Solanum lycopersicum (tomato)-Botrytis cinerea interaction and examined the potential of plant sRNAs to silence their predicted mRNA targets in the fungus. Almost a million unique plant sRNAs were identified that could potentially target 97% of all fungal genes. We selected three fungal genes for detailed RT-qPCR analysis of the correlation between the abundance of specific plant sRNAs and their target mRNAs in the fungus. The fungal Bcspl1 gene, which had been reported to be important for the fungal virulence, showed transient down-regulation around 20 hours post inoculation and contained a unique target site for a single plant sRNA that was present at high levels. In order to study the functionality of this plant sRNA in reducing the Bcspl1 transcript level, we generated a fungal mutant that contained a 5-nucleotide substitution that would abolish the interaction between the transcript and the sRNA without changing the encoded protein sequence. The level of the mutant Bcspl1 transcript showed a transient decrease similar to wild type transcript, indicating that the tomato sRNA was not responsible for the downregulation of the Bcspl1 transcript. The virulence of the Bcspl1 target site mutant was identical to the wild type fungus.Previous studies have suggested that plants can modulate gene expression in pathogenic fungi by producing small RNAs (sRNAs) that can be translocated into the fungus and mediate gene silencing, which may interfere with the infection mechanism of the intruder. We sequenced sRNAs and mRNAs in early phases of the  Plants are continuously exposed to fungal pathogens but they are usually able to combat these pathogens and remain healthy. To achieve this, plants have evolved an array of defense mechanisms. One of the most extensively studied defense mechanisms is conferred by host programmed cell death (PCD) at the host-pathogen interaction site, which can subsequently restrict the invasion by biotrophic and necrotrophic pathogens. However, PCD does not necessarily stop the invasion of necrotrophic pathogens effectively. By contrast, necrotrophic fungal pathogens take advantage of the induction of host cell death for their infection as they feed on dead tissue. Plant PCD triggers a series of reactions in the host, such as fortification of the plant cell wall , local production of pathogenesis-related (PR) proteins, phytoalexins and production of reactive oxygen species (ROS) . DespiteBlumeria graminis genes that encode either effector Avra10 or 1,3-β-glucanosyltransferases involved in haustoria formation. The successful deployment of HIGS suggested the occurrence of trafficking of RNA molecules from a plant into a fungus that could induce RNAi in the fungus, and thus HIGS can be considered as an example of cross-kingdom RNAi (ckRNAi).A novel insight in plant defense mechanisms has emerged in the past 15 years, demonstrating that plant transgene-derived double-stranded RNAs (dsRNAs) could induce gene silencing by RNA interference (RNAi) in invading pathogens and pests , in a prVerticillium dahliae interaction, in which the production of two cotton microRNAs (miRNAs) increased upon infection by V. dahliae and the miRNAs were translocated to the fungal hyphae where they silenced specific V. dahliae genes  that selectively accumulate in fungal cells and are predicted to target B. cinerea genes that are involved in vesicle-trafficking pathways. A study by Phytophthora capsici. They observed that Arabidopsis mutants that are impaired in secondary siRNA biogenesis exhibited hyper-susceptibility to P. capsici. In several pathosystems, the naturally delivered host sRNAs have been proposed to contribute to plant immunity (likely) via silencing genes in the invading pathogens  can be naturally translocated to parasitic organisms and trigger silencing of genes in the invaders through RNAi. This phenomenon provided an example of the natural occurrence of ckRNAi, and it was proposed to be an effective host defense strategy by several studies. The first case was in the cotton-ae genes . Cai et athogens . This prSolanum lycopersicum (tomato) is an important crop species that can be parasitized by many pathogenic microbes, resulting in considerable economic losses. The necrotrophic pathogen B. cinerea can infect different organs of the above-ground part of tomato plants, causing devastating damage for the crop during both pre- and post-harvest stages. B. cinerea was shown to be able to take up both sRNA and dsRNA molecules from the environment (B. cinerea has been exploited in plant protection with spray-induced gene silencing (SIGS) by the application on plant surfaces of synthetic RNA molecules targeting genes involved in growth or in signaling required for virulence  - sequence dataset were generated within the first 24 hours post inoculation (hpi). This dataset was described in a previous study  were grown in a greenhouse at 20°C for five to six weeks, and detached compound leaves were used for inoculation assays.B. cinerea conidia were diluted in Potato Dextrose Broth  medium to 1000 conidia/μl and 2 μl droplets containing the conidia suspension were inoculated on detached tomato leaves essentially as described by B. cinerea strain. For the sampling of B. cinerea (or mock) - infected tomato leaves that was used for mRNA and sRNA sequencing, 10 droplets of 2 μl conidia suspension (or only PDB) were inoculated on both leaf halves at ~1 cm from the central vein. Four leaflets of one compound leaf were inoculated at the same time, and one leaflet was sampled by excising the central vein and collecting the remaining leaf tissue at each defined time point . For the sampling B. cinerea (or mock) - infected tomato leaves that was used for the quantitative reverse transcription PCR (RT-qPCR), the leaflets were inoculated in six to eight circular areas and area included five 2 μl droplets of conidia suspension (or only PDB). The inoculated areas were excised by a cork borer with a diameter of 15.6 mm at each time point . More details on the inoculation and sampling design were described in ® microRNA Isolation Kit (Sigma-Aldrich) while mRNA was isolated using the Maxwell® 16 LEV Plant RNA Kit (Promega).Fungal mycelium or tomato leaf samples were frozen in liquid nitrogen and used for extraction of small RNA using the mirPremierSingle-end Illumina sequencing was applied to all sRNA and mRNA samples by Vertis Biotechnologie AG  on a strand-specific library with read length of 75 nt. Sequence processing and bioinformatic analyses of data are described in B. cinerea liquid culture, as described in our recent study . Reads that had a perfect match to the B. cinerea genome were also mapped to the S. lycopersicum genome and vice versa. Reads mapping perfectly on both genomes were labelled as ‘shared reads’ and were discarded, while reads that mapped to only one genome were labelled as ‘unique reads’. Target prediction of sRNAs was performed using sRNAs extracted from the ‘unique reads’. The tool psRNATarget was used to predict the plant sRNA targets on the fungal mRNA population  in the fungus were identified by comparing fungal mRNA levels in the infected tomato leaf tissues with a nt study . Differepulation , with seBcrpl5 from B. cinerea was used to normalize fungal mRNA levels. A sRNA of the tomato U6 spliceosomal RNA component was used to normalize plant sRNAs. The threshold cycle (CT) values were determined by Bio-Rad CFX Manager 3.1 and fold-changes calculated using the 2−ΔΔCt method (Synthesis of cDNA from mRNA was performed using M-MLV reverse transcriptase (Promega). For reverse transcription of sRNA, the qScript microRNA cDNA Synthesis kit (Quanta Bioscience) was used. RT-qPCR was performed using SensiMix SYBR Hi-ROX Kit (Bioline). Primer combinations described in t method .B. cinerea mutant strains used in this study were generated by PEG-mediated protoplast transformation as described by (® G2 DNA Polymerase (Promega). The coding region of the Bcspl1 gene from the mutants used in this study was sequenced to verify whether or not the sRNA target site contained the 5-nucleotide substitution.ribed by  with minB. cinerea liquid culture, as described in B. cinerea genes (one quarter of the annotated genes) were differentially expressed at 24 hpi, as compared to growth in liquid culture, of which 1724 were up-regulated and 1282 were down-regulated , 51 proteases, 63 membrane transporters, 5 proteins involved in signaling and 12 putative transcription factors. Moreover, the cluster of Bcboa genes encoding biosynthetic enzymes for the production of the polyketide phytotoxin botcinic acid were upregulated at 24 hpi, as compared to the in vitro liquid culture. Meanwhile, the in planta down-regulated genes (at 24 hpi) included genes encoding 36 CAZymes, 27 proteases, 39 membrane transporters, 33 proteins involved in signaling and 93 putative transcription factors. Moreover, there was significant down-regulation in planta at 24 hpi of melanin biosynthetic genes  secondary metabolites  that were predicted targets of the tomato sRNAs indeed displayed significant down-regulation in at least one of the time points  as compared to the liquid culture  and their matching target mRNAs in B. cinerea (Bc-mRNAs) by reverse transcription-quantitative PCR (RT-qPCR). We selected three Sl-sRNAs and their predicted target Bc-mRNAs  in the dataset; the transcript should be (predicted to be) targeted by a single tomato sRNA, in order to minimize the impact of multiple sRNA-mRNA interactions; the gene should participate in virulence of B. cinerea. The Bcspl1 gene was selected as it is the predicted target of a single tomato sRNA (sRNA1187) and encodes a cell-death inducing effector protein with a role in virulence  to -11 kCal/Mol (mutant), without changing the encoded protein (Bcspl1 target site would not result in a sequence that could inadvertently be targeted by different tomato sRNAs in the dataset. A gene replacement construct was generated that encompassed the entire Bcspl1 gene (containing the desired five-nucleotide substitution) and a part of the downstream gene Bcin03g00510 with a hygromycin-resistance cassette (hph) inserted in the intergenic region . Transformation of this construct to wild type B. cinerea strain B05.10 yielded seven transformants of which two contained the hph cassette in the target locus and five were ectopic transformants  in the entire dataset. Fungal hyphae that penetrate the plant surface and enter the interior plant tissues are vastly outnumbered by plant cells. Furthermore, the fungal sRNA population consists of thousands of distinct unique sequences and their potency in silencing target mRNAs in a host plant is essentially defined by random chance  is distinct, as the abundance of plant RNAs largely exceeds that of fungal RNAs. In an early phase of the plant-fungus interaction, fungal hyphae that penetrate the host surface and enter the plant tissue are surrounded by a large number of plant cells, each of which may produce and release small RNAs. al cells . Fungal B. cinerea-tomato interaction, numerous fungal genes were downregulated over the course of the infection process. Based on observations of B. cinerea undergoes infection-related developmental transitions during penetration of the host tissue, by forming either appressoria or infection cushions, each with their specific developmental and transcriptional program  genes are knocked out. There are seven DCL genes in tomato   , and SlDvailable . Phenotyrphology . BesidesThe natural occurrence and function of the ckRNAi from both sides of the plant-parasite interaction is a relatively young research topic. A number of studies concluded that ckRNAi is a naturally occurring phenomenon. There were also observations that specific plant sRNAs can play a role in plant defense against pathogens, however, mostly through indirect methods by either overexpressing these sRNAs in plants  or knockwww.ncbi.nlm.nih.gov/bioproject/ with accession number PRJNA496584. Raw sequence reads are deposited in the Sequence Read Archive at www.ncbi.nlm.nih.gov/sra/ project SRP166089 under accession numbers SRX4902781-SRX4902782, SRX4902789, SRX4902791-SRX4902800, SRX15228005-SRX15228010, SRX15231189, SRX15231190, SRX19002251 (sRNAs) SRX4902771-SRX4902780, SRX4902783-SRX4902787, SRX4902790, SRX15231192, SRX15231193, SRX19002219 (mRNAs).The datasets presented in this study have been deposited in the NCBI database under Bioproject at SQ, JV, and JK designed the study. SQ, JV, and GP performed the experiments. JV performed the bio-informatic analyses. SQ, JV, and JK interpreted the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.


Mixed allogeneic chimerism has considerable potential to advance the achievement of immune tolerance to alloantigens for transplantation and the restoration of self-tolerance in patients with autoimmune disease. In this article, I review evidence that graft-versus-host (GVH) alloreactivity without graft-vs-host disease (GVHD), termed a lymphohematopoietic graft-vs-host reaction (LGVHR), can promote the induction of mixed chimerism with minimal toxicity. LGVHR was originally shown to occur in an animal model when non-tolerant donor lymphocytes were administered to mixed chimeras in the absence of inflammatory stimuli and was found to mediate powerful graft-vs-leukemia/lymphoma effects without GVHD. Recent large animal studies suggest a role for LGVHR in promoting durable mixed chimerism and the demonstration that LGVHR promotes chimerism in human intestinal allograft recipients has led to a pilot study aiming to achieve durable mixed chimerism.  Achievement of immune tolerance to alloantigens and restoration of self-tolerance are major goals in transplantation and autoimmune diseases, respectively. In this article, I discuss the ability of mixed allogeneic chimerism to achieve these goals and the potential role of lymphohematopoietic graft-vs-host reactions in achieving mixed chimerism with minimal toxicity.Responses to allogeneic major histocompatibility (MHC) antigens pose the single greatest barrier to organ allograft acceptance and promote severe graft-versus-host disease (GVHD) in hematopoietic cell transplantation (HCT). Approximately 1–3% of an individual’s T cell repertoire recognizes an allogeneic donor’s HLA [Only two approaches have been used successfully to achieve allograft tolerance so far in humans. The first is planned weaning and withdrawal of immunosuppressive therapy, which has been more successful in liver than any other type of transplant and ultimately succeeds in only a fraction of highly selected long-term liver allograft recipients. Success rates are approximately 22% in the small subset of patients who qualify for withdrawal , but thiThe second approach that has been used successfully to withdraw immunosuppression is HCT, which is the only strategy that has been used successfully in renal allograft recipients. The first trials were carried out at Massachusetts General Hospital, where we achieved allograft tolerance without GVHD using combined HLA-mismatched donor kidney and bone marrow transplantation (CKBMT) in a small group of patients , 9. Tria+ and CD4+ T cells in the DLI were critical for this effect and donor CD4+ T cells given without CD8+ cells could, paradoxically, incite donor allograft rejection through an apparently cytokine-dependent mechanism that activated residual recipient T cells [The MGH clinical trial that achieved renal allograft tolerance across HLA barriers via non-myeloablative HCT built on work we had carried out in murine models aiming to achieve graft-vs-leukemia/lymphoma effects without GVHD. We had observed that mixed chimerism could serve as a platform for donor lymphocyte infusions (DLI) that converted mixed to full donor chimerism  and coul T cells . We show T cells . We subs T cells  and HLA- T cells  settings T cells  and othe T cells . Our mur T cells , 20, 22. T cells . Results T cells . Importa T cells , 30.One of the first regimens tested in the HLA-mismatched setting using the above approach resulted in only transient chimerism without GVHD. NHP studies had meanwhile shown that transient MHC-mismatched mixed chimerism could be sufficient to achieve renal allograft tolerance in a large fraction of animals . FurtherAnimal studies have demonstrated that mixed chimerism also has the potential to reverse autoimmune diseases . TreatmeBased on the potential of durable mixed chimerism to simultaneously reverse autoimmunity and allow donor islet replacement without the need for chronic immunosuppression, we have developed an NHP model aiming to achieve durable mixed chimerism with minimal conditioning. We have achieved this in a cohort of animals  and demonstrated tolerance to donor kidney allografts in an MHC haplotype-mismatched setting. Although mechanistic studies have not yet been carried out, it seems likely that the large number of GVH-alloreactive donor T cells carried in the PBSC graft plays a significant role in the achievement of durable chimerism and tolerance in this model. This regimen is based on a similar one, involving only low dose (100 cGy) TBI, partial peripheral T cell depletion, and a short course of calcineurin inhibitor, that achieved low levels of durable mixed chimerism across MHC barriers in a porcine model. Unlike the monkey model, in which high levels of chimerism persisted in both the myeloid and lymphoid cell lineages, the myeloid chimerism in the porcine model was minimal, while the durable chimerism was seen mainly in the T cell lineage . GVH-reade novo donor-specific antibody production compared to patients lacking macrochimerism [de novo in the recipient after engrafting in the recipient’s bone marrow. Using a high throughput TCRβ CDR3 sequencing approach that we have developed to identify and track alloresponses in human transplant recipients, we were able to demonstrate that donor T cells pre-existing in the intestinal allograft mucosa and mediating an LGVHR appear to play a major role in allowing these beneficial outcomes [Recently, our group has demonstrated that multilineage mixed chimerism develops spontaneously in patients receiving intestinal allografts, and particularly in those receiving multivisceral transplants that include liver, stomach, and pancreas in addition to the intestine. ‘Macrochimerism’, which we defined as a peak level of T cell chimerism >4%, is associated with reduced rates of graft rejection and himerism . The chihimerism . Consequhimerism , respectoutcomes . We haveoutcomes . These Goutcomes . In mostoutcomes . This diThe demonstrated ability of LGVHR to promote durable mixed chimerism in humans suggests an additional potential benefit, namely the destruction of pre-existing autoreactive recipient T cells in patients with autoimmune disease. As summarized above, LGVHR destroys recipient hematopoietic cells and we suspect that the association between reduced allograft rejection and LGVHR in intestinal transplant recipients reflect that ability of GVH-reactive T cells to destroy or suppress donor-reactive (HVG) recipient T cells. Likewise, pre-existing autoreactive T cells might be destroyed or attenuated by an LGVHR, particularly one that converts mixed into full donor chimerism, as demonstrated in the murine model. Although this mechanism would not be expected to exhibit specificity for autoreactive T cells but instead to globally destroy recipient hematopoietic cells, it could achieve a functioning donor immune system without GVHD.In summary , LGVHR,


Both substance-related as well as non-substance-related addictions may include recurrent engagement in risky actions despite adverse outcomes. We here apply a unified approach and review task-based neuroimaging studies on substance-related (SRAs) and non-substance related addictions (NSRAs) to examine commonalities and differences in neural correlates of risk-taking in these two addiction types. To this end, we conducted a systematic review adhering to the PRISMA guidelines. Two databases were searched with predefined search terms to identify neuroimaging studies on risk-taking tasks in individuals with addiction disorders. In total, 19 studies on SRAs  and 10 studies on NSRAs  were included. Risk-related brain activation in SRAs and NSRAs was summarized individually and subsequently compared to each other. Results suggest convergent altered risk-related neural processes, including hyperactivity in the OFC and the striatum. As characteristic for both addiction types, these brain regions may represent an underlying mechanism of suboptimal decision-making. In contrast, decreased DLPFC activity may be specific to SRAs and decreased IFG activity could only be identified for NSRAs. The precuneus and posterior cingulate show elevated activity in SRAs, while findings regarding these areas were mixed in NSRAs. Additional scarce evidence suggests decreased ventral ACC activity and increased dorsal ACC activity in both addiction types. Associations between identified activation patterns with drug use severity underpin the clinical relevance of these findings. However, this exploratory evidence should be interpreted with caution and should be regarded as preliminary. Future research is needed to evaluate the findings gathered by this review.The online version contains supplementary material available at 10.1007/s11065-022-09552-5. Addictive disorders are among the most common psychopathologies with lifetime prevalence rates ranging from around 1.2% for gambling disorder  striatum seem to be more related to emotional arousal or salience during risk-taking  are altered in both SRAs and NSRAs. Specifically, altered (anticipatory) signaling in the amygdala and striatum seems to underlie aberrant reward processing in addictive disorders  guidelines  of the reviewed studies examined risk-taking in individuals with stimulant addiction . Of the remaining studies, k = 3 studies investigated cannabis addiction, k = 2 studies investigated alcohol addiction and k = 5 studies did not report the exact addiction type or included mixed samples. Table Our review identified 19 eligible functional imaging studies which investigate risk-related decisions in SRAs compared to controls. A total of 648 individuals participated across all SRA studies. The number of included SRA individuals per study ranged from 11 to 75. Among the included studies, four studies were identified which did not include a healthy control group, but assessed the  relationship between clinical characteristics  and altered neural correlates during risk-taking  was decreased in individuals with SRAs  and also identified increased risk-related dorsal ACC activity (k = 2). Both studies used the Cambridge Risk Task which measures decision-making under risk.Conversely, the ventral ACC showed decreased risk-related activity in the context of similar  in a sample of individuals with SRAs who exhibited increased risk-taking behavior after experiencing losses under uncertainty  studies that included a HC group reported risk-related alterations in the temporal lobe in SRAs. All used the IGT task. Alterations included decreased activity in the middle temporal gyrus  striatum and the dorsal ACC point to elevated risk-related brain activity. Increased dorsal ACC activity was mostly reported by studies which could also show trends towards increased behavioral risk-taking or could show associations to clinical symptoms. In contrast, activity in the ventral ACC may be decreased in SRAs. Findings on the temporal lobe are mixed but point to a positive relationship between activity in the middle temporal lobe and a longitudinal increase in substance use.k = 6 studies on individuals with Internet Gaming Disorder (IGD) and k = 4 studies on Pathological Gambling. Of those, seven studies did find elevated levels of behavioral risk-taking in individuals with NSRAs, whereas three studies could not find any behavioral differences regarding risk-taking behavior in NSRAs relative to controls. A total of 187 individuals participated across all NSRA studies. The number of included NSRA individuals per study ranged from 8 to 41. Table In total, 10 studies investigated neural correlates of risk-related decisions in individuals with NSRAs, with k = 1; Wang et al., k = 2; Lin et al., Decreased risk-related brain activity in the DLPFC could only be identified by one study  revealed increased OFC/vmPFC activity in individuals with NSRAs. Of those, two studies investigated decision-making under uncertainty  showed increased activity in k = 3 studies  reported decreased activity in occipital brain regions for decisions under risk  striatal hyperactivity was only reported by studies that also found increased behavioral risk-taking. Additional preliminary evidence points to hyperactivity of the thalamus, the dorsal ACC and hypoactivity of the ventral ACC. Findings regarding further brain areas are rather mixed.Compared to studies on SRAs, this review identified both overlapping and specific brain activation patterns for NSRAs. These preliminary findings suggest concordant alterations in SRAs and NSRAs in the OFC/vmPFC and the striatum. These regions show increased activity in both addiction types during risk-related decisions. Across studies, increased activity could be identified in both SRAs and NSRAs, irrespective of behavioral task performance. Increased dorsal ACC activity was also reported for both addiction types, but only in studies which also revealed relationships to behavioral risk-taking. In contrast, studies that identified neural alterations in the ventral ACC revealed decreased activity in both addiction groups. Interestingly, the postcentral gyrus, posterior cingulate and precuneus activity seem to be increased in SRAs. In contrast, activity in these regions seem to be decreased in NSRAs. We could also identify differing risk-related patterns of brain activity for the two addiction types. A finding specific to SRA studies is decreased right DLPFC activity, whereas left IFG activity was mostly reported by NSRA studies. To clarify if frontal areas that altered in NSRAs are distinct from the DLPFC, we extracted reported brain coordinates and overlaid them with a DLPFC mask . Compared to HCs, neural activity during risk-taking in this brain region is reduced in individuals with NSRAs. Our systematic review could not find any evidence for altered patterns of IFG activity in SRAs. However, we did find reduced activity in the anterior insula, a region adjacent to the IFG, in SRAs. The anterior insula is often coactivated with the IFG and reviewed studies may have detected and reported peak activation only for one of the regions. Activity in the IFG/anterior insula is modulated by individual risk processing and more specifically by risk/harm avoidance  variability in methods (utilized tasks and neuroimaging approaches) and 2) heterogeneity in included disorders. Specifically, we compared different kind of tasks in different populations. While most SRA studies employed standard decision-making under uncertainty tasks , which we could not identify in this review.Moreover, most reviewed studies were cross-sectional in nature. More longitudinal studies are needed in order to investigate whether alterations in risky decision-making processes are a cause or a consequence of addictive disorders. In addition, within-subject designs may also reveal predictors for relapse or recovery as shown by the study conducted by Blair et al. .It should also be noted that our conclusions are based on a small number of studies. We identify a lack of studies specifically with regard to investigating neural correlates of NSRAs. Therefore, meta-analytic techniques in order to quantitatively support our conclusions could not be applied. Future single studies as well as study syntheses may focus on brain networks related to risk-related decision-making in addiction. For example, several single fMRI studies as well as reviews on brain structure point to an aberrant frontostriatal network in SRAs . Here, we gather exploratory evidence of altered risk-related processes, including hyperactivity in the OFC and the striatum in both addiction types. As characteristic for both addiction types, these brain regions may represent an underlying mechanism of suboptimal decision-making. In contrast, decreased DLPFC activity may be specific to SRAs and decreased IFG activity could only be identified for NSRAs. While findings regarding the precuneus and posterior cingulate point to elevated risk-related activity in SRAs, findings regarding these regions are mixed but rather point to attenuated activation patterns in NSRAs. Additional scarce evidence suggests decreased ventral ACC activity and increased dorsal ACC activity in both addiction types. However, these preliminary findings should be interpreted with caution due to a lack of studies is this field. Future research is needed to evaluate the findings gathered by this review.Supplementary file1 (DOCX 20 KB)Supplementary file2 (DOCX 644 KB)Below is the link to the electronic supplementary material.


Cocaine use disorder (CUD) is a significant public health issue for which there is no Food and Drug Administration (FDA) approved medication. Drug repurposing looks for new cost-effective uses of approved drugs. This study presents an integrated strategy to identify repurposed FDA-approved drugs for CUD treatment.Our drug repurposing strategy combines artificial intelligence (AI)-based drug prediction, expert panel review, clinical corroboration and mechanisms of action analysis being implemented in the National Drug Abuse Treatment Clinical Trials Network (CTN). Based on AI-based prediction and expert knowledge, ketamine was ranked as the top candidate for clinical corroboration via electronic health record (EHR) evaluation of CUD patient cohorts prescribed ketamine for anesthesia or depression compared with matched controls who received non-ketamine anesthesia or antidepressants/midazolam. Genetic and pathway enrichment analyses were performed to understand ketamine’s potential mechanisms of action in the context of CUD.The study utilized TriNetX to access EHRs from more than 90 million patients world-wide. Genetic- and functional-level analyses used DisGeNet, Search Tool for Interactions of Chemicals and Kyoto Encyclopedia of Genes and Genomes databases.A total of 7742 CUD patients who received anesthesia (3871 ketamine-exposed and 3871 anesthetic-controlled) and 7910 CUD patients with depression (3955 ketamine-exposed and 3955 antidepressant-controlled) were identified after propensity score-matching.EHR analysis outcome was a CUD remission diagnosis within 1 year of drug prescription.Patients with CUD prescribed ketamine for anesthesia displayed a significantly higher rate of CUD remission compared with matched individuals prescribed other anesthetics . Similarly, CUD patients prescribed ketamine for depression evidenced a significantly higher CUD remission ratio compared with matched patients prescribed antidepressants or midazolam . The mechanism of action analysis revealed that ketamine directly targets multiple CUD-associated genes  and pathways implicated in neuroactive ligand-receptor interaction, cAMP signaling and cocaine abuse/dependence.Ketamine appears to be a potential repurposed drug for treatment of cocaine use disorder. As of 2020, more than 1.3 million adults suffer from cocaine use disorder (CUD) in the United States , 5. The The traditional drug discovery process for medication development is lengthy and costly , 8. DrugThe National Drug Abuse Treatment Clinical Trials Network (CTN) is conducting the ‘Drug Repurposing for cocaine use disorder (CUD)’ study using a combined strategy of AI-based prediction and retrospective clinical corroboration (CTN-0114). In this study, we developed a drug repurposing strategy that combined AI-based drug prediction, expert panel review, clinical corroboration through EHR-based testing and data-driven mechanisms of action analysis. Our drug repurposing strategy is highly generalizable and dynamic, and can be used for drug discovery for other substance use disorders. The present paper describes the outcome for a top candidate that completed the process: ketamine, a small synthetic organic molecule used clinically as an anesthetic and as a depression treatment.Our study consisted of the following steps . (1) We Knowledge graph, a powerful AI technology to abstract, organize and integrate knowledge extracted from multiple data sources, has become an emerging technology for biomedical discovery. We constructed a knowledge graph-based prediction system, KG-Predict, that prioritizes candidate drugs for a given input disease by modeling the interconnections between drugs, genes, diseases and phenotypical annotations from publicly available phenome-level databases, genome-level databases and text-mined knowledge bases , 27. In In this study, the input to KG-Predict is a list of CUD-associated genes. The output is a list of candidate drugs prioritized based on their genetic, genomic and phenotypical relevance to CUD. To collect CUD-associated genes, we first obtained 383 genes that are associated with cocaine-related diseases  from DisGeNet , a discoThe names of the CTN-0114 advisory committee members are listed in the Acknowledgements section. The committee includes experts in data science, addiction psychiatry and addiction treatment. All seven members are seasoned CTN investigators, with the majority boasting more than 20 years’ experience conducting addiction trials, including multiple trials testing medications for the treatment of CUD. The role of the advisory committee was to provide feedback on the potential clinical utility of the identified candidates with the investigative team making the decision about which candidates to submit to EHR analysis. A REDCap  survey wThis study utilized TriNetX , a federKetamine exposure information was obtained from pharmacy data in TriNetX, including the drug name, issue dates of prescription and order records. Ketamine was coded as National Drug Code  6130. In addition, we collected drug information for 10 widely utilized anesthetics for analysis as comparison drugs: propofol (NDC 8782), methohexital (NDC 6847), thiopental (NDC 10493), isoflurane (NDC 6026), desflurane (NDC 27340), etomidate (NDC 4177), sevoflurane (NDC 36453), fospropofol (NDC 828682), enflurane (NDC 3920) and midazolam (NDC 6960). Midazolam and the class of antidepressants (NDC CN600) were also used as comparison drugs in a subsequent analysis.The status of CUD was based on the International Classification of Diseases 10th revision (ICD-10) diagnosis code for ‘cocaine-related disorder’ (ICD F14). The status of anesthesia was based on the Current Procedural Terminology (CPT) relevant codes for ‘Anesthesia’ (CPT 1002796) and the ICD-10 diagnosis code for ‘Anesthesia of skin’ (ICD R20.0). The status of depression was based on the ICD-10 diagnosis code for ‘depressive episode’ (ICD F32) and ‘major depressive disorder, recurrent’ (ICD F33). The outcome ‘remission from CUD’ was based on the diagnosis of ‘cocaine abuse in remission’ (ICD F14.11) or ‘cocaine dependence in remission’ (ICD F14.21).Using the TriNetX database, we identified a total of 379 409 patients diagnosed with CUD between January 2007 and June 2022. Among the population with CUD, 16 754 patients were prescribed ketamine.We first investigated if patients with CUD who were prescribed ketamine for anesthesia have a higher CUD remission rate compared with patients prescribed other anesthetics. The exposure group was composed of patients who were prescribed ketamine within 1 year of their initial CUD diagnosis but who were never in remission from CUD prior to ketamine prescription. The control group included patients who were never prescribed ketamine but who were prescribed another anesthetic within 1 year of their initial CUD diagnosis; they had also never experienced remission from CUD prior to this anesthetic prescription.We investigated if CUD patients with unipolar or major depression who were prescribed ketamine (75% of patients received a single prescription and 25% received two or more prescriptions) displayed higher CUD remission rates compared with individuals prescribed other drugs to treat depression, such as antidepressants or midazolam . TheAs there is a 25% overlap between CUD patients who received anesthesia and those with unipolar or major depression, we performed additional analyses to examine potential effects of ketamine on CUD remission in two non-overlapping cohorts of CUD patients. (a) The exposure group included CUD patients who were never diagnosed with depression but prescribed ketamine for anesthesia. The control group included patients who were never prescribed ketamine and never diagnosed with depression but who were prescribed another anesthetic for anesthesia within 1 year of their initial CUD diagnosis; they had also never experienced remission from CUD prior to this anesthetic prescription. (b) The exposure group was composed of CUD patients who never received anesthesia and were prescribed ketamine for depression. The control group included CUD patients with depression who were never prescribed ketamine and never received anesthesia, but who were prescribed either midazolam or antidepressants within 1 year of their initial CUD diagnosis; they also never experienced remission from CUD before their midazolam or antidepressant prescription.The following studies were conducted:The index event for each study was the date of drug prescription . Patients who experienced remission from CUD prior to drug prescription or those whose initial CUD diagnosis was more than 1 year before drug prescription was excluded .To balance the cohorts, the TriNetX built-in propensity score-matching function was used, which involves 1:1 matching using a nearest-neighbor greedy matching algorithm with a caliper of 0.1 standardized mean differences (SMD) to account for potential confounding variables , 46, 60.The list of covariates, as well as their standardized name codes and data types used in TriNetX, is described in the Comparisons between cohorts were made using Cox’s proportional hazards model (a built-in function in TriNetX). The diagnosis of remission from CUD within 1 year of drug prescription was the outcome of interest. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated to compare the outcome of interest between cohorts based on comparison of time to event rates. The proportional hazard assumption was tested using the generalized Schoenfeld approach . We examWe used the STITCH  database to obtain the protein-coding genes associated with ketamine. STITCH contains data on the interactions between 500 000 small molecules and 9.6 million proteins from 2031 organisms . The scoP < 0.01. Genetic targets of ketamine were also obtained from the STITCH database. Genetic pathways that are significantly associated with ketamine-associated genes were identified from the KEGG database [P-values ≥ 0.01 were discarded. Finally, we intersected ketamine-associated pathways with CUD-associated pathways to determine which pathways are implicated in both CUD and ketamine.We identified the CUD-associated pathways that ketamine directly targets to more clearly understand the potential causal relationship between ketamine use and CUD remission. Genetic pathways for each CUD gene , and clozapine was implicated as CUD treatments in published literature [The top 10 drugs ranked by our knowledge graph-based drug discovery system are listed in terature . All 10 terature .https://clinicaltrials.gov/) and evidence from PubMed, the top 35 (2.5% of 1430) FDA-approved drugs identified by our knowledge graph-based drug discovery system were provided to the advisory committee (Based on the enrichment of potential anti-CUD drugs from clinical trials (ommittee . The advThe cohort of CUD patients prescribed ketamine differed from the unmatched sample prescribed other anesthetics in age, race, comorbidities and socio-economic and psychosocial status. As can be seen in The characteristics of CUD patients prescribed ketamine versus antidepressants or midazolam are shown in To further clarify the impact of overlap between the anesthesia and depression patient samples, we stratified the analysis by dividing CUD patients into two non-overlapping cohorts . The patWe analyzed how ketamine relates to CUD at the genetic and functional levels. At the genetic-level, we obtained 154 genes associated with ketamine, among which 10 genes are implicated in CUD, including BDNF, CNR1, DRD2, GABRA2, GABRB3, GAD1, OPRK1, OPRM1, SLC6A3 and SLC6A4. The complete list of CUD- and ketamine-associated genes is provided in We then analyzed the functional relationship between ketamine and CUD. We identified 13 genetic pathways that are significantly enriched for the 24 CUD-associated genes from the KEGG database. The most enriched CUD pathways involve neuroactive ligand–receptor interaction, alcoholism, cAMP signaling and cocaine addiction (see In this study, we implemented a drug repurposing strategy for CUD treatment by combining AI-based drug prediction, expert panel review, large-scale clinical corroboration based on EHRs and data-driven mechanism of action analysis. The AI system generated a list of candidates based on a large amount of genetic, genomic and phenotypical data from both human and mouse models. Experts refined the list of candidates based on probable clinical utility and unanimously selected ketamine for further EHR and mechanism of action analyses, the results of which suggest that ketamine has the potential to improve remission rates in patients with CUD.Ketamine is a rapid-acting general anesthetic that is typically administered as a single injection . We idenThe present results, which suggest a role for ketamine in the treatment of CUD, are consistent with several clinical trials evaluating ketamine in patients with CUD –59, 72. The mechanisms by which ketamine may impact cocaine use have not been delineated, although there is some evidence that the mystical-type experience that can arise from single-dose ketamine exposure might be important for ketamine’s impact , 73. It We previously developed KG-Predict for general purpose drug discovery and disease genetic prediction , 27. In Our study has several limitations. First, information on drug usage duration, dosage and patient adherence is limited in the EHR database; for example, prescriptions may be obtained from outside providers that were not recorded in TriNetX. Due to these limitations, we could not evaluate how the duration, dosage and compliance of drug use affect CUD remission. Secondly, although widely used and accepted for observational studies on health-care utilization, drug utilization, epidemiology, risk factors and safety surveillance, the observational, retrospective nature of this study could introduce selection, information, testing and follow-up biases. Patient EHR data may suffer from underdiagnosis/overdiagnosis/misdiagnosis and do not include all possible confounding factors. One potential limitation may come from propensity score-matching. Although patient matching can make distributions of covariates more balanced to avoid many confounders, we could still miss some unobserved and unmeasured confounders due to the observational nature of the study design, which could increase data imbalance and bias. Thirdly, while our study used standard statistical analyses for cohort studies, it would benefit from additional sensitivity analysis as well as negative control analysis. Fourthly, although the TriNetX EHR database collects de-identified data from 90 million unique patients across the United States, patients in the EHR database represent people who had medical encounters with healthcare systems and do not necessarily represent the entire US population. While patients with CUD probably have medical encounters, the generalizability of the results from the TriNetX platform remains unknown and needs to be validated in other populations. Finally, the characterization of the genes and pathways involved in the pathogenesis, etiology and biochemistry of CUD is probably incomplete. In the future, newly identified CUD-related genes and pathways will improve the identification of potential drugs for CUD.In conclusion, the results from our drug repurposing strategy combining AI-based drug prediction, expert panel review, clinical corroboration and mechanisms of action analysis support ketamine as a potential repurposed drug to treat CUD. These findings, taken together with findings from the small clinical trials that have tested ketamine for CUD, suggest that additional clinical trials are warranted.Supporting information


A growing body of research has placed the ventral striatum at the center of a network of cerebral regions involved in anticipating rewards in healthy controls. However, little is known about the functional connectivity of the ventral striatum associated with reward anticipation in healthy controls. In addition, few studies have investigated reward anticipation in healthy humans with different levels of schizotypy. Here, we investigated reward anticipation in eighty-four healthy individuals  recruited based on their schizotypy scores. Participants performed a variant of the Monetary Incentive Delay Task while undergoing event-related fMRI.Participants showed the expected decrease in response times for highly rewarded trials compared to non-rewarded trials. Whole-brain activation analyses replicated previous results, including activity in the ventral and dorsal striatum. Whole-brain psycho-physiological interaction analyses of the left and right ventral striatum revealed increased connectivity during reward anticipation with widespread regions in frontal, parietal and occipital cortex as well as the cerebellum and midbrain. Finally, we found no association between schizotypal personality severity and neural activity and cortico-striatal functional connectivity. In line with the motivational, attentional, and motor functions of rewards, our data reveal multifaceted cortico-striatal networks taking part in reward anticipation in healthy individuals. The ventral striatum is connected to regions of the salience, attentional, motor and visual networks during reward anticipation and thereby in a position to orchestrate optimal goal-directed behavior. To elicit reward anticipation, the MID presents participants with cues that inform them about the amount of money they can win per trial. These cues are followed by a simple discrimination task and with a percentage of the cued money amount if they respond accurately in the discrimination task. The MID has been modified by Simon, Cordeiro5 to adapt to participants’ mean response time and thereby allow every participant to receive a similar amount of money at the end of the task.Reward processing is a core element of goal-directed behavior. It provides organisms with a probabilistic framework to direct attention and guide behavior towards more valued actions and away from less valuable actions. Reward processing includes a phase of reward anticipation, which precedes the consumption phase, elicited by the reception of reward. Correctly anticipating a reward enables motivated behaviors and adaptively initiating actions. The monetary incentive delay (MID) task7. The interplay between these regions is thought to facilitate the detection of salient upcoming events and prepare goal-directed actions9.Studies using the MID in healthy participants have identified a network of areas underpinning reward anticipation and preparing an individual for action. Robust brain-wide activations have been shown in the ventral striatum, bilateral insula, cingulate cortex, thalamus, premotor cortex and occipital cortex, amongst others11, as well as individual differences in healthy participants14 only one study specifically described mean cortico-striatal functional connectivity in healthy individuals15. In their study, Cao, Bennett15 demonstrated functional connectivity between the ventral striatum and the supplementary motor area, the dorsal cingulate cortex, the anterior insula and the medial occipital lobe. However, these results come from adolescents and cannot be directly generalized to adults, as adolescents have been shown to process reward anticipation differently than adults16.However, the functional cortico-striatal networks of reward anticipation in healthy participants remain to be established. While many studies have assessed task-based functional connectivity deficits in pathological populations18, and correlates negatively with negative symptoms20. In individuals high in schizotypal personality traits (SPT), ventral striatal activity to reward predicting cues seems preserved at the group level22, although Yan, Wang22 found less ventral striatal activity in participants with mainly negative symptoms. Similarly, resting-state functional magnetic resonance imaging (rs-fMRI) studies have shown cortico-striatal functional connectivity disturbances in schizophrenia24 and in individuals high on SPT25.Interestingly, ventral striatal activity during reward anticipation is lower in patients with schizophrenia than in healthy controlsOur study aimed to replicate previous studies on healthy individuals’ whole-brain activations during reward anticipation. In addition, we sought to describe for the first time cortico-striatal functional connectivity during reward anticipation specifically in healthy adult participants. Having access to schizotypy and negative symptom scores for all of our participants, we performed exploratory analyses on correlations between these scores and whole-brain activity. We also explored potential activity and connectivity differences between participants with high schizotypy and participants with low schizotypy scores. Since very few published studies have investigated reward anticipation deficits in schizotypy, the hypotheses we formulated are speculative. Because schizotypy is thought to lie on a continuum between health and schizophrenia, we expected to find similar results for participants with high schizotypy scores and patients with schizophrenia in terms of activity and functional connectivity.ρ = 0.3, at α = 0.05 and β = 0.8). This showed that we needed to include 84 participants in our sample. 928 individuals were screened by phone from the general population at the University of Zurich. Amongst the individuals that passed the screening process  we selected participants based on their SPT scores . In total, 86 participants were recruited . Two participants from the mid SPT group were excluded from analyses due to non-processable fMRI data. In total, we analyzed the data of 84 participants . The project was approved by the Ethics Committee of the Kanton (KEK) of Zurich. All methods were implemented following the relevant guidelines and regulations. All participants provided written informed consent to take part in the study, in accordance with the Declaration of Helsinki.We conducted a power analysis for the correlation tests we were planning to perform  indicated the maximum amount of reward winnable  and was followed by a fixation cross (2.5-3 s). Participants were then asked to discriminate the incongruent target within a set of three circles . Next, a feedback screen (2 s) was presented. In case of a correct response, the feedback screen showed the amount of reward won for that trial. If there was no response, the feedback screen asked participants to respond faster. The amount of reward won was calculated as a percentage of the cued amount based on the difference between the response time in the present trial and the mean response time in the previous 15 trials. The mean response time up to the 15th trial was calculated using a pre-defined fixed array of response times from previous piloting . Finally, the feedback screen was followed by a jittered intertrial interval .Reward anticipation was assessed using a modified version of the Monetary Inventive Delay task . Six movement parameters were modelled as covariates of no interest and outlier scans discovered by the Art toolbox were added as covariates to be scrubbed. We removed low-frequency noise using a high-pass filter with a cut-off of 0.008 Hz. We also corrected the time-series for serial autocorrelations using an autoregressive AR(1) model.32 using “reward anticipation” as a search term . This approach was selected to render our results as generalizable as possible.Left  and right  ventral striatum seed regions were defined as the activity cluster within the second level whole-brain probability map masked with meta-analytic ROIs extracted from Neurosynth33. We defined the psychological factor as the contrast between high and no reward conditions. We then used the PPI toolbox in SPM12 to calculate the interactions between the physiological and psychological factors. We modelled PPI interaction, seed activity and onset regressors of the activation GLM for each seed region in an individual GLM for each participant together with two session constants.The connectivity maps of lVS and rVS during reward anticipation were assessed separately using a PPI analysisp < 0.05 FWE, and a cluster-level threshold of p < 0.05 FWE), with all groups taken together. We used a stringent primary threshold of p < 0.05 FWE instead of the classical p < 0.001 uncorrected due to the highly significant whole-brain one-sample t-test (i.e. large clusters spanning multiple regions). A more stringent threshold was necessary to extract clusters we could clearly define.We performed a whole-brain analysis on reward anticipation [high reward > no reward] using a one-sample t-test  with each regressor of interest.We performed regression analyses to assess the relationship between whole-brain activity and total negative symptoms and apathy scores using the SNS and schizotypal personality using the SPQ. To do so, we used one-sample t-tests . We also assessed categorical differences in ventral striatal activity between high SPT and low SPT using both rVS and lVS seeds taken together as a mask on a two-sample t-test.Group differences between high SPT and low SPT in whole-brain localized activity were assessed using a two-sample t-test , analyzing all groups together. Note that this methodology does not allow the localization of the connectivity signal within a cluster.We performed a one-sample t-test on individual connectivity maps based on the first-level reward anticipation contrast [high reward > no reward] using lVS and rVS seeds separately . Regressors of interest included the SNS total and apathy scores and the total score and negative factor of the SPQ for schizotypal personality.We assessed the link between cortico-striatal functional connectivity and regressors of interest by performing a one-sample t-test on individual connectivity maps [high reward > no reward] using both lVS and rVS seeds .Group differences between high SPT and low SPT in cortico-striatal connectivity during reward anticipation were assessed using a two-sample t-test on individual connectivity maps with lVS and rVS seeds  on average more quickly than to no reward targets  = 14.49, p < 0.001, dCohen = 1.58). However, there were no group differences between participants with high schizotypy and participants with low schizotypy (F(58) = − 1.07, p = 0.29). Additionally, reward-related speeding did not correlate with SNS total and apathy scores, nor with SPQ total scores . In addition, neither SNS total and apathy scores, nor SPQ total scores, correlated with mean Framewise Displacement .The main characteristics of our sample can be found in Table Whole-brain analyses showed robust reward anticipation activations in the bilateral VS, dorsal striatum, anterior insula (AI), thalamus, precuneus and cerebellum Crus I. Moreover, activity occurred in the right mid cingulum/anterior cingulate cortex, superior frontal gyrus, ventral tegmental area, left precentral gyrus, cerebellum VI, dorsolateral prefrontal cortex and inferior parietal gyrus Fig. , Table 2Our exploratory analysis on the contrast [high reward > low reward] showed increased activity in similar cortical and subcortical regions, although effect sizes were smaller than those of the main contrast, as one would expect. To illustrate this, we extracted the activity from the left and right ventral striatum for all three conditions and found that anticipating rewards of increasing magnitude was associated with corresponding increases in BOLD response Fig. .Figure 3We found no correlations between whole-brain activity and SNS total and apathy scores, nor with SPQ scores.We found no categorical difference in whole-brain localized activity between high SPT and low SPT groups. Additionally, no categorical difference was found between high SPT and low SPT when looking solely at ventral striatal activity.Our analyses showed increased cortico-striatal functional connectivity for high compared to no reward conditions between the lVS and the bilateral precuneus, anterior insula, precentral gyrus, right dorsal anterior cingulate cortex, mid frontal gyrus, caudate nucleus, inferior operculum, supramarginal gyrus, and the left mid occipital gyrus. Additionally, we found increased striato-striatal connectivity between the lVS and the bilateral putamen. We also found stronger reward anticipation-related functional connectivity between the rVS and the bilateral precentral gyrus, the right putamen/anterior insula, calcarine gyrus, supplementary motor area, inferior operculum, the left mid occipital gyrus, superior frontal gyrus and mid frontal gyrus Fig. , Table 3No correlations were found between cortico-striatal functional connectivity and SNS total and apathy scores, nor with SPQ scores.We found no categorical difference in cortico-striatal functional connectivity between high SPT and low SPT, with both rVS and lVS seeds.We designed this fMRI study to assess whole-brain activity and functional connectivity between the ventral striatum and the rest of the brain during reward anticipation in a large sample of healthy individuals. Our analyses showed robust whole-brain activations during reward anticipation. Importantly, our data revealed functional connectivity related to reward anticipation between the ventral striatum and components of the salience, attention, visual and motor networks, in line with the attention enhancing and motor facilitating functions of reward. In addition, we assessed associations between activity and functional connectivity and schizotypal personality and negative symptom scores. Contrary to our hypotheses, we found no correlation between activity or functional connectivity and schizotypal personality and negative symptom scores. Our exploratory analyses also showed no categorical differences between the high and the low schizotypy groups. Taken together, our results suggest that reward anticipation is affected differently in the various stages of the psychosis continuum.1, comprising the ventral striatum, dorsal striatum and anterior insula. Among these regions, the ventral striatum processes the expected (subjective) value of future rewards and helps compute reward prediction errors9. The dorsal striatum’s role in reward anticipation is the integration of ventral striatal information to select future actions based on the best outcome possible34. Finally, as a part of the salience network, the anterior insula helps integrate motivational signals with attentional processes36. The anterior insula also processes outcome uncertainty37, which applies to the cues but not the rewards in the modified version of the MID we used.Our whole-brain analyses revealed activity in the traditional regions dedicated to reward anticipation, already described by Knutson, Westdorp39 and processes reward prediction errors40 and incentive salience41 in animal studies. Cortical and cerebellar activations closely matched those described in previous meta-analyses42. These included regions dedicated to motor functions, including the primary motor cortex, the supplementary motor area, thalamus and cerebellum6, which could facilitate motor preparation when facing highly rewarded trials.Additionally, we found activations in the ventral tegmental area (VTA). The VTA is known to have strong connections with the ventral striatum15, who described functional connectivity in healthy adolescents. First, we found functional connectivity of the ventral striatum to the salience network, particularly the anterior insula, again suggesting integration of motivation and attention36. In line with this interpretation, we found connections to attentional network regions such as the supramarginal gyrus and the inferior frontal cortex44. Increased connectivity with these regions is also compatible with stronger reward anticipation-related communication of the ventral striatum with the visual network, including the calcarine and mid occipital gyrus. Thus, anticipated reward can facilitate visual attention towards reward-predicting cues45. We also found connections to motor networks, with the dorsal anterior cingulate cortex and supplementary motor area, indicating once again motor preparation for highly rewarded trials46.We observed cortico-striatal networks similar to Cao, Bennett47 that the ventral striatum is at a crossroads of networks that act together to favor rewarded actions over non-rewarded ones. The recruitment of salience and attentional networks during reward anticipation might help disrupt other ongoing processes to focus more specifically on the rewarded stimuli. In contrast, the recruitment of visual and motor networks might prepare humans to perceive and react to rewarded stimuli as fast and accurately as possible.Taken together, these results corroborate the well-established notion22. It is possible that the reward anticipation impairments might appear solely in sub-populations, for example those with high negative schizotypy22. However, in our population, the positive and negative factors of the SPQ were highly correlated (r = 0.77) and therefore no selective correlation between whole-brain activity during reward anticipation and negative schizotypy was found.Contrary to our hypothesis, we found no correlation between local activity or functional connectivity and schizotypal personality or negative symptom scores. We also found no difference in local activity or functional connectivity between participants with high schizotypy and participants with low schizotypy. These results converge with those of previous studies reporting unimpaired activity at the group level in participants with comparably high schizotypy scoresThere are limitations to this study. First, the population we assessed mostly comprised young students, which does not represent the full extent of the variability of people experiencing schizotypy. Additionally, the size of our categorical samples might not be big enough to detect subtle differences in reward anticipation in schizotypy. For example, for a two-tailed t-test, with a strong effect size of 0.7 , an alpha of 0.05, and beta of 0.8, 34 participants are required per group. However, a more modest effect size  significantly increases the required group size. For example, for an effect size of 0.5 (medium), 64 participants are required per group. These limitations could partly explain why we did not find any categorical difference in localized activity and functional connectivity analyses. Additional analyses based on a more extensive population could address these limitations.Our analyses confirmed the central role of the ventral striatum during reward anticipation. On the one hand, we replicated previous findings showing activations in the ventral and dorsal striatum, as well as in regions dedicated to salience and motor processing. On the other hand, we identified the functional networks orchestrated by the ventral striatum during reward anticipation in healthy adults. The widespread network of regions interacting with the striatum included components of the salience, attention, visual and motor networks, which conjointly may optimize goal-directed actions. Finally, we showed that reward anticipation might not be equally affected in the psychosis continuum, but instead seems to reflect the gravity of pathology.


Herpes simplex virus type II (HSV-II) with superimposed bacterial skin infection is an uncommon presentation of cutaneous necrosis in the setting of infective endocarditis. This case reflects a unique presentation of an immunosuppressed patient with infective endocarditis complicated by septic emboli and cutaneous skin lesions attributable to HSV-II and superimposed bacterial skin infection. The patient presented from an outside hospital with symptoms consistent with acute onset heart failure and skin lesions. Transthoracic and transesophageal echocardiography performed there demonstrated focal thickening of the anterior mitral valve leaflet with severe mitral regurgitation. The patient then underwent extensive infectious work-up and was put on broad-spectrum antibiotics. Further work-up demonstrated greater than three DUKE minor criteria and reiterated the focal thickening of the anterior leaflet of the mitral valve, making infective endocarditis the most likely etiology. Biopsies of the skin lesions were performed which stained positive for HSV-II and grew methicillin-resistant Staphylococcus aureus and Bacteroides fragilis. The cardiothoracic surgery service ultimately decided not to perform any surgical intervention to the mitral valve during her hospitalization as she was deemed to be too high of a risk due to her thrombocytopenia and significant comorbidities. She was later discharged in hemodynamically stable condition on long-term intravenous antibiotics with repeat echocardiography demonstrating significant reduction in the mitral regurgitation and the focal thickening of the anterior leaflet of the mitral valve.  Infective endocarditis (IE) is a rare but serious condition with an estimated incidence of 3-10 cases per 100,000 people per year in the United States. It typically occurs in patients with underlying heart disease or prosthetic heart valves and is associated with an elevated risk of morbidity and mortality [The cutaneous manifestations typically associated with IE are Janeway lesions and Osler nodes - nontender macules or papules on the soles and palms of the feet and painful immunogenic skin lesions, respectively. Other manifestations of IE include splinter hemorrhages, which often manifest as painless erythematous lesions in the nailbed . Rarely,A 47-year-old Caucasian female with a past medical history of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) on chronic anticoagulation therapy presented to an outside hospital for shortness of breath and significant dyspnea on exertion. A transesophageal echocardiogram (TEE) there demonstrated findings consistent with severe mitral valve regurgitation and focal thickening of the anterior leaflet of the mitral valve. Given her autoimmune history and initial negative cultures for bloodstream infection, there was some suspicion for Libman-Sacks endocarditis. There was additional concern for an active SLE flare and catastrophic antiphospholipid syndrome (CAPS); therefore, the patient was treated with steroids, intravenous immunoglobulins (IVIG), and plasmapheresis. On transfer, she was evaluated by the cardiothoracic surgery service, who postponed immediate mitral valve surgical replacement given her severe thrombocytopenia with overall pancytopenia. Her hospital course was complicated by a variety of infectious processes, including bacteremia caused by Klebsiella oxytoca, Enterococcus faecium, Klebsiella pneumonia, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), as well as possible CMV colitis. She was placed on cefepime, daptomycin, and intravenous ganciclovir. The patient began to develop cutaneous lesions consistent with splinter hemorrhages and Janeway lesions. Repeat blood cultures were negative - though this was in the setting of multiple days on broad-spectrum antibiotics. A repeat TEE was ordered which re-demonstrated the mitral valve thickening previously seen from the outside hospital's TEE along with mildly dilated left atrium, and a left ventricular ejection fraction of 55%. As the patient had persistent fevers, vascular phenomenon, previously positive blood cultures from other organisms, and echocardiographic abnormalities, there was now increasing concern for infectious endocarditis since that fulfills three or more of the DUKE minor criteria for infective endocarditis.CT-angiogram of the aorta with intravenous contrast demonstrated new tiny focal hypodensity in the left common iliac artery, possibly representing thrombus/embolus. There was also, of note, a focal wedge-shaped hypodensity of the posterior spleen suspicious for developing infarct. CT of the chest demonstrated new bilateral soft tissue pulmonary nodules, greater in the lower lobes, that were suspicious for septic emboli vs. metastatic disease. The cardiothoracic surgery service was re-consulted, given these new findings, and ultimately postponed surgical intervention to the mitral valve citing her significant comorbidities, thrombocytopenia, and her being too high risk for intra- and post-operative complications.An erythematous lesion was noted on the dorsal aspect of her right wrist on hospital day 8 Figure . There wThis case demonstrates a unique presentation of cutaneous skin necrosis caused by polymicrobial infection, including HSV-II. Bacteroides and MRSA are well-established organisms that can cause necrotizing fasciitis with abscess formation . HoweverHSV-II is commonly associated as the leading causative agent for anogenital lesions and is known to reactivate in times of physiological stress and immunosuppression. Cutaneous manifestations outside of the anogenital region usually manifest as herpetic whitlow vesicles . AlthougThis cause further demonstrates the need for timely diagnosis of causative organisms in cutaneous necrosis to treat appropriately. There is also further elucidation of HSV-II, causing yet another cutaneous manifestation of the virus. The medical team acted quickly by performing a punch biopsy that revealed underlying HSV-II and an anaerobic infection. This case demonstrates a rare multi-factorial complication of infective endocarditis in a patient with immunosuppression and subsequent HSV-II reactivation. Further, it demonstrates a range in skin infection manifestations that can be used to increase suspicion for multiple contributing organisms in this population subgroup. Previous literature seems lacking in describing and attributing causal organisms to this type of lesion. This patient population, as well as their providing physicians, would benefit from more research and classification of these types of lesions as this would promote increased expediency in appropriate antibiotic use.


Depression in Parkinson’s disease (PD) is common, disabling and responds poorly to standard antidepressant medication. Motivational symptoms of depression, such as apathy and anhedonia, are particularly prevalent in depression in PD and predict poor response to antidepressant treatment. Loss of dopaminergic innervation of the striatum is associated with emergence of motivational symptoms in PD, and mood fluctuations correlate with dopamine availability. Accordingly, optimising dopaminergic treatment for PD can improve depressive symptoms, and dopamine agonists have shown promising effects in improving apathy. However, the differential effect of antiparkinsonian medication on symptom dimensions of depression is not known.We hypothesised that there would be dissociable effects of dopaminergic medications on different depression symptom dimensions. We predicted that dopaminergic medication would specifically improve motivational symptoms, but not other symptoms, of depression. We also hypothesised that antidepressant effects of dopaminergic medications with mechanisms of action reliant on pre-synaptic dopamine neuron integrity would attenuate as pre-synaptic dopaminergic neurodegeneration progresses.We analysed data from a longitudinal study of 412 newly diagnosed PD patients followed over five years in the Parkinson’s Progression Markers Initiative cohort. Medication state for individual classes of Parkinson’s medications was recorded annually. Previously validated “motivation” and “depression” dimensions were derived from the 15-item geriatric depression scale. Dopaminergic neurodegeneration was measured using repeated striatal dopamine transporter (DAT) imaging.Linear mixed-effects modelling was performed across all simultaneously acquired data points. Dopamine agonist use was associated with relatively fewer motivation symptoms as time progressed  but had no effect on the depression symptom dimension (p=0.6). In contrast, monoamine oxidase-B (MAO-B) inhibitor use was associated with relatively fewer depression symptoms across all years . No associations were observed between either depression or motivation symptoms and levodopa or amantadine use. There was a significant interaction between striatal DAT binding and MAO-B inhibitor use on motivation symptoms: MAO-B inhibitor use was associated with lower motivation symptoms in patients with higher striatal DAT binding . No other medication effects were moderated by striatal DAT binding measures.We identified dissociable associations between dopaminergic medications and different dimensions of depression in PD. Dopamine agonists may be effective for treatment of motivational symptoms of depression. In contrast, MAO-B inhibitors may improve both depressive and motivation symptoms, albeit the latter effect appears to be attenuated in patients with more severe striatal dopaminergic neurodegeneration, which may be a consequence of dependence on pre-synaptic dopaminergic neuron integrity. As depression in PD often occurs early in the condition and predicts increased caregiver burden5, greater impairment in activities of daily living6 and higher costs of care7, effective treatment of depression in PD has the potential to achieve significant health and economic benefits. However, current treatment guidelines for depression in PD advise the same approach as for depressed patients with other long-term conditions, despite evidence suggesting that standard antidepressant drugs are ineffective for these patients9.Depression in Parkinson’s disease (PD) is common, affecting up to one-third of patients10. One study of non-motor fluctuations in PD found that two-thirds of patients experience depressed mood exclusively during off states11. This raises the possibility that depression in PD may be related to dopaminergic deficit, and have a specific aetiology, explaining why treatment recommendations for depression may not generalize to PD10.Mood changes in PD are frequently associated with motor fluctuations, or “on/off” states, that begin as end-of-dose deterioration of the effect of dopaminergic medications, and later progress to unpredictable fluctuations12 This degree of clinical heterogeneity has stimulated efforts to define subtypes based on symptom profiles.12 In PD, motivational symptoms of depression such as apathy (diminished initiation of and engagement in activities) and anhedonia (inability to experience pleasure and loss of motivation to act in order to seek pleasure) are particularly prevalent, occurring in 40% and 46% of patients, respectively13. Motivational symptoms are also particularly challenging to treat. The “interest-activity” symptom dimension in depression that includes loss of interest, diminished activity, fatigue, and difficulty making decisions has been associated with poor outcome of antidepressant treatment in large prospective clinical studies.14 People with PD report worse apathy and anhedonia when off dopaminergic medication16 and loss of dopaminergic innervation of the striatum is associated with emergent motivational symptoms in PD.17Depression is a heterogeneous and aetiologically complex syndrome. There are at least 256 possible unique symptom profiles that meet DSM-V criteria for a diagnosis of major depressive disorder.20 The effect of dopamine signalling depends on the dynamics of its release and clearance from the synapse.21 Short-latency phasic firing of dopaminergic neurons in the striatum is thought to encode reward prediction errors, crucial for reinforcement learning, while tonic levels of activity are thought to signal average reward valuation.22 Understanding how dopamine signalling regulates adaptive behaviour and motivation at different timescales and in different brain regions has important implications for the mechanisms underlying motivation and different depressive symptoms, and their diverse responses to different dopaminergic agents.22Dopamine activity plays a crucial role in goal-directed behaviour, signalling how much better or worse an event is than predicted (a “reward prediction error”), as well as the valuation of reward and effort costs of actions.24 Lab-based studies in both animals and humans utilising dopamine depletion have consistently implicated mesolimbic dopamine activity as a key modulator of motivation.25 However, it remains unclear whether the antidepressant effects of dopamine agonists are primarily caused by improvement in motivational deficits rather than mood. Therefore, we aimed to perform the first comprehensive analysis of the dimensional symptom predictors of antidepressant effects of dopamine agonists.Prescription of different dopaminergic medications in PD is currently largely driven by consideration of motor symptoms and side-effect profile, including impulse control disorders that most commonly occur with dopamine agonists. However, dopaminergic medications are also known to have antidepressant effects. Double-blind randomised controlled trials have shown that dopamine agonists can improve depression and apathy in patients with PD, and pramipexole, a relatively selective D3 receptor agonist, has shown promise as a treatment in chronic and severe depression in patients without PD.26 Selective type-A monoamine oxidase (MAO-A) inhibitors, such as moclobemide, are primarily used in depression to prevent the metabolism of serotonin and noradrenaline.26 In contrast type-B monoamine oxidase (MAO-B) inhibitors, such as rasagiline and selegiline, were developed for treatment in PD due to their ability to inhibit dopamine metabolism, thereby increasing striatal dopamine levels.26 However, MAO-B inhibitors such as selegiline have also been shown to improve depression in PD28 and, at high doses, they are non-selective, additionally affecting serotonergic transmission which may represent a complementary pathway to improving mood.29 There remains limited understanding of the effects of MAO-Is on different symptom dimensions of depression in PD, and no study has examined how these effects change over time or their relationship with progression of neurodegeneration within dopaminergic pathways.Other treatments, such as monoamine oxidase inhibitors (MAO-Is), are also clinically used for both their antidepressant and antiparkinsonian effects.30 Our secondary hypothesis was that dopaminergic medications with mechanisms of action reliant on pre-synaptic dopamine neuron integrity, such as MAO-B inhibitors, would have attenuated antidepressant effects as pre-synaptic dopaminergic neurodegeneration progressed.We hypothesised that there would be dissociable effects of dopaminergic medications on different depressive symptom dimensions. Considering lab-based findings of the behavioural effects of dopamine agonists, we predicted that dopamine agonist treatment would improve motivational symptoms, but not other depression symptom dimensions; whereas MAO-B inhibitors may improve mood due to their concurrent serotonergic action at higher doses.31 Launched in 2010, PPMI enrolled untreated, newly diagnosed PD patients and age- and sex-matched healthy controls. All participants underwent a standard battery of assessments including the MDS-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS), the 15-item Geriatric Depression Scale (GDS-15) and single photon emission computed tomography (SPECT) dopamine transporter (DAT) imaging with Ioflupane [123-I] yearly, over five years.We used data from the Parkinson’s Progression Markers Initiative (PPMI) cohort, an international multicentre cohort study.In order to avoid capturing chronic symptoms in the context of depression with onset prior to the development of PD, all participants who had received a diagnosis of major depressive disorder more than five years prior to diagnosis with PD were excluded (n=11).32, and a nine-item “depression” factor33  inhibitors.Striatal DAT imaging was used as a measure of pre-synaptic dopaminergic neurodegeneration, indexing the established decrease in striatal DAT specific binding ratio (SBR) in PD as the disease progresses, owing to a loss of presynaptic dopaminergic projections from the substantia nigra and ventral tegmental area.We used linear mixed-effects modelling to examine the relationship between GDS-15 factor score (dependent variable) and medication state for each class of PD medication , which were acquired contemporaneously, and how this relationship changed over the progression of illness. This involved fitting both the main effects of medication class state  and their interactions with time. This allowed inter-individual heterogeneity and unequal follow-up intervals to be accommodated by incorporating random effects. Random intercept terms at the participant level were tested.The interaction term between medication class state and time allowed us to assess how the relationship between each GDS-15 factor and specific medication changed over time, using all available data.Two sets of regressions for each GDS-15 factor were conducted: 1) unadjusted and 2) adjusted for age, sex, years of education, duration of PD ; plus cognition , impulsivity (Questionnaire for Impulsive-Compulsive Disorders in Parkinson’s Disease), severity of motor symptoms , stage of disease/functional disability , levodopa equivalent dose (LED), antidepressant medication status and the other GDS-15 factor to ensure specificity . Model fit was tested using the Akaike information criterion (AIC).Secondary analysis was conducted to assess moderation of pharmacological effects by dopaminergic neurodegeneration, replacing the time interaction with an interaction with DAT SBR. The interaction term between medication class state and DAT SBR enabled analysis of how the relationship between each GDS-15 factor and specific medication changed with striatal pre-synaptic dopaminergic neurodegeneration.All statistical analyses were performed in R version 4.1.2. The R package ‘lme4’ was used for mixed effects modelling.In total, 412 participants with PD were included at baseline with loss to follow-up of one-quarter of participants by year five . Over haTotal depression symptom scores and dimension scores both increased over time. However, only 13% of patients reported a GDS-15 score >5 at baseline  and less than 10% of participants with PD were taking antidepressant medication across all years of follow up, though indication for treatment was not available.17In almost all (98.8%) patients, DAT imaging was available at baseline and in years one, two and four, with only 17 participants imaged in years three and five. As reported previously in this sample, DAT SBR in PD participants was on average around half that of healthy controls; and as expected there was evidence of a marked decline over time .We identified dissociable relationships between different PD dopaminergic medication classes and specific dimensions of depression .Longitudinal analysis revealed that dopamine agonist treatment was associated with relatively lower motivation symptoms as the disease progressed , but this was not the case for depressive symptoms  . Both thIn contrast, MAO-B inhibitor treatment was associated with significantly lower depression symptoms , but not motivation symptoms , on average across all years of follow up. However there were no significant interactions with time  .Treatment with COMT inhibitors was associated with relatively improved depression symptoms as the disease progressed , but worse symptoms on average across all years of follow up  . This inNo significant associations were observed between levodopa or amantadine treatment and either depression or motivation symptom factor scores .Mixed-effects model analysis of the relationship between striatal DAT SBR and depression symptoms across all timepoints revealed that the effect of MAO-B inhibitor treatment on motivation was moderated by striatal DAT SBR  . PD patiStriatal DAT SBR did not moderate the effects of dopamine agonist or amantadine treatment on individual depression symptom dimensions.34We performed the first longitudinal analysis of how different types of dopaminergic medication for PD are associated with different symptom dimensions of depression, and how these relationships are influenced by neurodegeneration of dopaminergic pathways. We found dissociable associations of PD dopaminergic medications with different dimensions of depression. Dopamine agonist treatment was associated with relatively lower motivation symptoms beyond the second year following diagnosis, but not depressed mood. In contrast, MAO-B inhibitor treatment was associated with fewer depression, but not motivation, symptoms. However, MAO-B inhibitor use did appear to improve motivation symptoms in patients with relatively preserved striatal pre-synaptic dopamine projections, indexed by higher striatal DAT binding. A potential explanation for this pattern is that the MAO-B inhibitor mechanism of action is dependent on pre-synaptic striatal dopamine neuron integrity, whereas other medication classes, such as dopamine agonists, act post-synaptically. Our results support existing evidence that striatal dopamine dysfunction plays a crucial role in motivation, and highlights the need for further understanding of the effect of different dopaminergic therapies on depressive symptoms as PD pathology progresses over time.35 However, at present the choice of dopaminergic therapy is largely guided by motor symptoms and potential side effects.36 Our findings suggest that depressive symptom profile may also have a role to play in guiding dopaminergic treatment therapy selection. For example, dopamine agonist treatment may be an effective strategy to treat motivational symptoms beyond two years following diagnosis, while alternative therapies such as MAO-B inhibitors may be indicated in patients with prominent depressed mood.Optimisation of dopaminergic therapies is often the first strategy in the treatment of depression in PD.14 The dopamine agonist pramipexole is currently used as a treatment for refractory depression and our findings support the potential for further repurposing of PD dopaminergic therapies for treatment of motivational symptoms.37Though our study was conducted in patients with PD, these results also reveal mechanistic insights and potential treatment strategies for depression in patients without PD, specifically for those with prominent motivational symptoms that are resistant to standard antidepressant medications.38 However, the role of dopaminergic therapies on motivation and mood depends on dopaminergic receptor profile, pharmacodynamics, brain region and progression of underlying PD pathology.22 For example an animal study comparing D1-, D2- and D3-specific agonists in rescuing motivational deficits induced by lesions to the substantia nigra, found that only D3 agonists were effective39. D3 receptors have a narrow distribution and are primarily located within the ventral striatum, a region implicated in reward processing and the cognitive mechanisms underlying motivation. Agonism of D3 receptors has been identified as a potential treatment of apathy in PD, and further research is needed to investigate whether dopamine agonists with high affinity for D3 receptors, such as pramipexole, are more effective options for the treatment of the motivational symptoms of depression 25.Dopamine agonists have been identified as an effective treatment of disorders of motivation in PD previously.40. Dopaminergic therapies modulate the complex dynamics of dopamine signalling via inhibition of synaptic clearance mechanisms, post-synaptic agonism or potentiating synaptic release. Owing to the variation in metabolism, signalling and receptor distribution, manipulating dopamine can have paradoxical consequences for cognitive processing, often depending on basal levels of dopamine in different brain regions22. For example, dopamine synaptic clearance varies substantially across different brain regions. In the ventral striatum, rapid recycling via DAT predominates.21 In contrast, in the prefrontal cortex DAT recycling is minimal, and enzymatic degradation by catechol-O-methyltransferase (COMT) is the primary mechanism for clearance, modulating evoked dopamine release measured over minutes42. Though in our sample too few patients were taking COMT inhibitors to draw definitive conclusions, functional polymorphisms in COMT have been associated with motivational and mood disorders.43Improved understanding of the dynamics of dopamine neurotransmission underlying mood and motivation may also guide novel treatment strategies for depression in PD. Optogenetic studies have confirmed that phasic midbrain dopamine firing encodes reward prediction errors, which are crucial for learning, whereas tonic dopamine signals encode reward valuation and effort costs that drive motivation44Investigating the effects of different dopaminergic therapies on depression at the symptom dimension level provides insight into the potential utility of specific dopaminergic medications for the treatment of depression. However, large-scale randomised controlled trials of dopaminergic therapies for depression in PD are needed before any firm clinical recommendations can be made. Effective development of future treatments for depression in PD will depend on refining phenotypes, and integrating our understanding of the evolution of depressive symptoms over time with the progression in PD pathology.The associations we observed between different classes of treatment and depression symptom dimension could be a consequence of clinician treatment selection bias. However, this is unlikely as depression symptom dimension severity did not predict treatment selection at year one.The PPMI cohort only includes recently diagnosed patients with PD and may not be applicable to individuals in the later stages of the condition where the spread of neurodegeneration and systems involved are likely more complex.The associations we observed between dopaminergic medication, striatal DAT binding and the depression symptom dimension could potentially be a consequence of other PD symptoms or functional disability. However, this is unlikely as all models were adjusted for cognition, motor symptoms, disease duration and functional disability.Though all medication classes were incorporated into single model, with the sample size available in PPMI it was not feasible to account for all the interactions of the various combination therapies patients were on.Finally, depressive symptoms were analysed as a continuous trait measure due to limited numbers of patients meeting a cut-off for clinical depression based on GDS-15 score. Future replication of our findings in a large cohort studies of PD patients with an established clinical diagnosis of depression is needed.We identified dissociable effects of PD dopaminergic medications on different dimensions of depression. Dopamine agonists are a potentially effective treatment for motivational symptoms of depression in PD, especially beyond the second year following diagnosis. In contrast, MAO-B inhibitors may improve both depression and motivation symptoms, though the latter effect may be dependent on the severity of striatal dopaminergic neurodegeneration, potentially as a consequence of their mechanism of action depending on pre-synaptic dopaminergic neuron integrity. Further clinical trials of different dopaminergic treatment regimes are needed to identify clinically efficacious treatments for depression in PD.Supplement 1