Drosophila melanogaster exhibits similar psychostimulant responses to humans, a high degree of gene conservation, and allow performance of behavioral assays in a large population. Additionally, amphetamine and methylphenidate reduce impairments in fly models of ADHD-like behavior. Therefore, Drosophila represents an ideal translational model organism to tackle the genetic components underlying the effects of psychostimulants. Here, we break down the many assays that reliably quantify the effects of cocaine, amphetamine, methamphetamine, and methylphenidate in Drosophila. We also discuss how Drosophila is an efficient and cost-effective model organism for identifying novel candidate genes and molecular mechanisms involved in the behavioral responses to psychostimulant drugs.The addictive properties of psychostimulants such as cocaine, amphetamine, methamphetamine, and methylphenidate are based on their ability to increase dopaminergic neurotransmission in the reward system. While cocaine and methamphetamine are predominately used recreationally, amphetamine and methylphenidate also work as effective therapeutics to treat symptoms of disorders including attention deficit and hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). Although both the addictive properties of psychostimulant drugs and their therapeutic efficacy are influenced by genetic variation, very few genes that regulate these processes in humans have been identified. This is largely due to population heterogeneity which entails a requirement for large samples.  Psychostimulants with abuse potential, such as cocaine, amphetamine, methamphetamine, and methylphenidate, are drugs that increase central nervous system activity and arousal . These dDrosophila melanogaster enables high-throughput analysis of behavior in assays that can scale to accommodate thousands of flies while supporting targeted genetic and molecular manipulations [Drosophila can help elucidate the genetic basis of behavioral response to psychostimulant drugs to improve our understanding of human addiction and psychopathology.One approach to answering these questions is to take advantage of model organisms that are experimentally amenable and frequently used for their powerful genetics. Genes are a primary determinant of variation in behavioral responses to the addictive or therapeutic effects of psychostimulant drugs ,12, and ulations ,19. ThisDrosophila is a powerful model organism for studying the genetic and molecular basis of behavioral responses to drugs of abuse. A fast generation time, excellent genetic tractability, and a broad range of molecular tools enable precise spatiotemporal manipulation of gene expression, protein function, and cellular activity in Drosophila [Drosophila research [Drosophila is a valuable model for studying complex addictive disorders such as alcoholism [Drosophila alcohol responses have helped identify corresponding genes that contribute to alcohol use disorder (AUD) in humans [Drosophila alcohol responses [Drosophila to identify novel genes and mechanisms involved in human AUD provides the rationale for performing similar studies to investigate other addictive drugs. Psychostimulants increase dopaminergic signaling in flies as well as humans, but little is known about the genes, molecules, and pathways that influence the behavioral response to psychostimulants, making Drosophila a great model organism to answer these questions and provide valuable translational information.osophila ,21,22,23osophila , highligresearch . Additiocoholism . Studiescoholism , and numn humans . Additioesponses , includiesponses  and in hesponses . The sucExperiences that benefit biological fitness, such as food or sex, are perceived as rewarding . The assDrosophila is a model with high face validity for studying the behavioral response to psychostimulants.Psychostimulant administration in rodents leads to dose-dependent changes in motor behaviors including grooming, locomotion, dyskinesia, and seizures . Flies eDrosophila dopamine transporter (dDAT) and vesicular dopamine transporter (dVMAT) [Drosophila with 3-iodo-tyrosine reduces the motor-activating effects of cocaine [Drosophila reduces self-administration of methamphetamine [Drosophila as a model organism for studying the genetic underpinnings of response to psychostimulant drugs.Dopaminergic signaling is central to psychostimulant-induced behavioral responses. In mammals, the locomotor activating effects of psychostimulants require increased dopaminergic signaling in the neural substrates associated with reward . Disrupt (dVMAT) . Disrupt cocaine , blockin cocaine , and muthetamine . These eDrosophila.Exposure to psychostimulants impacts many behaviors, including locomotion, sleep, arousal, attention-like processes, learning, memory, and social interactions. These behaviors have been studied using assays that quantify motor behaviors, feeding behavior, and attention-like processes. Psychostimulant-induced changes in fly motor behavior produce motor-activating and motor-impairing effects. Assays corresponding to each of these responses allow measurement of sensitivity and sensitization to the activating or impairing effects of psychostimulants. Sensitization involves the development of increased sensitivity to the effects of a drug across subsequent exposures, and is associated with neuroadaptations that increase the salience of stimuli associated with drug reward . In assaDrosophila behavior, is time-intensive, and subjective. One automated option that provides objectivity and can decrease the time spent scoring the motor activating effects of psychostimulants is the Drosophila activity monitor (DAM).One of the most obvious effect of psychostimulants is the activation of motor-related behaviors, an effect that is highly conserved across model organisms. Flies exposed to increasing concentrations of cocaine transition through phases of increased grooming behavior, hyperlocomotion, loss of negative geotaxis, stereotyped circling behaviors, erratic movements such as twirling, akinesia, seizures, and even death. One of the first ways this behavior was quantified was assigning a behavioral severity score using a behavioral scoring assay. Experimenters rated the activity of drug exposed flies from 0 to 7 using an ordinal scale of defined behavioral responses a. This bThe DAM system is a high-throughput automated method of behavioral analysis that uses an infrared beam to detect motor activity . Flies iWhile each psychostimulant administration method produces measurable changes in behavior, it is important to consider tradeoffs in speed of delivery, precision of dosing, and how disruptive each method is to fly behavior. Drug-supplemented food provides the least control of speed and precision, but does not disrupt fly behavior. Drug injection is time consuming and disturbs normal fly behavior, but provides strict control of the dose. The FlyBong , designeMultiple methods of drug administration can be combined with the DAM system, as demonstrated in a modified DAM where flies in each cuvette have access to capillary tubes filled with feeding solutions of 100 mM sucrose and 100 mM sucrose supplemented with methamphetamine . Out of Drosophila. Locomotor responses to psychostimulant drugs in adult or larval Drosophila can be measured by video recording responses in an observation chamber. Adult Drosophila [While the constrained analysis of locomotion in the DAM is a reliable automated method for measuring the motor activating effects of psychostimulant drugs, some behaviors cannot be measured within the interior of a glass cuvette. Several assays that address this issue allow measurement of locomotion in freely moving osophila  and 3rd osophila  fed cocaosophila ,74,75.Drosophila have also been studied by analysis of repetitive startle-induced hyperactivity (ReSH), which is based on locomotor response to recurrent puffs of air [Psychostimulant responses in s of air . Air pufs of air ,78. Recos of air . Activits of air . Cocaines of air .Drosophila courtship is a complex social behavior, with successful copulation requiring the integration of audible [Drosophila behaviors, such as response to visual stimuli, optimal responses occur within a specific range of dopaminergic activity and are disrupted when dopamine levels are outside of that range [While psychostimulants activate some motor behaviors, they can impair others. This is the case for negative geotaxis  and some audible  visual [ audible  olfactor audible  and gust audible  stimuli.at range . It is lDrosophila, dating back to Seymour Benzer’s experiments in the 1960s [Drosophila offer. Untreated control flies quickly climb to the top of the tube, whereas flies exposed to cocaine exhibit dose-dependent impairments in negative geotaxis [Startle-induced negative geotaxis (SING) was one of the first behavioral assays developed in he 1960s . Benzer geotaxis   [Assays of voluntary consumption are indispensable techniques for studying addiction and can be used to measure increases in self-administration over time. The CApillary FEeder (CAFE) assay  is a rel at 24 h . When th at 24 h . A simil at 24 h . In contl (DGRP) ,93. Addil (DGRP)  allow mel (DGRP) . TogetheDrosophila, attention-like processes have been studied by measuring responses to visual stimuli in the optomotor maze , latrophilin-3 (LPHN3), and neurofibromin-1 (NF1). These three genes are associated with ADHD in humans, and were chosen to test whether disruption of the fly ortholog would mimic ADHD-like behaviors in Drosophila [Drosophila genes with phenotypic descriptors related to hyperactivity, excitability, or attention. This search produced a list of 78 genes, with 69 conserved in humans. Five of those conserved genes, including DAT, were also identified in a list of 91 human ADHD-associated genes. The Drosophila DAT null mutant fmnDAT was identified based on its sleep phenotype—fmn stands for fumin, Japanese for sleepless [fmnDAT flies using the DAM system showed hyperactivity and reduced sleep, with hyperactivity exacerbated in the absence of light, e.g., at night, but also during the subjective daytime when housed in dark:dark conditions. Feeding fmnDAT flies methylphenidate [Drosophila DAT, the orthologue of a well-known ADHD risk gene highly represented in human GWAS studies, recapitulates two common symptoms of ADHD observed in humans that can be rescued by psychostimulant drugs used to treat the symptoms of human ADHD.Hyperactivity and reduced sleep are common behavioral symptoms of ADHD ,119,120.osophila . The dopleepless . Observahenidate  or amphehenidate  rescued DAT knockout is useful for modeling the effects associated with loss of function, there is significantly more variability in the DAT mutations that contribute to the prevalence of ADHD in humans [DAT variants exhibit heterogenous molecular phenotypes, including differences in DA uptake kinetics, reverse transport, and altered binding to psychostimulant drugs [Drosophila is an efficient model for studying the effects of human DAT mutants in vivo and have shown that DAT mutations associated with early-onset Parkinson’s [Drosophila is a feasible system for performing similar experiments to unravel the behavioral and molecular nature of DAT variants associated with ADHD, offering a practical model to identify the molecular mechanisms involved in response to psychostimulant drugs.While n humans ,123,124.nt drugs . Drosophkinson’s  as well kinson’s  lead to LPHN3 is an ADHD-risk gene that was identified in a linkage study based on a prevalence of ADHD with large generational families of an isolated population in the Paisa region of Colombia [LPHN3 variants that lead to haploinsufficiency are associated with the development of ADHD in humans [LPHN3 risk alleles respond to stimulant medication, a molecular mechanism linking reduced LPHN3 expression to dopaminergic dysfunction has not been unraveled in human studies [LPHN3 fly ortholog have also been studied in Drosophila using the GAL4/UAS binary expression system. This method uses a cell type or tissue-specific enhancer to drive expression of the yeast transcriptional activator GAL4. Transgenes coupled to the GAL4 upstream activation sequence (UAS), are expressed wherever GAL4 is transcribed. The pan-neuronal driver elav-GAL4 was used to drive expression of UAS-Cirl-RNAi, the single Drosophila orthologue for the Latrophilin family of genes. This reduced complexity can be beneficial when attempting to characterize a gene’s molecular function. In mammals, the effects of mutating a gene that is a member of a multi-gene family can be difficult to observe because of genetic redundancy. In flies, the reduction in gene copy number makes characterizing loss of function phenotypes more straightforward. Neuronal knockdown of the Drosophila orthologue of LPHN3 was sufficient to produce the hyperactivity and decreased sleep associated with dopaminergic dysfunction [DAT null mutants, hyperactivity was more pronounced at night and could be exacerbated during the day by turning off the lights. Both the hyperactivity and sleep loss were rescued by feeding Drosophila methylphenidate. Tyrosine hydroxylase staining in fly brains showed that knockdown of the Drosophila orthologue of LPHN3 did not alter the distribution or survival of dopaminergic neurons, indicating that LPHN3 regulates dopaminergic signaling directly [LPHN3 in dopaminergic signaling and variation in response to psychostimulant drugs is poorly understood. Drosophila is a useful model for studying neurotransmission [LPHN3 disruption.In contrast to DAT, the influence of the G-Protein coupled receptor and cell adhesion protein Latrophilin-3 on dopaminergic signaling is poorly understood. Colombia . LPHN3 vn humans . While p studies . The behfunction . As withdirectly . The rolsmission ,130 and NF1 is associated with the autosomal dominant disease neurofibromatosis type I. Neurofibromatosis type I is a multi-system disorder involving tumors of the nervous system that lead to complications such as eye disease, musculoskeletal disorders, and epilepsy, among others. In addition to the physical impairments associated with neurofibromatosis type I, most patients display cognitive deficits [NF1 would lead to ADHD-like behavior in flies, NF1 was knocked down in all neurons of the Drosophila by using elav-GAL4 to drive expression of UAS-NF1-RNAi. Knockdown of NF1 in Drosophila neurons induced a hyperactivity and sleep-deficiency phenotype, with a noticeable increase in nighttime hyperactivity. Feeding NF1 knockdown flies methylphenidate rescued both hyperactivity and sleep dysregulation [deficits , includideficits . In somedeficits . To invegulation .DAT, LPHN3, and NF1 orthologue mutants in the DAM system indicate that (1) hyperactivity along with reduced sleep is an endophenotype for ADHD in flies, (2) flies are a model with mechanistic validity for studying ADHD such that genes whose disruption leads to ADHD in humans also produce ADHD-like behavior in flies, and (3) flies are a model with predictive validity for studying ADHD since the psychostimulant drugs that reduce the behavioral symptoms of ADHD in humans also reduce ADHD-like impairments in flies. While DAT, LPHN3, and NF1 all respond to treatment with methylphenidate, the molecular basis for the psychostimulant-mediated rescue of ADHD-like behaviors is unknown. Targeted knockdown of DAT, LPHN3, or NF1 specifically in Drosophila dopamine neurons or subsets of dopamine neurons might help unravel how mutations with opposite effects on dopaminergic signaling can display similar response to psychostimulant drugs such as methylphenidate.The psychostimulant-mediated rescue of hyperactivity as well as sleep-deficiency phenotypes observed for Drosophila can model more complex ADHD-like symptoms associated with dysregulation of attention-like processes [Drosophila memory mutant radish1 also displays impaired responses to visual-stimuli in the optomotor maze and experiments of tethered flight [radish1 mutants flies [radish1 mutants are successful in operant visual learning [radish1 mutants, generating responses to visual stimuli similar to those observed for wild-type flies in the optomotor maze [radish1 mutants flies also exhibit altered brain activity in response to visual stimuli. Normally, flies display an increase in 20–30 Hz local field potentials (LFP)—observed by brain recordings—in response to visual stimuli. Electrophysiological recordings from flies suspended in a flightless arena show that flies repeatedly shown the same shape will display an increase in 20–30 Hz response when presented a novel shape. In contrast, radish1 mutants have a diminished 20–30 Hz response to novel visual stimuli. Treatment with methylphenidate also rescues the 20–30 Hz response to novelty in radish1 [In addition to the locomotor signature observed with ADHD-like changes in sleep and hyperactivity, rocesses . Humans rocesses ,134,135 d flight . Wild-tyts flies . This detor maze . In addi radish1 .radish1 mutants and pharmacological rescue with methylphenidate demonstrate how the optomotor maze and tethered flight experiments can identify genetic and molecular determinants of attention span. Moreover, these assays can uncover the molecular basis of psychostimulant-mediated rescue in attention-like processes such as visual fixation, novelty response, and distractibility. However, with radish1 as the sole example of psychostimulant-mediated rescue of attention-like processes, these assays would benefit from validation using other known ADHD-linked mutants. The observation of similar deficits in ADHD-associated mutants, along with improvement following treatment with psychostimulants, would provide support for impaired responses in the optomotor maze and tethered flight experiments as endophenotypes for attention-like processes dysregulated in ADHD. Because ADHD is predominantly a polygenic disorder, with impairments occurring on a spectrum, it is unlikely that a single behavioral assay will be effective in studying every putative risk gene. The current models of ADHD-like behavior in flies address two broad features of ADHD: dysregulation of attention-like processes and dysregulation of arousal. Complementary use of the DAM system, optomotor maze, and flight loop experiments may be useful in unraveling how specific behaviors are improved with psychostimulant treatment, supporting the identification of genes and pathways that determine treatment efficacy.The impairment of visual attention-like processes observed in LPHN3 and NF1 have a large effect size, meaning a large portion of observed phenotypic variance can be attributed to these individual genes. In contrast, common genetic variants associated with ADHD have a smaller effect size, meaning their individual contribution to observed phenotypes is less severe. Rare disease variants, because of their increased severity, can be helpful in elucidating molecular mechanisms that can help understand the deficits observed in mild cases disease. However, identifying the common variants that contribute to mild-cases of diseases has been more difficult. Because these common variants have a small effect size, it is difficult to perform studies with enough subjects to reach the statistical power necessary to identify common risk genes. The first successful identification of common variant risk was performed in a 2019 GWAS that used data from more than 50,000 subjects to identify of 12 ADHD risk loci [Drosophila offer a model where additional genetic tools can circumvent the limitations associated with underpowered research. A recent study combined transcriptome and behavioral analysis across hundreds of methylphenidate exposed flies, to behavioral analysis of 172 lines from the DGRP to identify genetic variants that contribute methylphenidate response [Drosophila is a model that enables identification of novel candidate genes that mediate response to treatment with psychostimulant drugs, information that might also help elucidate differences in genetic etiology that contribute to treatment resistance in humans.Rare genetic variants associated with ADHD such as isk loci . In contresponse . The sturesponse , a proceresponse . This trDrosophila [Most cases of ASD are polygenic and 80% of cases involve multiple genes, making it difficult to unravel how each gene contributes disease-related impairments . Additioosophila ,108,109.Drosophila to functionally characterize an ASD-associated de novo mutation in the human dopamine transporter (hDAT) at residue 356 (hDAT T356M). [Drosophila DAT knockout background (fmnDAT). hDAT-T356M expressing flies displayed increased baseline locomotor activity compared to control flies expressing hDAT. Additionally, while amphetamine increases locomotor activity in hDAT expressing flies, Drosophila expressing the ASD-associated mutant do not display an amphetamine-induced increase in locomotion [Hamilton and colleagues used  T356M).  Cell culcomotion .DAT transgene was expressed in DAT knockout flies (fmnDAT), and compared to hDAT expressing controls. Flies expressing hDAT-ΔN336 displayed hyperactivity and increased grooming behavior compared to hDAT expressing control flies [A similar behavioral phenotype was observed in flies during an experiment involving the first characterization of an ASD-associated in-frame deletion in the human dopamine transporter (hDAT), which eliminates asparagine residue 336 (hDAT-ΔN336) . Cell cuol flies . Flies eol flies .DAT mutant that impacts psychostimulant response contains an arginine to tryptophan substitution at N-terminal position 51 (hDAT-R/W), a residue important for interaction with the membrane protein Syntaxin-1A (STX1A) [fmnDAT flies expressing the ASD-associated variant were used to characterize how this mutation impacts behavior. No difference in basal locomotion was observed between hDAT-R/W expressing flies and hDAT expressing control flies. However, hDAT-R/W expressing flies exhibited a significant reduction in amphetamine-induced locomotion compared to hDAT expressing control flies [Another ASD-associated  (STX1A) . Murine  (STX1A) . Experim (STX1A) . DATfmn ol flies .Drosophila display broad repertoire of social behaviors that range from population scale changes in group dynamics [Drosophila, and both respond to changes in dopamine. As social deficits are common in fly models of ASD, Drosophila could provide an effective model to study whether psychostimulant drugs can rescue social responses. These experiments could help identify specific genes and molecules required for psychostimulant-mediated improvements in social behavior.These experiments demonstrate how flies be used to study the behavioral impact of mutations, linking the behaviors in question to molecular mechanisms characterized in cell culture. These experiments cannot be performed in humans, and can help characterize how specific mechanisms contribute to the pathogenesis of complex polygenic disorders such as ASD . While tdynamics  to pairwdynamics  and courdynamics . Quantifdynamics  and analdynamics  are bothDrosophila, and highlight their relevance in modeling psychostimulant addiction.Addiction is an etiologically complex polygenic disorder that develops in an experience-dependent manner. In humans, features of addiction include increases in drug preference and consumption. Individual differences in sensitivity to the behavioral activating effects of psychostimulant drugs, the development of sensitization, initial preference, and patterns of drug consumption are all important factors predictive of future psychostimulant response and propensity to develop substance use disorder. Studies of psychostimulant-induced behavior in mammals show that simple locomotor responses can be predictive of more complex addiction-related behaviors such as the acquisition of self-administration. In rats, for example, sensitivity to the locomotor activating effects of cocaine predict the development of cocaine-conditioned place preference . Here, wDrosophila, including locomotion, attention-like processes, memory, and reward [While the intoxicating effects of commonly abused psychostimulants vary in intensity and duration, they all act to increase dopaminergic signaling . Dopamind reward ,150,151.Drosophila larvae ablates locomotor response to amphetamine [pale because of their discoloration [pale mutants have reduced brain dopamine, are hypoactive [Tyrosine hydroxylase is the rate limiting enzyme involved in the conversion of L-tyrosine to dopamine . RNAi-mehetamine . Tyrosinloration . pale mupoactive ,155, andpoactive . Reducinpoactive .dDAT codes for the Drosophila plasma membrane dopamine transporter (DAT) that functions to remove extracellular dopamine from the synaptic cleft [Drosophila and humans [Drosophila, the activating effects of psychostimulants on arousal are dopamine transporter dependent. Both amphetamine and methylphenidate increase locomotion in Drosophila larvae, a response that is blunted in fmnDAT flies or flies expressing dDAT RNAi in dopaminergic neurons. Expression of the human dopamine transporter (hDAT) in fmnDAT mutants rescues the hyperlocomotive response to both amphetamine and methylphenidate [Drosophila Ric, encodes a Ras-related small GTPase involved in dopamine transporter trafficking [Rit2, the mammalian orthologue of Ric, has been shown it impact cocaine sensitivity in mice [Drosophila, dopaminergic expression of a constitutively active Ric-GTPase mutant (RicQ117L) increases cell surface expression of DAT and enhances sensitivity to the locomotor activating effects of amphetamine [ic cleft . DAT strd humans ,40. In Dhenidate , demonsthenidate . Drosophfficking . Rit2, t in mice . In Droshetamine .dDAT and reducing uptake of synaptic dopamine [Drosophila [Flotillin 1 mutant (02554Flo1) larvae or larvae expressing Flo1 RNAi in dopamine neurons did not display an increase in locomotive speed after administration of amphetamine [FLOT1 in dopaminergic neurons of 02554Flo1 mutant larvae rescued the hyperlocomotive response to amphetamines [Flo1 in amphetamine-induced reverse transport might be the role of lipid rafts in acting as a scaffold for other signaling proteins such as CamKII [fmnDrosophila DAT mutants, where dopaminergic expression of an hDAT construct with serine to alanine substitutions that prevent phosphorylation ablates amphetamine-induced hyperlocomotion [hDAT, as well as a mutant hDAT where N-terminal serine residues are replaced with phospho-mimetic aspartate residues. Flies were also used to demonstrate DAT phosphorylation requires CAMKII in dopamine neurons, and that expressing CaMKII inhibitory peptide in the dopamine neurons of larvae ablates amphetamine-induced hyperlocomotion [hDAT mutant rescues the locomotor response to amphetamine in flies expressing CaMKII inhibitory peptide [Drosophila were also the first model organism to show that the membrane phospholipid Phosphatidylinositol -bisphosphate (PIP2) interaction with the human dopamine transporter is important for amphetamine-induced dopamine efflux [hDAT-K/A) do not alter DAT cell surface expression, but to reduced amphetamine-induced dopamine efflux. To test the behavioral impact of the hDAT-K/A mutation, fmnDAT mutant flies expressing hDAT or hDAT-K/A in dopaminergic neurons were fed amphetamine and monitored for changes in locomotor behavior. While hDAT expressing flies fed amphetamine showed an significant increase in locomotion, this effect was abolished in hDAT-K/A expressing flies [K337 and R443 lead a reduction in DAT cell surface expression of more than 80% [hDAT-R443A), which did not affect surface localization, fmnDAT mutant flies expressing hDAT or hDAT-R443A in dopaminergic neurons were observed in the DAM system. No difference was observed when comparing baseline locomotion of hDAT and hDAT-R443A expressing flies. However, a difference in locomotion was observed after exposure to 1 mM amphetamine, and fmnDAT flies expressing hDAT displayed an increase in cumulative locomotion while no significant change was observed in fmnDAT flies expressing hDAT-R443A [fmnDAT flies expressing hDAT-R443A abolishes preference of 1 mM amphetamine that is observed in fmnDAT flies expressing wild-type hDAT. In contrast to the preference observed at low doses of amphetamine (1 mM), fmnDAT flies expressing hDAT avoid amphetamine at higher doses (10 mM amphetamine). fmnDAT flies expressing hDAT-R337A also display avoidance for 10 mM amphetamine in the CAFE, suggesting the interaction of PIP2 with DAT is required for amphetamine preference, but not amphetamine avoidance [While the activating effects of cocaine, methylphenidate, and amphetamine-like psychostimulants are all DAT-dependent, they do not increase dopaminergic signaling through the same mechanism of action . Cocainedopamine , while adopamine ,162,163.dopamine ,153 and dopamine . The firosophila . In conthetamine . Expressetamines . One exps CamKII ,166. In comotion . The hypcomotion . Dopamin peptide . Drosophe efflux . In siling flies . The DATng flies . In vitrthan 80% . To testAT-R443A . In addivoidance .VMAT2) regulates the storage and release of dopamine, and is a key mediator of behavioral responses to psychostimulants of abuse [Drosophila homologue of VMAT2 (dVMAT) has two splice variants, with dVMAT-A identified to play a role in dopamine transport similar to VMAT2 [Drosophila dopamine neurons leads to increased release of DA, along with an increase in grooming behavior, an increase in locomotion, and a decrease in negative geotaxis [dVMAT-A transgene. Interestingly, flies that overexpress the dVMAT-A transgene in dopaminergic neurons display a decrease in behavioral sensitivity to cocaine in assays of locomotion, grooming, and negative geotaxis. One explanation for this blunted response to cocaine might be a ceiling effects related to the overexpression of dVMAT-A, suggesting that cocaine mediated increases in synaptic dopamine are behaviorally inert in a system that is already saturated with dopamine. This explanation does not, however, justify the response to acute cocaine exposure in flies overexpressing dVMAT-A, where increasing dVMAT-A expression actually reduced the sensitivity to the impairing effects of cocaine on negative geotaxis [dVMAT-A transgene displayed the greatest reduction in cocaine induced impairment [Drosophila in uncovering molecular interactions that cannot be determined in humans. Such experiments might be especially useful for identifying the behavioral effects VMAT2 polymorphisms associated with an increased risk of addiction [The vesicular monoamine transporter 2 , one D2-like dopamine receptor (DD2R), and one dopamine and ecdysone hormone receptor (DopEcR) [Drosphila dopamine receptors are integral to the effects of psychostimulants on arousal [dumb1 mutant flies, where Dop1R1 expression is significantly reduced. Expressing Dop1R1 in the mushroom body of dumb1 mutant flies restores the methamphetamine-induced reduction in sleep [dumb2 mutant flies, who show no reduction in sleep after cocaine exposure [dumb2 mutant flies also display an increase in repetitive startle induced hyperactivity (ReSH), where active flies subjected to repeated bouts of mechanical stimulation display an increase in locomotion and a protracted period of hyperactivity greater than that observed in wild-type flies [dumb2 mutant flies, where ReSH is the same in the presence and absence of cocaine [All psychostimulants of abuse act to increase synaptic dopamine, and their locomotor activating as well as rewarding effects similarly depend on the activation of dopamine receptors along with their downstream signaling pathways. In (DopEcR) . Drosphi arousal , locomot arousal , and rew arousal . In wild arousal  and coca arousal  increasein sleep . Dop1R1 exposure . dumb2 mpe flies . Interes cocaine .Drosophila dopamine receptors regulate psychostimulant preference, and mushroom body knockdown of Dop1R1 or Dop1R2 both suppress acute methamphetamine preference in the CAFE [Drosophila will aid in discovery of the genes that modify each pathway, and bias behavior for or against the development of addiction.In addition to regulating psychostimulant-induced arousal, the CAFE . In contthe CAFE . These rthe CAFE . IdentifDrosophila have been useful in molecular characterization of genes known to impact the psychostimulant response in mammals, they have also been instrumental in identifying novel genes that regulate response to psychostimulant drugs. Research in flies was the first to show that mutation of the circadian gene period increased initial sensitivity to the motor activating effects of cocaine, but disrupted the development of sensitization [clock, cycle, and doubletime were also identified in mutant flies that failed to develop sensitization to the motor activating effects of cocaine [Period [Clock [Drosophila also demonstrated that period is involved in the regulation of dopamine receptor responsiveness [period null mutants do not develop preference and do not self-administer methamphetamine [While tization . The rol cocaine . Subsequ [Period  and Clocd [Clock  in resposiveness . Additiohetamine .Drosophila were also used to identify a novel role of Lim-only (dLmo) gene in modulating response to cocaine [dLmo regulates the sensitivity of primary pacemaker neurons to cocaine-induced increases in synaptic dopamine, with loss of function mutants exhibiting an increase in sensitivity to the motor-activating effects of cocaine on locomotion and the motor-impairing effects of cocaine on negative geotaxis [dLmo gain of function mutants are resistant to the effects of cocaine, and display a reduction in cocaine-induced locomotion and a decrease in impairment of negative geotaxis [Drosophila as a translational genetic model for identifying novel regulators of drug response that can inform our understanding of human disease. cocaine . The expgeotaxis . In contgeotaxis . While tgeotaxis , the iniDrosophila circadian pacemaker cells [Drosophila type II cAMP-dependent protein kinase (PKA-RII) is also involved in circadian regulation, and acts downstream of circadian pacemaker cells. PKA mutants display reduced sensitivity to the motor activating effects of cocaine in the behavioral scoring assay, require more than double the amount required to generate a response in control flies, and do not develop sensitization to repeat exposures [Drosophila are a promising model organism to help identify potential druggable targets that might be useful in the development of therapeutics for the treatment of addiction [Protein kinase (PKA) is involved in signaling molecular targets downstream of the er cells . The Droxposures . D1 recexposures , and chrxposures . Severalddiction . Drosophila, and several whir mutants have reduced sensitivity to the motor impairing effects of cocaine on negative geotaxis behavior [tao mutants are resistant to the motor-impairing effects of cocaine on negative geotaxis [Drosophila GSK-3β homologue shaggy is another molecule implicated in the regulation of synaptic plasticity that is immediately downstream of PKA, and regulates the activity of multiple proteins including cAMP-response element binding protein (CREB), timeless, and the microtubule-binding protein tau [EP1379shaggy mutant, missing the serine 9 site regulated by PKA, produces flies sensitive to cocaine-induced effects on negative geotaxis [TAOK1, the mammalian orthologue of tao, is transcriptionally regulated in mammalian models of cocaine and methamphetamine addiction [Tau enhances cocaine conditioned place preference in mice while Tau overexpression in the hippocampus suppresses cocaine conditioned place preference [Drosophila provide the ability to test hypotheses related to the development of targeted therapies for addiction, which is specifically relevant for Rho family GTPase and GSK-3β signaling pathways which are promising drug targets for the treatment of neuropsychiatric disorders [In addition to PKA, several other molecules associated with regulating synaptic plasticity have also been shown to impact psychostimulant response in flies. The Rho family GTPase activating protein RhoGAP18B regulates actin dynamics and response to alcohol in behavior . The Stegeotaxis . The Drotein tau . The shageotaxis . While tgeotaxis , and aregeotaxis  and the geotaxis  in mice.ddiction ,189. GSKddiction , and cocddiction . Knockoueference . The ideeference ,194. Addisorders ,196.Drosophila do not completely recapitulate the features of psychostimulant abuse in humans, they effectively capture features of drug response that can be used to identify novel genes that predict the risk to develop addiction. While there is currently no Drosophila equivalent of the operant-controlled psychostimulant self-administration paradigms commonly used in rodent models of addiction, such as the lever press, analysis of simple behaviors such as voluntary consumption are an equally effective measure of hedonic value [Drosophila such a powerful model organism. The benefits of this model are especially relevant to the study of complex polygenic disorders such as addiction, which are difficult to study in human populations. While the heritability of risk genes for the development of addiction has been established with family studies and genome-wide association studies (GWAS), it is difficult to collect enough human data to identify all of the genes involved in psychostimulant abuse. In addition solving the problem of sample size, tools such as the Drosophila melanogaster Genetic Reference Panel (DGRP) enable characterization of the relationship between genetic variation and quantitative traits [While the assays used to measure psychostimulant response in ic value . The dece traits . The DGRe traits .Drosophila in effectively identifying genes the determine consumption and preference for psychostimulant drugs [The DGRP has been used to study the genetic basis of variation in cocaine and methamphetamine consumption in a 2-choice CAFE . Voluntant drugs .Drosophila in performing unbiased screens genes that regulate psychostimulant consumption, but also demonstrates the ability to identify the contribution of common intergenic SNPs—analogous to many human SNPs associated in various GWAS analyses—to phenotypic variation.A similar experiment was performed to identify the genetic basis of variation in cocaine and methamphetamine consumption in single-fly CAFE assays with 18,000 individuals flies generated from an outbred advanced intercross population (AIP) derived from 37 highly diverse, fully sequenced inbred lines from the DGRP . This deDrosophila have human orthologues, and several of the identified genes are known to play a role in cocaine response in humans [Drosophila.One question that is not possible to investigate in human studies is what genes are transcriptionally regulated in response to acute psychostimulant drug exposure. Analysis of single-cell transcriptional responses in psychostimulant-exposed flies enables investigation of this question at scale and resolution that is not feasible in other models organisms. To characterize the impact of cocaine on transcriptional response, 114 female along with 128 male flies were selected for analysis after ingesting equal amounts of cocaine solution, and similar number of control flies were selected after ingesting an equivalent volume of sucrose . The acun humans . The ideDrosophila in unveiling novel regulators psychostimulant response, demonstrating how flies can be used to identify the genes that regulate psychostimulant response in complex polygenic disorders such as addiction and ADHD [Drosophila offers, enabling analyses that would be prohibitive in a mammalian model organism. Similar utility is showcased in analysis of single-cell transcriptional response to cocaine [With the increasing accessibility of high-throughput sequencing techniques, multiple methods of genetic analysis might be combined to identify novel genes involved in response to psychostimulant. The recent experiments using AIPs generated from inbred lines of the DGRP exemplify the capability of and ADHD ,91. Thes cocaine . While tDrosophila is in the evolving stages as a model for studying the biological basis of response to psychostimulant drugs of abuse, but has unparalleled potential for identifying novel genes and molecules with an efficiency that could not be achieved in other models. Genetic and behavioral accessibility of Drosophila contribute to flexible manipulation of genes in high-throughput experiments that recapitulate many of the complex behavioral responses observed in humans. There are over 200 human disease models in Drosophila, including diseases that respond to psychostimulant treatment such as ADHD and ASD. Additionally, Drosophila is a powerful translational model for studying addiction, and have proven successful in identifying genes that regulate alcohol consumption in humans. In contrast to the substantial amount of research on AUD in flies, there are still relatively few studies on psychostimulant response. The majority of psychostimulant experiments in Drosophila focus on sensitivity to motor-activating or motor-inhibiting drug effects, and the development of behavioral sensitization. While these assays of sensitivity and sensitization have been used for more than 20 years [Drosophila.20 years ,63, studIn the context of addiction, several newer assays of consumption and preference such as the Fly Liquid-Food Interaction Counter  and Fly Drosophila that display behavioral features of ADHD [These assays can also be used to investigate the mechanisms of psychostimulant response in ADHD models. While psychostimulant medication is effective in ameliorating the cognitive and behavioral deficits associated with ADHD, the mechanism of this behavioral response is poorly understood. The unbiased generation of candidate genes that impact psychostimulant response from screens using AIPs of the DGRP ,93 provi of ADHD ,105,121 Drosophila to study behavioral responses to psychostimulant drugs we have shown several ways that flies can provide new insights into the biological basis of psychostimulant addiction. Additionally, we have reviewed how Drosophila is an effective model organism for identifying genes that impact the therapeutic efficacy of psychostimulant drugs. Together, the implementation of improved behavioral assays combined with high-throughput next generation analysis methods will help identify the distinct gene networks and mechanisms that mediate psychostimulant responses in Drosophila models of ADHD, ASD, and substance abuse. Due to the translatability of Drosophila research, these experiments have the potential to uncover novel druggable targets relevant for human therapeutics.In examining the use of


Prognosis is significantly determined by the stage of diagnosis where survival for stages I, II, III and IV is 73–92%, 45–55%, 21% and <6%, respectivelyOvarian cancer (OC) is a disease that most often affects post-menopausal women who present abdominal discomfort and bloating over a few months prior to detection. The majority of patients are diagnosed at advanced stages of the disease as the early stages are commonly asymptomatic . AccordiThere are four main types of OC: epithelial, Germ cell, sex chord and stromal, with each having different epidemiological statistics, and with epithelial ovarian cancer (EOC) accounting for approximately 90% of all cases . In EOC Cancer Antigen-125 (CA-125) and trans-vaginal imaging are currently routinely used as part of ovarian cancer diagnosis. Blood level of CA-125 is the most widely used serum biomarker, but it lacks the sensitivity or specificity to be used alone as a screening test . It is aSeveral different approaches have been proposed to combat the shortcomings of current techniques. Studies focused on smaller molecules, such as circulating miRNA and cell-free DNA (cf-DNA) have gained significant attention in recent times . These mEXs are extracellular vesicles, ranging between 40 and 160 nm in size , releaseIt is now well established that the proteomic profile of EXs can act as a useful tool or biomarker for diagnosing various types of cancer . For exavia exosomal and anti-apoptotic activities  where antibody-coated-magnetic beads are used for EXs capture and labelling following detection via electrochemical sensing . Doldan  samples . This te samples  and skin samples  cancer s samples , while a samples . QCM tec samples , potenti samples  and even samples . Recentl samples . Thus, tAchieving early detection of ovarian cancer remains the greatest hope for improved patient survival outcomes. Therefore, investigation of these novel tools and technologies for early ovarian cancer diagnosis is a high priority.


MRI is routinely used for rectal cancer staging to evaluate tumor extent and to inform decision-making regarding surgical planning and the need for neoadjuvant and adjuvant therapy. Extramural venous invasion (EMVI), which is intravenous tumor extension beyond the rectal wall on histopathology, is a predictor for worse prognosis. T2-weighted images (T2WI) demonstrate EMVI as a nodular-, bead-, or worm-shaped structure of intermediate T2 signal with irregular margins that arises from the primary tumor. Correlative diffusion-weighted images demonstrate intermediate to high signal corresponding to EMVI, and contrast enhanced T1-weighted images demonstrate tumor signal intensity in or around vessels. Diffusion-weighted and post contrast images may increase diagnostic performance but decrease inter-observer agreement. CT may also demonstrate obvious EMVI and is potentially useful in patients with a contraindication for MRI. This article aims to review the spectrum of imaging findings of EMVI of rectal cancer on MRI and CT, to summarize the diagnostic accuracy and inter-observer agreement of imaging modalities for its presence, to review other rectal neoplasms that may cause EMVI, and to discuss the clinical significance and role of MRI-detected EMVI in staging and restaging clinical scenarios. T2WI demonstrates EMVI as an expanded, irregular vessel with intermediate tumor-signal intensity.CT can sometimes depict EMVI as a heterogeneously enhancing, serpentine cord-like structureInter-observer agreement in assessing the presence or absence of EMVI is variable.EMVI is a predictor of poor prognosis and indicates biologic aggressiveness.EMVI may be seen in association with rectal  tumors other than adenocarcinoma.Colorectal cancer is the third most common neoplasm in adults, with approximately 43,000 patients diagnosed with rectal cancer in the United States in 2020 . MRI proDirect tumor invasion into the extramural veins on histopathology, known as extramural venous invasion (EMVI), has been recognized as an indicator of poor prognosis , 7. BrowThis article reviews the imaging findings of EMVI on MRI and CT, summarizes the diagnostic accuracy and inter-observer agreement for detection of EMVI, and reviews the clinical significance and role of MRI-detected EMVI in staging and restaging scenarios with an aim to disseminate a common understanding of EMVI and its varied appearances.Understanding the rectal venous plexus anatomy can be helpful when aiming to accurately identify EMVI on MRI. The venous drainage of the anorectum occurs via two interconnected plexuses, the inner submucosal plexus of the anorectum  and the outer muscularis plexus . Venous drainage of the upper 2/3 of the rectum is via the superior rectal/hemorrhoidal veins, which drain into the inferior mesenteric vein . Venous EMVI is defined histopathologically as the presence of tumor cells within blood vessels located beyond the muscularis propria of the rectal wall Fig. a; howeveInvasion of tumor into blood vessels is considered to be an initial step in hematogenous metastasis , and hisThe MERCURY study evaluated high-resolution rectal MRI with an in-plane resolution of 0.6 × 0.6 mm and reported that MRI could predict surgical margin status  and extrEMVI at MRI is usually associated with primary tumor extending beyond the muscularis propria and into the mesorectal fat. The leading edge of the tumor with such extramural extension and associated EMVI is typically nodular or sawtooth  rather than smooth. The infiltrated vessel is contiguous with the primary tumor, may be expanded, and has lost the normal black signal flow void, which is replaced with intermediate signal T2 tumor intensity. The vessel often has irregular margins and may appear beaded or nodular on T2 and post contrast imaging Fig.  5, 21]., 21.5, 2To help in assessing EMVI, Smith et al. developed a 5-point scoring system on T2WI based on the aforementioned MR imaging features . This scBeyond the 5-point grading, additional features of MR detected EMVI have been investigated, including the significance of the number and diameter of involved vessels, the location , and the causes of vascular involvement . EMVI aDiffusion-weighted images (DWI) often depict the intravenous tumoral component as intermediate or high tumor-signal intensity within normal or slightly expanded extramural vessels adjacent to the primary tumor Fig. . DespiteOn contrast-enhanced T1 weighted images (CE-T1WI), EMVI can be identified as either enhancing tumor within the vessel or as a non- or hypoenhancing intraluminal filling defect  within aBrown et al. showed that rectal cancer MRI revealed 83% of histopatologically-detected EMVI with a diameter greater than 3 mm . Table 2Data regarding the value of DWI in detecting EMVI are conflicting. Ahn et al. reported that the addition of DWI reduced inter-observer agreement with no additional diagnostic benefit . ConversJhaveri et al. showed that the sensitivity and specificity of rectal MRI for EMVI did not significantly change with the addition of contrast-enhancement , but that interobserver agreement remained good . SimilarCompared with MRI, the role of CT in assessing EMVI is limited because of its lower contrast resolution. However, CT can be an alternative to assess EMVI in patients who have a contraindication to MRI or who are unable to undergo MRI due to claustrophobia. On CT, EMVI is often seen as a heterogeneously enhancing, serpentine cord-like structure connecting veins with the irregular, contiguous margins of the primary tumor Fig. . Ortega Dilation of the superior rectal and inferior mesenteric veins may help to predict EMVI Fig. . Wu et aMultiple studies have demonstrated the clinical significance of EMVI detected on MRI as a predictor of poor prognosis or biologic aggressiveness in rectal cancer. MRI-detected EMVI is generally present in patients with T3 and nodal disease and is consequently, at least in North America, used to determine which patients will benefit from neoadjuvant therapy. Conversely, the overuse of neoadjuvant therapy in rectal cancer patients does not improve survival and can result in bowel and sexual dysfunction , and a rMRI-detected EMVI is reported to be an independent significant prognostic factor for overall disease-free survival and systemic recurrence in rectal cancer . In locaRectal cancer MRI can also demonstrate in situ evidence of response or progression of EMVI after neoadjuvant therapy. Also, a change in MRI-detected EMVI status from positive to negative has been shown to predict histopathologic response . AdditioIn one study, EMVI score correlated with histologic lymph node stage . In otheRectal neoplasms other than usual adenocarcinomas such as squamous cell carcinoma, mucinous adenocarcinoma, and neuroendocrine tumor also can invade perirectal vessels Figs. , 10, 11.Mucinous rectal carcinoma is a distinct pathologic subtype of rectal cancer defined as a tumor composed of greater than 50% extracellular mucin and neoplastic epithelium surrounded by extracellular mucin lakes on histopathology . The priRectal squamous cell carcinoma is a rare tumor, which accounts for less than 1% of colorectal malignancies . Risk faThe rectum is a common site of neuroendocrine neoplasms. Rectal neuroendocrine tumor usually appears as a submucosal nodule or focal area of plaque-like wall-thickening; however, less commonly it can present as a large ulcerating, avidly enhancing, invasive mass . The incMRI-detected EMVI correlates closely with histopathological EMVI and is a predictor of lymph node and distant metastases, tumor recurrence, and poor prognosis. Therefore, it is important to evaluate for the presence of EMVI on rectal cancer MRI examinations before and after neoadjuvant therapy to determine risk-stratification and therapeutic options. Despite the clinical significance of MRI-detected EMVI, inter-observer variability in assessing its presence or absence is problematic both at initial staging and after neoadjuvant treatment. Radiologists should therefore be familiar with the imaging features of EMVI and its implications for patient management. Findings of EMVI include expanded vessel caliber adjacent to the primary tumor, intermediate tumor signal intensity in the vessel, and irregular vessel margin. DWI and contrast enhanced T1 weighted images may be helpful adjuncts to T2WI and may help improve reader confidence in select cases. Further investigation is necessary to determine if multi-parametric MRI improves diagnostic performance without compromising interobserver agreement. In addition, further investigation is needed to assess the clinical importance of CT-detected EMVI and to determine whether detailed features of EMVI, including location, vessel diameter, and the number of involved vessels can improve risk-stratification.


Cardiovascular disease is the leading cause of mortality worldwide, with atherosclerotic coronary artery disease (CAD) accounting for the majority of cases. X-ray coronary angiography and computed tomography coronary angiography (CCTA) are the imaging modalities of choice for the assessment of CAD. However, the use of ionising radiation and iodinated contrast agents remain drawbacks. There is therefore a clinical need for an alternative modality for the early identification and longitudinal monitoring of CAD without these associated drawbacks. Coronary magnetic resonance angiography (CMRA) could be a potential alternative for the detection and monitoring of coronary arterial stenosis, without exposing patients to ionising radiation or iodinated contrast agents. Further advantages include its versatility, excellent soft tissue characterisation and suitability for repeat imaging. Despite the early promise of CMRA, widespread clinical utilisation remains limited due to long and unpredictable scan times, onerous scan planning, lower spatial resolution, as well as motion related image quality degradation. The past decade has brought about a resurgence in CMRA technology, with significant leaps in image acceleration, respiratory and cardiac motion estimation and advanced motion corrected or motion-resolved image reconstruction. With the advent of artificial intelligence, great advances are also seen in deep learning-based motion estimation, undersampled and super-resolution reconstruction promising further improvements of CMRA. This has enabled high spatial resolution (1 mm isotropic), 3D whole heart CMRA in a clinically feasible and reliable acquisition time of under 10 min. Furthermore, latest super-resolution image reconstruction approaches which are currently under evaluation promise acquisitions as short as 1 min. In this review, we will explore the recent technological advances that are designed to bring CMRA closer to clinical reality. Coronary artery disease (CAD) is the leading cause of cardiovascular morbidity and mortality worldwide , 2. CAD The clinical manifestations of CAD can present either as an acute process or a more stable but chronic deterioration of symptoms. The European Society of Cardiology has recently categorised CAD as either acute coronary syndromes (ACS) or chronic coronary syndromes (CCS) . Whilst Chronic coronary syndromes can present clinically in a variety of ways; symptoms of stable angina , symptoms of heart failure or asymptomatic left ventricular impairment, symptomatic and asymptomatic cardiac arrhythmias . The earX-ray coronary angiography is the reference standard imaging modality for the assessment of CAD with unrivalled temporal and spatial resolution, as well as the versatility to enable real time coronary intervention for patients presenting with ACS. The addition of functional physiological assessment of CAD with pressure wiring is particularly helpful in patients presenting with CCS and has been shown to be prognostically significant , 8. PlaqComputed tomography coronary angiography (CCTA) has emerged as a credible non-invasive alternative for the assessment of patients with CCS. It offers high diagnostic accuracy in terms of comprehensive assessment of luminal stenosis, fractional flow reserve and plaque characterisation –15. HoweCoronary magnetic resonance angiography could potentially offer a non-invasive alternative for the early detection and long-term monitoring of CCS, which is free of ionising radiation and iodinated nephrotoxic contrast agents. It could also be combined with volumetric assessment of left ventricular function, myocardial perfusion, myocardial tissue characterisation, valvular assessment and coronary plaque characterisation to brand CMR as one of the most comprehensive imaging modalities for cardiovascular assessment. Here we discuss the current status quo and potential future role of CMRA as a viable alternative for the assessment of patients with CCS.The potential of CMRA to exclude significant CAD was first demonstrated two decades ago in a multicentre study of 109 patients, which compared non-contrast CMRA against invasive X-ray angiography . In thisHowever, despite the early promise of CMRA, clinical application is currently confined to a few specialised centres within a set of niche indications e.g., suspected anomalous coronary arteries, coronary artery aneurysms and assessment of the proximal coronary arteries. Reasons for the slow uptake of CMRA in clinical practise include lower spatial resolution compared to CCTA and invasive X-ray angiography, significantly longer and unpredictable acquisition time, complicated scan planning, and motion related (cardiac and respiratory) image quality degradation.Significant technological strides in CMR image acquisition and image processing are now enabling some of these technical challenges to be overcome. The most notable breakthroughs are in the fields of respiratory motion compensation and image acceleration. Further recent advances in deep learning-based motion correction and image reconstruction could potentially enable 3D whole-heart CMRA with similar spatial resolution and acquisition time as CCTA in the future. We will discuss some of these recent developments in the following sections.In order to acquire a high spatial resolution 3D whole-heart CMRA, patients are required to be continuously scanned for several minutes with data acquired from numerous heat beats under free breathing. This scenario produces a challenging multi-dimensional cardiac and respiratory motion environment, which needs to be compensated through image acquisition and reconstruction techniques. Cardiac motion artefacts are typically minimised by prospective electrocardiographic (ECG) triggering to acquire data during mid-to-late diastole. However, this may be susceptible to image degradation as a result of cardiac arrhythmias, heart rate variability or user dependent estimation of the resting period. Systolic image acquisition is less sensitive to arrhythmias and heart rate variability. However, longer acquisition times may be needed due to a shorter resting period compared to diastolic imaging. Alternatively, data can be continuously acquired and then retrospectively reconstructed into multiple cardiac phases, thus selecting the phase with the minimum cardiac motion artefact, similar to CCTA , 22.Various techniques have been proposed to compensate for respiratory motion artefacts during CMRA acquisition. The most rudimentary techniques utilise breath-holding to minimise respiratory motion artefacts –26, whicSeveral novel respiratory motion compensation frameworks have recently been proposed to overcome some of these drawbacks, principally to deal with the 3D nature of respiratory motion, improve image quality, increase respiratory scan efficiency to 100% and therefore significantly reduce acquisition times. Using the so called “1D self-navigation” approach , the acqHowever, with 1D self-navigation, it is difficult to separate moving  from static  tissue, which introduces artefacts . Image-bDespite offering 100% respiratory scan efficiency, self-navigation or iNAV frameworks on their own are not sufficient. It can take up to 30 min to acquire a fully sampled high resolution (≈1 mm isotropic) CMRA. Here we will discuss the novel techniques in development which incorporate self-navigation or iNAV systems with a combination of image acceleration techniques  to achieve high-spatial resolution CMRA within a clinically feasible acquisition time. We will also touch on more advanced motion correction frameworks which factor in the more complex 3D non-rigid motion of the heart within the thoracic cavity. Finally, we will briefly discuss emerging deep learning super resolution CMRA reconstruction frameworks that could potentially enable 3D whole-heart CMRA with similar spatial resolution and acquisition time as CCTA.It is possible to combine an undersampled 3D radial trajectory acquisition with 1D navigators (diaphragmatic or self-navigation) to allocate the acquired data into specific phases in the respiratory cycle (respiratory bins). Registration algorithms subsequently assess the motion between each bin and a reference end-expiratory bin. Images from all respiratory bins are then motion corrected to the common respiratory position and averaged to produce a respiratory motion corrected image. This methodology allows for 100% respiratory scan efficiency with a drastically shorter acquisition time in comparison to respiratory gating alone with similar image quality  22, 40,, 53. Res3 spatial-resolution free-breathing whole-heart 3D CMRA with <10 min predictable acquisition time with 100% scan efficiency. In a validation cohort of healthy subjects, image quality was comparable to the fully sampled acquisition and significantly improved compared to both iterative SENSE and compressed sensing reconstruction methods. This framework has been extended to include bin-to-bin non-rigid respiratory motion correction and has been compared against CCTA in patients with suspected CAD ..3 spatiacted CAD  66). Th. Th3 spaWhilst an acquisition time of ~10 min for sub-1 mm isotropic spatial resolution is clinically feasible, it is nevertheless considerably longer than a full CCTA scan . It is particularly disadvantageous if image quality is sub-optimal on first attempt and repeat imaging within the same scan session is required. Furthermore, reconstruction times of iterative methods are relatively long and computationally demanding, especially if combined with non-rigid motion correction or non-Cartesian trajectories. Finally, highly undersampled acquisitions are vulnerable to residual aliasing or staircasing and blurring artefacts, putting a realistic limit on the extend of conventional image acceleration methods. Recently, deep-learning based image reconstruction networks have been proposed to address some of these shortcomings –71. The Our understanding of CAD and CCS is rapidly evolving. The COURAGE trial highlighted the limitations of coronary luminography as a standalone prognostic measure of CAD, with no significant difference in revascularisation vs. best medical therapy on hard end point outcomes such as death or myocardial infarction in patients that were angiographically identified to have significant CAD . The FAMDespite this, MR coronary plaque imaging is constrained by comparable technical challenges to CMRA, e.g., respiratory motion, prohibitively long and unpredictable acquisition time, plaque to coronary artery misalignment due to the sequential nature of data acquisition. Recently, a novel framework (CATCH) has been proposed to overcome some of these limitations for MR coronary plaque imaging. This framework assembles advanced motion correction techniques outlined above to facilitate simultaneous multi contrast bright and black blood coronary artery angiography and visualisation of vulnerable coronary plaque  96, 97), 9796, 9It is also possible to directly target specific components of the atherosclerotic plaque –101. VarChronic coronary syndrome is a progressive and multifaceted condition associated with coronary artery atherosclerosis, which manifests clinically as either angina, heart failure and gradual left ventricular dysfunction, arrhythmia and/or acute myocardial infarction. The timely recognition of CCS enables bespoke risk assessment of patients, prompt initiation of therapeutical intervention and long-term monitoring of potential complications. Amongst a maelstrom of diagnostic imaging modalities for CAD, CMRA could potentially emerge as a viable, versatile, reproducible and non-invasive imaging modality for the assessment of CCS, which is free of ionising radiation, iodinated contrast agents and can be performed without gadolinium contrast enhancement. These characteristics would be ideally suited for repeat imaging of patients to monitor disease progression and gauge response to treatment. Furthermore, it can be combined with myocardial volumetric assessment, tissue characterisation, perfusion and coronary plaque assessment to enable comprehensive assessment of ischaemic heart disease.RH drafting and editing of the manuscript. CM, GC, RK, WK, CP, and RB reviewing and editing of manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The brain encompasses a complex network of neurons with exceptionally elaborated morphologies of their axonal  and dendritic  parts. De novo actin filament formation is one of the major driving and steering forces for the development and plasticity of the neuronal arbor. Actin filament assembly and dynamics thus require tight temporal and spatial control. Such control is particularly effective at the level of regulating actin nucleation-promoting factors, as these are key components for filament formation. Arginine methylation represents an important post-translational regulatory mechanism that had previously been mainly associated with controlling nuclear processes. We will review and discuss emerging evidence from inhibitor studies and loss-of-function models for protein arginine methyltransferases (PRMTs), both in cells and whole organisms, that unveil that protein arginine methylation mediated by PRMTs represents an important regulatory mechanism in neuritic arbor formation, as well as in dendritic spine induction, maturation and plasticity. Recent results furthermore demonstrated that arginine methylation regulates actin cytosolic cytoskeletal components not only as indirect targets through additional signaling cascades, but can also directly control an actin nucleation-promoting factor shaping neuronal cells—a key process for the formation of neuronal networks in vertebrate brains. G-monomethylated arginine (MMA), ω-NG, NG-asymmetric dimethylarginine (ADMA) and ω-NG, N’G-symmetric dimethylarginine (SDMA)  resulting in ω-Ne (SDMA)  1]. Pro. ProG-moe (SDMA) .All subtypes catalyze the monomethylation of guanidino nitrogen atoms of arginine residues utilizing the methyl donor S-adenosylmethionine. Additional dimethylation of the monomethylated arginine is brought about solely by type I and II PRMTs, whereas the only known type III PRMT, PRMT7, solely exhibits monomethylation catalytic activity because of its distinctive structural characteristics. Type I  PRMT enzymes can additionally catalyze the formation of asymmetric dimethylarginine as an end-product, while type II PRMTs (PRMT5 and PRMT9) form in a second, subsequent step a symmetric dimethylarginine ..42].Cobl has been identified as a crucial cytoskeletal component for dendrite and dendritic branch formation . Via funInterestingly, PRMT2 has been identified as a direct interaction partner for the actin nucleator Cobl . Thus faAmong the family of arginine protein methyltransferases, only PRMT2 comprises a Src Homology 3 (SH3) domain , and it Molecular mechanistic studies unveiled that arginine methylation controls Cobl’s actin binding abilities—the crucial prerequisite for Cobl-mediated actin filament formation . InhibitThus, arginine methylation of Cobl was shown to represent a key requisite for Cobl’s actin binding and its functions in the formation of the specialized neuronal architecture underlying neuronal network formation .Kif5b was ablated only after birth to avoid lethality, revealed defects in dendritic spine morphogenesis, synaptic plasticity and memory formation, thereby confirming the functional importance of KIF5B in controlling excitatory synaptic plasticity [The original view that microtubules are not present in dendritic spines was changed by studies revealing the invasion of microtubules itself, and additionally kinesin motor proteins from the dendritic shaft to dendritic spine heads ,80. A reasticity . It willSeveral aspects of arginine methylation represent urgent topics to be explored in the future. First, it will be key to identify further direct cytoskeletal targets. This will help to clarify whether also for cytosolic effector proteins arginine methylation is an important common mechanism of functional control. Subsequently, the molecular mechanistic details of the effect of arginine methylation on the functional properties of the cytoskeletal effectors need to be exploited and followed up by cellular analyses. Together, such studies will provide important new insights into the cytoskeletal functions of cells in general and into crucial aspects of cytoskeleton-driven development and plasticity of neuronal shape, compartmentalization and function in particular.Furthermore, it is critical to reveal whether arginine methylation is a post-translational modification that is rather constitutive, or whether it is also temporally reversed and thus represents a dynamic post-translational modification. Yet, the existence of arginine demethylases is still controversial ,83,84,85It will also be of interest to analyze putative crosstalk and interconnections between arginine methylation and additional post-translational modifications. As an example, Akt-mediated serine phosphorylation and arginine methylation of polyglutamine-expanded androgen receptors occur at the same consensus site and were reported to have opposing effects on neurotoxicity .


Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The “classic” quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 “next-generation” viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA.Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy–suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples.Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation  for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold . Frozen storage did not substantially alter infectious units per million values .The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays. We demonstrated similar estimates of variation for classic and next-generation quantitative viral outgrowth assays (QVOAs), with enhanced sensitivity supporting the use of next-generation QVOAs as a proxy for classic QVOAs in measuring the latent human immunodeficiency virus reservoir. The human immunodeficiency virus (HIV) reservoir of latently infected cells is established early in infection and primarily exists in long-lived memory T cells, which have the capacity to be induced to produce new rounds of replication if suppressive anti-retroviral therapy (ART) is interrupted , 2. This+ cells in serial dilution coculture with repeated addition of target and feeder cells, and requires sensitive monitoring for the detection of HIV outgrowth. Furthermore, in vitro stimulation to reverse latency and induce outgrowth may be incomplete or insufficient and may not recapitulate the in vivo context of latency reversal. After 20 years of use, QVOA remains laborious, and only recently have there been rigorous assessments of its performance compared with other reservoir measurement strategies  One HIV-uninfected control was included, and all 6 participants underwent leukapheresis collections of mononuclear cells. Isolated peripheral blood mononuclear cells from each collection were divided into identical replicate aliquots for testing in blinded fresh and frozen panels with multiple classic QVOA and next-generation assays. Five laboratories participated in the study and performed 9 HIV reservoir assays: University of Pittsburgh (QVOA M) , UniversWe used methods described elsewhere  to accouThese 4 levels of variation are modeled as random effects , whereas systemic assay scale differences and frozen storage were both modeled as fixed effects. We used Markov chain Monte Carlo methods to obtain posterior medians and 95% CIs for all effects. In addition to modeling all 9 assays together, we compared estimates obtained by modeling subsets separately: the 3 classic QVOAs, the 2 enhanced -sensitivity QVOAs, the 4 next-generation QVOAs, and the 3 classic QVOAs plus each of the other 5 assays (sets of 4 assays modeled together). Finally, we modeled every possible pair of different assays (36 different sets of 2) to obtain the most direct estimates of variation between pairs of assays. Each model assumed that the aliquot and batch sources of variability, along with the effect of frozen storage, were the same for all assays in the model. For reporting, random effect sizes (standard deviations) were exponentiated to obtain fold increases in variation.10 terms) was computed, as in reference We used the above estimates to project a “bottom line” expected error in each next-generation or enhanced sensitivity assay, relative to the classic QVOAs. Specifically, median absolute error  than QVOA M and QVOA SR, owing to detection of more abundant targets (0.1 IUPM)  . They di.1 IUPM) .Seven of the 9 assays studied had correlated readouts  . These aIntervention strategies designed to reduce or eliminate the latent reservoir require accurate, reproducible, and scalable assays to detect changes in reservoir size and estimate the efficacy of eradication efforts. Understanding the precision of assays in the context of latency-reducing interventions is critical both to assessing how well changes in latent reservoirs can be measured and how to design clinical study protocols to estimate reduction in reservoir size with desired precision. The classic QVOA, though historically the reference standard, is not scalable for routine use in clinical studies of latent reservoir–reducing interventions. In addition, the low readout scale of classic QVOA limits its sensitivity and dynamic range, and it is not routinely practical to obtain the larger peripheral blood mononuclear cell inputs needed to improve these assay characteristics. The greater sensitivity of next-generation assays reduces cell input requirements and increases the available dynamic range for measuring reductions in the size of the latent reservoir.+ cells harboring inducible provirus-derived p24 Ag or RNA—there is variation among experimental approaches, even within assay categories [+ cells (thus determining input cell numbers) [Although these assays measure related targets—CD4tegories , 22. Evenumbers) , 9, 23. numbers) .A single, complete measurement of the clinically relevant latent reservoir remains elusive . AlthougOnce systematic differences in assay scale were accounted for, we found that next-generation assay readout both correlated with classic QVOA and exhibited similar levels of random variation. In some cases, the excess variation associated with using a next-generation assay as proxy for classic QVOA was found to be similar to that of batch-to-batch variation . This fiIn experimental practice, combined aliquot and batch variation may be more relevant than variation at a single level, because aliquot- and batch-level variation would be combined when samples are tested in different batches. In theory, if a research study could batch samples from a participant, then only aliquot variation (the first source of excess variation described in Methods) would contribute to extra variability above Poisson variability. Batch size may be limiting because most laboratories cannot set up large batches, this might not be a factor if one needs to assay only 2–3 longitudinal samples from a participant to measure the efficacy of an intervention.Assays using ultrasensitive means of monitoring QVOA culture supernatants tend to report approximately 4.5–28-fold higher IUPM values than classic QVOA when normalized to QVOA SR . The incInterestingly, newer modifications of the QVOA can increase the sensitivity by as much as 20-fold . In addiIn the current study, we found that cryopreservation had a <2-fold effect on IUPM estimates. The cryopreservation process, however, is complex and quality of the procedure can vary dramatically in different study sites. It is important to note that experienced researchers carried out all laboratory procedures, and thus our results likely represent a best-case scenario. Our analysis also assumes that freezing causes a fixed fold change in all samples. If cryopreservation had opposite effects on different subsets of the reservoir, it could decrease assay reliability in a manner not captured by our study.Outgrowth assays using culture-based methods tend to underestimate the true size of the latent reservoir, and measurement of outgrowth depends on the capacity of infected cells to produce infectious virus on stimulation , 22. FurSpecifically, it will be important to determine whether induced viral RNA or virus with low-level growth kinetics at concentrations not detectable by ELISA are replication competent, and thus relevant to the reservoir that would rebound when treatment is interrupted. This would be informed by genetic characterization of the HIV transcripts detected and virus present at low levels in culture wells, and by assessment of the intactness of provirus producing such transcripts and virions. Preliminary studies have shown that examination of longitudinal outgrowth kinetics and single-genome sequencing analyses verified replication competence of reactivated virus in some cases . HoweverOverall, our results offer cautious support for applying next-generation assays with systematically higher readouts as proxies for the more laborious and less sensitive classic QVOA. Analytical tools introduced by Rosenbloom et al . allow rThe Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiaa089_suppl_Supplementary-Table-1Click here for additional data file.jiaa089_suppl_Supplementary-Table-2Click here for additional data file.


Data on the clinical characteristics, electrocardiogram (ECG) findings and outcomes of patients with acute myocardial infarction (AMI) due to total unprotected left main (ULM) artery occlusion is limited.Between 2009 and 2021, 44 patients with AMI due to total ULM occlusion underwent primary percutaneous coronary intervention (PCI) at our institution. The ECG, collateral circulation, clinical and procedural characteristics, and in-hospital mortality were retrospectively evaluated.Twenty five patients presented with shock and 18 patients had in-hospital mortality. Nineteen patients presented with ST-segment elevation myocardial infarction (STEMI), while 25 presented with non-ST-segment elevation myocardial infarction (NSTEMI). ST-segment elevation (STE) in I and STEMI were associated with the absence of collateral circulation, while STE in aVR was associated with its presence. In the NSTEMI group, patients with STE in both aVR and aVL showed more collateral filling of the left anterior descending coronary artery (LAD) territory, while patients with STE in aVR showed more collateral filling of the LAD and the left circumflex artery territory. Compared with total ULM occlusion, patients with partial ULM obstruction presented with more STE in aVR, less STE in aVR and aVL, and less STEMI. Shock, post-PCI TIMI 0–2 flow, non-STE in aVR, STEMI, and STE in I predicted in-hospital mortality. STEMI and the absence of collateral flow were significantly associated with shock.STE in the precordial leads predicted the absence of collateral circulation while STE in aVR and STE in both aVR and aVL predicted different collateral filling territories in ULM occlusion. STE in I, non-STE in aVR, and STEMI predicted in-hospital mortality in these patients. Acute total occlusion of the unprotected left main (ULM) is an uncommon but clinically catastrophic event. In the last two decades, several contemporary registries/cohorts  presentation is variable in patients with total occlusion of ULM in the setting of acute myocardial infarction (AMI). Several ECG patterns have been reported to be associated with ULM occlusion : ST-segmAccordingly, this study surveyed the clinical characteristics, collateral circulation, and ECG features of acute total ULM occlusion (TIMI 0) and sought to explore the relationship between these factors and the clinical outcomes in these patients.A retrospective cohort analysis was performed on all patients with AMI due to total ULM occlusion who underwent emergent myocardial revascularization in a single center  between November 2009 and June 2021. The inclusion criteria were as follows: patients presenting with AMI defined according to the Fourth Universal Definition of Myocardial Infarction (2018)  and ULM Baseline clinical and angiographic data were retrieved from the patients’ hospital charts. All available ECGs obtained after symptom onset and before primary percutaneous coronary intervention (PCI) were included in the analysis. The ECG closest to angiography was used for analysis when more than one ECG was available. ST elevation or depression was considered if the ST deviation at the most prominent point at the J point was > 0.1 mV as previously described. , 8 The ECoronary angiography was reviewed by two independent operators (JXZ and YCH) and collateral circulation was classified into four types according to the territory supplied by the collateral flow: left anterior descending coronary artery (LAD), left circumflex artery (LCX), both LAD and LCX and no collateral circulation. Primary PCI was performed using standard interventional techniques, and adjunctive pharmacological treatments were administered according to STEMI guidelines. The primary endpoint of the study was in-hospital mortality, while the long-term major adverse cardiovascular events  were assessed as the secondary endpoint.p < 0.05. The SPSS software  was used for all statistical analyses.Data are presented as mean ± SD for continuous variables and as the proportion of valid cases for discrete variables. Differences in the baseline clinical, procedural characteristics, and ECG features were compared between the survival and mortality groups using the unpaired t-test (for continuous variables) or the Fisher exact test . Fisher’s exact test was used to evaluate the relationship between the ECG features and collateral circulation. Chi-square test was used to compare differences in ECG characteristics between total ULM occlusion and partial ULM obstruction; Survival curves were generated using the Kaplan–Meier method. Receiver operating characteristic curve (ROC) analysis was used to evaluate prognoses in patients with ULM-AMI. Statistical significance was set at Between November 2009 and June 2021, 48 patients with AMI due to total ULM occlusion underwent emergent coronary angiography at our institution (one patient with in-stent restenosis and one patient with acute in-stent thrombosis in ULM). Among them, 44 had primary PCI, two underwent fibrinolytic therapy followed by immediate PCI and two underwent emergent coronary artery bypass graft after coronary angiography. Collateral circulation was not assessed in two patients who underwent LM PCI before right coronary artery (RCA) angiography. So, 44 patients had LM PCI (RCA angiography prior to LM PCI) and were included in our study. Over the same period, 11, 343 patients were treated with primary PCI for AMI, and 58 patients with partial ULM obstruction (> 90% stenosis) were enrolled for ECG analysis.The baseline clinical and angiographic data of the 44 consecutive patients with acute total ULM occlusion are listed in Table p > 0.05). STEMI and absent collateral flow were significantly associated with shock . However, non-STE in lead aVR and STE in lead I were not associated with shock.Nineteen patients presented with the STEMI pattern, while 25 patients presented with the NSTEMI pattern Fig. A. Interep < 0.05). The sensitivities of STEMI, non-STE in lead aVR, and STE in lead I for collateral flow absence were 73%, 58%, and 62%, respectively. The specificities of these characteristics for collateral flow absence were 100%, 100%, and 83%, respectively.Eighteen patients presented with collateral circulation from the contralateral right coronary artery (RCA) (Rentrop score ≥ 1). Two patients with STE in aVL, aVR and V2-V3 had collateral flow (this ECG presentation was categorized as NSTEMI pattern due to no STE in V4-V5). Fisher’s exact test indicated that collateral flow presence/absence was associated with the characteristics of STE in ULM occlusion. STE in lead I and the STEMI pattern were associated with collateral flow absence, while STE in lead aVR was associated with its presence . The sensitivity of STE in lead aVR + aVL for collateral filling of the LAD territory was 90%, and the sensitivity of STE in lead aVR for collateral filling of the LAD and LCX territory was 100%. The representative ECG findings in patients with different collateral filling territories were shown in Fig. Furthermore, Fisher exact test indicated that STE characteristics in NSTEMI were associated with collateral filling territory. Patients with STE in both leads aVR and aVL showed more collateral filling of the LAD territory, while patients with STE in lead aVR showed more collateral filling of the LAD and LCX territory . The specificity of STE in leads aVR and aVL for total ULM occlusion was 97%, while the specificity of STE in lead aVR for partial ULM obstruction was 76%.The ECG characteristics of patients with partial ULM obstruction were shown in Fig. p < 0.05). The use of an intraaortic balloon pump before PCI did not improve clinical outcomes compared with insertion during the procedure. Similarly, mechanical ventilation did not improve the clinical benefits in these patients. The ECG findings of STE in lead I, non-STE in lead aVR, and STEMI were significantly associated with in-hospital mortality (Table p < 0.05). However, there was no difference in STE in lead aVL between the survival and mortality groups. Moreover, our study demonstrated that STE in lead I plus right bundle branch block predicted in-hospital mortality with a specificity of 96%.Eighteen 41%) patients died in the hospital due to cardiac rupture (3/18), refractory cardiogenic shock (10/18), or multiorgan failure (5/18). Fisher exact test indicated that cardiogenic shock, collateral flow absence, and post-PCI TIMI 0–2 flow were associated with in-hospital mortality , the overall incidence of MACE was 45%. The majority of mortality occurred within the first month post-procedure with near flattening of the survival curve afterward . Many previous studies on ULM were vague about the degree of ULM stenosis and included patients with occlusion and partial obstruction, leading to non-specific ECG findings , 11, 12.There is uncertainty regarding the ECG features in patients with AMI due to ULM occlusion. Previous studies with small samples reported several different ECG patterns associated with acute ULM obstruction: STE in aVR with less STE in V1 , STE in STE in lead aVR combined with diffuse ST depression  was considered to be representative of ULM occlusion in many previous studies , 18. HowPrevious studies have reported that some ECG characteristics are associated with in-hospital prognosis in ULM-related AMI. Yamaji et al.  reportedDespite improvements in the PCI technique in recent years, the mortality of these patients remains high. Emergent revascularization is the only effective therapy to reduce mortality in patients with cardiogenic shock complicating AMI . Two impIn the current study, we found that cardiogenic shock, post-PCI TIMI 0–2 flow, and collateral perfusion absence were associated with early mortality, which is in line with previous studies , 26. TheOur study had several limitations. First, this was a retrospective study with a small number of patients in a single center. Second, selection bias could have influenced the enrollment of patients with ULM occlusion because most of them had died before undergoing coronary angiography. Lastly, the number of participants in our study was too small to perform a multivariable-adjusted analysis of risk factors, so the predictive value of ECG patterns for in-hospital mortality may be limited. Further study is needed to evaluate these risk factors in LM occlusion, using a larger, prospective study.The present study clarified the association between ECG patterns and collateral filling territories in patients with total ULM occlusion: STE in the anterior leads indicated the absence of collateral circulation, STE in both leads aVR and aVL predicted the presence of collateral circulation from the RCA to the LAD territory, STE in aVR predicted the presence of collateral circulation from the RCA to both LAD and LCX territory. In-hospital mortality was associated with the presentation of cardiogenic shock, post-PCI TIMI 0–2 flow, collateral flow absence, STEMI, non-STE in lead aVR, and STE in lead I. STE in both leads aVR and aVL is a useful indicator for discriminating total ULM occlusion from partial ULM obstruction. Patients presenting with STE in both leads aVR and aVL should be recognized as high-risk individuals and have emergent coronary angiography.