers following identification keys available (Mangifera indica L.) fruits were used for oviposition and larval development, whereas adults were fed on a mixture of hydrolyzed protein, vitamins, and sucrose. Populations were maintained by sampling over 100 mating pairs of adults to generate nonoverlapping generations and reduce inbreeding.Crosses were derived from established lab populations of vailable . These l♀ A. fraterculus x ♂ A. fraterculus and ♀ A. obliqua x ♂ A. obliqua), whereas reciprocal F1 hybrids were derived from interspecific crosses . Because the ♀ A. fraterculus × ♂ A. obliqua cross only produces female progeny . Entire male and female reproductive tissues were obtained from 10-day-old mature virgin progeny from each of the crosses. These tissues were chosen because they are likely to express genes that play important roles in species differences as they tend to evolve more rapidly than background genome  was retained for each profile, and this assembly was used as a template to generate the “common diploid Anastrepha reference” used in further analyses. Allim can automate multiple rounds of SNP identification and GSNAP mapping (with or without the SNP-tolerant option). After mapping the reads from both species to the selected reference assembly, divergent SNPs were identified. Polymorphism information was then used in a subsequent round of SNP-tolerant mapping. SNPs were identified using SAMtools  and regulatory divergence . An FDR correction . The read count matrix was filtered for a minimum count cutoff of 3 cpm for each parental species and hybrids over at least two of three replicates per comparable group. All zero values were then adjusted to one to satisfy the binomial tests used below for ge edgeR  after TMrrection  using a Log2(P1/P2) and between hybrids and each parental species: Log2(P1/F1) and Log2(P2/F1) while ignoring allelic information . A negative-binomial GLM analysis and implemented in edgeR software was used to evaluate pairwise comparisons of gene expression between hybrids and their parental species. Based on these comparisons, each transcript was classified according to a commonly used system to describe additive and nonadditive modes of gene expression (a) .Modes of inheritance were investigated by comparing global expression levels of a given gene between the parental species pression a (McManusion (a) . Nondiffcis-acting or trans-acting effects (b). The contribution of regulatory effects on gene expression was investigated by comparing the extent of allele imbalance in hybrids :Gene expression can be regulated by  effects b. The coand the relative expression of these alleles when homozygous in the parental species :trans-effects, which means that any allele imbalance will be the result of a cis-regulatory effect . Consequently, any deviation on the degree of ED between parents and the degree of allele imbalance in hybrids indicates the occurrence of trans-regulatory effects. Hence, the trans effect is given by:In the genetic regulatory background of the hybrid, both parental alleles are exposed to the same cis-effects, based on allelic imbalance in the hybrids. Trans-effects were identified through a binomial GLM comparing the ratios of ASE between hybrids (hR) and parents (pR). Thus, nondifferential ratios are evidence of cis-only regulatory divergence, whereas any significant deviation between ratios is evidence of additional trans-effects favoring the expression of one allele.A negative-binomial GLM analysis was used to evaluate significant conserved if there was no significant differential expression between parents, between alleles in hybrids, and between their ratios. Transcripts show cis-only regulation when there is significant differential expression between parents, and that pattern is retained for the alleles in hybrids, but no significant differences between their ratios. Transcripts show trans-only regulation when there is significant differential expression between parents, but not between alleles in hybrids, with significant differences between their ratios. Transcripts may also show a mixture of these, with significant differential expression between parents, between alleles in hybrids, and significant differences between their ratios. In the case where there is an expression difference between parental and hybrid alleles that goes in direction of the same allele, transcripts are classified as cis + trans, whereas when the expression differences are biased toward different alleles, transcripts are classified as cis x trans. Transcripts are classified as compensatory when there is no significant differential expression between parents, but there are significant differences between alleles in hybrids and significant differences between their ratios. In this case, opposite changes between cis and trans-effects compensate each other, resulting in no expression differences between species. Finally, transcripts are classified as ambiguous when expression patterns did not follow any clear expectations according to these criteria.Based on the extent and direction of expression change, we classified transcripts in seven different patterns. Transcripts were classified as cis-acting versus trans-acting mechanisms for each transcript. For this, we created an index that measures the size of the cis component relative to the total cis and trans effects:To characterize the overall pattern of regulation, we quantified the relative effect of This index was used to investigate the relative role of these mechanisms concerning expression inheritance and molecular and EDs.Anastrepha species, we inferred potential X linkage from the expression data. Transcripts were designated as putatively X-linked if they showed significant AI with the complete absence of read counts from the A. fraterculus allele in hybrid males . Because these transcripts cannot be unambiguously assigned to the X chromosome, the counts generated by this method provide an upper-limit estimate to the number of X-linked transcripts that could potentially be contributing to the patterns observed in each category of transcripts.To evaluate the potential contribution of X-linked transcripts to patterns of regulatory divergence and gene expression inheritance observed in males in the absence of a reference genome for mpileup option, and then allele frequencies were obtained using PoPoolation II software with a minimum quality base of Q = 20 and minimum allele frequency MAF = 2%. We then calculated the interspecific differentiation index (d) as the absolute difference in allele frequencies between species (|frat—obliq|) for each SNP and then estimated the molecular divergence as the percentage of fixed variation per transcript . Then, protein coding sequence (CDS) sequences were predicted using the TransDecoder software (http://transdecoder.github.io), and evolutionary rates were estimated with the KaKsCalculator program , and Ka/Ks estimates between species was generated. GLM analyses were performed using sex, direction of the cross (OF and FO) categories of regulatory divergence, inheritance modes, and tissues as independent variables, whereas both the number of transcripts detected and the ED were considered dependent variables. Then, the response variables were compared across categories of inheritance, reciprocal hybrids (OF and FO). Because postzygotic incompatibilities follow Haldane's rule, hybrid males are not viable when crossing A. obliqua females with A. fraterculus males , GLM analyses were performed after square-root transformation.Because the number of transcripts detected under each category depends on the transcriptome length (number of assembled transcripts), we normalized all transcript counts to the number of assembled contigs per library. Because several variables are calculated as proportions . For this, we used predicted CDS regions from TransDecoder to perform homology searches against known sequence databases  using the programs BLASTX (transcripts) and BLASTP (predicted CDS) (Assembled transcriptomes were annotated using the Trinotate framework (ted CDS) . HMMER sted CDS)  against ted CDS) . Finallyted CDS)  was usedted CDS) . All theted CDS) .evad071_Supplementary_DataClick here for additional data file.


Background. Streptococcus gallolyticus (previously known as Streptococcus bovis type-1) bacteremia has a well-established, almost pathognomonic association with colorectal carcinoma, with the most common hypothesized mechanism being ulceration of polyps leading to hematologic dissemination. There are few reported cases of streptococcus bacteremia from other, seemingly benign sources like cellulitis or colonic adenomas. Hence, there is limited focus on skin and soft tissue infections leading to potentially fatal infective endocarditis. Case Presentation. We present a novel case of streptococcus bacteremia from uncommon sources like abdominal wall cellulitis or colonic adenoma leading to infective endocarditis as well as other manifestations, including osteomyelitis and discitis. This report highlights a unique case of streptococcus bacteremia with an uncommon origin, arising from abdominal wall cellulitis or colonic adenoma, ultimately resulting in the development of infective endocarditis. Furthermore, the patient presented with additional clinical manifestations, including osteomyelitis and discitis. Conclusions. Through our case report, we emphasize the importance of investigating uncommon sources like cellulitis when initial malignant workup is negative in streptococcus bacteremia and further elucidate the pathophysiology of streptococcus bacterial dissemination from nonmalignancy-related sources. Streptococcus gallolyticus, previously known as Streptococcus bovis type 1, is a colonizer of human skin in 2-15% of the human population [Streptococcus gallolyticus bacteremia and colorectal carcinoma has been well established through the adenoma-carcinoma sequence [Streptococcus gallolyticus enters into the bloodstream in patients without polyps. We present a case of infective endocarditis with Streptococcus gallolyticus bacteremia who presented with abdominal wall cellulitis. The data correlating Streptococcus gallolyticus with particularly abdominal wall cellulitis is sparse. Thus, through this case report, we aim to assist clinicians in considering alternative causes of underlying infective endocarditis, such as gastrointestinal diseases, as well as hepatobiliary and pancreatic pathologies, which can help in earlier recognizing and improving patient-centered outcomes.Streptococcus gallolyticus, and the patient was found to have infective endocarditis of the aortic and mitral valves. At that time, he was discharged with a ceftriaxone infusion for six weeks.This is a 53-year-old male with a past medical history of uncontrolled type 2 diabetes mellitus who presented for evaluation of one week of new-onset worsening and persistent exertional shortness of breath associated with bilateral leg edema, orthopnea, and paroxysmal nocturnal dyspnea. Three weeks prior, while investigating for abdominal wall cellulitis, blood cultures were positive for On physical examination, jugular venous distension and hepatojugular reflux were noted; a systolic murmur in the second intercostal space, right parasternal area radiating to the right carotid artery, and a diastolic murmur in the fifth intercostal space of the midaxillary line, bilateral basilar rales, and pitting edema were present. A 2 × 2 cm soft, nontender, immobile mass was appreciated in the right side of the neck. He had a brain natriuretic peptide of 1406 pg/ml (<100) and hemoglobin A1C of 13.6%. The chest X-ray was consistent with pulmonary edema . CT chesDue to the severity of valvular involvement, the patient was transferred emergently to our higher level of care facility for aortic and mitral valve replacement. Subsequently, he underwent a C5/C6 corpectomy, C5-6 and C6-7 bilateral discectomy and foraminotomies, placement of vertebral body cage, and cervical plate spanning of C4-C7 and fusion. The repeat blood cultures remained negative for twenty days before valve replacement surgery. Colonoscopy was unremarkable for colon cancer, demonstrating two benign colon polyps consistent with tubular adenoma. The patient was treated for endocarditis for a total of 6 weeks on ceftriaxone 2 grams daily. Following the patient six months after discharge, he has been stable with no active medical issues while following up with the cardiologist.Streptococcus gallolyticus, formerly known as Streptococcus bovis type 1, is a gram-positive coccus. It is commonly associated with infective endocarditis, particularly in patients with underlying gastrointestinal pathology and colorectal tumors [Streptococcus gallolyticus accesses the bloodstream in patients without polyp [Streptococcus gallolyticus bacteremia has been identified in cases of acute cholangitis and hepatic abscesses [Streptococcus gallolyticus with colonic adenoma [Streptococcus gallolyticus in the colonic adenoma and later mainly found in colorectal cancer demonstrates its possible role in the pathogenesis and oncogenesis in the progression from colonic adenoma to adenocarcinoma [l tumors , 4. It iut polyp . One accbscesses . There i adenoma , 7. The Streptococcus gallolyticus bacteremia originating from abdominal wall cellulitis. In our case, no association was found between the source of Streptococcus gallolyticus and colon cancer. However, we hypothesize that the Streptococcus gallolyticus-causing bacteremia and infective endocarditis in our patient were either related to the abdominal wall cellulitis or the benign colonic adenomas. The most common microbes in cellulitis are identified as Streptococcus pyogenes and Staphylococcus aureus [Streptococcus gallolyticus endocarditis with lower extremities cellulitis and no macroscopic colonic pathology [Streptococcus gallolyticus as a cause of abdominal wall cellulitis is still possible.We presented a case of infective endocarditis secondary to s aureus . Howeverathology . TherefoStreptococcus gallolyticus can manifest in conditions beyond infective endocarditis, including osteomyelitis and discitis [Streptococcus gallolyticus with not only malignant colonic polyps but also benign polyps should be emphasized, warranting careful monitoring during the management of patients presenting with similar presentation.Several studies showed that discitis , 11. Mandiscitis . The sigStreptococcus gallolyticus bacteremia with colorectal carcinoma is well established in the literature. However, there are only a few reported cases in the literature of Streptococcus gallolyticus bacteremia associated with other conditions like abdominal wall cellulitis or benign colonic adenoma resulting in infective endocarditis. This bacterium can also potentially cause life-threatening complications beyond infective endocarditis, such as septic emboli, osteomyelitis, or discitis. This case report highlights an uncommon cause of infective endocarditis secondary to Streptococcus gallolyticus and its serious complications. The aim is to broaden the spectrum of possibilities in diagnosing and managing infections caused by these bacteria.The association of


Survivors of out-of-hospital cardiac arrest (OHCA) experience significant mortality rates and neurological impairment, potentially attributed to the hypoxic-ischemic injury sustained amid the cardiac arrest episode. Post-resuscitation care plays a crucial role in determining outcomes for survivors of OHCA. Supportive therapies have proven to be influential in shaping these outcomes. However, targeting higher blood pressure or oxygen levels during the post-resuscitative phase has not been shown to offer any mortality or neurological benefits. In terms of maintaining hemodynamic instability after resuscitation, it is recommended to use norepinephrine rather than epinephrine. While extracorporeal cardiopulmonary resuscitation has shown promising results, targeted temperature management has been found ineffective in improving outcomes despite its previous potential. This review also investigates various challenges and barriers associated with the practical implementation of these supportive therapies in clinical settings. The review also highlights areas ripe for future research and proposes potential directions to further enhance post-resuscitation supportive care for OHCA survivors. An out-of-hospital cardiac arrest (OHCA) refers to an abrupt stoppage of mechanical heart activity outside a hospital environment, which is confirmed by the lack of signs of circulation . OHCA isNeurons are very susceptible to hypoxic-ischemic insults and are therefore easily damaged due to hypoperfusion thus neuronal injury is the major cause of mortality and morbidity in OHCA survivors , followeEvidence indicates that TTM has the potential to reduce brain damage and improve neurological outcomes. It is recommended especially in comatose OHCA adults who remained unresponsive after the return of spontaneous circulation (ROSC) . CardiacThis review seeks to offer a comprehensive examination of the present condition of supportive care following successful resuscitation for individuals who OHCA. The review focuses on recent breakthroughs and advancements in post-resuscitation care tailored to OHCA survivors, analyzing their impact on survival rates, functional outcomes, and overall quality of life. Additionally, it examines challenges and barriers associated with the practical implementation of these breakthroughs in clinical settings. The review also highlights areas ripe for future research and proposes potential directions to further enhance post-resuscitation supportive care for OHCA survivors, contributing to the refinement of clinical practices and ultimately improving patient outcomes in this critical field.MethodologyVarious databases such as MEDLINE (via Pubmed), Cochrane Library, and Embase were searched to retrieve relevant studies. The literature search used terms related to “cardiac arrest,” “targeted temperature management,” “supportive management,” and “post-resuscitation.” Two authors independently screened the search results and any conflict among them was resolved by the third author.Targeted temperature management (TTM)TTM, or protective hypothermia, can reduce cerebral metabolic rate after resuscitation by lowering the demand for cerebral oxygen . By reduPreviously, many clinical trials have shown that TTM can significantly improve neurological outcomes and overall survival rates in post-resuscitation care in patients who have suffered OHCA ,17. HoweA meta-analysis of seven randomized controlled trials by Aneman et al. aimed to assess the impact of TTM in post-resuscitation care . They foThe latest guideline advocates the optimization of TTM during the post-resuscitation phase to improve outcomes and long-term neurological rehabilitation . FollowiIn conclusion, while clinical guidelines advocate for the use of TTM, its suboptimal performance in recent trials beckons for a profound re-evaluation and prompts the need for large-scale randomized controlled trials to determine its role in cardiac arrest survivors.Hemodynamic optimizationHemodynamic instability often follows successful resuscitation from OHCA, arising due to the complex interplay of various pathophysiological mechanisms such as reperfusion injury, myocardial dysfunction, and systemic inflammatory response syndrome (SIRS) . This caGuidelines advocate for keeping MAP equal to or greater than 65 mm Hg in the post-resuscitation period . The latA recent meta-analysis by Cheema et al. did not find any significant difference regarding mortality between higher versus lower BP target goals  in post-OHCA patients . Both grFluid resuscitation is the initial intervention to restore intravascular volume and improve cardiac output . HemodynVentilation targetsOxygen TargetsHypoxic ischemic encephalopathy (HIE) stands as the predominant cause of impairment and fatality subsequent to OHCA. It is common for patients to experience hypoxic brain damage upon the restoration of spontaneous circulation . More thAlthough oxygen is commonly administered as a treatment for individuals revived from OHCA, there exists limited evidence to provide healthcare practitioners with definitive guidance on the optimal utilization of oxygen for this particular patient group . An exceIn a study by Kuisma et al., individuals who were successfully resuscitated after an OHCA were divided into two groups . These gAccording to a meta-analysis conducted by Young et al., conservative oxygen therapy, as opposed to liberal oxygen therapy, led to a significant decrease in mortality  . HoweverBased on a comprehensive review of multiple studies conducted over time, the current guidelines from ERC-ESICM for post-resuscitation care recommend maintaining pulse oximetry levels within a designated “safe range” of 94%-98%. This approach is advocated to prevent both hypoxia and hyperoxia .2 TargetsCO2 after ROSC, which typically returns to baseline in an hour. PaCO2 is an important regulator of cerebral blood flow. After a cardiac arrest, CO2 derangements can have detrimental effects, necessitating careful CO2 monitoring in comatose patients during the post-resuscitation period after ROSC for improved neurological outcomes [2 should be in the range of 4.5-6.0 kPa or 35-45 mm Hg in patients requiring mechanical ventilation after ROSC [Patients who suffer from cardiac arrest frequently experience high PaCOoutcomes . CurrentModerate hypercapnia improves brain oxygenation, minimizing damage caused by ischemia-reperfusion injury . A prospIn a retrospective cohort study, Okada et al. aimed to understand the relationship between carbon dioxide levels and neurological function after one month in OHCA patients . They fo2 levels through arterial blood gas (ABG) analysis and end-tidal CO2 monitoring [Observational studies have indicated that survivors of OHCA who undergo TTM are susceptible to developing hypocapnia, a condition that can be averted by monitoring COnitoring .Glycemic targetsDerangements in glucose levels following cardiac arrest are common in diabetic and non-diabetic patients . The durThe European Resuscitation Council proposes that blood glucose levels should be kept above 180 mg/dL, and measures should be taken to prevent hypoglycemia . A studyEvidence regarding glucose control in the post-resuscitation period highlights the need for more trials to evaluate the most optimal glucose management strategies after resuscitation in OHCA survivors.Extracorporeal life support (ECLS) in post-resuscitation careECLS is a highly advanced medical procedure designed to offer temporary mechanical support to patients who have experienced cardiac arrest that is refractory to traditional CPR . By tempA meta-analysis by Alfalasi et al. studied the impact of extracorporeal CPR (ECPR) compared to traditional CPR in 18,620 patients with OHCA . PatientSeizure controlSeizures manifest in OHCA patients as a result of the hypoxic-ischemic brain injury sustained during the cardiac arrest episode . SeizureAnticonvulsants' role in OHCA patients has not been extensively researched. Kim et al. evaluated the impact of anticonvulsants on 930 patients who have survived OHCA . They foAdditional research is necessary to gain deeper insights into the impact of anticonvulsant drugs on individuals who have survived OHCA. Additionally, it could help establish guidelines regarding the use of these drugs in clinical practice when caring for OHCA survivors.Role of antibioticsInfections frequently complicate recovery and increase ICU stays for OHCA survivors. More than half of survivors suffer from pneumonia, and around 10% develop sepsis . These pAlthough beneficial, therapeutic hypothermia (TH) can impair the immune response, increasing the risk of infections. TH further challenges the situation by masking pyrexia . A retroA meta-analysis by Couper et al. compared the effects of early antibiotic initiation and delayed or clinically driven antibiotics in survivors . The resPatients who were administered prophylactic antibiotics showed a markedly reduced incidence of pneumonia (12.6% versus 54.9%) and sepsis (1.2% versus 5.7%) in comparison to those who did not . One hunDespite the advances in resuscitative efforts, individuals who survive OHCA are still faced with significant risks of morbidity and mortality. To improve outcomes for these survivors, several supportive therapies have been explored, including TTM, aggressive hemodynamic control, maintenance of specific ventilation targets, control of glucose derangements and seizures, treatment of infections, and the use of ECPR. These strategies have shown promise in various trials and are now integrated into post-resuscitation care guidelines.Recent clinical trials have produced conflicting results on the effectiveness of certain therapeutic interventions. TTM, higher blood pressure, and oxygen targets did not demonstrate significant benefits in terms of mortality or morbidity for OHCA survivors. Further research is needed to clarify their role in improving patient outcomes after OHCA. Ongoing studies may also require revisions to existing guidelines. It is crucial to optimize the use and implementation of these supportive therapies in order to enhance post-resuscitation care for OHCA survivors and ultimately improve their long-term outcomes and quality of life.


Maize is one of the most important crops, but its production is threatened by drought stress worldwide. Thus, increased drought tolerance has been a major goal of maize breeding. Conventional breeding strategies have led to significantly increase of maize yields; however, these strategies often fail to meet the need for drought stress tolerance enhancement. Here, we focus on progress related to the genetic dissection of drought tolerance in maize at different developmental stages achieved through linkage mapping and association mapping. Moreover, recent molecular breeding systems, including transgenic, genome-wide marker-assisted selection, and genome editing technologies, have provided a more direct, efficient, and accurate approach for trait improvement. We also provide perspectives on future directions regarding multi-omics studies and maize improvement. Overall, the application of acquired knowledge will facilitate maize breeding to meet the challenges. Zea mays L.) is a primary cereal crop and is cultivated for food, feed, and industrial materials. Although maize yields have increased significantly in modern times, its production in all stages of plant growth, especially flowering and grain filling, is threatened globally by drought  when plants subjected to water stress is the final target trait used to assess the degree of drought tolerance, correlated traits, such as ASI and kernel number per row (KNR), are considered to have a higher heritability and thus may be more suitable as target traits for improving maize drought resistance  are the two major strategies used in plants to identify QTLs for complex traits. A major objective in genetic mapping is to identify the causative gene(s) responsible for the phenotypic variation. Since the release of the maize B73 reference genome, GWAS and linkage mapping analyses in maize have substantially increased, resulting in the dissection of many agriculturally important traits. However, to identify the causal gene or variant is still challenging. To address this problem, new approaches, including Mendelian randomization (MR) analysis and transcriptome-wide association studies (TWAS), have been developed to analyze expression-trait associations and prioritize candidate genes associated with a trait based on variations in gene expression . Populations adopted for linkage mapping are usually derived from a bi-parental cross with clear ancestry. QTL mapping allows a researcher to determine if a chromosomal fragment between two specific breakpoints is associated with a specific phenotype. Molecular markers, such as restriction fragment length polymorphisms (RFLPs), random amplified polymorphism DNA (RAPD), sequence-characterized amplified regions, and simple sequence repeats (SSRs), have been developed and used for QTL analysis beginning in the last century  and IABO78 (low L-ABA parent) genotypes  in maize is ~ 5.5 kb in maize hapmap2 , however, is a major factor affecting the resolution achieved by association mapping. Three versions of the maize haplotype map have been published, and the number of SNP variants on each of them were 3.3 million for 27 lines  that was significantly associated with natural variation in maize drought tolerance. The MITE insertion is correlated with lower ZmNAC111 expression in maize and suppresses ZmNAC111 expression via the RNA-directed DNA methylation and H3K9 dimethylation pathway when it is expressed in Arabidopsis   was identified to contribute to the seedling drought tolerance.Regulation of gene expression is fundamental aspect of stress response and adaptation. Thus, a transcriptomic approach has been widely used in studies of drought stress response in maize  were crossed with two testers (CML254 and CML274) and evaluated under different water regimes. Mean grain yield of the test hybrids was consistently at least 50% higher than control hybrids under severe water-stressed conditions (Ribaut and Ragot ZmVPP1 from drought-tolerant inbred lines (CIMBL70 and CIMBL91) was introgressed into a drought-sensitive inbred line (Shen5003) through four generations of successive backcrossing of F1 plants (Shen5003 × CIMBL70 and Shen5003 × CIMBL91). For each generation, the ZmVPP1-heterozygous plants were selected and backcrossed with Shen5003. The homozygous-tolerant plants, NIL-ZmVPP1CIMBL70and NIL-ZmVPP1CIMBL91, were more tolerant than the NIL-ZmVPP1Shen5003 siblings  using CRISPR-Cas9 technology. The survival rate of all three mutants under drought stress was significantly higher than wild-type plants  than for grain yield (0.58) using random regression best linear unbiased prediction in combination with five-fold cross-validation (Zhao et al. With the advent of new genomic tools, genome wide selection has also emerged as an important approach for complex trait improvement (Zhao et al. Over the past several decades, researches have shed light on the complex genetic architectures and regulatory mechanisms involved in drought resistance in maize. Drought-related QTL analyses have identified genomic regions that can be used for the direct selection of specific alleles. Meanwhile, GWAS has been used to identify hundreds of genetic variants in maize that are associated with drought-related traits. However, the candidate genes through which the identified genetic variants exert their effects on traits are remained largely unknown. Therefore, it is important to integrate multi-omic studies into linkage and association mapping to bridge the knowledge gap. Metabolomics provides a comprehensive high-throughput quantification of a broad range of metabolites and has been valuable for both phenotyping and diagnostic analyses in maize (Fernie and Schauer