Chronic intestinal inflammation is characteristic of Inflammatory Bowel Disease (IBD) that is associated with the exaggerated infiltration of immune cells. A complex interplay of inflammatory mediators and different cell types in the colon are responsible for the maintenance of tissue homeostasis and affect pathological conditions. Gene expression alteration of colon biopsies from IBD patients and an in vivo rat model of colitis were examined by RNA-Seq and QPCR, while we used in silico methods, such as Ingenuity Pathway Analysis (IPA) application and the Immune Gene Signature (ImSig) package of R, to interpret whole transcriptome data and estimate immune cell composition of colon tissues. Transcriptome profiling of in vivo colitis model revealed the most significant activation of signaling pathways responsible for leukocyte recruitment and diapedesis. We observed significant alteration of genes related to glycosylation or sensing of danger signals and pro- and anti-inflammatory cytokines and chemokines, as well as adhesion molecules. We observed the elevated expression of genes that implies the accumulation of monocytes, macrophages, neutrophils and B cells in the inflamed colon tissue. In contrast, the rate of T-cells slightly decreased in the inflamed regions. Interestingly, natural killer and plasma cells do not show enrichment upon colon inflammation. In general, whole transcriptome analysis of the in vivo experimental model of colitis with subsequent bioinformatics analysis provided a better understanding of the dynamic changes in the colon tissue of IBD patients. Inflammatory bowel disease (IBD) is an umbrella term of chronic inflammatory disorders of the gastrointestinal tract . Its maiThe molecular background of IBD is extremely complex because of its multifactorial pathogenesis . PreviouAccordingly, instead of focusing only on certain molecules or mutations, the comprehensive investigation of gene expression patterns is more informative in defining the consequences of the interactions between these various factors. Whole transcriptome analysis of an in vivo model of experimentally induced colitis represents a snapshot of the inflamed colon, and enables the simultaneous observation of thousands of mRNA molecules. In this work we examined different aspects and participants of inflammatory response by following an axis from the gut lumen through the network of tissue resident and infiltrating immune cells to the endothelial cells of the vascular wall, by the measurement of gene expression pattern of affected genes in rat and human colon tissue samples. Furthermore, we applied several bioinformatics tools in order to decipher more precisely the detected gene expression signatures. We determined the activated signaling pathways by applying Ingenuity Pathway Analysis (IPA); in addition, we used the ImSig package of R to define the immune cell composition of colon tissues based on gene expression signatures.n = 2), samples were taken from random colon sections, while samples from colitis-induced animals were taken from the inflamed colon region (n = 6) as well as from a non-adjacent uninflamed region (n = 6). All samples were kept in TRIzol reagent  at −80 °C.To investigate the molecular background of IBD we used the 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced rat model of experimental colitis. Male Wistar rats (180–220 g) were randomly divided into two groups as detailed in : the firColonic biopsies were obtained from 21 consenting patients with IBD (Samples from rat colons were homogenized in TRIzol reagent by ULTRA-TURRAX T-18 (IKA) instrument as described previously . In tota−ΔΔCT method.Reverse transcription was performed by SuperScript VILO Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. Gene expression was measured by quantitative real-time PCR using the StepOne PCR Systems (Thermo Fisher Scientific). SybrGreen technology based QPCR reactions were performed by SYBR Select Master Mix (Thermo Fisher Scientific) with specific exon spanning primer sets , while TRNA-Seq was performed by SOLiD total RNA-Seq Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions, and sequenced on SOLiD 5500xl instrument using the 50-base sequencing chemistry. Raw sequence data was size-selected discarding reads shorter than 50 bp. Rnor_5.0 (GCA_000001895.3) from ENSEMBLE release 77 was used as a reference to align RNA-Seq data. Legacy RNA-Seq engine of CLC Genomic Workbench tool  was used to obtain gene expression estimates (mapped read counts) for each annotated gene in all samples with the following mapping parameters: maximum number of mismatches: 2, minimum alignment length fraction: 0.8, minimum similarity fraction: 0.8, unspecific match limit: 10. Reads mapping to rRNA genes were removed. Subsequently, “calcNormFactors” from package “edgeR” was used to perform data normalization based on the “trimmed mean of M-values” (TMM) method. Common and tagwise negative binomial dispersion values were estimated in edgeR using “estimateCommonDisp” and “estimateTagwiseDisp” functions of edgeR package, respectively. Lists of differentially expressed genes were obtained via the “exactTest” function of edgeR. After data normalization, genes showing at least two-fold gene expression change with a false discovery rate (FDR) value below 0.05 were considered significant. Multi-dimensional scaling was also applied to visually summarize gene expression profiles, revealing similarities between samples and unsupervised cluster analysis was carried out on the normalized data. Function plotMDS from package Limma was used to generate http://www.ingenuity.com). Database of IPA (Knowledge Base) is based on published experimental results. Significantly activated canonical pathways in the in vivo model of experimental colitis were determined by core analysis function of IPA by using log fold change and FDR values. Determination of significantly activated canonical pathways was based on two different methods. The most significantly altered canonical pathways between sample groups of RNA-Seq analysis were calculated using a Right-Tailed Fisher’s Exact Test; it reflects the likelihood that the association or overlap between a set of significant molecules from the experiment and given process/pathway transcription neighborhood is due to random chance. The smaller the p-value, the less likely that the association is random; for a clear visualization, values were transformed into negative log scale, where a higher “–log” means it is less likely that the association is random. By using the downstream effect activation of the z-score, a statistical measure of the correlation between the relationship direction and the resulting gene expression were performed. Z-score > 2 or < −2 is considered significant, which can be applied in some analysis types, and provides predictions about upstream or downstream processes. It takes into account the directional effect of one molecule on another molecule or on a process, and the direction of change of molecules in the dataset.For the interpretation of transcriptomic data and for the definition of functional relationship between significantly changed molecules, we used the Ingenuity Pathway Analysis (IPA) application   to identAccording to the recommendations of the original manuscript , we usedp value of less than 0.05 was considered significant.Statistical evaluations were performed using the IBM SPSS Statistics program for Windows. Graphs were plotted with GraphPad Prism 6 software or by ggplot package of R. Quantitative data are presented as the mean ± SEM and the significance of difference between sets of data was determined by one-way analysis of variance (ANOVA) following LSD post-hoc test; a http://www.ncbi.nlm.nih.gov/geo under accession GSE149517.Gene Expression Omnibus (GEO) archive of the three sequenced libraries was deposited in NCBI’s GEO Archive at In the widely used 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced rat model of IBD the intracolonically-administered TNBS destroys the integrity of intestinal epithelium and generates sporadically located lesions . Hence, Based on two independent methods, TNBS treated uninflamed and inflamed regions of the colon significantly differ from each other at transcriptional level. To quantify the transcriptomic data, we compared gene expression values of sample groups to each other. The expression of ~3800 transcripts changed significantly in at least one of the three different ways of comparisons, namely TNBS treated –uninflamed vs. control (abbreviated as UI-C), TNBS-treated–inflamed vs. control (abbreviated as I-C) and TNBS treated-inflamed vs. TNBS treated–uninflamed (abbreviated as I-UI).To validate our transcriptome data, first we compared the RNA-Seq results to our previous data on gene expression changes related to epithelial-to-mesenchymal transition (EMT) ,15,19. UIn order to better understand results obtained by RNA-Seq and to identify possible relationship/s between molecules with altered expression, next, we performed in silico pathway analysis by using IPA. Functional analysis of our data sets revealed significant alteration of canonical pathways related to communication between immune cells, immune cell recruitment, signaling through pattern recognition receptors and regulation of inflammatory response at the site of inflammation; in line with this, we detected the activation of NF-κB signaling that interconnects several inflammation regulating pathways . Based oThe relationship of the intestine and the microbiome in the lumen of the bowel is tightly regulated. In the healthy colon, the double mucin layer precludes the direct contact of bacteria and epithelium and protects the surface against hazardous agents . The maiThe glycosylation in IBD does not only affect mucins, but also the formation of apical surface of intestinal epithelial cells (IECs) and immune cell tethering . In addiDownregulation of FUT2 may lead to diminished IEC fucosylation and decreased protection against bacteria in the colon lumen, thereby promoting the progression of chronic inflammation.Perturbation of the commensal flora or the disintegration of glycosylated mucus barrier results in the direct contact of IECs and microbiome. The innate immunity, as a first line of defense, monitors environmental or host-derived factors and distinguishes between dangerous and safe signals. Pattern recognition receptors (PRRs) serve as sensors of pathogen associated molecular patterns (PAMPs) from microbes in the colon lumen or damage/danger associated molecular patterns (DAMPs) derived from dysfunctional cells of colonic tissue. NOD-like receptors (NLRs) belong to PRRs, whose significant activation were observed in the TNBS treated rat colons .Cytosolic NLRs are classified by their NATCH nucleotide-binding domains and leucine-rich repeats, and it is also the basis of their standard nomenclature . NLRs acLigand binding of PRRs induces the expression of chemotactic molecules, such as cytokines and chemokines from resident cells, resulting in the infiltration of immune cells to the damaged tissues .Members of the IL-1 family, such as IL-1α, IL-1β and IL-18 have an important role in the regulation of tissue homeostasis, inflammatory response and immune cell activation ,44. AlthAbnormal overexpression of pro-inflammatory TNF plays a role in the development of inflammatory diseases, such as IBD or psoriasis. As a pleiotropic cytokine, under homeostasis, it contributes to tissue regeneration, tumor control and defense against infections. However, uncontrolled TNF expression triggers inflammation, activation of endothelial cells, recruitment and survival of inflammatory cells and tissue destruction . Both inTNF exerts its function through TNFRI and TNFRII (tumor necrosis factor receptor I and II) signaling that activates NF-κB pathway, thereby regulating the transcription of both pro- and anti-inflammatory molecules. Important negative regulators of inflammation are TNFAIP3  and TNFAIP6  ,49. UbiqMembers of the IL-6 cytokine family include IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotropin-1 (CTF1) ,51. IL-1Activation of canonical IL-10 signaling pathway was also observed in the inflamed colon of TNBS treated rats . As an aIn addition to the anti- and pro-inflammatory cytokines, chemokines are important regulators of inflammations by influencing leukocyte recruitment . These sLactobacillus lactis is a promising approach that is in phase I/II clinical stage [Novel therapeutic strategies are based on the suppression of pro-inflammatory cytokines or enhancement of anti-inflammatory mediators that targets a certain molecule. Many of the abovementioned molecules are already therapeutic targets for the reduction of IBD symptoms . Currental stage ,60.Even though the above examples represent a great leap forward in the treatment of IBD, targeting a certain molecule is not always fully successful. In line with this, our transcriptome analysis revealed the complex interplay of thousands of molecules, including cytokines and chemokines that are able to corporate, regulate or even replace each other’s function. Our genome wide transcriptome analysis revealed that upon inflammation many molecules belonging to a given class of effectors have synchronous alteration in their expression profile . From this perspective, targeting common regulator/s—such as microRNAs—of inflammation-associated molecules may have the potential to moderate the symptoms in most IBD patients more generally. Taken together, our data on the expression profile of cytokines and chemokines in both in vivo rat model of experimental colitis and inflamed colon samples of IBD patients are in line with numerous reports  highlighSecretion of cytokines and chemokines promotes the activation of endothelial cells that, in turn, facilitate the transmigration of leukocytes. After inflammatory stimuli, endothelial cells express E-selectin (SELE) and p-selectin (SELP), while ligands, L-selectin (SELL) and SELPLG ) are located on the surface of activated leukocytes . Their iChemoattractant-triggered leukocytes express integrin subunits , which assemble into ligands of ICAM1 and VCAM1 on the surface of endothelial cells promoting their rolling and arrest, while fibronectin (FN1) enhances this tight adherence by complex formation with integrins ,64. MoesRolling of leukocytes is necessary for finding the exit site from the blood vessel that is supported by the endothelial ligand ICAM2 . Dockingx) antigens [x, and as a cancer marker, elevated level of sialyl-Lex is associated with high chance of metastasis [The fucosyltransferases mentioned earlier are responsible for the synthesis of CD15  and Translation signature genes (61/84), the median correlation has not reached the required 0.7 correlation threshold value ; hence, their average expression unambiguously increased after TNBS treatment. Interestingly, activation of this gene signature was detectable not just in the inflamed but also in the TNBS-treated uninflamed colon samples , the majority (30 out of 33) of the gene expression changes were identical, demonstrating that animal models—in this case TNBS-induced colitis—are still useful tools in deciphering molecular mechanisms underlying human diseases, such as IBD.In the life of IBD patients inactive and active disease phases fluctuate, while the localization of inflamed and uninflamed colon regions alternates along the digestive tract. This course is coupled with the fluctuation of the expression of genes regulating inflammation. Here, we report a genome-wide alteration in the expression pattern of inflammation-related gene sets in the TNBS-treated rat model of colitis, and the validation of a subset of these genes on samples derived from IBD patients. As a barrier tissue, the intestinal epithelium is constantly exposed to the colonizing bacteria from the lumen; in line with the perturbation of commensal gut bacteria, termed as dysbiosis, that is characteristic in IBD patients. Here, we measured the expression pattern of genes related to glycosylation and recognition of PAMPs/DAMPs. Absence of fucoslytransferases (FUT2) may affect the protection of colonic epithelium, while damaged cell-derived alarmins activate various immune cells through pattern recognition receptors, such as NLRs. Induction of inflammatory cytokines and chemokines leads to the recruitment and homing of leukocytes.Colon tissue is composed of several cell types, and all of them contribute differently to the maintenance of mucosal homeostasis or to the regulation of immune response. In the present work, we monitored the actual gene expression alteration of a heterogenic cell population present in the uninflamed or inflamed regions of rat and human tissue samples. The rapid development of single-cell sequencing methods allows for increased resolution of our transcriptome analysis by applying single-cell RNA-Seq (scRNA-Seq) and determine the gene expression alteration of specific cell types in the inflamed vs. uninflamed colon tissues.


IntroductionWalk-in and after-hours clinics are being increasingly utilized in orthopedics and are especially beneficial for patients with simple sprains, fractures, or overuse injuries that might otherwise require an emergency room visit. To meet the increased patient load, additional staffing often is required, which might include a family medicine physician, nurse practitioner, or physician assistant. Few studies have evaluated the performance of these non-surgeon providers in an orthopedic clinical setting. This study compared the time to definitive care of pediatric patients with forearm and elbow injuries between non-surgeon providers in a walk-in clinic, orthopedic surgeons in a walk-in clinic, and a pediatric orthopedic surgeon in a regular clinic.MethodsChildren who had closed reduction and fixation of an elbow or forearm injury from January 2010 to December 2017 were identified. The patients were divided into groups: patients initially evaluated in a walk-in clinic by a non-surgeon provider; patients initially evaluated in a walk-in clinic by an orthopedic surgeon; and patients initially seen by a fellowship-trained, pediatric orthopedic surgeon in a regular clinic (control group). Neither type of provider (non-surgeon or surgeon) in the walk-in clinics definitively treated any injury but rather transferred care of the patient to a pediatric orthopedic surgeon. The number of clinic visits until surgery, the number of providers seen, the days before evaluation by a pediatric orthopedic surgeon, and the number of days before definitive surgical treatment were documented.ResultsOf the 162 patients identified, 36 (22%) were initially seen by an orthopedic surgeon and 62 (38%) by a non-surgeon provider in a walk-in clinic. The remaining 64 (40%) (control group) were initially seen in a regular office visit by a pediatric orthopedic surgeon. There were no significant differences noted for patients treated by orthopedic surgeon and non-surgeon providers in days before a referral visit to the pediatric orthopedic surgeon  or days to surgery for definitive treatment . The average number of providers seen  and average number of clinic visits before surgery  also were similar when comparing the two groups. The control group had significantly fewer days from evaluation to surgical treatment than the surgeon walk-in group  and the non-surgeon walk-in group .ConclusionThere was no difference in the number of days to transfer patient care to a pediatric orthopedic surgeon between non-surgeon providers and orthopedic surgeons in the walk-in clinic. However, there was a one-day delay reaching definitive treatment when initial evaluation occurred in a walk-in clinic, regardless of whether the patient was initially seen by a surgeon or non-surgeon, when compared to an initial evaluation by a pediatric orthopedic surgeon. Providing pediatric orthopedic care to patients in the current healthcare climate commonly involves practitioners with a variety of backgrounds and training. The surgeon leads the healthcare team, but this team may be augmented by family medicine providers, nurse practitioners, or physician assistants -3. The gComplaints of in-toeing, overuse injuries, and simple fractures comprise a significant proportion of services provided in a pediatric orthopedic clinic, but these complaints may not always require evaluation by a surgeon to be treated safely and effectively ,4. In a Despite the significant role that non-surgeon providers already play in many orthopedic practices, there is little objective evidence concerning their performance in clinical settings compared to orthopedic surgeons -3,6-9. IThe purpose of this study was to compare the plan of care between non-surgeon providers and orthopedic surgeons in a walk-in clinic in regard to the number of provider visits required before surgery, the number of providers seen during the episode of care, the time until the first office visit with a pediatric orthopedic surgeon, and the number of days to surgery for treatment of pediatric elbow or forearm injuries. We hypothesized that initial evaluation by non-surgeon providers in a walk-in clinical setting would not significantly delay treatment.This retrospective cohort study was approved by our Institutional Review Board (IRB no. 16-05056-XP). Pediatric patients (younger than 18 years of age) who were evaluated at one institution from January 2010 to December 2017 were identified in the electronic medical records system using the Current Procedural Terminology (CPT) code for elbow or forearm injury. The patients were subsequently placed into three groups: patients who were seen by a non-surgeon provider in a walk-in clinic; patients who were seen by an orthopedic surgeon in a walk-in clinic; and patients who were seen by a fellowship-trained pediatric orthopedic surgeon at a scheduled appointment (control group). Orthopedic surgeons in the walk-in clinic included fellowship-trained providers from a variety of subspecialties as well as current orthopedic fellows at the study institution. Non-surgeon providers included family medicine and physical medicine physicians, nurse practitioners, and physician assistants. All patients with injuries requiring surgical treatment were transferred to a pediatric orthopedic surgeon for definitive care. Patients in the control group were initially evaluated and subsequently underwent management by one fellowship-trained pediatric orthopedic surgeon. These patients had been scheduled for a regular appointment with the pediatric provider and scheduled for procedural or surgical care after that visit by the same provider.All providers were employed by the same orthopedic practice, and only patients who were evaluated on a walk-in basis, without a scheduled appointment or prior evaluation of the injury in question, were included in the study. Only acute injuries, defined as having occurred within seven days of presentation, were included. Patients who were referred from the walk-in clinic setting directly to a hospital or emergency room for any procedure were excluded from analysis. Those patients in whom a trial of non-operative management failed before surgical treatment also were excluded.Demographic data  were collected for all cohorts. The primary endpoints were number of days to initial evaluation by the treating pediatric orthopedic surgeon, number of days to definitive care, total number of providers seen during the episode of care, and total number of clinic visits required before surgery.Comparative analysis was undertaken using descriptive statistics, and comparisons between cohorts were done using Student’s t-tests and Analysis of Variance (ANOVA). P-values less than 0.05 were considered significant.A total of 162 patients with elbow or forearm injuries requiring surgery were identified from the medical record system. Ninety-eight patients who were evaluated initially in a walk-in clinic and subsequently had procedural or surgical treatment for an elbow or forearm injury were identified. Of these, 36 of 98 (37%) were initially evaluated by an orthopedic surgeon not fellowship-trained in pediatrics, and 62 of 98 (63%) were seen by a non-surgeon provider. The remaining 64 patients seen over the same time period at a regularly scheduled appointment and solely treated by a pediatric orthopedic surgeon served as the control group. Demographic information is listed in Table Non-surgeon versus orthopedic surgeon providersNo statistically significant differences were found between the two groups (non-surgeon versus orthopedic surgeon) of walk-in patients when comparing the time between initial evaluation and first visit with the treating pediatric orthopedic surgeon. Care of patients was transferred to a treating pediatric orthopedic surgeon an average of 3.9 days after initial walk-in evaluation by an orthopedic surgeon and 3.7 days by a non-surgeon provider (p = 0.631). Also, no significant differences were noted when comparing the time between initial walk-in evaluation and surgical treatment between the non-surgeon and surgeon groups. Walk-in patients seen by an orthopedic surgeon underwent surgery an average of 5.2 days after initial evaluation compared to 4.8 days for patients seen by a non-surgeon provider (p = 0.622). In both groups most of the patients had two visits with two different providers prior to their definitive procedure, with no significant differences noted between providers.Control groupSixty-four patients who were seen over the same time period at a regularly scheduled appointment and treated solely in a pediatric orthopedic surgeon’s clinic underwent a definitive procedure significantly faster than either walk-in group . Additionally, patients solely seen by a pediatric orthopedic surgeon were significantly more likely to undergo their surgery after a single clinic visit. Twenty patients (56%) in the surgeon walk-in group and 24 patients (61%) in the non-surgeon walk-in group had two or more clinic visits prior to surgical treatment compared to the control group in which all 64 patients (100%) underwent their procedure after a single visit.Analysis by injury typeIn the walk-in group, distal radial and forearm shaft fractures were the most common diagnoses in 33 patients (34%) and 28 patients (29%), respectively. In contrast, the most common injuries seen in the pediatric orthopedist's group (control) were supracondylar fractures of the humerus . The complete distribution of injuries is listed in Table Patients with supracondylar fractures seen in the walk-in clinic underwent their procedures an average of 4.6 days after evaluation, compared to an average of 2.6 days for patients seen and treated by a pediatric orthopedic surgeon (p = 0.004). Times to treatment for the remaining diagnoses were not significantly different Table .Demand for pediatric musculoskeletal care is high due in part to the fact that approximately 25% of the population in the United States is under 18 years of age and that this segment of the population has been shown to have increasing rates of musculoskeletal injury ,11. In aAdvanced practice providers are increasing in orthopedic settings; however, data comparing the care they provide with that of orthopedic surgeons are lacking. A recent study by Althausen et al. analyzed the financial and clinical effects of adding a physician assistant to an orthopedic trauma service at a level-2 community trauma hospital . They foThis study directly compared the operative treatment of pediatric patients with forearm and elbow trauma initially seen by orthopedic surgeons and non-surgeon care providers in a large, single-specialty orthopedic walk-in clinic. No significant differences between surgeon and non-surgeon providers were found in days before appointments with the treating pediatric orthopedic surgeon or total days elapsed between clinic evaluation and definitive surgery. This suggests that advanced practice providers can be safely integrated into orthopedic clinical treatment teams without introducing a significant delay in care.Although no differences were found when comparing orthopedic surgeons with non-surgeon providers in the walk-in setting, patients seen and treated initially by a pediatric orthopedic surgeon underwent their procedures significantly sooner than patients seen in the walk-in clinic. This may be attributable to the pediatric surgeon’s ability to diagnose the injury and schedule surgery in a single encounter. Additionally, the control group contained a higher proportion of supracondylar fractures, and these injuries were treated significantly faster by the pediatric orthopedic surgeon.Aside from being retrospective in nature, this study has other limitations, and our findings may not be generalizable to advanced practice providers seeing patients with musculoskeletal complaints in non-orthopedic clinics. This study was completed at a high-volume, academic orthopedic practice where non-surgical providers are well integrated into the treatment team. Non-surgical team members can easily communicate with orthopedic surgeons either in person or by telephone, which is unlikely to be the case in smaller orthopedic practices, multi-specialty clinics, or urgent care facilities. The results of this study are most applicable to larger orthopedic groups with multi-specialty representation. Also, selection bias potential exists as a result of excluding patients who were prescribed a trial of non-operative management because they may represent the group with the most variability in treatment between different types of providers. Larger, prospective studies will be needed to better assess patient care by advanced practice and non-surgeon providers as their participation in the orthopedic workforce continues to expand.In this retrospective study of 162 pediatric patients with upper extremity injuries, the time from initial evaluation to definitive treatment was assessed under three different outpatient scenarios: initial evaluation by a non-surgeon provider in a walk-in clinic with subsequent referral for definitive treatment; initial evaluation by an orthopedic surgeon in a walk-in clinic with subsequent referral for definitive treatment; and initial evaluation with definitive treatment by a fellowship-trained pediatric orthopedic surgeon at a regularly scheduled office appointment. No significant differences were found between the non-surgeon providers and orthopedic surgeons in the walk-in clinic, suggesting that advanced practice providers do not introduce significant delays in care. A statistically significant difference was noted between the walk-in clinic (regardless of provider) and the regularly scheduled office appointment; however, this delay of just over 1 day may not be clinically significant.


NO-producing stents are particularly promising in future clinical use due to their ability to store NO resources or to generate large NO flux in a controlled and efficient manner. In this review, we first introduce NO-releasing and -generating coatings for prevention of thrombosis and restenosis. We then discuss the advantages and drawbacks on releasing and generating aspects, where possible further developments are suggested.Cardiovascular stenting is an effective method for treating cardiovascular diseases (CVDs), yet thrombosis and restenosis are the two major clinical complications that often lead to device failure. Nitric oxide (NO) has been proposed as a promising small molecule in improving the clinical performance of cardiovascular stents thanks to its anti-thrombosis and anti-restenosis ability, but its short half-life limits the full use of NO. To produce NO at lesion site with sufficient amount, NO-producing coatings (including NO-releasing and NO-generating coatings) are fashioned. Its releasing strategy is achieved by introducing exogenous NO storage materials like NO donors, while the generating strategy utilizes the  Cardiovascular diseases (CVDs) are a common cause of morbidity worldwide, accounting for 18 million deaths per year, and serve as a third of all global deaths  with unacceptable levels of thrombosis and restenosis  produce NO to achieve thrombotic homeostasis by preventing platelet activation ] are pharmacologically active substances that carry NO for localized release , requires reactions between nitrosating agents and thiols, and NO can be exhausted from SNAP by heat, light (340 and 590 nm), or copper ions , For example, Joung et al. fabricated NONOates containing liposome stent coatings via layer-by-layer (LBL) method to control the release of NO  after testing by ECC over 4 h. Although this strategy achieved long-term NO release, the low NO levels in the later release stages limited the long-lasting anti-thrombotic effect.However, the NO release period in these studies was limited. To extend the NO release time, Hopkins et al. designed a highly stable NO-releasing coating by covalently attaching SNAP with poly(dimethylsiloxane) (PDMS)  to maintain the stability of donors and control the release  immobilized surface coating and prepared glycosylated NONOate  as NO prodrug  consist of hydrophobic tails coupled to hydrophilic functional peptide sequences that are attractive in biomimetic scaffolds, since enzyme-mediated degradable sites and cell adhesion ligands can be incorporated into PAs to mimic some biochemical properties  or copper ions to stimulate NO production. These strategies can achieve sufficient and long-term NO release depending on the endogenous regulation and continuous supply of NO resources.in vitro, and exhibited a non-thrombogenic surface compared to unseeded controls in vivo.In the cardiovascular system, nitric oxide synthase (NOS) especially endothelial NOS (eNOS) and inducible NOS (iNOS) play a very important role. eNOS is highly related to the function of the endothelium, which can catalyze the production of NO via enzymatic effect  vectors  by regulating the SeCA–DA molar ratio. The study showed that the SeCA/DA coating could release NO for more than 60 days, which achieved long-lasting prevention of thrombosis and restenosis. Additionally, the simple operation avoided the tedious process and toxic chemicals. This coating is believed to be universally formed on diverse types of materials with great clinical potential. We further improved the NO production ability by employing CuII instead of SeCA, since CuII possesses superior NO catalytic efficacy  by adjusting the dose of CuII with NO flux ranging from 0.4 to 6.5 × 10−10 mol × cm−2 × min−1. After implantation for 3 months, the DA-CuII-coated stents not only inhibited thrombosis, but also promoted re-endothelialization and reduced neointimal formation. We then additionally endowed our stents with anti-bacterial properties  with DA-CuII coating  in the framework of dopamine (DA) via co-immobilization  can be more efficient for biomedical applications, but the NO donors such as SNAP have been shown to release NO near the low end of the physiological levels. Based on this, Handa et al. incorporated SNAP in a medical grade polymer coated with copper nanoparticles to achieve better regulation of NO release  particles with EPT  Smart NO production system can be further developed to respond to the microenvironment changes and to produce NO flux on-demand during different pathologic process. (5) Most researchers use healthy animals to evaluate the effect of NO; however, differences between the healthy and the CVD individuals should be assessed. Researchers should also combine the effects of NO-producing coatings and the implanted materials as a whole to investigate the therapeutic effect in their corresponding disease models, which will have much more clinical significance.The progress of the NO-producing stent coatings are highly inspiring in the prevention of thrombosis and restenosis, and researchers have also attempted to improve the fabrication process and committed to developing simple materials for future clinical application. However, the immune reaction, inflammation, anaphylaxis, and biocompatibility of the coatings need to be further investigated. Although these problems cannot be solved temporarily, in the long term, what can be done is to promote the development of NO production strategies from those achievable directions: (1) Combination strategies of NO and other pathway regulation need to be carefully considered about the dosage ratio between the drugs to avoid the antagonism effect and ensure synergistic effect. (2) The toxicity and metabolism of the NO reaction, by-products [like N-Acetyl-D-penicillamine (NAP) and disulfide] should be taken into account. (3) Scientists should develop precise methods of NO quantitative detection In summary, NO-releasing and NO-generating strategies still have significant room for improvement, and a thorough understanding of NO production and solutions for the corresponding pathological reaction will ensure the safe clinical practices for the next generation of technological devices. We hope that this review can be helpful for the further development in this research field.XZ and HL supervised the whole review. JR and HP wrote the manuscript. YY and YL performed literature search and revised the manuscript. All the authors approved the review for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The LRR-domain containing Slits and their Robo receptors are one of the best characterized examples of ligand-receptor pairs that regulate midline crossing and axon guidance in both Drosophila and vertebrates . There are 66 eLRR proteins in Drosophila, many of which are expressed in the nervous system and exhibit strikingly specific expression patterns, often labeling distinct subpopulations of neurons . The binding partners and functions of many of these eLRR proteins remain unknown.Leucine-rich repeat (LRR) domain-containing proteins play central roles in organizing neural connectivity. The LRR is a protein-recognition motif and proteins with extracellular LRR (eLRR) domains mediate intercellular communication and cell adhesion, which in turn regulate neuronal processes such as axon guidance, target selection, synapse formation and stabilization of connections . This method involves using fusion proteins containing the extracellular domain (ECD) of a protein fused to a pentamerization domain (COMP), followed by human placental alkaline phosphatase (AP). These AP fusion proteins are used to stain live-dissected stage 16 Drosophila embryos. The resulting staining patterns can be used as a template to identify expression patterns of the binding partners of the AP fusion protein. Using this technique, we have identified ligands for the receptor tyrosine phosphatases Ptp10D, Lar and Ptp69D . Here, we describe novel binding patterns for 5 eLRR proteins using their respective AP fusion proteins.We have previously described a novel method to identify ligands and/or binding partners for extracellular proteins . Studies of trn and caps single and double mutants suggest that the two genetically interact and may function through a common binding partner . Tartan may be a substrate for the receptor tyrosine phosphatase Ptp52F . We stained wild-type live-dissected stage 16 Drosophila embryos with trn-AP and caps-AP fusion proteins separately, and found distinct as well as overlapping staining patterns for both fusion proteins. Both trn-AP and caps-AP bind to longitudinal axons in the ventral nerve cord (VNC), with stronger binding seen in one particular axon bundle close to the midline . Both also show binding to muscles , indicating that they interact with a binding partner expressed on the surface of muscles. trn-AP shows binding to a subset of sensory neurons , which caps-AP does not. In addition, caps-AP binds to the transverse nerve, which emanates from the midline and is located on the dorsal side of the VNC .Tartan (trn) and Capricious (caps) are two closely-related eLRR proteins with known functions in embryonic motor axon guidance and the innervation of antennal lobe glomeruli by olfactory sensory axons  and olfactory receptor neuron (ORN) targeting in the antennal lobe . It is expressed at moderately-high levels during embryonic stages 12 – 17 and during 24 – 48 hours after puparium formation . These developmental stages correspond to peak synaptogenesis times, implying a developmental role of Fili in regulating synaptogenesis. Thus, identification of binding partners of Fili is crucial to understand its roles in CNS development. Staining of wild-type stage 16 embryos with Fili-AP fusion protein shows a restricted binding pattern in the CNS, indicating a similar restricted expression pattern of its binding partners. It binds to a set of dorsal midline neurons  and a subset of longitudinal axons in the VNC . A subset of midline cells, putatively glial cells are also labeled with Fili-AP. Strong binding is seen to the transverse nerve in the VNC (c1) and in the periphery , while no labeling is seen to the SNa in the same focal plane .Fish-lips (Fili) is an eLRR with roles in the regulation of apoptosis  and encodes an eLRR protein of unknown function. Caldwell et al. 2007 showed a broad expression pattern of the encoded protein in the adult nervous system. We performed staining of wild-type stage 16 embryos with rdo-AP fusion protein and found a very strong binding signal in the longitudinal and commissural axons of the VNC . This binding was limited to the VNC, and no binding was observed to the muscles (data not shown), indicating that the eLRR encoded by rdo interacts with neuronal-specific ligands. We also observed binding in a subset of midline glial cells in the VNC .Reduced ocelli (rdo) is a gene that regulates ocelli development . No other information is known about this eLRR. We stained wild-type stage 16 embryos with 2mit-AP fusion protein and saw a wide pattern of binding by this fusion protein, unlike the other restricted patterns observed above. Both longitudinal, commissural as well as exiting motor axons in the VNC are labeled by 2mit-AP . Moreover, we observed a pan-cellular pattern of labeling in the periphery as well as in the VNC, where 2-mit-AP binding signal is seen on the surface of cells, resulting in a cell-membrane staining pattern . This implies that the eLRR encoded by 2mit is capable of interacting with ligands expressed on neuronal as well as non-neuronal cell types.2mit is another gene encoding a putative eLRR and is expressed in the developing nervous system. It has a putative role in regulating short-term memory y


In this article, we focus on the latest progress in defining the mechanisms of chemotherapy resistance in hyperglycemic patients including the abnormal behaviors of cancer cells in the hyperglycemic microenvironment and the impact of abnormal glucose metabolism on key signaling pathways. To better understand the advantages and challenges of breast cancer treatments, we explore the causes of drug resistance in hyperglycemic patients that may help to better inform the development of effective treatments.Female breast cancer is a complex, multifactorial disease. Studies have shown that hyperglycemia is one of the most important contributing factors to increasing the risk of breast cancer that also has a major impact on the efficacy of chemotherapy. At the cellular level, hyperglycemia can promote the proliferation, invasion, and migration of breast cancer cells and can also induce anti-apoptotic responses to enhance the chemoresistance of tumors  Breast cancer (BC) is the most common malignancy in women all over the world  and has The “Warburg” effect is considered to be the most important feature of glucose metabolism in tumors . Under hde novo synthesis of fatty acids  , 14 throLong-term hyperglycemia results in insulin resistance, hyperinsulinemia, and dysfunctional signaling through the insulin-like growth factor-1 pathway . It has It has also been reported that hyperglycemia promotes the proliferation of malignant BC epithelial cells by increasing activity of the leptin/insulin-like growth factor-1 receptor signaling pathway and causing activation of the Protein Kinase B/mechanistic target of rapamycin (AKT/mTOR) pathway . Luey etThe regulation of key transcription factors and inflammatory mediators in the glycolytic phenotype of BC remains an area of intense investigation . HypoxiaHypoxia-inducible factor 1α-induced glycolysis is essential for the activation of inflammatory macrophages. For example, the high infiltration of M2 tumor-associated macrophages is an extremely important feature of inflammatory BC . HIF-1 rEpithelial-mesenchymal transformation (EMT) is an important mechanism that promotes the migration, invasion, and metastasis of cancer cells , 34. ZieP53 inhibits cellular transformation and activates tumor cell responses to chemotherapy drugs. Homeodomain-interaction protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that mediates p53-dependent apoptotic pathways in tumor cells . HyperglRac1 is a small GTP binding protein. Li et al. found it is overexpressed and associated with multidrug resistance to neoadjuvant chemotherapy (NAC) . Rac1 acAdriamycin (ADR) resistant BC cells have increased glucose metabolism . It has Paclitaxel is a commonly used chemotherapy drug in the treatment of TNBC to which patients commonly develop resistance . StudiesCisplatin is a common chemotherapy drug to which BC patients often develop resistance mediated by the altered expression of glycolytic enzymes and glucose transporters . ConsideTamoxifen is the most commonly used endocrine therapy in BC and patients often develop drug resistance due to enhanced glycolysis in ER-positive BC. He et al. demonstrated that activation of the EGFR signaling pathway and its downstream glycolytic genes play an important role in tamoxifen-resistant BC cells . TamoxifTrastuzumab (Herceptin) is an antibody targeted against HER2. Aerobic glycolysis can be inhibited by the ErbB2 (HER2)-heat shock factor1 (HSF1)-LDHA pathway. The sensitivity to trastuzumab has been associated with LDHA activity . PKM2 isBC cells undergo metabolic reprogramming that usually includes enhanced glycolysis and increased activity of the tricarboxylic acid cycle . ScholarThe TME is mainly composed of tumor cells, surrounding immune and inflammatory cells, fibroblasts, interstitial tissue, capillaries, various cytokines, and chemokines. The TME is characterized by high levels of H2O2 and glutathione (GSH), low pH, and hypoxia that have important implications for responses to treatment .The TME plays an important role in the metabolic adaptation and survival of tumor cells. The inflammatory microenvironment can directly induce aerobic glycolysis . Studiesin vivo and in vitro -sugars are generated as intermediate products of glucose metabolism. The levels of these sugars are significantly increased in BC and have been shown to promote the accumulation of hyaluronic acid which is a known promoter of the disease. The metabolism of hyaluronic acid within the TME a key factor that drives invasive growth and metastasis , 70. In Cancer cells within the TME have higher levels of reactive oxygen species (ROS) compared to normal cells. In basal-like and BRCA1-associated BC, it has been shown that ROS levels are associated with the expression and activity of the transcription factor aryl hydrocarbon receptor (AHR). These changes promote the transcription of antioxidant enzymes, epidermal growth factor receptor (EGFR) ligands, and the bidirectional regulatory factor AREG. AHR can attract monocytes into the TME and activate macrophages to promote angiogenesis , 74. ParThe highly acidic TME formed by glycolysis may affect the infiltration of immune cells eventually leading to immune escape and cancer progression . The immvia the transmission of extracellular vesicles (EV) that contain a myeloid-specific HIF-1α-stabilizing long noncoding RNA (HISLA). Lactic acid released by glycolytic tumor cells upregulates macrophage HISLA and forms a feed-forward loop between TAMs and tumor cells in the TME  comprise B and T cells. Cytotoxic CD8+T lymphocytes (CTL) are the most abundant TILs in the TME of BC but helper CD4+T cells and NK cells are also present . Tumor cMyeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immunity. In TNBC mouse models, studies have found glycolysis inhibits the expression of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) resulting in the decreased expression of MDSCs and promotion of tumor immunosuppression . AlthougIn BC patients, hyperglycemia during chemotherapy will increase the resistance to treatment  and so rMetformin was developed in the late 1970s as an anti-hyperglycemia drug . It has Additionally, studies have shown that the thiazolidinediones are insulin sensitizers that can also inhibit the growth of BC cells. Pioglitazone acts as an agonist of the tumor suppressor PPAR. When PPAR is activated, the levels of free fatty acid (FFA) and eicosanoid are reduced, and VEGF-induced angiogenesis is inhibited. Also, the proliferation and migration of cancer cells are inhibited through the JAK2/STAT3 pathway .Insulin and insulin analogs such as sulfonylureas and glitinides have powerful hypoglycemic effects. Studies have reported that insulin downregulates IGF-BPs and sex hormone-binding proteins (SHBGs) leading to IGF and hormone-dependent BC . Type 2 The antidiabetic drugs described above may also have potential roles as anti-cancer drugs. A total of 46 studies of metformin in BC patients have been registered in ClinicalTrials.gov. 17 studies have completed, 14 studies are recruiting, 4 studies were terminated due to inapplicable factors such as long rest intervals, changes in treatment methods, slow accumulation of patients’ number, and data loss. And we also have listed completed in Combining the above table, we can see that the role of metformin on its anti-tumor effects are researched continuously. In addition, there are reported retrospective studies in the past have shown that the use of metformin combined with neoadjuvant chemotherapy can enable BC patients to obtain a higher pCR rate . The METAlthough metformin has shown good antitumor activity in BC, there remain many challenges concerning how best to further optimize treatment. The efficacy of metformin as an anticancer drug depends largely on the glucose concentration in the TME and so treating diabetes in cancer patients may be necessary. There are relatively few studies concerning glitazones and other antidiabetic drugs. The heterogeneity and differences in diseases mean that it is necessary to adopt personalized treatments and using precision medicine approaches to tailor treatments at the individual patient level.Patients with hyperglycemia are resistant to chemotherapy drugs. Metformin resistance is no exception . ScherbaReports have shown that glycolysis inhibitors such as 2-deoxy-d-glucose (2DG) combined with metformin can also significantly reduced the survival of BC cells . It has PI3K/AKT/mTOR and CDK4/6 inhibitors are emerging drugs for the treatment of ER-positive and human epidermal growth factor receptor-2 (HER2) negative metastatic BC . The PI3In vitro experiments have shown that ER+/HER2- BC cells are sensitive to palbociclib under conditions of enhanced aerobic glycolysis whilst ER+/HER2+ cells show enhanced glycolytic catabolism with the development of palbociclib resistance. These metabolic phenotypes may have potential prognostic value , the Key Construction Project Co-sponsored by Province and Ministry (WKJ-ZJ-2116 to XM), the Key Project for Medical and Health in Zhejiang Province (2016ZDA004 to XM), Zhejiang Medicine and Health Technology Plan Project(WKJ-ZJ-1803-01-XM), the Medical and Health Projects in Zhejiang Province (2020KY495 to QZ) and Zhejiang Traditional Chinese Medicine Project(2020 ZQ 009-QZ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A dominant vibrational cooling time constant of ~4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a ~30 and 12% increase of the fluorescent quantum yield vs. the parent sfGFP. Similar time constants were also retrieved from the transient absorption spectra, substantiating the correlated electronic and vibrational motions on the intrinsic molecular timescales. Key carbon-halogen stretching motions coupled with phenolate ring motions of the deprotonated chromophores at ca. 908 and 890 cm−1 in Cl-GFP and Br-GFP exhibit enhanced activities in the electronic excited state and blue-shift during a distinct vibrational cooling process on the ps timescale. The retrieved structural dynamics change due to targeted site-specific halogenation of the chromophore thus provides an effective means to design new GFP derivatives and enrich the bioimaging probe toolset for life and medical sciences.Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the  After photoexcitation, the neutral chromophore A* undergoes a picosecond (ps) excited-state proton transfer (ESPT) reaction to reach a deprotonated intermediate state (I*) within an unrelaxed protein environment preceding green emission  has been widely used for biolabeling and bioimaging due to its characteristic bright green emission, high fluorescence quantum yield (FQY), and stability  mutant contains a strong electron-withdrawing group capable of forming additional hydrogen bonds in addition to an ~30 Å3 increase in residue volume   and 5.5 . As control samples, the monohalogenated 4-hydroxybenzylidene-1,2-dimethylimidazolinone (HBDI) chromophores were synthesized according to literature by combining an iminoglycine methyl ester with a Schiff base  is observed (max = 488 nm) dominates  and 1,558 cm−1 (Br-HBDI) in −1 mode of HBDI in basic solution  stereoisomer, but the halogen substituent on tyrosine can occupy two distinct atropisomeric positions with the probability of each determined by the properties of the substituent itself as well as the local environment supplied by the protein interior , C–Br stretch (500–700 cm−1), and NO2 bend (~820 cm−1) are all expected to occur in this region , but generally approaches the fluorescence lifetime (~3 ns) of the photoexcited deprotonated chromophore  relaxation dynamics other than fluorescence (vide infra), especially with the excess energy provided by the 480 nm pump. The excellent match between these TA time constants and the aforementioned Raman mode intensity decay time constants , and DFT calculations of normal mode frequencies. In particular, the single-site halogenated proteins display improved properties that include the red-shifted absorption and emission, increased concentration of deprotonated emissive species, and an increased fluorescence quantum yield. Such desirable application properties of the halogenated GFP mutants stem from a solid biophysical chemistry foundation in that they are a direct consequence of the engineerable molecular structure and dynamics of the photosensitive unit inside a protein matrix. The nitro-GFP provides a useful contrasting sample that will be further investigated.We focused on the structural aspects of single-site halogenation at the protein active site to examine key conformational preference and elucidate the excited-state energy dissipation pathways in Cl-GFP and Br-GFP. Such a targeted analysis using a well-known series of electron-withdrawing groups with sufficient temporal and spectral resolution paints a more complete picture of chemically modified chromophores reacting to the incoming photons, thus enabling future rational design of functional molecular machines (Fang et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RM and CF conceived and designed the research and acquired funding. BO and LZ performed spectroscopic experiments. BO, NR, AC, and JS performed data curation including calculations. HW, JP, and KS contributed new protein and chromophore samples. LZ and CF contributed advanced non-linear spectroscopic tools. BO and CF wrote the manuscript. All the authors have edited the final manuscript and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Colorectal cancer (CRC) is one of the most common malignant tumors in the world. Lymph node metastasis (LNM) is a common mode of metastasis of CRC. However, the combined mRNA biomarkers associated with LNM of CRC that can effectively predict CRC prognosis have not been reported yet.Methods: To identify biomarkers that are associated with LNM, we collected data from the The Cancer Genome Atlas (TCGA) database. The edgeR package was searched to seek LNM-related genes by comparisons between cancer samples and normal colorectal tissues and between LNM and non-LNM (NLNM) of CRC. Univariate and multivariate regression analysis of genes in the intersection to build gene signature associated with independent prognosis of CRC, and then verified by Kaplan-Meier curve and log-rank test, receiver operating characteristic (ROC) curve was used to determine the efficiency of survival prediction of our four-mRNA signature. Finally, the potential molecular mechanisms and properties of these gene signature were also explored with functional and pathway enrichment analysis.Results: 329 mRNAs were up-regulated in CRC tissues with LNM, and 8461 mRNAs were up-regulated in CRC tissues, the intersection is 100 mRNAs. After univariate and multivariate Cox regression analysis of 100 mRNAs, a novel four LNM related mRNAs  were screened as independent prognostic indicators of CRC. Surprisingly, the four-mRNA signature can predict the prognosis of CRC patients independently of clinical factors andthe area under the curve (AUC) of the ROC is 0.730. The novel four-mRNA signature was used to identify high and low-risk groups. Stratified analysis indicated the risk score based on four-mRNA signature was an independent prognostic indicator for female, T3+T4, N1+N2 ,stage III+IV and patients with no new tumor event. Functional annotation of this risk model in high-risk patients revealed that pathways associated with neuroactive ligand-receptor interaction, estrogen signaling pathway, and steroid hormone biosynthesis.Conclusions: By conducting TCGA data mining, our study demonstrated that a four-mRNA signature associated with LNM can be used as a combined biomarker for independent prognosis of CRC. Colorectal cancer (CRC) is one of the common malignant tumors of the digestive tract Currently researches on CRC-related biomarkers continue to grow In this work, we analyzed 614 patients with CRC in The Cancer Genome Atlas (TCGA) database and found that 100 mRNAs were up-regulated both in CRC patients and CRC patients with LNM. By further analysis we found that based on four-mRNA signature patients with high risk scores have a poorer prognosis and four-mRNA signature associated with LNM can effectively predict the prognosis of CRC patients.The expression of CRC mRNA and the corresponding clinical data were downloaded from the TCGA database. According to the inclusion criteria: (a) complete gene expression and survival information (b) the CRC patients with LNM were filtered by the criteria that N stage of patients was I-IV, and the exclusive criteria were as follows: (a) survival information or gene expression is incomplete (b) N stage in clinical pathological parameters is not available, we finally obtained a total of 614 CRC tissues and 51 normal colorectal tissues of mRNA expression profiles for further research, among them, 614 tissues included 264 tissues with LNM and 350 tissues with non-LNM (NLNM)  to find the intersection of up-regulated genes in CRC tissues and CRC tissues with LNM.Using Venn digram web-tool (p < 0.05), followed by multivariate Cox regression analysis to identify mRNAs as independent prognostic indicators. Subsequently, based on the expression level of each mRNA and the regression coefficient obtained from multivariate Cox conduct a risk score, risk score =In our work, 614 CRC patients were randomly divided into two groups  (Table (Exp represents the expression level of each mRNA and β represents the regression coefficient of each mRNA).https://david.ncifcrf.gov/). GO terms and KEGG pathways with a false discovery rate (FDR) < 0.05 were statistically significant.In this study, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed for these up-regulated mRNAs in patients with high risk score by using the Database for Annotation, Visualization, and Integrated Discovery online tool  of the receiver operating characteristic (ROC) curves used to determine the predicted power of the prognostic gene signature. Moreover, we applied univariate and multivariate Cox analysis to evaluate whether or not the risk score was an independent factor of the other clinical variables including age, T, N, M, stage, residual tumor and neoplasm cancer status in patients with CRC. We used Pearson test or Fisher's exact test to analyze the correlation between LNM and clinical pathological parameters. All statistical analysis was using SPSS 16.0 and GraphPad Prism7.p < 0.05) in 614 CRC tissues and 51 normal colorectal tissues and found that 8461 genes were up-regulated in CRC tissues, while 264 CRC tissues with LNM and 350 CRC tissues with NLNM for differential mRNA mining under the same conditions , test set (N=322) and validation set (N=292) respectively were divided into low-risk and high-risk groups according to the median of the prognosis risk score , indicating that they can be used as independent prognostic indicators for CRC patients , among them, 112 genes were up-regulated in high risk group  and high risk group (N=307) according to the median risk score and screened for differential genes  was closely related to the prognosis of patients with CRC. We further compared the four mRNAs expression between patients with LNM and with NLNM. The results showed that EPHA8, KRT85, and GABRA3 and CLPSL1 were highly expressed in patients with LNM (EphA8 is one of the receptors in Ephs subfamily of receptor tyrosine kinases, it is associated with angiogenesis, cell adhesion and migration. Overexpression of EphA8 enhances the invasive ability of oral squamous cell carcinoma For the first time, our study reported a four-mRNA signature associated with LNM for prognosis of CRC using bioinformatics methods. In this work, patients with high risk scores have a poorer prognosis. Independent of other clinic pathological parameters, four-mRNA signature may become a combined biomarker for predicting the prognosis of CRC patients. However, the four-mRNA signature should be validated in a larger sample size database as well as in clinical samples. Moreover, our results warrant further studies of the mechanisms by which the four-mRNA signature affects prognosis of CRC.The results of this study indicate that a four-mRNA signature related with LNM can effectively predict the prognosis of patients with CRC, but the specific molecular mechanism of the four-mRNA acting on colorectal cancer and whether this four-mRNA signature can be successfully applied to the clinic still need further research.Supplementary figures and tables.Click here for additional data file.


Single-cell RNA sequencing has become a standard technique to characterize tissue development. Hereby, cross-sectional snapshots of the diversity of cell transcriptomes were transformed into (pseudo-) longitudinal trajectories of cell differentiation using computational methods, which are based on similarity measures distinguishing cell phenotypes. Cell development is driven by alterations of transcriptional programs e.g., by differentiation from stem cells into various tissues or by adapting to micro-environmental requirements. We here complement developmental trajectories in cell-state space by trajectories in gene-state space to more clearly address this latter aspect. Such trajectories can be generated using self-organizing maps machine learning. The method transforms multidimensional gene expression patterns into two dimensional data landscapes, which resemble the metaphoric Waddington epigenetic landscape. Trajectories in this landscape visualize transcriptional programs passed by cells along their developmental paths from stem cells to differentiated tissues. In addition, we generated developmental “vector fields” using RNA-velocities to forecast changes of RNA abundance in the expression landscapes. We applied the method to tissue development of planarian as an illustrative example. Gene-state space trajectories complement our data portrayal approach by (pseudo-)temporal information about changing transcriptional programs of the cells. Future applications can be seen in the fields of tissue and cell differentiation, ageing and tumor progression and also, using other data types such as genome, methylome, and also clinical and epidemiological phenotype data. Genome-wide single cell transcriptomics experiments provide snapshot data, which resolves the molecular heterogeneity of cell cultures and tissues with single cell resolution under static conditions ,2. TheseHence, pt trajectories refer to ordered series of cell states. Alterations of activities of selected genes or gene sets along these trajectories then provide pt profiles of gene expression, which represent x-y plots depicting the expression levels as a function of pt . They chWe previously developed the expression portrayal method that visualizes multidimensional, whole genome expression landscapes in terms of two-dimensional images making use of self-organizing map (SOM) machine learning ,23. SuchWe have previously shown that the arrangement of spots indeed reflects dynamics of different processes. For example, melanoma progression from naevi to metastatic tumors has been analyzed by combining SOM portrayal and pt-scaling of bulk melanoma transcriptomes  and singIn this publication, we aim at further developing the idea of constructing developmental trajectories in SOM-feature state space from cross-sectional data in order to supplement pt trajectories in cell-state space with such trajectories describing developing molecular programs. Our efforts should be understood as a first step in this direction as a proof of principle approach. It extends previous work by explicitly extracting “similarity paths” in the expression landscape provided by the SOM method. In other words, we supplement our portrayal of static molecular landscapes by (pseudo-)dynamical views in analogy to pt approaches in cell-state space.Making use of topological features of the expression landscape we also process so-called RNA-velocities as an independent approach. RNA-velocity analysis directly “forecasts” the transcriptional state of a cell based on the relation between spliced and unspliced mRNA ,32. We hWe selected “flatworm” single cell RNAseq data as an illustrative example. Planarian transcriptomics was well studied as a first application of scRNAomics to developmental dynamics in a whole complex animal . This daOur publication is organized as follows , and three wild-type regeneration samples (6451 cells) [Single-cell RNA Seq data of a whole planarian Schmidtea mediterranea were taken from the publication of Plass et al.  as read-1 cells) ,35. As i1 cells) , we anal1 cells) . We appl1 cells)  throughoFor transcript-related analysis we applied the “Single-Cell R Analysis Toolkit” available as R-package “scrat” ,23,27. IMetagenes of mutually correlated profiles cluster together forming “spot-like” red and blue areas of over- and under-expression in the portraits because of the self-organizing properties of the SOM. These “spot”-modules were detected using distance-metrics (D-map) clustering . They exThe spot-patterns obtained represents a characteristic fingerprint of each particular sample. This “logFC” scale is used as default color scale of the portraits . It enaFor an overview, we visualized similarities between transcriptomes of planarian single cells using t-distributed stochastic neighbor embedding (tSNE) algorithm . PseudotGene-state trajectories were calculated by applying the maximum flow algorithm  to transThe so-called RNA-velocity is the time derivative of gene expression. It is positive for increasing and negative for decaying transcript abundance. It can be evaluated from the relative amounts of unspliced and spliced mRNAs of each gene taken from RNAseq data, their ratio under steady state conditions and assuming a constant splicing rate of all genes . The mRNWe applied gene set analysis to the lists of genes located in each of the modules to discover their functional context using the planMine database . planMinhttps://github.com/mschmidt000/oposSOM.PT). It should be used together with the R-package oposSOM [https://github.com/hloefflerwirth/oposSOM) in order to generate trajectories upon running the program.Trajectory generation and visualization is implemented as R-package oposSOM.PT  terms A. For exNote that spots and underlying cellular programs are activated in different cell types  as argument shows the decline of expression of the stem cell module “N” and the gain of the expression of the spot-modules referring to differentiated tissues as expected D. DifferNext, we made use of previous stratification of planarian genes into 48 clusters of co-expressed genes, which were assigned to different tissues and tissue-combinations . The treRNA-velocity analysis offers an independent approach of studying developmental dynamics. It calculates the change of mRNA abundance of each gene per unit of time in every single cell making use of the relation between spliced and unspliced mRNA and assumptions about the reaction kinetics of splicing . RNA velHere we apply this concept also to expression space, where RNA velocity refers to the rate change of mRNA expression in each metagene-pixel of the SOM-space. It results in a vector whose components are given by rate changes of all individual genes contained in the metagene-cluster. The resulting RNA-velocity vector points in the direction of local mRNA rate changes in SOM space. Overall mRNA velocity analysis thus generates a metagene-based vector-field pointing toward increasing transcript abundance. It consequently points in the direction of “high” expression spot areas containing either genes upregulated in differentiated tissues such as epidermal (spot “J”), neuronal , and muscular (“L”) tissues or genes upregulated in stem cells (module “N”). Hence, overexpression spots represent attractors of RNA-velocity  dimensionality reduction to extract most relevant axes of variability, (2) cluster and diversity analysis to extract most relevant groups of cells and similarity relations between them, and (3) feature selection to extract most relevant genes as markers characterizing the different cell types. Assumptions about temporal development enabled to perform diversity analysis of cross sectional data in such a way that one can deduce information about the  dynamics of cellular development and adaptation to changing environmental conditions. State-of-the-art methods accomplish this task in cell-state space, i.e., in the multidimensional manifold spanned by all possible states that cells can adopt upon development and changing environmental conditions. Hereby, i.e., by using scRNAseq data, the particular “state of the cell” is given by its transcriptional phenotype, i.e., the entirety of transcripts produced by a cell in a certain developmental and environmental situation and measured by the scRNAseq technology applied. We here “transpose” this view from the state space of the cells into the space of transcriptional patterns, which are derived from the scRNAseq data without linking gene expression directly to cell states.From a methodical perspective, we complemented our SOM portrayal providing static transcriptomic landscapes by this dynamic aspect in order to deduce (pseudo-)temporal information from snapshot data obtained in cross sectional studies. Using SOM-portrayal we identified tissue-specific clusters of activated genes and linear trajectories linking the stemness-source and tissue-related sink clusters expressing transcriptional programs, which reflect the specific functional requirements of the respective tissue types. In addition, part of the sink spot-patterns reveal common activation of cellular functions in different tissues in support of our initial statement that cell- and gene-states are closely related but do not match in an one-to-one fashion. For example, morphogenesis-related genes (spot F) were activated in neuronal and muscle tissues as well, meaning that one and the same transcriptional program can contribute to different cell states. Our approach explicitly supports identification of such common gene states. Overall, the gene-state landscape decomposes into a directed graph describing development via linear and partly via branched paths. RNA velocity constitutes an independent approach to forecast future expression states. In the SOM landscape RNA velocity vectors point in the direction of the local slope of gene expression. This result seems reasonable and confirms this approach as an independent calculus for transcriptomic trajectories where the generated vector field provides the detailed shape of the local attractor fields pointing toward the overexpression spots.We have chosen the Planarian example because it provides a multibranched lineage tree of tissue differentiation and also because for the first time it demonstrated the impact of single cell transcriptomics for establishing such lineage trees in a multicell organism . MeanwhiNovel developments further extend single cell technologies toward capturing other molecular features such as chromatin accessibility , DNA-metWe expect tumor progression as an important potential application of developmental trajectories where in addition to cell types  further diversity is expected to reflect distinct tumor stages and/or clonal lineages ,52,53. SWe here developed a method to “transpose” the concept of developmental trajectories in phase space of the cells into their phenotype space spanned by phenotype variables such as the expression of ten thousands of genes. After our proof-of-principle study using RNAseq data describing tissue development in the flatworm as example we see applications in different fields of biology and medicine, to other than transcriptomics data and also further needs for refinements of the method, e.g., to better disentangle the topology of the feature landscape and to link both, cell- and gene-state space trajectories.


Oral potentially malignant disorders (OPMDs) encompass histologically benign, dysplastic, and cancerous lesions that are often indistinguishable by appearance and inconsistently managed. We assessed the potential impact of test-and-treat pathways enabled by a point-of-care test for OPMD characterization.We constructed a decision-analytic model to compare life expectancy of test-treat strategies for 60-year-old patients with OPMDs in the primary dental setting, based on a trial for a point-of-care cytopathology tool (POCOCT). Eight strategies of OPMD detection and evaluation were compared, involving deferred evaluation (no further characterization), prompt OPMD characterization using POCOCT measurements, or the commonly recommended usual care strategy of routine referral for scalpel biopsy. POCOCT pathways differed in threshold for additional intervention, including surgery for any dysplasia or malignancy, or for only moderate or severe dysplasia or cancer. Strategies with initial referral for biopsy also reflected varied treatment thresholds in current practice between surgery and surveillance of mild dysplasia. Sensitivity analysis was performed to assess the impact of variation in parameter values on model results.Requisite referral for scalpel biopsy offered the highest life expectancy of 20.92 life-years compared with deferred evaluation (+0.30 life-years), though this outcome was driven by baseline assumptions of limited patient adherence to surveillance using POCOCT. POCOCT characterization and surveillance offered only 0.02 life-years less than the most biopsy-intensive strategy, while resulting in 27% fewer biopsies. When the probability of adherence to surveillance and confirmatory biopsy was ≥ 0.88, or when metastasis rates were lower than reported, POCOCT characterization extended life-years (+0.04 life-years) than prompt specialist referral.Risk-based OPMD management through point-of-care cytology may offer a reasonable alternative to routine referral for specialist evaluation and scalpel biopsy, with far fewer biopsies. In patients who adhere to surveillance protocols, POCOCT surveillance may extend life expectancy beyond biopsy and follow up visual-tactile inspection. Worldwide, there are over 500,000 new cases of oral cavity and pharyngeal cancer diagnosed each year . In the The American Dental Association recommends screening for OPMDs as a part of routine dental care in order to improve the chances of early intervention for cancerous lesions. Still, examination and management remain inconsistent in dental care, where most OPMDs are typically encountered., 10 SomeA novel point-of-care cytological test called the Point-of-care Oral Cytopathology Tool (POCOCT) offers precision diagnostic information to guide lesion management and mitigate the potential harms of both under- or over-testing. This computer-vision based technology rapidly scans and categorizes cells collected by mucosal brushing, and combines these features with other clinical risk predictors to determine the likelihood of dysplasia or malignancy. In a diagnostic performance trial involving 714 prospectively recruited patients with OPMDs with matched histopathologic diagnoses, lesion categories were evaluated across the spectrum of disease from benign to dysplasia to carcinoma; POCOCT pFig 1).A state-transition model was developed using TreeAge Pro version 2019 . A state-transition (or Markov) model is a type of mathematical model for simulating patients through a sequence of particular health states [The aim of opportunistic screening for oral cancer/OPMDs is to have a high sensitivity for detecting significant disease (e.g. early cancers or severely dysplastic lesions). Having stated that, mildly or moderately dysplastic OPMDs can progress and transform. As such, the threshold/cut-off that we set for POCOCT can have trade-offs. A high threshold results in treatment of only higher grade disease and monitoring lower grade disease that could transform to cancer. A low threshold of treatment risks over-referral for unnecessary specialist evaluation/biopsies and possibly minimal improvement in cancer-specific outcomes. The overall performance of POCOCT for each histopathologic cut-off (or split) was generated from a prior trial . As suchFig 2). These POCOCT pathways were compared against alternative strategies: prompt referral for specialist evaluation and biopsy of all OPMDs visually detected by primary care dentists (i.e. representing the recommended practice pattern), or deferral of further evaluation of OPMDs by a specialist unless frankly malignant (i.e. representing a less common practice pattern) [Using each diagnostic split to create multiple possible test-treat pathways for OPMDs, we compared life expectancies using the model. This assessment included varied thresholds for further evaluation or treatment based upon the different diagnostic evaluations. In the standard care test-and treat pathways the decision to treat is based on the various histopathological diagnoses rendered by specialist biopsy. In the non-invasive pathways, the decision to refer and treat is based on a positive POCOCT test outcome and given that the threshold or cut-off for a positive POCOCT test is an accurate surrogate for histopathology, it is possible to set these test thresholds across multiple diagnostic splits. The sensitivity and specificity of POCOCT for each diagnostic split was defined in a prior diagnostic accuracy trial , 18. Thepattern) . StrategThe complete list of strategies included: (1) deferral of further evaluation of visible OPMDs by a specialist; (2) prompt referral for specialist evaluation and biopsy for OPMDs, surgery if biopsy is positive for any grade of dysplasia or carcinoma, and no option for surveillance; (3) prompt referral for specialist evaluation and biopsy for OPMDs, surgery if biopsy is positive for any grade of dysplasia or carcinoma, and surveillance for benign lesions; (4) prompt referral for specialist evaluation and biopsy for OPMDs, surgery if biopsy is positive for moderate or severe dysplasia or carcinoma, and surveillance for benign lesions and mild dysplasia; (5) POCOCT benign|mild dysplasia diagnostic split, with surgery for any dysplasia or carcinoma, and surveillance for benign lesions; (6) POCOCT mild|moderate dysplasia diagnostic split, with surgery for moderate or severe dysplasia or carcinoma, and surveillance for benign lesions and lesions with mild dysplasia; (7) POCOCT “low risk|high risk” diagnostic split, with surgery for “higher-risk” dysplasia (encompassing more severe degree of moderate dysplasia and severe dysplasia) or carcinoma, and surveillance for benign and “lower-risk” dysplasia; and (8) POCOCT moderate|severe dysplasia diagnostic split, with surgery for severe dysplasia and carcinoma, and surveillance for benign lesions or lesions with mild or moderate dysplasia.To compare against strategies representing prompt screening and evaluation for OPMDs, one model pathway represented deferral of OPMD management and characterization and its consequences. In the deferral strategy, only symptomatic lesions were referred .In the strategies with prompt referral for biopsy of OPMDs (strategies 2–4), the indications for surgery were varied according to the histopathology of the lesion. In one biopsy strategy (#2), the patients with benign results did not undergo visual surveillance, in order to model effects of possible sampling error with biopsy. In strategy 3, patients underwent surgery if biopsy was positive for any dysplasia or carcinoma and benign results warranted visual examinations every six months for the first year and thereafter annually up to five years. Likewise, other strategies involving visual exam-based surveillance of biopsy findings below the treatment threshold applied a schedule of every six months for the first year and thereafter annually up to 5 years. In the case of deferred oral cavity screening, patients with localized stage (stage 1 or 2) malignant lesions had 13% probability of presenting for a visual-tactile exam followed by scalpel biopsy and treatment , 22.In the four strategies with OPMD management based on POCOCT results, we used the diagnostic risk categories to vary the treatment threshold, such that in the most conservative treatment approach OPMDs were referred for scalpel biopsy and treated if any degree of dysplasia or carcinoma was indicated by POCOCT (Strategy 5), and alternatively, only progressively higher thresholds for biopsy and treatment were pursued (Strategies 6–8). Surveillance in these point-of-care testing strategies entailed repeat POCOCT evaluation in 6 months, then at 1 year, and then yearly up to 5 years. In the model’s surveillance strategies, patients were subject to the accuracy of the follow up test  and then assumed to receive recommendation for scalpel biopsy for a positive exam.Table 1). The sensitivity and specificity of POCOCT for each diagnostic split was informed using diagnostic trial findings [The model incorporated patient age, comorbidity status, misclassification of histology along the spectrum of dysplasia and malignancy, and risks of cancer progression. All-cause mortality rates were derived from U.S. life tables . Comorbifindings . The basfindings . The profindings .Table 2) according to the classification [We also calibrated the progression of mild to moderate dysplasia and early stage oral cancer to late stage oral cancer using similar methods; early stage represented a combination of American Joint Committee on Cancer stage I and II while the late stage combined AJCC stage III and IV . Likewise, the POCOCT strategies that entailed referral and treatment of POCOCT positive results at the benign|mild dysplasia split (with surveillance of benign results by the primary care dentist), or at the mild|moderate dysplasia split (with surveillance of mild dysplasia and benign results) resulted in the same life expectancy of 20.90 years. Therefore, there was a small difference in life expectancy with use of POCOCT to monitor mild dysplasia as compared with biopsy of all OPMDs . Other POCOCT-based strategies with higher thresholds for treatment at the low|high split or the moderate|severe dysplasia split offered lower life expectancy. Finally, the pathway modeling deferred evaluation yielded a life expectancy of at least 0.25 years less than any of the active evaluation strategies. Similar results were found for 60-year-old women  in the POCOCT strategy that referred patients with POCOCT + results at the mild|moderate split to specialists, including 29% fewer biopsies for benign lesions over the same period (721 vs. 513 per 1000 patients).The number of oral cavity carcinomas diagnosed and treated in early stage and late stage cancer were estimated in each strategy for 1 million patients The uncertainty around key model inputs was examined using a sensitivity analysis. Within the tested ranges of variable values, model results were most sensitive to the probability of adherence to surveillance protocols, adherence to biopsy recommendations, probability of death from metastatic OSCC and the probability of developing distant metastasis with untreated OSCC. When the monthly probability of metastasis from untreated OSCC was less than 0.01, the most favorable strategy was the use of POCOCT to refer only those with a POCOCT + test at the mild|moderate split. In two-way sensitivity analysis, the adherence to surveillance and to biopsy recommendations were varied and favored the use of POCOCT to refer and treat only those with a POCOCT + test at the mild|moderate split when adherence to both was ≥0.88.Because late-stage diagnosis of OSCC has been a barrier to improving cancer-specific outcomes, emphasis has been placed on the need for more effective screening and specifically, adjunctive diagnostic testing to aid OPMD characterization and management. The advantage of a cytopathological test is that lesions can be retested at follow-up visits, thereby allowing the monitoring of evolving OPMDs in a primary dental setting. We built a decision-analytic model based on a diagnostic accuracy trial of a point-of-care test for precision OPMD characterization and compared the effectiveness of this risk-assessment tool with common clinical strategies . A key fFurthermore, when adherence to surveillance was higher (probabilities ≥0.88), POCOCT-based management for OPMDs extended life expectancy compared with referral, due to the possibility of surgical mortality . This further supports the potential of risk-based OPMD management using this point-of-care test in the primary dental clinics, since patients could elect through informed decisions to serially monitor visible lesions non-invasively and receive call-backs if surveillance visits were missed. The model results were also driven by the prevalence of severely dysplastic or malignant OPMDs at the time of initial detection. Although POCOCT’s imperfect sensitivity meant that more dysplasia or cancers were initially missed than with initial biopsy, repeat visits for surveillance served to capture early-stage OSCC before progression to late-stage cancer, substantially lowering the rates of metastatic disease development compared with deferred evaluation.In 2014, the United States Preventive Services Task Force concluded that there was insufficient evidence to recommend routine screening for oral cavity cancer by primary care physicians even for high-risk patients . HoweverThe primary limitations of our study include the simplifying assumptions inherent to any decision analytic model. Retrospective studies on the progression of dysplastic lesions may not reflect accurately the natural progression of dysplasia to carcinoma, though we estimated the likely transformation rates using calibration targets from patient studies . We addrIn conclusion, we assessed the projected long-term clinical outcomes of point-of-care characterization of OPMDs with a brush-based, cytological artificial intelligence-assisted device. Using POCOCT, a surveillance protocol for POCOCT negative results (both benign|mild and mild|moderate dysplasia splits) in visible lesions extended life expectancy compared to deferred evaluation of the oral cavity for OPMDs. Even with suboptimal patient adherence, POCOCT offered only slightly less life expectancy benefit than routine specialist referral for biopsy while substantially reducing referrals and biopsies for benign lesions or mild dysplasia. The major potential risk of a surveillance program using POCOCT is the possibility for a false negative result, particularly in patients who do not adhere to surveillance and have limited opportunity for retesting. One solution may be to offer patients bifurcated options based on their preferences for non-invasive monitoring versus immediate intervention. These results may guide design of clinical validation trials that establish the benefit-risk profiles of more tailored management, including benefits of more frequent detection of early-stage OSCC and high-grade dysplasia, and substantially fewer specialist referrals for low-risk lesions.S1 Table(DOCX)Click here for additional data file.


Background: The QRS-T angle from the surface EKG is a promising prognostic marker in patients with coronary artery disease. Cardiovascular magnetic resonance (CMR) imaging with late gadolinium enhancement (LGE) offers high resolution imaging of myocardial damage. We investigated the association of the QRS-T angle and the extent of myocardial damage as assessed by LGE in patients with acute ST-segment myocardial infarction (STEMI)Methods: 169 patients with STEMI obtained a standardized digital 12-lead EKG on admission for the calculation of the QRS-T angle and underwent CMR imaging for analysis of infarct size by LGE within the first week. Patients were divided into groups: (1) abnormal QRS-T angle ≥ 90 degree and (2) QRS-T angle < 90 degree.Results: Patients with a QRS-T angle of 90 degree or more had larger infarcts  and lower ejection fraction .Conclusion: The extent of myocardial damage as measured by the gold standard LGE is associated with a larger QRS-T angle calculated from the surface EKG. Early restoration of epicardial blood flow by percutaneous coronary intervention (PCI) or thrombolysis is the gold standard of ST-segment elevation myocardial infarction (STEMI) treatment Apart from clinical risk scores, biomarkers and imaging it would be extremely helpful to establish a simple parameter from the routine surface ECG for risk stratification in acute STEMI ≥90°) seems to correlate with electrical instability and has been related to cardiac prognosis in several studies The QRS-T angle represents the spatial angle between the QRS loop (depolarization) and T loop (repolarization) Cardiovascular Magnetic Resonance (CMR) imaging following gadolinium contrast administration allows for high-resolution imaging of myocardial damage This study aims to investigate the correlation of the QRS-T-angle on the 12-lead surface ECG and the extent of myocardial damage in patients with acute STEMI as assessed by late gadolinium enhancement CMR.Consecutive patients admitted to the Department of Cardiology and Angiology at Elisabeth Hospital Essen from February 2015 until March 2017 with acute ST-segment elevation myocardial infarction (STEMI) were enrolled into the study. Patients obtained a standardized 12-lead digital ECG immediately on admission using a Schiller Cardiovit AT 102 plus®. Computerized values of QRS and T-wave axis were given by the Schiller AT 102 plus® software. The frontal QRS-T-angle was calculated as the absolute difference between the frontal QRS- and frontal T-wave axes. With a value greater than 180°, 360° were subtracted in order to give a continuous variable ranging from 0° to 180°.CMR imaging was performed on a 1.5 Tesla MR System  not later than 7 days after admission. MR sequences were acquired using a phased-array receiver coil during end-inspiratory breathholding and in line with the recommendations of the Society of Cardiovascular Magnetic Resonance (SCMR) Cine and LGE images were evaluated blinded to the clinical data by two experienced observers in consensus. LV function was analyzed by outlining epicardial and endocardial borders on the short axis SSFP sequences. Papillary muscles were excluded from analysis. Left ventricular volumes and ejection fraction were derived from contour summation. The extent of late gadolinium enhancement was assessed on long and short axis contrast images by using the 17-segment model of the LV. Typical expansion of LGE in patients with acute myocardial infarction (STEMI) is defined as areas of high signal intensity progressing from the subendocardial myocardial layer. Areas with ischemic infarct scar were defined as enhanced with a signal intensity above at least five SD as compared to non-infarcted remote myocardium. The presence and transmurality of infarcts were scored using the 17-segment model and a 5-point scala per segment . The total infarct size as expressed as percentage of LV mass was calculated as follows: sum of enhanced segments and their transmurality score (LGE score) divided by 17 segments multiplied by the maximum transmurality score Statistical analyses were performed with the Excel™ software . Depending on the distribution continuous variables were expressed as means with standard deviation. Patients were divided into two groups . The two-sample t-test was chosen to analyze differences between these two groups. Pearson correlation and one-sample t-test were applied to investigate the correlation of QRS-T angle and CMR parameters in each group. P values <0.05 were considered as statistically significant.From February 2015 to March 2017, 169 patients  admitted for acute STEMI  were enrolled.Patients were divided into two groups: 1) patients with a frontal QRS-T angle of 90 degree or more (n=54), (2) patients with a frontal QRS-T angle of less than 90 degree (n=114)  and the heart rate on admission EKG was higher . There was no difference between the two groups with regards to maximum creatinkinase  and maximum troponine .On CMR imaging Figure , Table 1Our study shows that in a population of STEMI patients QRS-T angle on the surface EKG correlates with infarct size on contrast-enhanced CMR.Previously several studies have been able to demonstrate the prognostic value of QRS-T angle in different patient populations. In a general Rotterdam population of 6,134 patients Kors et al. found an odds ratio of 5.6 for sudden cardiac death, which was superior to standard EKG indicators of adverse events such as ST depression and T wave inversion Several ECG signs have been evaluated against distribution, transmurality and extent of late gadolinium enhancement on CMR following gadolinium contrast administration: In acute STEMI the resolution of ST-segment elevation, a marker of successful reperfusion therapy, relates to LGE and prognosis The concept of validating EKG parameters by scar (LGE) rather than left ventricular morphology, hypertrophy and function has also been investigated in the setting of non-ischemic cardiomyopathies: In hypertrophic cardiomyopathy late gadolinium enhancement is a strong and independent prognosticator of cardiac death For a prognostic EKG marker that represents myocardial depolarization and repolarization in the way the QRS-T angle does, the correlation to myocardial pathology on CMR is particularly useful. To the best of our knowledge, this is the first study that relates QRS-T angle in patients with acute myocardial infarction (STEMI) to CMR function and scar.Several non-invasive parameters that could help risk stratify patients with acute myocardial infarction are used in clinical routine. These include left ventricular ejection fraction, left ventricular volumes, several ECG parameters and detection of myocardial necrosis by contrast-enhanced cardiovascular magnetic resonance imaging. The ultimate goal in risk stratification is to identify patients who will suffer from sudden cardiac death and who need further protection, e.g. the implantation of cardioverter-defibrillator (ICD). However, up till now no single parameter has been successful to fulfill the task. In fact, due to the complex pathophysiology of ventricular arrhythmias a combination of non-invasive parameters might improve the prediction of ventricular arrhythmias in post acute MI patients. The QRS-T angle in patients with acute MI can be ubiquitously assessed and might add to clinical risk stratification in the future.In conclusion, a QRS-T angle of 90 degrees or more is associated with lower ejection fraction and greater infarct size as assessed by contrast-enhanced CMR. Prospective studies in a larger STEMI patient population with clinical follow-up should be carried out in order to further investigate the prognostic impact of the QRS-T angle and its potential implication on clinical workflow.