Relationships to global and individual subdomains of cognition were also assessed. 5C-CPT and CPT-IP measures of performance (d-prime) were strongly correlated (r = 0.60). In a regression model, the 5C-CPT and CPT-IP collectively accounted for 54% of the total variance in MCCB total scores, and 27.6% of overall cognitive variance was shared between the 5C-CPT and CPT-IP. These results indicate that the reverse translated 5C-CPT and the gold standard CPT-IP index a common attentional construct that also significantly overlaps with variance in general cognitive performance. The use of simple, cross-species validated behavioral indices of attentional/cognitive functioning such as the 5C-CPT could accelerate the development of novel generalized pro-cognitive therapeutics for SZ and related neuropsychiatric disorders.Attentional dysfunction contributes to functional impairments in schizophrenia (SZ). Sustained attention is typically assessed via continuous performance tasks (CPTs), though many CPTs have limited cross-species translational validity and place demands on additional cognitive domains. A reverse-translated 5-Choice Continuous Performance Task (5C-CPT) for human testing—originally developed for use in rodents—was designed to minimize demands on perceptual, visual learning, processing speed, or working memory functions. To-date, no studies have validated the 5C-CPT against gold standard attentional measures nor evaluated how 5C-CPT scores relate to cognition in SZ. Here we examined the relationship between the 5C-CPT and the CPT-Identical Pairs (CPT-IP), an established and psychometrically robust measure of vigilance from the MATRICS Consensus Cognitive Battery (MCCB) in a sample of SZ patients ( Current pharmacological treatments, however, have limited efficacy for treating cognitive deficits. The link between cognition and functional outcomes, combined with lack-of-effective treatments, has galvanized research to identify pro-cognitive therapeutics for SZ patients6. While pre-clinical research has yielded insights that may inform the future development of pro-cognitive treatments, profound translational gaps across pre-clinical and clinical studies exist. These gaps remain in large part due to the limited use of cognitive paradigms with cross-species translational validity and relevance9.Attentional dysfunction is a core deficit in schizophrenia (SZ) that negatively impacts functional outcomes10. The Cognitive Neuroscience Treatment Research to Improve Cognition in Schizophrenia (CNTRICS) initiative was established to identify cross-species relevant behavioral paradigms of cognitive constructs such as attention11.Over the past 15 years the National Institutes of Mental Health (NIMH) has sponsored two projects intended to improve the measurement of cognitive deficits in SZ and identify promising tasks with translational validity. The first, the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) initiative, was an effort to standardize cognitive measurement for approval of any pro-cognitive compounds. The product of this initiative, the MATRICS Consensus Cognitive Battery (MCCB), was designed for use in clinical trials and now represents the “gold standard” assessment of cognitive functioning in SZ. Given that the purpose of this battery was to inform clinical trials, limited effort was made to develop this battery in conjunction with cross-species available non-human tests10, and was highlighted by the CNTRICS initiative as a promising cross-species paradigm for assessing the control of attention11. The 5C-CPT has shown good cross-species validity, including (a) 36 h sleep deprivation-induced deficits12; (b) amphetamine-induced improvement13; and (c) vigilance decrement observations across time10. Importantly, patients with SZ exhibit deficient 5C-CPT performance14, consistent with other CPTs16. To-date however, no studies have validated the 5C-CPT against a consensus gold standard attentional measurement nor evaluated how 5C-CPT performance is related to cognition in SZ.The 5-choice continuous performance test (5C-CPT) was developed for use in mice15. In their simplest form, CPTs involve subjects’ being presented with a string of rapidly occurring stimuli and asked to identify targets from among background or non-target stimuli. Common variants of the CPT include CPT-Identical Pairs version (CPT-IP- chosen as part of the MCCB), degraded stimulus CPT (DS-CPT), the AX-CPT (AX-CPT), and the Connors’ CPT (C-CPT). Whereas, all CPTs are designed to quantify sustained attention/vigilance, each CPT emphasizes a unique balance between target detection and response inhibition, which may have important implications for cognition. Important methodological differences also exist across tasks and place demands on other cognitive domains17. For example, the CPT-IP has been referred to as a “memory load” CPT given that responses are required when two sequentially presented numbers are identical, likely indexing both attention and working memory. The DS-CPT has been referred to as a “perceptual load CPT” since target stimuli with varying degrees of degradation are presented to subjects and therefore is sensitive to both perceptual and attentional deficits19. The AX-CPT requires responding when the letter X follows an A in sequence (ignoring B and Y stimuli presentations), likely requiring modest working memory functioning in addition to attentional control20. In contrast, the Connors’ CPT simply requires responses when target stimuli (letters other than X) are presented, and response inhibition when non-target stimuli (letter X) are presented21. Despite these task differences, CPT paradigms have demonstrated clinical sensitivity in quantifying attentional deficits in first episode and chronic SZ patients, unmedicated SZ patients, and unaffected first-degree relatives of SZ patients25.Continuous performance tasks (CPTs) are the most common paradigms used for quantifying attentional functioning in neuropsychiatric patients including SZ26. Attention is thought to represent a core domain of cognitive functioning, which might explain large correlations between measures of attentional performance and overall cognition as measured by the MCCB27. Furthermore, attention-dependent cognitive measures have been used as exemplars to demonstrate applicability as important targets of medications used to treat SZ2.Psychometric theories of cognition suggest that cognitive abilities are hierarchically structured such that variance (and covariance) in specific abilities can be explained by a smaller number of general abilitiesAlthough, CPTs are established measures of attentional functioning, they are often multidimensional (as described above), potentially confounding attentional dysfunction with deficits in perceptual or other cognitive domains, e.g., working memory. Moreover, lack-of-cross-species CPTs further limits opportunities for novel treatment development. Quantifying attentional abilities underlying normal and impaired cognitive performance in SZ patients on the 5C-CPT could accelerate the development of pro-cognitive therapeutics. The availability of tasks for measuring similar attentional abilities across species would enable researchers to develop agents for targeting attentional systems in animals and begin spanning the translational gap to human findings.This study aimed to characterize SZ patients’ attentional task performance on the reverse-translated 5C-CPT in comparison to a “gold standard” measure, the CPT-IP, and to assess its relationship with a global measure of cognition (MCCB). We hypothesized that measures of 5C-CPT and CPT-IP task performance would be significantly correlated and that attentional functioning (as measured by both CPTs) would predict cognitive test performance . To determine the effects of symptom severity on attentional and cognitive functioning, we assessed the extent to which 5C-CPT, CPT-IP, and MCCB scores were related to positive and negative symptom ratings.29, and cognitive assessment using the MCCB . The CPT-IP score was omitted when calculating the MCCB total score to avoid biasing predictive relationships. The MCCB neurocognitive composite score was calculated using the mean of the domain T-scores, as is consistent with prior publications30. The 5C-CPT was completed following the diagnostic and cognitive assessments.Thirty-five SZ patients between the ages of 18–61 years old were recruited from a transitional care facility that primarily serves people with diagnoses of SZ or schizoaffective disorder . All participants performed a practice block prior to initiating the session and indicated they understood the task. The full task consisted of 270 trials, 225 target and 45 non-target stimuli, presented pseudorandomly so that no >3 presentations of a specific stimulus appeared consecutively. Responses were recorded and include hits and misses to target trials, and false alarms (FAs) and correct rejections (CRs) to non-target trials. Composite metrics of task performance were used in the analysis of performance, including hit rate (HR), false alarm rate (FAR), task accuracy, d′, and responsivity index (RI) as indicated in our previous work6.The 5C-CPT requires responses to targets and inhibition of responses to non-target trials. Participants were instructed to move the joystick in the direction a circle (target stimuli) appears, but inhibit from responding if five circles appeared simultaneously (non-target stimuli) Fig. 14. Stimu31, the CPT-IP version is a computerized measure of sustained, focused attention. The task involves monitoring a series of numbers  and responding when two identical stimuli occur consecutively . Multivariate linear regression models using 5C-CPT and CPT-IP d′ measures as predictors, were used to determine the unique contribution of each behavioral measure to cognition . Estimates of variance explained (R2), standardized regression slopes (β), and Pearson correlations between predictors are reported32. Statistical significance for correlations was adjusted for multiple comparisons using the Bonferroni method34.All statistical analyses were conducting using SPSS . Paired sample r = 0.60, p < 0.001). Additional correlations between each CPT d′, MCCB total score and MCCB subscales are reported in Table r’s < 0.31, ps > 0.08)  = 18.7, p < 0.001). Comparisons of model parameter estimates (standardized regression slopes) indicated that 27.6% of the variance in global MCCB performance was shared attentional variance between the 5C-CPT and CPT-IP. The 5C-CPT further accounted for a unique but non-significant 2.0% of the variance in cognition , while the CPT-IP uniquely accounted for an additional 24.3% of the variance  in MCCB composite scores. A Venn diagram visualizing the relationships among the three variables is shown in Fig. To assess the shared and unique contributions of each CPT to cognition, MCCB composite scores were regressed onto 5C-CPT and CPT-IP d′ scores. A model including both CPTs accounted for 53.9% of the variance in MCCB composite score , and whose combined variance accounted for more than half of the variance MCCB total scores (53.9%), underscores the importance of attentional functioning to cognition. Regression analyses indicated that, of the 36% of variance shared between the 5C-CPT and the CPT-IP, 27.6% is likely attention-specific variance that contributes to global cognition   of the variance in MCCB performance after accounting for shared variance with 5C-CPT, indicating that CPT-IP performance may reflect not only attentional ability but also broader cognitive functioning. This finding is further supported by significant correlations between CPT-IP performance and MCCB subdomains of speed of processing and visual learning  were significantly better than those of the CPT-IP, suggesting that 5C-CPT may generally be less difficult than CPT-IP. The CPT-IP explicitly manipulates task difficulty across blocks using two-, three-, or four-digit strings, compared to the single difficulty level of the 5C-CPT. Supplementary analyses revealed a linear decrease in d′ with increased difficulty on the CPT-IP, consistent with prior reports38. The current data suggest good convergent validity among CPTs for attention measures, but also highlights the challenge of measuring a cognitive domain in isolation. The pattern of correlations between the 5C-CPT, CPT-IP, and MCCB subdomains indicates increased specificity in attentional measurement between the 5C-CPT and cognition compared to the gold standard CPT-IP. In contrast, correlations between CPTs and other cognitive domains ranged from small to moderate  all suffer from various levels of interpretive ambiguity by including additional cognitive task demands in the measurement of attentional functioning17. As attention is thought to be a necessary component of higher-order cognitive functioning, inter-correlations amongst related cognitive domains are to be expected. Thus, future research should utilize reverse-translated tasks targeting specific cognitive domains to better disentangle specific cognitive deficits in psychiatric patients. Given the availability of the mouse42 and rat44 5C-CPT versions, treatments that improve task performance in animals could potentially be used to improve global cognitive functioning in humans.Attentional functioning (in both CPTs) was also significantly correlated with other cognitive subdomains, and outlines attentional functioning as a necessary but insufficient construct underlying global cognition. Recent research has indicated the importance of attentional functioning for other cognitive processes , as well as its direct and indirect relationships with functional outcomes in SZ45. Beyond cognitive control, however, given the specific task demands of the 5C-CPT and low correlations with non-attentional MCCB subdomains, it is unlikely that other cognitive abilities account for such high-overlapping variance.A few study limitations deserve discussion, most notably the lack of a non-psychiatric comparison group. Future studies will investigate both SZ and healthy control samples to ascertain differential behavioral relationships between CPT task performance and cognitive functioning across groups. Additionally, as in the vast majority of SZ studies, all participants were medicated at the time of testing, with most treated on a combination of typical and atypical antipsychotic mediation along with other psychotropics. Although, the heterogeneity of medication regimens in our cross-sectional sample precluded the examination of medication effects, our behavioral measures were still sensitive to cognition with this medication-heterogeneous sample. Nonetheless, we cannot rule out an impact of antipsychotic medications on our findings, and future randomized controlled trials should prospectively examine medication effects on subjects. Finally, it is possible that the overlapping variance between 5C-CPT and CPT-IP represents a latent construct other than attention that is relevant to cognitive test performance in SZ. For example, these tasks may also provide a measurement of cognitive control that has been proposed to be consistently disturbed across psychiatric conditions5. This translational approach may provide further utility by identifying individuals likely to benefit from treatment and identifying those who may benefit from additional targeted pharmacological or psychosocial pre-treatments to help boost treatment gains and long-term functional outcomes. Finally, as attentional deficits may be present prior to full disease onset and signal poor functional outcomes, this approach may facilitate early identification of individuals at elevated risk for developing pathologies with primary attentional dysfunction.In conclusion, we demonstrate a novel reverse translated behavioral measure of attention is robustly related to an established measure of attention and cognitive test performance in a group of transitionally housed SZ inpatients. By validating novel reverse-translated laboratory measures like the 5C-CPT, together with existing gold standard measures of attention and cognition, we can provide more direct cross-species relationships to aid the development of pro-cognitive therapeutics, and ultimately improve functional outcomes in SZSuppl MaterialSuppl Fig IP2Supplemental legends


The Northwestern University SuperAging Program studies a rare cohort of individuals over age 80 with episodic memory ability at least as good as middle-age adults to determine what factors contribute to their elite memory performance. As psychological well-being is positively correlated with cognitive performance in older adults, the present study examined whether aspects of psychological well-being distinguish cognitive SuperAgers from their cognitively average-for-age, same-age peers.Thirty-one SuperAgers and 19 cognitively average-for-age peers completed the Ryff 42-item Psychological Well-Being questionnaire, comprised of 6 subscales: Autonomy, Positive Relations with Others, Environmental Mastery, Personal Growth, Purpose in Life, and Self-Acceptance.The groups did not differ on demographic factors, including estimated premorbid intelligence. Consistent with inclusion criteria, SuperAgers had better episodic memory scores. Compared to cognitively average-for-age peers, SuperAgers endorsed greater levels of Positive Relations with Others. The groups did not differ on other PWB-42 subscales.While SuperAgers and their cognitively average-for-age peers reported similarly high levels of psychological well-being across multiple dimensions, SuperAgers endorsed greater levels of positive social relationships. This psychological feature could conceivably have a biological relationship to the greater thickness of the anterior cingulate gyrus and higher density of von Economo neurons previously reported in SuperAgers. Age-related cognitive decline is widely considered to be a “normal” consequence of aging. However, evidence from the Northwestern University SuperAging Program suggests that such decline is not inevitable . SuperAgInitial results suggest that SuperAgers represent a rare cognitive phenotype with unique physiological brain features. SuperAgers are selected for having above-average-for-age episodic memory at the time of study entry and most maintain this high level of performance over at least 18-months, suggesting resistance to age-related memory decline . On struThis idea is furthered by post-mortem histopathologic analyses of the anterior cingulate, which demonstrated a lower frequency of Alzheimer disease pathology and a higher density of von Economo neurons (VENs) in SuperAgers compared to their cognitively average same-age peers . VENs arPsychological well-being is a positive psychological construct related to subjective views of one’s self and life. Higher levels of psychological well-being have been positively correlated with cognitive performance in older adults. For example, Llewellyn and colleagues (2008) demonstrated that among individuals over age 50, those who scored in the highest quintile on the CASP-19 psychological well-being scale scored higher on neuropsychological tests than those who scored in the lowest quintile . PreviouTable 1. Inclusion criteria for SuperAgers is as follows: 1) performance at or above normative values for average 50 to 65-year-olds  on delayed recall of the Rey Auditory Verbal Learning Test (RAVLT), a 15-word list-learning episodic memory test [Data were prospectively collected from participants enrolled in the Northwestern University SuperAging Program, an ongoing, longitudinal investigation of aging in community-dwelling adults over age 80 who have been described previously . Brieflyory test ; 2) perfory test , Trail Mory test , and Catory test . In contAll participants were required to have preserved activities of daily living and lack clinical evidence of significant neurological or psychiatric illness. In the present study, participants were required to maintain their cognitive status (SuperAger or cognitively average elderly) from enrollment to the time questionnaires were completed to minimize the unintended inclusion of individuals with emerging Alzheimer’s dementia and maintain the integrity of our elite SuperAger sample.The Northwestern University Institutional Review Board approved study procedures. All participants provided written informed consent.. This six-factor model has support for use in healthy elderly adults [The Ryff 42-item Psychological Well Being questionnaire  and 19 Cognitively Average Elderly adults  were actively enrolled in the SuperAging Program, met inclusion criteria, and completed the PWB-42 questionnaire. All are included in this analysis.p’s>0.05; Table 3). Consistent with inclusion criteria, SuperAgers had significantly better RAVLT performance compared to their cognitively average peers (median delay raw score (interquartile range (IQR)) SuperAgers: 11 (10–13); Cognitively Average Elderly: 7 (6–7)); p<0.001). The groups did not differ on other neuropsychological measure, including estimates of premorbid intellectual ability (p’s>0.05).There were no significant group differences in age, gender, race, or education : 40 (36–41); mean item score: 5.5) compared to Cognitively Average Elderly adults : 36 (33–39); mean item score: 5.1; p = 0.005). There were no other significant between-group differences on PWB-42 subscales (p’s>0.0083). There were no significant gender differences within either group on the PWB-42 subscales .Scores from the PWB-42 are shown in p = 0.013) and between RAVLT performance and Positive Relations with Others , but were not significant with Bonferroni correction.Spearman correlation showed low positive associations between RAVLT performance and Environmental Mastery ; , suggestThe presence of gender differences in measures of psychological well-being are inconsistent across studies, with some studies showing that women tend to rate themselves higher on Positive Relations , 34 and In summary, SuperAgers endorse higher levels of Positive Relations with Others compared to their cognitively healthy but average-for-age same-age peers suggesting that this aspect of psychological well-being may be an important factor for exceptional cognitive aging. Investigation of the longitudinal effects of psychological well-being on subsequent cognitive performance and investigation of the conceivable relationship between psychological well-being and von Economo neurons in SuperAgers represent intriguing future directions.S1 FileThis file contains the raw data used for analyses.(XLSX)Click here for additional data file.


Adipose tissue lipolysis occurs during the development of heart failure as a consequence of chronic adrenergic stimulation. However, the impact of enhanced adipose triacylglycerol hydrolysis mediated by adipose triglyceride lipase (ATGL) on cardiac function is unclear. To investigate the role of adipose tissue lipolysis during heart failure, we generated mice with tissue-specific deletion of ATGL (atATGL-KO). atATGL-KO mice were subjected to transverse aortic constriction (TAC) to induce pressure-mediated cardiac failure. The cardiac mouse lipidome and the human plasma lipidome from healthy controls (n = 10) and patients with systolic heart failure  were analyzed by MS-based shotgun lipidomics. TAC-induced increases in left ventricular mass (LVM) and diastolic LV inner diameter were significantly attenuated in atATGL-KO mice compared to wild type (wt) -mice. More importantly, atATGL-KO mice were protected against TAC-induced systolic LV failure. Perturbation of lipolysis in the adipose tissue of atATGL-KO mice resulted in the prevention of the major cardiac lipidome changes observed after TAC in wt-mice. Profound changes occurred in the lipid class of phosphatidylethanolamines (PE) in which multiple PE-species were markedly induced in failing wt-hearts, which was attenuated in atATGL-KO hearts. Moreover, selected heart failure-induced PE species in mouse hearts were also induced in plasma samples from patients with chronic heart failure. TAC-induced cardiac PE induction resulted in decreased PC/ PE-species ratios associated with increased apoptotic marker expression in failing wt-hearts, a process absent in atATGL-KO hearts. Perturbation of adipose tissue lipolysis by ATGL-deficiency ameliorated pressure-induced heart failure and the potentially deleterious cardiac lipidome changes that accompany this pathological process, namely the induction of specific PE species. Non-cardiac ATGL-mediated modulation of the cardiac lipidome may play an important role in the pathogenesis of chronic heart failure. Chronic heart failure (CHF) is still one of the leading causes of morbidity and mortality worldwide, and new therapeutic approaches are required. Here we show that perturbation of adipose tissue lipolysis by tissue-specific deletion of the lypolytic enzyme ATGL (adipose trigylceride lipase) protects against heart failure development. By using MS-based lipidomics we show that the lack of ATGL in adipose tissue protects the heart against deleterious cardiac lipidome changes such as increases of phosphatidylethanolamines. Changes of cardiac phosphatidylethanolamines may contribute to adipose ATGL-mediated heart failure pathophysiology by an impairment of cell membrane integrity. These data point towards a crucial function of ATGL in adipose tissue by regulating processes outside of the primary target tissue such as the heart. This is a new concept which may lead to alternative studies of gene function with a focus on non-target tissue and tissue/ organ interactions. Furthermore, therapeutic targeting of non-cardiac tissue may provide new opportunities for future heart failure therapy. The development of chronic systolic heart failure is marked by a continuous incline in adrenergic activity that results in the typical clinical signs of this disease, including tachycardia and increased peripheral vascular resistance , 2. In aTriacylglycerol (TAG) hydrolysis in adipose tissue occurs in a stepwise process and involves three major lipases: adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoglyceride lipase (MGL), the cumulative effect of which is an efflux of glycerol and FFA from adipocytes into the circulation . ATGL, aMice carrying a constitutive deletion of ATGL showed a marked reduction of FA release from white adipocytes and of TAG accumulation in adipose tissue . SimilarIn this study, we report that perturbation of adipose tissue lipolysis by conditional deletion of ATGL protects against pressure-induced cardiac failure. By applying an untargeted shotgun lipidomics approach, we identified cardiac lipidome changes significantly associated with LV failure and demonstrated that these were prevented by ATGL deficiency in adipose tissue. In particular, failing wild-type (wt) hearts exhibited a marked increase of multiple phosphatidylethanolamines (PE)  whereas ceramides (Cer) , and cardiolipin (CL76:12) were decreased. These changes were prominently attenuated in atATGL-KO hearts. Moreover, distinct heart-failure-regulated PE species in mouse hearts were modified in a similar manner in plasma samples from patients with chronic heart failure. These findings strongly suggest that adipose tissue lipolysis determines cardiac morphology and function during heart failure development. This inter-organ crosstalk likely involves an alteration of the cardiac lipidome which may mediate functional impairment by mechanisms such as induction of apoptosis or mitochondrial dysfunction.We previously described that fabp4-Cre-driven ATGL deletion resulted in a lack of ATGL in white adipose tissue but not in the heart or in bone marrow-derived macrophages, and in an attenuation of adipose tissue lipolysis . To exclMice were challenged with transverse aortic constriction (TAC) to induce pressure-mediated cardiac enlargement or failure. Pressure gradients (pre-/ post-stenotic) in TAC mice assessed by Doppler echocardiography did not differ between wt- and atATGL-KO mice, indicating similar degree of stenosis, LV-pressure load and aortic vascular function in both genotypes . Eleven weeks post TAC, wt hearts increased markedly in size and weight compared to wt-sham mice . TAC-indReduced adipose tissue lipolysis in atATGL-KO mice led to improved insulin sensitivity and glucose tolerance compared to their wt littermates . In addiWe hypothesized that dysregulated serum FAs may serve as the biochemical bridge between the lack of ATGL in adipose tissue and the observed cardiac phenotype. Thus, we next investigated whether modifications of the circulating FA profile are accompanied by changes in the cardiac lipidome using MS-based shotgun lipidomics.A total of 225 lipid species from 18 different lipid classes were analyzed in LV samples, and included glycerolipids, glycerophospholipids, sterol lipids and sphingolipids . The mosAmong the identified lipid species, the most significant change was an increase in individual PE species in failing wt-hearts (TAC) compared to wt-sham hearts . This inIn summary, lack of ATGL in adipose tissue and reduced lipolysis associated with the reduction of selected circulating FAs mediates pressure-induced modifications of distinct cardiac lipid species among which PEs display the most apparent regulation.In order to better understand the degree and relevance of cardiac lipid changes during LV failure, we performed an in-depth analysis of all lipid classes in which the log2-fold change (sham vs. TAC), and the concentration of each lipid species (mean mole %) was included in a pairwise comparison (sham vs. TAC) in wt and atATGL-KO mice. Recently, a decrease of the PC/ PE ratio has been identified to regulate cell membrane integrity in hepatocytes, and was accompanied by pronounced cell damage . Thus, wIn summary, these data demonstrate that pressure-mediated LV failure is associated with an increase of distinct cardiac PE-species leading to a reduction of cardiac PC/PE ratios. The decline of PC/ PE ratios is accompanied by the enhanced expression of cardiac apoptosis markers. Deficiency of adipose tissue ATGL prevents cardiac PE upregulation in an FA-specific manner and protects the heart against LV failure.To explore the relevance of our findings to LV failure in humans, we performed shotgun lipidomics analysis using plasma samples obtained from male patients with HFrEF (n = 13) and male control subjects with normal LV function (n = 10). The clinical characteristics of both groups are outlined in We analyzed 147 lipid species from 13 lipid classes in human plasma . PCs, TATaken together, our data from human plasma samples demonstrate that the presence of HFrEF is accompanied by a dysregulation of individual lipid classes and lipid species. Interestingly, comparable changes in PE species were observed in failing mouse hearts and in plasma from HFrEF patients.In this study, we demonstrated for the first time that pressure-induced cardiac failure is accompanied by marked changes in the cardiac lipidome. The most robust alteration in lipid species was an upregulation of distinct PE species in failing wt-hearts. Perturbation of adipose tissue lipolysis in atATGL-KO mice led to an improvement of pressure-induced heart failure accompanied by the prevention of cardiac PE induction. Pressure-induced cardiac PE induction resulted in a decreased PC/ PE-ratio and a concurrent increase in apoptotic marker expression in failing wt-hearts, a process which was absent in atATGL-KO hearts. Taken together, this study demonstrates that the cardiac lipidome undergoes profound changes during heart failure development. The absence of these changes in atATGL-KO mice suggests that non-cardiac tissues such as adipose tissue, and in particular ATGL-mediated adipose tissue lipolysis, participate in the regulation of the cardiac lipidome.The role of ATGL in heart failure development and cardiac metabolism has been previously studied, with most studies focusing mainly on cardiac ATGL function –20. In tCommunication between the heart, the vasculature and other organs has been recently described as an important pathophysiological mechanism involved in the development of cardiovascular disease . Cross-tLipidomic analysis using advanced MS-technologies has been recently utilized in cardiovascular research for the identification of new biomarkers in the context of risk prediction . Stegema2), a crucial generator of two second messenger molecules, DAGs and inositol 1,4,5-triphosphate (IP3) [In addition, we found an increase of cardiac PIs in wt-TAC mice compared to sham mice, a regulation absent in atATGL-KO mice. PIs are important precursors of PI  biphosphate (PIPte (IP3) . Both site (IP3) , 33. ThuThe results described in this study are the first to demonstrate an induction of cardiac PE species during the development of LV failure. Furthermore, we showed that these cardiac lipidome changes were dependent on adipose tissue lipolysis. PEs are the most abundant phospholipids in mammalian cells, and are predominantly located in plasma membranes and mitochondrial membranes . DisruptHow does the increase in PE species affect the development of LV failure during pressure overload, and can the absence of this regulation explain why atATGL-KO mice are protected from LV failure? PEs are major constituents of plasma membranes . PreviouAn additional mechanism of how enhanced ATGL-activity/ AT-lipolysis and increased cardiac PE-levels may impair cardiac function might be the modulation of β-adrenergic signaling. Cardiac β2-adrenergic stimulation has been recently shown to be protective in CHF . In paraWe also identified changes in cardiac sphingolipid and CL levels. In particular, CLs have been implicated in the regulation of cardiac energy metabolism and function , 46. A dFinally, changes in distinct plasma lipid classes and individual lipid species were shown in a small group of HFrEF patients when compared to a non-HFrEF control group. These data allude to the presence of lipidomic plasma changes in human heart failure which have been previously described in a metabolomics study . In our Taken together, the results of this study demonstrate for the first time that perturbation of adipose tissue lipolysis by deletion of AT-specific ATGL leads to an improvement in pressure-induced heart failure, and can prevent potentially deleterious cardiac lipidome changes such as PE induction. Furthermore, we showed that the development of LV failure is associated with a distinct lipidomic profile in human plasma and mouse hearts, suggesting a process of cardiac FA partitioning into different cardiac lipid classes. Further studies are required to improve our understanding of the underlying mechanisms and functional consequences of cardiac FA partitioning during heart failure, and to identify novel therapeutic targets for the treatment of this disease.Male adipose tissue specific adipose triglyceride lipase (ATGL) deficient mice (atATGL-KO) and control littermates (wt) were generated, as described previously . Mice weAll animal procedures were performed according to the guidelines of the Charité Universitätsmedizin Berlin and were approved by the Landesamt für Gesundheit und Soziales  for the use of laboratory animals and according to the current version of the German Law on protection of animals.Echocardiographic analysis was performed 11 weeks after sham/TAC-intervention, using Vevo 770 high-resolution imaging system with a 30-MHz transducer  as described previously .Animals were metabolically phenotyped after 10 weeks. Intraperitoneal glucose tolerance tests (ipGTT) and intraperitoneal insulin tolerance tests (ipITT) were performed using a dose of 1 g/kg body weight (BW) glucose and by injecting 0.25 U/kg BW insulin , respectively, as described previously . Tail veMouse HL-1 cardiomyocytes, kindly provided by W.C. Claycomb  were cultivated and stimulated, as described previously . BrieflymRNA analysis and western immunoblotting were performed as previously described . The detCardiac tissue samples were formalin-fixed, paraffin-embedded and stained with Hematoxylin/Eosin, and Picrosirius red, and analyzed, as previously described , 56.Cardiac myocyte cross sectional area was assessed using H/E-stained sections of myocardium. Perimeter of the cell borders of 50 myocytes cut transversally were measured using cellSens (Olympus). Myocytes were selected in up to ten 400x microscopic fields each separated into 20 equally sized squares. Only one transversally myocyte per square was measured to increase representativeness. Fibrotic content (collagen fibers stained with picrosirius red) was assessed from the total cross section by a blinded expert in veterinary pathology using an analysis software (Olympus) and a semi-quantitative scoring system , 2 = several collagen fibers (moderate), 3 = massive accumulation of collagen fibers (severe)).Human fasting plasma samples were collected from healthy subjects and patients with systolic heart failure (HFrEF) at the Division of Cardiology, University Hospital RWTH Aachen, Germany. Human plasma was isolated from EDTA blood samples by centrifugation at 2000rpm for 10min. Supernatant was collected and stored at -80°C as 200μL aliquots. Left ventricular (LV) ejection fraction was assessed by standard echocardiography. Blood sampling and clinical data collection and analysis was approved by an institutional review board , and all participants provided written informed consent.Mouse serum was isolated from whole blood. Blood samples were placed for 60min at room temperature for induction of coagulation. Afterwards, samples were centrifuged at 5000rpm for 5min at 4°C, and supernatant (serum) was collected. For FA serum profiling, 40μl samples of murine serum were hydrolyzed under alkaline-methanolic conditions, neutralized and diluted 1:10 in methanol containing internal FA standards. The HPLC measurement (Agilent 1200 HPLC system), coupled with an Agilent 6460 triple quad MS was performed, as described previously [Lipids from adipose tissue were extracted using a two-step chloroform/ methanol procedure . SamplesLipid extraction and analysis of mouse LV samples (3mg per sample) and human plasma (15μl per sample) were performed as described previously , 59 at LSamples were analyzed by direct infusion on a QExactive mass spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in both positive and negative ion modes with a resolution of Rm/z = 200 = 280000 for MS and Rm/z = 200 = 17500 for MSMS experiments, in a single acquisition. MSMS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1 Da increments . Both MSMeasured lipid amounts were normalized to total lipid amount in samples and log-transformed to approach a symmetric and approximative normal distribution. Lipid species with missing values across many samples were removed from further analysis, and in total, we evaluated the results of 225 lipid species in the mouse and 147 in the human data set. Normal distribution was checked visually by using Q-Q plots. Depending on their scale and distribution, results are given in proportions, mean, standard deviation or standard error of the median with 25% to 75% quartiles as indicated. Statistical tests for significance were performed by using the tow-tailed t-test or Mann-Whitney-U-test as appropriate. Multivariable analysis was done by linear and robust linear regression with log-transformed relative lipid amounts as dependent and experimental or clinical conditions, age and BMI, as independent variables. For class level comparisons only, we used regression or median imputation of missing values. To avoid alpha inflation, p-values were adjusted by the Benjamini-Hochberg procedure . AdjusteTo compare differences between groups of mice phenotypes, we performed two-way ANOVA (Bonferroni posttest), two-way ANOVA with repeated measures (Bonferroni posttest) or t-test, as appropriate, and data was analyzed with GraphPad Prism Software. Statistical significance was assumed at p<0.05. Vertical lines in the histograms indicate means ± standard error of the mean (SEM). The n-number is indicated for each experiment.S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableShown are raw data lipid concentrations in pmole.(CSV)Click here for additional data file.S3 Table(CSV)Click here for additional data file.S4 Table(CSV)Click here for additional data file.S5 TableShown are raw data lipid concentrations in pmole.(CSV)Click here for additional data file.S1 FigA: ATGL mRNA expression in cardiac endothelial cells. Cardiac endothelial cells were isolated from cardiac tissue of atATGL-KO- and wt-mice using a Langendorff perfusion system, collagenase Type II digestion and CD31 microBeads Macs Miltenyi Biotec System, as described in methods. qRT-PCR studies of ATGL expression, relative to 18S were carried out using total RNA isolated from those cells . B: MS-based shotgun lipidomics analysis of Triacylglycerol (TAG) species in white adipose tissue samples (WAT) isolated 11 weeks after intervention (sham) from wt- and atATGL-KO mice. *p<0.05 vs. wt sham. C: Profile of selected serum FAs in sham-operated mice analyzed by rapid resolution HPLC/ Tandem MS. . *p<0.05 vs. wt sham. D: WB analysis of HL-1 cells cultivated in starving (0.5% FBS) or full medium (10% FBS), using antibodies against cleaved caspase 3, loading control: beta-Actin (βActin).(TIF)Click here for additional data file.


Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE) with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay) for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria . 53  Enterobacteriacae (CPE) [A. baumannii OXA-23 is the most prevalent carbapenemase in Europe [Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed and considered to be a significant threat to patient safety –3. Carbaae (CPE) ,4. Amongn Europe ,6.Treatment of patients suffering from infections with carbapenemase-producing MDR-GNB is difficult as only a few alternative treatment options remain . Being oEnterobacteriaceae  and 10 carbapenemase-negative Enterobacteriaceae were selected for analysis. One VIM-1-positive isolate was excluded, as on retesting it no longer showed decreased carbapenem susceptibility. All 27 members of the A. baumannii-complex  were subjected to analysis by both assays. After incubating overnight at 36 °C on blood agar , a cell suspension (McFarland 0.5–1) was prepared using UltraPure™ DNase/RNase-Free Distilled Water  and subjected to both tests.Between August 2012 and August 2016 146 MDR-GNB with decreased carbapenem susceptibility were isolated from various clinical specimens in our laboratory and carbapenemase-production was confirmed at the molecular level at the German National Reference Laboratory (NRL) for Multidrug-resistant Gram-negative bacteria . Copy strains were excluded . Isolate®, Straßberg, Germany) allows the genetic detection of various carbapenemases by reverse hybridization , 0.5 μl (= 2.5 units) HotStarTaq Plus DNA Polymerase  and 5 μl sample DNA. Thermal cycling conditions were used as proposed by AID. Detection of the biotinylated amplicons was performed by reverse hybridization with the carbapenemase probes on the provided nitrocellulose strips. Results were interpreted per the AID instructions. The total time to perform AID line probe assay from cultural isolates to result is approximately 300 minutes.The AID carbapenemase line probe assay RDB2290  detects the presence of the carbapenemases KPC, NDM, VIM and OXA-48 by multiplex real-time PCR . The assEnterobacteriaceae OXA-48 was the most frequently detected carbapenemase. Among A. baumannii-complex OXA-23 was the predominant carbapenemase. Both assays missed IMP-14 with IMP-14 being in the proposed spectrum of AID line probe assay but not Check-Direct CPE  were confirmed after analysing the organisms from fresh stock cultures.Among rect CPE . ID and Enterobacteriaceae-strains. One of twenty-five (4%) carbapenemase-positive members of A. baumannii-complex were detected by AID line probe assay, while Check-Direct CPE detected none (Both assays correctly confirmed 42/43 (97.7%) of the carbapenemase-producing ted none . Both teThe main goal of the current study was to find a reliable method for confirmation and typing of carbapenemases in phenotypically suspicious cultured isolates in order to provide guidance for antibiotic therapy and to rapidly institute infection control measures for these difficult-to-treat bacteria. Thus, we compared the commercially available AID line probe assay with the Check-Direct CPE multiplex-PCR in well-characterized cultured MDR-GNB-isolates.Our results show, that the tested assays are highly reliable to confirm CPE as respective sensitivities were 97.7%. Both assays failed to detect IMP-14 with IMP-14 being in the proposed spectrum of AID line probe assay but not Check-Direct CPE.A. baumannii-complex. It appears highly desirable to augment these assays with the capability of detecting OXA-23, as this enzyme may also emerge in Escherichia coli [A. baumannii-complex strains was very low, the A. baumannii strains with carbapenemases not targeted by the assays are negative specificity controls for this line of bacteria.In line with current epidemiology both tests appeared unsuitable for analysis of members of the hia coli ,11. HoweAssuming that both assays are able to detect their predicted carbapenemase targets, we calculated expected sensitivities using the nationwide German data of the NRL Bochum of the year 2015 . ExpecteA recently published study showed also excellent diagnostic reliability using AID line probe assay for cultured isolates. In contrast, direct molecular testing of urine samples revealed problems with specificity/positive predictive values as positive AID line probe assay-results could not be confirmed by culture methods . This faIn general, false-positive results seem to be appearing in many studies performing molecular carbapenemase testing in stool samples or rectal swabs ,15,16. IThe major strength of our study was the utilization of well-characterized MDR-GNB-isolates that were analysed by the German NRL for Multidrug-resistant Gram-negative bacteria as reference standard. There, confirmation of carbapenemase-production was performed on a molecular level. By intentionally using cultured isolates our aim was to focus not primarily on the hygiene and contact isolation issue using the assays for screening of primary specimens, but on confirmation of carbapenemase-production in phenotypically suspicious isolates of a relevant clinical sample , i.e. reduced carbapenem susceptibility and/or positive modified hodge test. So, we suggest that a combination of culture plus susceptibility testing and molecular methods is the ideal workflow.A. baumannii-complex was low as prevalence of carbapenemase-production (92.6%) was high in these MDR-GNB  NDM and VIM because both are detected by the same fluorochrome. In regard to the goal concerning the current needs, differentiation between class A, B and D will be sufficient, as the newly introduced β-lactamase inhibitor avibactam shows no activity against class B and partial activity against class D . Current


Revealing the cause-and-effect mechanism behind drug-disease relationships remains a challenging task. Recent studies suggested that drugs can target microRNAs (miRNAs) and alter their expression levels. In the meanwhile, the inappropriate expression of miRNAs will lead to various diseases. Therefore, targeting specific miRNAs by small-molecule drugs to modulate their activities provides a promising approach to human disease treatment. However, few studies attempt to discover drug-disease causal relationships through the molecular level of miRNAs. Here, we developed a miRNA-based inference method miRDDCR to comprehensively predict drug-disease causal relationships. We first constructed a three-layer drug-miRNA-disease heterogeneous network by combining similarity measurements, existing drug-miRNA associations and miRNA-disease associations. Then, we extended the algorithm of Random Walk to the three-layer heterogeneous network and ranked the potential indications for drugs. Leave-one-out cross-validations and case studies demonstrated that our method miRDDCR can achieve excellent prediction power. Compared with related methods, our causality discovery-based algorithm showed superior prediction ability and highlighted the molecular basis miRNAs, which can be used to assist in the experimental design for drug development and disease treatment. Finally, comprehensively inferred drug-disease causal relationships were released for further studies. Therefore, it is of critical importance to investigate how drugs and diseases form their causal relationships in the molecular level. Many evidences suggest that a drug can act on multiple targets rather than one target3. More recently, increasing studies revealed that drugs can target microRNAs (miRNAs), which are short (~22 nucleotide) non-coding RNAs, and regulate their expressions. For instance, Rossi et al.4 detected that the expression levels of 22 miRNAs were altered with the treatment of 5-fluorouracil in human colon cell line.Most drugs achieve their therapeutic functions by binding to specific molecular targets which are relevant to an abnormal state, thereby changing the biochemical and/or biophysical activities of these molecules7. Thus, accumulating researches indicated that miRNAs are involved in a broad range of biological processes, such as cellular signaling8, proliferation10 and metabolism11. As such, changes in the expression levels of particular miRNAs are related to many kinds of critical diseases. For example, the reduced expression level of let-7 was shown to be associated with lung cancer progression12. In addition, miRNAs are suitable to be drug targets as they have several attractive features, such as specific secondary structures and conserved sequences13. As a result, restoring miRNA expression levels with small-molecule drugs offers an innovative and promising approach to human disease treatment13, which has brought forward to a new research field of miRNAs in pharmacogenomics14.miRNAs are single-stranded RNAs with post transcriptional regulatory functions. They regulate gene expressions by base pairing to complementary sequences of their target mRNAsWith this understanding, studying drug-disease causal relationships under the perspective of their genetic basis miRNAs could not only help to unveil mechanisms of action of drugs but also advance public health. However, few researches address this question in a systematic view.et al.15 created computational models to predict drugs’ biological activities on miRNAs. Jiang et al.16 applied transcriptional responses to identify associations between drugs and cancer-related miRNAs. Depending on gene expression signatures of bioactive small molecule perturbation and Alzheimer’s disease (AD)-related miRNA regulation, Meng et al.17 presented a systematic computational approach to constructing a drug-miRNA association network in AD. By integrating features of drugs and miRNAs, Lv et al.18 proposed an algorithm of Random Walk with Restart (RWR) to infer new associations between small molecules and miRNAs. The above four methods all identify only drug-miRNA associations, but do not comprehensively provide therapeutic potential for drugs.Based on chemical descriptors and machine learning algorithms, Jamal 33 have been devoted to detecting potential miRNA-disease associations for understanding the molecular mechanisms of diseases. These methods mainly relay on the assumption that miRNAs tend to show similar dysfunctional evidences for similar disease clusters34. Generally, for these researches various features were first integrated for miRN-miRNA and disease-disease similarity calculation. Network-based or machine learning-based algorithms were then developed to rank the most promising disease-related miRNAs or miRNA-related diseass for further biomedical tests. These studies provide reliable guidance for in vivo experiment design. However, one limitation lied in these methods is that they could not provide information of disease treatment.In the meantime, as miRNAs are highly relevant to multiple complex diseases, many computational effortset al.35 applied hyper-geometric tests by combining existing drug-miRNA associations and miRNA-disease associations to predict drug-disease associations. This is the first computational model proposed to infer drug-disease associations, in which the molecular basis miRNAs is explicitly included. Even though a high AUC value could be received, this method is not workable for new drugs whose drug-miRNA associations cannot be obtained.For the above studies, predictions of drug-miRNA associations and miRNA-disease associations were treated separately. We argue that by incorporating information of target miRNAs, we can make more insightful drug-disease causal relationship predictions. Based on the assumption that drugs will form relationships with diseases when they share some significant miRNA partners, Chen miRNA-based method to extensively predict drug-disease causal relationships (miRDDCR). Based on two previous researches34, the proposed method miRDDCR relied on the hypothesis that similar small molecules tend to target similar miRNAs, and finally treat similar diseases. miRDDCR can predict drug-disease relationships in a large scale by combining similarity measurements, existing drug-miRNA associations and miRNA-disease associations. To evaluate the prediction performance of our method, leave-one-out cross validations (LOOCV) were conducted and satisfied AUC values could be received. Compared with existing methods, our method miRDDCR shows superior prediction ability. Moreover, case studies of two drugs demonstrated that our method is powerful in predicting drug-disease causal relationships with a high level of reliability. After validating the usefulness of our method, we used miRDDCR to comprehensively infer drug-disease causal relationships, which we hope will facilitate further drug discovery and disease treatment.In this paper, we developed a The datasets (Supplementary Dataset S1) used in our paper consisted of 831 drugs, 540 miRNAs, 341 diseases, 630 drug-miRNA associations and 6082 miRNA-disease associations. A whole view of the drug-miRNA bipartite graph and the miRNA-disease bipartite graph could be seen in Figs α1 and α2 were decay factors. The other 4 parameters 36 to set There exist 6 parameters involved in our algorithm. The parameters 37, DrugBank38 and Therapeutic Targets Database (TTD)39. We collected 13490 drug-disease relationships which were relevant to our study from the three databases. These confirmed relationships were used as a gold standard dataset for performance evaluation. Taking the known 13490 relationships as the positive instances, a receiver operating characteristics (ROC) curve Dipyridamole).Furthermore, we used leave-one-drug-out cross-validations (LOOCV) to evaluate the performance of miRDDCR in predicting drug-disease causal relationships. For the whole 831 drugs, each drug was considered as a test drug once and all its drug-miRNA association information was removed. The remaining 830 drugs were taken as the training dataset. For the 831 drugs, there existed 201 drugs whose experimentally confirmed drug-disease relationships were not available in the gold standard dataset. Therefore, we could not calculate their AUC values. For the remaining 630 drugs, the distribution of their AUC values, when leave-one-drug-out cross validations were implemented, could be seen in Fig. 35, in which drug-disease causality relationship predictions were only based on known drug-miRNA associations and miRNA-disease associations. For novel drugs, whose drug-miRNA associations were not available, their indications could not be inferred by this method. Our method miRDDCR overcame this drawback by taking advantage of similarity measurements. In the leave-one-drug-out cross-validation section, we considered each drug as a novel drug and reliable prediction ability could be obtained.Until recently, efforts made on causality discovery-based methods for drug-disease relationship predictions were rare. The most related study to ours is the inference model introduced in ref.43 have been put forward to predict potential drug–disease relationships. They first integrated multiple types of features to construct drug-drug and disease-disease similarity metrics. Depending on the similarity values and experimentally verified drug-disease relationships, machine learning-based or network-based methods were then developed to predict new indications for drugs. Different sources of information prevented a direct comparison between these methods and miRDDCR. Generally, these methods relayed heavily on known drug-disease relationships for new drug-disease association prediction. Obviously, experimentally confirmed drug-disease associations are scarce. The sparsity of data preparation might influence prediction accuracy. Our method did not need known drug-disease associations for prediction. Moreover, these methods, unlike our method miRDDCR, provided no molecular hints to help design experiments to test and confirm the results. Our method made full use of the information of target miRNAs and provided valuable resource for drug design and disease treatment.With the accumulation of biochemical data, several algorithmsAlmitrine) and CID:137 (Aminolevulinic acid), were analyzed for further evaluation of the ability of our method miRDDCR to predict potential drug-disease causal relationships.In this experimental scenario, case studies of the two drugs, CID:33887 (Almitrine), there was no target miRNA information in the prepared drug-miRNA dataset. After the first round of bi-random walks in miRDDCR on the drug-miRNA bipartite network, potential target miRNAs were ranked, in which the top 5 miRNAs were hsa-mir-21, hsa-mir-27b, hsa-mir-23a, hsa-mir-27a and hsa-mir-155. With the second round of bi-random walks on the miRNA-disease bipartite network, the indications of the drug Almitrine could be received. The top 10 predicted diseases were breast neoplasms, hepatocellular carcinoma, stomach neoplasms, colorectal neoplasms, melanoma, lung neoplasms, neoplasms, ovarian neoplasms, heart failure and prostatic neoplasms. The 9th (9/341) disease heart failure was the only indication available for the drug in the gold standard dataset and we successfully predicted this relationship with a high rank. It should be noted that 4  out of the top 5 target miRNAs, when expressed abnormally, were involved in the development of heart failure44.For the drug CID:33887 (Aminolevulinic acid), its potential indications were ranked according to the scores received by our algorithm miRDDCR. We selected the top 5, top 10, top 20, top 30 and top 40 predicted diseases and found that there were 5, 9, 17, 21 and 23 results supported by the gold standard dataset, respectively. We took the top 1 predicted disease breast neoplasms as an example to explain the inference ability of our algorithm. For the drug CID:137 (Aminolevulinic acid), there was no target miRNA information in the 630 drug-miRNA associations. After the first round of random walk, we chose the top 10 predicted target miRNAs and discovered that the 3rd target (hsa-mir-450a-2), the 5th target (hsa-mir-23a), the 6th target (hsa-mir-29b-2), the 7th target (hsa-mir-320b-1) and the 10th target (hsa-mir-375) were associated with the predicted disease breast neoplasms44.For the drug CID:137  was available for further experiment tests. For each of the 831 drugs, we published the top 50 predicted candidate indications for future studies. The full list of the whole inferred relationships can be obtained from the Supplementary Dataset S3 online. To be more accurate, we suggested taking the prediction results for the 376 drugs, whose AUC values were greater than 0.7 in leave-one-out cross validations, into consideration.Exploring the molecular mechanism behind the curative effects of drugs is crucial for drug development and disease treatment. Recent researches suggested that drugs can achieve their therapeutic functions by targeting miRNAs because the abnormal expression of miRNAs could lead to a lot of complex diseases and drugs can bind miRNAs to restore their expression levels. As a relatively new discipline in biomedical research, our understanding of drug-targeted miRNAs and miRNA-related diseases is lacking. Furthermore, few studies attempted to systematically discover drug-disease causal relationships through the target miRNAs.In this paper, we developed an approach miRDDCR to revealing the causal relationships between drugs and diseases under their molecular basis miRNAs. Our method makes full use of similarity measurements, known drug-miRNA and miRNA-disease associations to infer indications for drugs. As current drug-miRNA and miRNA-disease associations are insufficient, two rounds of bi-random walks are implemented to reveal the hidden associations in the drug-miRNA-disease heterogeneous network. We have applied our method to real datasets and the results showed that our method could successfully discover the causality underlying drugs and diseases. Compared with other algorithms, our method provided a straightforward strategy for causality discovery and showed superior prediction performance.As similarity values provide a vital role in the prediction procedure, we need to further investigate the types of features collected for similarity measurements. Meanwhile, the predicted drug-disease relationships might be biased as the numbers of known drug-miRNA associations and miRNA-disease associations were rare. We expect the prediction power of our method can be improved by integrating more experimentally confirmed drug-miRNA and miRNA-disease associations.45 for more details). At the same time, complex diseases are multi-factor driven. For example, the study conducted by Wang et al.46 suggested that DNA mutations and other genomic alterations, which were valuable biomarkers and could be used to construct disease hallmark networks, provided significant roles in cancer clonal evolution and associated clinical phenotypes. Therefore, more relevant biochemical information is needed for deepening our knowledge of drug-disease causal relationships, even though our study provided a feasible strategy for discovering the causality.It should be noted that the mechanism of action of drugs has not been completely investigated. The most recent studies indicated that drugs may also target other non-coding RNAs, including circular RNAs, long non-coding RNAs and Piwi-interacting RNAs 36 was successfully applied on both gene network and phenotype network simultaneously, with an averaged output from the two networks in each step, to infer potential gene-phenotype associations. Inspired by the successful application of BiRW, we extended the algorithm to the three-layer drug–miRNA-disease heterogeneous network to predict potential drug-disease causal relationships.Previously, random walkIn this study, a three-layer heterogeneous network, including a layer of causal factors of miRNAs, was constructed and the drug-disease causal relationship prediction process mainly included three steps. First, bi-random walks were applied on the two-layer drug-miRNA network. Second, another round of bi-random walks was established on the two-layer miRNA-disease network. Finally, the prediction results were received by combining the outcomes from the two previous steps. The whole pipeline of the algorithm miRDDCR could be outlined in Fig. Supplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3


Malignant cancers employ diverse and intricate immune evasion strategies, which lead to inadequately effective responses of many clinical cancer therapies. However, emerging data suggest that activation of the tolerant innate immune system in cancer patients is able, at least partially, to counteract tumor-induced immunosuppression, which indicates triggering of the innate immune response as a novel immunotherapeutic strategy may result in improved therapeutic outcomes for cancer patients. The promising innate immune targets include Toll-like Receptors (TLRs), RIG-I-like Receptors (RLRs), and Stimulator of Interferon Genes (STING). This review discusses the antitumor properties of TLRs, RLRs, and STING-mediated innate immune pathways, as well as the promising innate immune targets for potential application in cancer immunotherapy. The innate immune system is an evolutionarily ancient and dominant defense mechanism against pathogen infection . The innDefined innate immune mechanisms involving cancer immunotherapy include, but are not limited to antitumor immune responses elicited by recognition of tumor-derived antigens by Toll-like receptors (TLRs), as well as sensation of tumor-derived DNA by the stimulator of interferon gene (STING) and retinoic acid-inducible gene-I (RIG-I)-like Receptors (RLRs) ,8,9. TheTremendous advances in cancer immunotherapeutic strategies have been witnessed in recent years. Targeting the innate immune pathway has been considered as an effective auxiliary strategy to prevent tumor escape, and the enhancement of the sensitivity of innate immune effectors to tumor-derived antigens could facilitate evading tumor evasion . MeanwhiTLRs are mammalian homologues of drosophila Toll protein, which are regarded as critical PRRs in innate immunity. There are 10 TLRs (TLR1-10) that have been identified in human. TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed on the cell surface, while TLR3, TLR7, TLR8, and TLR9 are found on the membrane of endosomes. Each conserved TLR contains transmembrane domain, ectodomain (ligand binding domain with leucine-rich repeats motif), and cytosolic Toll/IL-1 receptor (TIR) domain . The ectTLRs exhibit specificity for ligand recognition. Different TLRs specifically recognize district PAMPs and DAMPs. TLR2 recognizes lipoproteins and peptidoglycans, TLR3 recognizes viral double-stranded RNA/viral RNA analogue polyinosinic-polycytidylic acid (poly I:C), TLR4 recognizes lipopolysaccharides (LPS), TLR5 recognizes bacterial flagellin, TLR7/8 recognizes single-stranded RNA, and TLR9 recognizes CpG-containing oligodeoxynucleotides (CpG-ODN) ,15,16. TRecent studies have suggested that TLRs expression is not confined to immune cells, but also found in various tumor cells ,38,39. ETo survive the host innate defense mechanisms, cancer cells have developed strategies to evade or counteract the signaling through the TLR pathways, which create an advantageous microenvironment for their propagation. Activation of certain TLRs on tumor cells enhances immune suppression through decreasing cytotoxicity of immune cells, increasing the production of pro-inflammatory factors, and triggering an abnormal form of tissue repair in tumor foci, and finally resulting in tumor evasion from immune surveillance ,46,47.The function of TLRs in immune and cancer cells seems complex, and the regulatory mechanisms on how to achieve targeted activation of TLRs on immune cells or cancer cells are still unclear. Perhaps distinct TLR signaling sub-pathways or unidentified downstream adaptor molecules are activated in cancer and/or innate immune cells during tumorigenesis; another possible explanation is that chronological order of TLR activation in immune cells or cancer cells markedly affect the subsequent activation and induced effectors ,21. The Inflaming metastasis has been proposed to be the seventh hallmark of malignant cancers . EvidencStreptococcus pyogenes and Serratia marcescens bacteria) and Bacillus Calmette-Guerin have become long-used therapeutic drugs against cancers [The strategies of promoting innate immune responses by TLRs against cancer do not require complex and patient-specific therapeutic agents; meanwhile, the increasingly successful applications of innate immunotherapy in the clinic have effectively targeted TLRs-induced immune response against cancers ,52. TLR2 cancers . Extract cancers .Combination therapy with TLR ligands and conventional radiation/chemotherapy is a promising strategy of cancer treatment, which has a much higher growth-inhibitory effect compared with single application and contributes to decrease the clinical dosage of chemotherapeutics ,56. TradRIG-I like receptors (RLRs) are a family of DExD/H box RNA helicases that play a major role in pathogenic RNA sensing for initiating antiviral immunity response . So far,Cell death after viral infection is considered to be an important antiviral mechanism that eliminates infected cells and prevents the virus from spreading. Activation of RLRs signaling pathway by using synthetic ligands or virus infection has been shown to induce the production of IFNs, which can induce ISGs (interferon-stimulated genes) for directly producing cell death or modulate apoptosis pathway ,70,71,72In consideration of the fact that immune responses against viruses and tumors share essential features, such as dependence on IFNs and CTLs (cytotoxic T lymphocytes), the idea arises that mimicking a viral infection using RLRs ligands to activate RLRs-signaling mediated immune responses could be exploited for tumor immunotherapy. While RLR ligands are not yet approved by the Food and Drug Administration (FDA) as a treatment for cancers, accumulating evidence strongly suggests that triggering MDA5 or RIG-I would provide a promising approach for cancer immunotherapy.+ T cells to the tumor, profound tumor cell apoptosis, as well as prolonged mice survival [Because ligands for RIG-I recognition are mostly independent of the RNA sequence and the gene silencing effect is not inhibited by the presence of a 5′-triphosphate (5′ ppp-) moiety in the small interfering RNA molecules (siRNA), several types of 5′ ppp-siRNAs were developed for inducing cell death through activation of the RIG-I signaling pathway and also silencing oncogenic or immunosuppressive targets. It has been reported that systemic administration of 5′ ppp-siRNA for B-cell lymphoma-2 (Bcl-2) silencing, results in melanoma tumor growth inhibition due to the downregulation of Bcl-2 and immune activation of the RIG-I signaling pathway, this anticancer response requires dendritic cells (DC)-dependent production of IFN-α and recruitment of natural killer (NK) cell at the tumor site . The 5′ survival   translocate to the cytoplasm after ionizing radiation (IR) treatment, thus stimulating the formation of RIG-I: RNA complexes and initiating downstream signaling events trigger IFN-β activity . It is k+ T cell-mediated antitumor immune responses and provides a promising approach for the immunotherapy of cancer  in ovarian cancer exhibits enhanced expression of HLA-class I, release of cytokines , as well as tumor cell apoptosis. This MDA5-mediated anticancer response requires DC-dependent engulfing of MDA5-activated cancer cells for DC activation and subsequently production of cytokines (CXCL10 and IFN-α) for providing a pro-inflammatory milieu to promote cytolytic activity and IFN-γ secretion of NK cells at the tumor site . Therefof cancer .Comparing with surgery, chemotherapy, or radiation therapy in cancer treatment, there are several benefits to targeting RLRs for cancer immunotherapy. Firstly, activation of RLRs by their specific ligands not only triggers antitumor immunity via IFN-dependent effector T cells and NK cells activation, but also can directly induce apoptosis of tumor cells in an IFN-independent manner. Secondly, it has been indicated that tumor cells are highly sensitive to RLR-induced apoptosis, whereas nonmalignant cells are protected via endogenous Bcl-xL ,76. TherIn addition to sensing of microbial RNAs by RLRs to activate innate immune responses against pathogen infection, the recognition of microbial DNA by DNA sensors is also a major defense mechanism for the detection of a wide variety of pathogens, because all microorganisms contain and need DNA in their life cycle. However, host cells also contain abundant self DNA. To avoid this self DNA recognition, the host has evolved specific mechanisms to distinguish self from non-self DNA based on the location of the DNA or sensors to avoid inappropriate activation of immune responses. After microbial infection, the pathogenic DNA is always presented in cytoplasm, which is different from the nuclear or mitochondrial localization of host DNA, and sensed by the cytosolic DNA sensors to initial innate immune responses. However, under certain pathological conditions, self DNA also can be recognized by innate immune sensors and cause several autoimmune and auto-inflammatory diseases .So far, several cytosolic DNA sensors have been discovered, such as DAI (DNA-dependent activator of IFN-regulatory factors) , RNA polAs RLRs signaling, activation of STING pathway results in production of IFNs for inducing ISGs to prompt cell death, it also can cause cell death via IFNs-independent manner, which is through STING-mediated IRF-3 interaction with Bcl-2-associated X protein (Bax) on mitochondria to activate the mitochondrial apoptosis pathway dependent on caspases 9 and 3 ,107,108.+ T cell-mediated antitumor immune responses. Recent studies have demonstrated that STING plays a key role in antitumor immune response [+ T cell priming against tumors was also defective in mice lacking STING, but not in those lacking TLRs, MyD88 or MAVS, suggesting cytosolic DNA sensing pathway is involved in controlling tumor growth. It has also been demonstrated that tumor-derived DNA is transferred to host DCs and induces IFNs production through cGAS-STING pathway for CD8+ T cell priming [+ T cells [IFNs play a multifaceted role in the induction of antitumor immunity. In addition to their direct cytotoxic effects on tumor, targeting low doses of type I IFNs to the tumor microenvironment also promotes the maturation and antigen presentation of DCs and boosts endogenous NK or CD8response ,111,112. priming . Other s+ T cell response and enhance antitumor immunity [Many kinds of cancers appear to induce a spontaneous adaptive T cell response. However, tumor cells often induce an immunosuppressive microenvironment favoring cancer development. Therefore, targeting of STING pathway by using STING agonists to produce IFNs for enhancing antitumor immune response may provide an alternative strategy for the improvement of cancer immunotherapy. A defined flavonoid compound known as a vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), was shown to have potent antitumor activity in mouse models due to its ability to induce type I IFNs in macrophages , the latimmunity ,118. In immunity ,120,121.These results indicate that STING activation by cGAMP and potentially other ligands may be an attractive treatment for antitumor immunotherapy. One of the STING ligands-MIW815, is now in phase I clinical trials. However, there are still several challenges yet to be fully addressed, because several studies found a risk of STING activation in cancer immunotherapy. It has been reported that activation of the cGAS-STING pathway can lead to tolerogenic responses and promote the growth of tumors with low antigenicity through the induction of indolamine 2,3-dioxygenase (IDO) ,123. A rMany cancer immunotherapeutic strategies enhancing distinct aspects of the immune system are showing clinical promise. Meanwhile, powerful synergistic effects in cancer immunotherapy have been evidenced when innate and adaptive immunity are combined in use, indicating that the engagement of both innate and adaptive immune responses is necessary for efficient cancer immunotherapy.The current barrier to effective cancer treatments is that cancer itself is highly immunosuppressive to both innate and adaptive responses . AlthougInitiation of innate immune responses can lead to subsequent long-term adaptive immunity against cancer. Thus, integration of innate immune activation strategies into combination therapies for cancer treatment will lead to more effective and long term clinical benefit. Evidence has demonstrated that manipulating tumor-associated macrophages (TAM) or myeloid derived suppressor cells (MDSC) to break the systemic and local tumor-induced immunosuppression potentially support cytotoxic T-cells (CTL)-mediated cancer immunotherapy ,127.+ T cells) and binds to its ligand PD-L1, which is expressed on multiple malignant cells [Monotherapy for most tumor patients is not sufficient for complete cancer eradication, perhaps due to compensatory immune evasion pathways. Negative regulatory receptor programed cell death-1 (PD-1) is an inhibitory molecule that is expressed on the surface of activated immune cells  to IL-2 could generate lymphokine-activated killer (LAK) cells that have the ability of spontaneous lysis of tumor cells. The IL-2-induced LAK activity has been successfully applied in immunotherapy against melanoma and renal cell carcinoma [Common γ chain cytokines, namely IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, produced by activated immune cells influence the activation of innate immune effector cells . IL-2 isarcinoma . IL-15 harcinoma .Antibody-based targeted therapy is one of the safest immunotherapeutics for cancer patients. Clinical studies have demonstrated that effective tumor-specific monoclonal antibodies (mAbs) induce direct tumor cell death involving innate immune pathways; some tumor-reactive mAbs function by the aid of complement dependent cytotoxicity, while most mAbs with antitumor efficacy involve innate effector cells via antibody dependent cell-mediated cytotoxicity (ADCC) . Thus, wThe classically activated (M1) macrophages play a tumor-destroying role and the M1 macrophage densities in the tumor islets are highly associated with the patient’s survival ,137. AntThe innate immune signaling pathways play a key role in tumor escape from immune surveillance, and thus triggering the innate immune pathways will likely be critical in strategies of cancer immunotherapy. Accumulating evidence has indicated that targeting certain innate immune signaling pathways, especially TLRs, RLRs, and STING signaling pathways, is a promising cancer immunotherapeutic approach. Therefore, further insight into the mechanisms of TLRs, RLRs, and STING-mediated innate immune signaling in cancer immune evasion, tumorigenesis, and cancer development may lead to discovery of novel therapeutic targets for cancer therapy. Besides this, considering the compensatory immune mechanism, together with integration of the strategies targeting both innate and adaptive immune systems for cancer treatment will contribute to more powerful synergistic effects and long term clinical benefits.


The imaging of infection and inflammation is lagging despite strong unmet medical needs. This review presents the potential routes for the development of 68Ga-based agents for the imaging and quantification of infection and inflammation in various diseases and connection of the diagnosis to the treatment for the individualized patient management. During the last decade, the utilization of  They taeceptors . Moreoveammation . [18F]FDsporters .67Ga-Citrate has been in clinical use for imaging of infection and inflammation for over 40 years. It is applicable, for example, for the diagnosis of lung infections, acute/chronic osteomyelitis, tuberculosis, sarcoidosis, and retroperitoneal fibrosis -fluorodeoxyglucose ([18F]FDG)/PET-CT reflecting the elevation of glucose transporter expression in tumour cells, and providing nearly universal application in the evaluation of various fast growing cancer types. [18F]FDG/PET-CT stands for over 90% of all PET-CT examinations [18F]FDG/PET is an established diagnostic means also in infection and inflammation, and the major indications for it are FUO, sarcoidosis, peripheral bone osteomyelitis, suspected spinal infection, metastatic infection, bacteremia, and vasculitis [18F]FDG fails to detect slowly growing tumours and to discriminate malignancy from sterile inflammation, infection, wound healing, tuberculosis, sarcoidosis, and reactive lymph nodes [18F]FDG in healthy organs such as brain and gut resulting in suboptimal image contrast and consequently potential risk for lesion detection failure.Most nuclear medicine applications worldwide (90%) stand for diagnostics with leading position for rdiology . The mosinations , 38. Ga-NOTA-c(RGDyK)/PET scanning with subsequent kinetic modeling studies in rats with forelimb ischemia showed higher uptake and distribution volume in the ischemic area as compared to that of sham operation and control regions [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-TRAP-(RGD)3 in rat model [68Ga]Ga-DOTA-E-[c(RGDfK)]2 was observed in the infarcted area while no accumulation was detected in the noninfarcted myocardium of the same rats [68Ga]Ga-DOTA-RGD in atherosclerotic plaques was studied in vivo in atherosclerotic mice with promising results [68Ga]Ga-NODAGA-RGD in injured myocardium as compared to viable ischemic areas in pig model presumably indicated increased expression of αVβ3 receptors associated with injury repair in the presence of coronary stenosis Ga-Siglec targeting VAP-1 demonstrated preclinical potential for imaging of synovial inflammation in patients with rheumatic diseases [68Ga]Ga-Siglec was found promising also for the detection of inflamed atherosclerotic lesions . Th. Th68Ga Cytokines are produced by macrophages, B-lymphocytes, T-lymphocytes, and mast cells and act through receptors modulating, for example, immune response to infection and inflammation. Cytokines include chemokines, interleukins, interferons, and lymphokines that can be classified in broad families exhibiting diverse functions, for example, IL-1 and IL-6 superfamilies and TNF/TNF receptor superfamily. Therapeutics targeting cytokines are in clinical use, for example, inhibiting TNF or IL-6 in rheumatic diseases.18F, 67Ga, 68Ga, or 64Cu Ga-pentixafor for detection and quantification of CXCR4 receptor density in ischemic heart diseases reflecting the role of the receptor in inflammatory and progenitor cell recruitment Ga-DOTA-PEG-FA comprising folic acid was investigated for the detection and quantification of inflammatory response to medical implants using mice with subcutaneously implanted polylactic acid and poly(N-isopropylacrylamide) particles as a model [68Ga]Ga-DOTA-PEG-FA with folate receptor expressed on activated macrophages. Another folic acid based agent, [68Ga]Ga-DOTA-folate, was successfully tested in an inflammatory paw rat model  are overexpressed on a variety of cancer cells and activated macrophages, but not on normal cells , 153. Thnografts –162. [68 a model . The ageat model  61]. Di. Di68Ga-and feet .68Ga-labelled somatostatin analogues demonstrated superior performance in terms of higher specificity and sensitivity, detection rate, shorter examination time, and quantification possibility and have become a golden standard for the detection of neuroendocrine tumours (NETs) taking over that title from [111In]-pentetreotide (OctreoScan®) and demonstrating specificity and sensitivity of over 90% [68Ga-labelled agents for the imaging of NETs demonstrated advantages also over other radionuclides and tracers such as [18F]FDG [123I-metaiodobenzylguanidine ([123I]MIBG) [18F]DOPA [99mTc]-dicarboxy propane diphosphonate [18F]NaF.Somatostatin receptor (SSTR) ligand analogues have found an extensive application in diagnosis and radiotherapy of neuroendocrine tumours. The elevated expression of SSTRs is known also in small cell lung cancer, breast cancer, renal cell carcinoma, prostate cancer, and malignant lymphoma. A number of somatostatin ligand analogues labelled with gamma- and positron-emitting radionuclides were used clinically for oncological cases , 164–174over 90% , 175–1803I]MIBG) , 182, [118F]DOPA , [99mTc]sphonate , and Ga-DOTA-TOC/PET-CT was found superior to 67Ga-Citrate/SPECT in detection of sarcoidosis lesions [68Ga]Ga-DOTA-TOC and SST2 mRNA expression and recorded the information in a database [68Ga]Ga-DOTA-NOC as compared to [18F]FDR-NOC G lesions . A clinidatabase  providin]FDR-NOC , overall68Ga-labelled molecules capable of blood-brain barrier penetration, several agents were suggested for the imaging of neuroinflammation, in particular Aβ plaques deposited on blood vessels Ga-NOTA-UBI29-41, and [68Ga]Ga-NOTA-UBI30-41 demonstrated possibility for the distinction between infection and inflammation in a rabbit model [ E. coli and S. aureus were labelled with 68Ga and their biodistribution in normal rats demonstrated fast clearance from liver [68Ga]Ga-DOTA-TBIA101, targeting bacterial lipopolysaccharides detected muscular E. coli-infection in mice  caused by Aspergillus fumigatus [68Ga]Ga-TAFC demonstrated superior characteristics in terms of specific target binding, metabolic stability, and fast blood clearance in a rat model of A. fumigatus infection. Seven analogues were developed in another study with TAFC and ferrioxamine E (FOXE) showing favorable binding, clearance, elimination, and stability characteristics [68Ga]Ga-triacetylfusarinine C and [68Ga]Ga-ferrioxamine E were investigated in rat model of A. fumigatus and demonstrated rapid uptake in the lungs (Desferri-triacetylfusarinine C (TAFC) and desferri-ferricrocin (FC) labelled with umigatus . [68Ga]Geristics  as well eristics . [68Ga]Ghe lungs  76]..68Ga wer18F localized specifically bacterial infection in mice Ga-citrate demonstrated high diagnostic accuracy of 90% of osteomyelitis and diskitis in clinical studies  binds to bacteria but also accumulates at the sites of fungal and viral infection as well as sterile inflammation due to binding to leukocytes. The improved specificity for bacteria was achieved for the fragments of HIG. It is feasible to develop specific antibodies to various antigens present on the bacterial cell surface . Monoclog NCA-95 . Variousl models , 211–214111In-labelled analogue of biotin was successfully utilized for diagnosis of vertebral infections in a clinical study [68Ga-labelled analogues given the advantages of 68Ga over 111In and promising [68Ga]Ga-DOTA-Biotin analogues -fluorodeoxysorbitol (18F-FDS) was demonstrated in a murine myositis model -FDG distribution sites. As mentioned above, the poor access to bacteria aggregated in a biofilm might make the imaging task challenging. Several peptide candidates with affinity for S. aureus biofilm were designed and labelled with 68Ga [Selective imaging of Enterobacteriaceae using 2-[is model . The uptolecules  it mightith 68Ga . The res68Ga was found superior to [18F]-FDG in infection detection in the rat model with diffuse osteomyelitis Ga-DOTA-NOC) demonstrated uptake in pathogenic areas in patients affected by idiopathic pulmonary fibrosis with potential for monitoring response to treatment and drug development [68Ga]Ga-pentixafor also showed potential of the agent for monitoring disease activity and response to treatment in idiopathic pulmonary fibrosis [2-c[CPGRVMHGLHLGDDEGPC] and [68Ga]Ga-NODAGA-PEG2-c[CPGRVMHGLHLGDDEGPC] for the imaging and quantification of fibrosis by PET were developed and characterized preclinically showing fast clearance from normal tissue and blood and binding specificity Ga-NOTA-MSA) was tested in a rat model of myocarditis targeting mannose receptors expressed on macrophages infiltrating myocardium [Mannosylated human serum albumin labelled with ocardium . The upt68Ga]Ga-DOTA was investigated for the quantification of increased blood flow which is one of the key events in inflammation [68Ga]Ga-DOTA in the site of inflammation in rats with induced inflammation correlated well with that of 15O-water, suggesting high relevance [68Ga]Ga-DOTA.[ammation . The upt68Ga is shorter than that of 64Cu, 67Ga, 99mTc, 89Zr, 111In, and 123,124,125I and thus in contrast to the latter it is not compatible with slow pharmacokinetics of large molecules such as antibodies and glycoproteins. The range of antigen-specific antibodies relevant to inflammation and infection is broad and a number of 99mTc-labelled antibodies were used clinically [68Ga-based agents could be similar. The solution to overcome the incompatibility of half-life time frames could be either the reduction of the antibody size or the application of the pretargeting concept.The half-life of inically , 21, 227The history of the pretargeting concept spans three decades, predominantly in the field of oncology –230. It 68Ga and ethylene glycol linker of various length demonstrated the influence of the latter on the affinity towards avidin.The high affinity of biotin to avidin and streptavidin proteins was utilized clinically and preclinically in pretargeting approach for the imaging and therapy of pancreatic adenocarcinoma , gliobla68Ga. Several analogues were developed for the imaging of carcinoembryonic antigen (CEA) pretargeted with anti-CEA bsmAb [68Ga-labelled hapten molecules and bsmAb were initiated [Particular example of hapten molecules is the ones comprising histamine-succinyl-glycine (HSG) motif and chelate moiety –253, 258EA bsmAb , 260, annitiated . trans-cyclooctene (TCO) and a radiolabelled tetrazine that can interact in vivo based on IEDDA reaction were studied [68Ga-labelled tetrazine dextran demonstrated favorable pharmacokinetics in a healthy mouse [111In and 64Cu-labelled tetrazine analogues. Anti-CA19.9 antibody-TCO in combination with 177Lu-labelled tetrazine demonstrated radiotherapeutic effect in pancreatic cancer murine model [Bioorthogonal reactions are fast, regioselective, requiring small reagent concentration, and occurring under mild conditions often in aqueous solution and temperature below 37°C , 263. Am studied –267. In hy mouse . Howeverhy mouse  and antihy mouse  antibodi68Ga providing the intrinsic advantages of 68Ga and PET. As mentioned above, most of the developed radiolabelled counterparts of pretargeting techniques have demonstrated promising results. There are a considerable number of potential antibody biomarkers that could be considered for the imaging of infection and inflammation.The pretargeted imaging techniques may contribute to the expansion of immuno-PET with 68Ga-labelled SST analogues [68Ga/PET wherein 68Ga-labelled analogues were used not only for localization of the lesions, but also for staging, patient stratification, prognosis, therapy selection, and monitoring of the response to the treatment of NETs and other cancer types [Theranostics  embracesnalogues –28, 271 nalogues , 272–274er types , 275–277 P. aeruginosa in mice was clearly and focally visualized within 0.6 h with retention for at least 24 h. These results obtained using analogues carrying dye for optical imaging can be translated to 68Ga-labelled counterparts for PET.The methodology can be translated to inflammation and infection allowing for accurate and specific selection of treatment regimen and for follow-up and evaluation of the response to therapy, resulting in improved treatment efficacy and decreased cost and side effects. The accommodation of both imaging function and antibiotic function in the same molecule is a novel example of a theranostic agent . A serieThe medical need for specific agents for noninvasive, quantitative, and whole-body imaging of inflammation and infection has not been met yet despite decades of research. However, the prerequisites in terms of identification of potential targets, design and synthesis of the respective ligands, and imaging technologies are evolving very fast. The potential of accurate and quantitative lesion localization as well as monitoring of the treatment response promises personalized patient management.68Ga in oncology is established proving the strong potential of 68Ga for the promotion of PET technology for effective and efficient diagnostics and personalized medicine. The experience of oncological 68Ga-based agents is getting translated to inflammation and infection. Pretargeted imaging technology opens wide possibilities based on antibody biomarkers.The use of