Ischemic stroke is a leading cause of morbidity and mortality worldwide. Thrombolytic therapy, the only established treatment to reduce the neurological deficits caused by ischemic stroke, is limited by time window and potential complications. Therefore, it is necessary to develop new therapeutic strategies to improve neuronal growth and neurological function following ischemic stroke. Membrane lipid rafts (MLRs) are crucial structures for neuron survival and growth signaling pathways. Caveolin-1 (Cav-1), the main scaffold protein present in MLRs, targets many neural growth proteins and promotes growth of neurons and dendrites. Targeting Cav-1 may be a promising therapeutic strategy to enhance neuroplasticity after cerebral ischemia. This review addresses the role of Cav-1 and MLRs in neuronal growth after ischemic stroke, with an emphasis on the mechanisms by which Cav-1/MLRs modulate neuroplasticity via related receptors, signaling pathways, and gene expression. We further discuss how Cav-1/MLRs may be exploited as a potential therapeutic target to restore neuroplasticity after ischemic stroke. Finally, several representative pharmacological agents known to enhance neuroplasticity are discussed in this review. Ischemic stroke is a common nervous system disease associated with high rates of disability and mortality. Ischemic stroke results from disruption of blood supply, resulting in hypoxic necrosis of brain tissue, and manifestation of corresponding neurological deficits This review will highlight the role of Cav-1 and MLRs in neuronal growth following ischemic stroke, with an emphasis on the mechanisms by which Cav-1/MLRs modulate neuroplasticity via related receptors, signaling pathways, and genes. Potential clinical applications will also be discussed.Recent studies have shown that new neurons are born during cerebral ischemia, and the underlying mechanisms of neuroplasticity may provide a basis for pharmacological enhancement of treatment of ischemic stroke MLRs are rich in cholesterol, sphingomyelin, and scaffold proteins. In addition, caveolin, a scaffolding and cholesterol-binding protein, is enriched in MLRs. Caveolin is a structural component of caveolae, which are components of lipid rafts, and are highly ordered microdomains located in the plasma membrane Neurite and dendrite outgrowth consists of protrusion, engorgement, and consolidation. Membrane lipid rafts are located at the leading edge of neuronal growth cones, providing an essential plasma membrane platform to establish cellular polarity and to compartmentalize pro-growth signaling components Following ischemic stroke, disruption of pro-survival and pro-growth signaling pathways limits neuroplasticity and subsequent recovery. Several receptors associated with Cav-1 and MLRs are crucial to neuroplasticity, and may be potential therapeutic targets to improve functional recovery after ischemic stroke.The Src family kinases (SFKs) are non-receptor tyrosine kinases involved in signal transduction that modulates cell morphology, adhesion, migration, invasion, proliferation, differentiation, and survival Neurotrophins are a family of growth factors that mediate development and survival of neurons and glial cells. Furthermore, NTs are essential effectors of neuroplasticity N-methyl-D-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and kainate receptors are the three classes of ionotropic glutamate receptors (iGluRs). These receptors are critical to neuronal development and synaptic plasticity G-protein coupled receptors (GPCRs) are a large family of transmembrane signaling receptors that bind to extracellular molecules to produce intracellular signals Modulation of neuroplasticity by Cav-1 may depend on several mechanisms, including angiogenesis. The role of vascular endothelial growth factor (VEGF) in angiogenesis following ischemia has been established c), a ubiquitous glycoprotein expressed strongly in neurons, acts as a cell-surface receptor and plays an important role in regulation of neuronal differentiation and neurite growth c that promote signal transduction and neurite outgrowth c-mediated neuritogenesis and cell differentiation occurred through increased phosphorylation of Cav-1 in PC12 cells Cellular prion protein  pathway is a well-characterized pathway that regulates endogenous neuroplasticity in response to ischemia Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase (MAPK) family, which transduces signals from the cell membrane to the nucleus to promote cell proliferation, migration, differentiation, and death -/- model, which provided a structural basis for facilitation of learning and memory Nuclear factor-kappa B (NF-κB) is a transcription factor comprised of p50, RelA/p65, c-Rel, RelB, and p52 subunits that can regulate growth and elaboration of neural processes, and protect neurons against ischemia-induced neurodegeneration The sonic hedgehog (Shh) signaling pathway mediates neuroprotection and neuroplasticity in various types of neurons in the central nervous system Cyclic adenosine monophosphate (cAMP) signaling in the brain has been shown to mediate numerous neural processes including development, synaptic plasticity, learning and memory, and motor function in response to neurodegeneration The Notch and Wnt/β-catenin pathways have been shown to play important roles in regulation of neuroplasticity following cerebral ischemia In addition to the classical non-coding RNAs (ncRNAs), transfer RNA (tRNA) and ribosomal RNA (rRNA), additional families of ncRNAs such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), and piwi-interacting RNAs (piRNAs) MicroRNAs (miRNAs) are a family of short non-coding RNA molecules that play important roles in gene expression via mRNA destabilization and translational repression MiR-124, the most abundant microRNA in the adult brain, positively modulated differentiation of SVZ stem cells to neurons The role of MiR-199 in ischemia-induced neuroplasticity has been studied extensively. A study showed that downregulation of miR-199a mediated neuroprotective effects in brain ischemic tolerance Studies have also indicated that other miRNAs may regulate caveolin and other signaling pathways involved in neuroplasticity. MiR-22 was reported to protect neurons against cerebral ischemia/reperfusion injury Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides without an open reading frame. More than half of lncRNAs are expressed in the central nervous system, and they play key roles in brain development and function Valproic acid (VPA), a classical anticonvulsive and antimanic agent, has been shown to promote neuronal regeneration in primary rat cortical neurons following hypoxia-reoxygenation via increased BDNF expression and activation in vitroResveratrol  is a phenol and phytoalexin found in grapes, red berries, and nuts Sildenafil (Viagra®) is an inhibitor of phosphodiesterase-5, and is used to treat erectile dysfunction Other agents may also target Cav-1 to enhance neuronal growth and neuroplasticity following ischemic stroke. Tanshinone I was shown to promote neuronal growth by increasing the expression of Wnt-3, p-GSK-3β, and β-catenin in mouse DG The effects of neuronal growth and neuroplasticity following stroke have received increased attention in recent years. Expansion of current studies might result in novel therapeutic options to restore neurological functions in patients that suffered strokes. Recent studies have shown that Cav-1 and MLRs are involved in regulation of neuronal growth and neuroplasticity after ischemic stroke. Many of these studies suggested that Cav-1 may be a promising molecular target to improve neuroplasticity. This review discussed the receptors, signaling pathways, genes, and treatment agents involved in Cav-1-mediated neuroprotection. Although more studies are needed to evaluate the efficacy and safety of the agents discussed in this manuscript, these agents should be evaluated further as treatment options to improve neuroplasticity following ischemic stroke. Further characterization of the roles of Cav-1 and MLRs in neuronal growth and neuroplasticity may provide for novel Cav-1/MLR-based therapies to treat cerebral ischemia. Additional studies are needed to determine whether findings in cells and rats can be translated to clinical applications in humans.


THK5351 and flortaucipir tau ligands have high affinity for paired helical filament tau, yet diverse off-target bindings have been reported. Recent data support the hypothesis that THK5351 binds to monoamine oxidase B (MAO-B) expressed from reactive astrocytes and that flortaucipir has an affinity toward MAO-A and B; however, pathological evidence is lacking. We performed a head-to-head comparison of the two tau ligands in a sporadic Creutzfeldt-Jakob disease (CJD) patient and performed an imaging-pathological correlation study.18F-THK5351 PET, but not 18F-flortaucipir PET showed high uptake in the left temporo-parieto-occipital regions, largely overlapping with the diffusion restricted areas. Cerebrospinal fluid analysis was weakly positive for 14–3-3 protein and pathogenic prion protein was found. The patient showed rapid cognitive decline along with myoclonic seizures and died 13 months after his first visit. A post-mortem study revealed immunoreactivity for PrPsc, no evidence of neurofibrillary tangles, and abundant astrocytosis which was reactive for MAO-B antibody.A 67-year-old man visited our clinic a history of 6 months of rapidly progressive dementia, visual disturbance, and akinetic mutism. Diffusion-weighted imaging showed cortical diffusion restrictions in the left temporo-parieto-occipital regions. Our findings add pathological evidence that increased THK5351 uptake in sporadic CJD patients might be caused by an off-target binding driven by its high affinity for MAO-B.The online version of this article (10.1186/s12883-019-1434-z) contains supplementary material, which is available to authorized users. THK5351 and flortaucipir tau ligands were developed with the anticipation that they would have specific high affinity for paired helical filament (PHF) type tau, the building blocks of neurofibrillary tangles (NFT) in Alzheimer’s disease. Yet, diverse off-target binding has been reported on positron emission tomography (PET) imaging for each ligand . FlortauSporadic Creutzfeldt-Jakob disease (sCJD), caused by prion protein, is a rapidly progressive neurodegenerative disorder that can have increased MAO-B activity as well as NFT. Previous imaging and pathology studies of CJD showed increased MAO-B expression by activated astrocytes and microglia , 6. MeanIn the present study, we performed a head-to-head comparison of two tau (THK5351 and flortaucipir) PET images in a patient with sCJD to find out whether the patient’s brain showed distinct uptake patterns. We further performed an imaging-pathological correlation study to determine whether increased uptake of tau ligands in this case represented increased PHF-type tau burden or MAO-B activity.A 67-year-old right-handed man with a history of hypertension visited the Memory Clinic at Samsung Medical Center for rapidly progressive dementia, visual disturbance, and akinetic mutism, which started six months prior to his first visit. On neurologic examination, he showed bilateral bradykinesia, parkinsonian gait, and postural instability. On neuropsychological tests, his Mini-Mental State Examination score was 21 . He showed poor performance on language , visuospatial, memory, and frontal/executive function.Sc) was found using a RT-QuIC assay; however, the PRNP mutation was not found. Diffusion-weighted imaging (DWI) showed cortical diffusion restrictions in the left temporo-parieto-occipital regions , and phosphorylated tau was mildly elevated . Amyloid-ß was within the normal range . Pathogenic prion protein (PrP18F-florbetaben) and PHF tau (18F-Flortaucipir and 18F-THK5351). 18F-florbetaben PET revealed amyloid negative. 18F-Flortaucipir PET showed focal uptake only in the left occipital white matter region. However, 18F-THK5351 PET showed diffuse high uptake on the left temporo-parieto-occipital regions, which largely overlapped with the diffusion restricted areas . We calculated the standardized uptake value ratio (SUVR) of each THK5351, flortaucipir, and florbetaben uptake in diffusion restricted and unrestricted voxels. Then, to analyze whether the diffusion restricted area showed higher tau uptake, we parcellated the whole cerebral cortex into 84 regions based on an AAL template. We manually classified each parcellated region as a diffusion restricted area when more than 50% of the region showed diffusion restriction or as a diffusion non-restricted area when less than 50% of the region showed diffusion restriction. We then compared the regional SUVR of THK5351, flortaucipir, and florbetaben between diffusion restricted and non-restricted areas. We found that the mean THK5351 SUVR of diffusion restricted voxels was 2.17 whereas the mean THK5351 SUVR of diffusion non-restricted voxels was 1.79. The mean flortaucipir SUVR of diffusion restricted voxels was 1.16 whereas the mean flortaucipir SUVR of diffusion non-restricted voxels was 1.20. The mean forbetaben SUVR of diffusion restricted voxels was 1.07 whereas the mean florbetaben SUVR of diffusion non-restricted voxels was 1.16. Quantitative analyses showed that THK5351 standardized uptake value ratio (SUVR) in diffusion restricted areas was higher compared to diffusion non-restricted areas, while flortaucipir SUVR and florbetaben SUVR did not show any difference . Neuropathological analysis was performed at Chuncheon Sacred Heart Hospital, Chuncheon, Korea. Autopsies were performed according to the standard protocols of National Neuropathology Reference and Diagnostic Laboratories for Dementia (NRD) supported by Korea National Institute of Health , 11. Neusc (3F4 and 1C5 antibody)  Fig. b-c. Therdy) Fig. d or NFT dy) Fig. e in the dy) Fig. f. MAO-B dy) Fig. g.Fig. 2We report the PET findings of two tau ligands (flortaucipir and THK5351) and the autopsy results in a patient with sCJD. Our novel finding was that THK5351 uptake was increased in regions similar to diffusion restricted cortical areas, while flortaucipir uptake was not. The post-mortem study revealed no NFT but severe astrocytosis which was reactive for MAO-B staining. Therefore, our findings add pathological evidence that flortaucipir is more specific to PHF tau, and increased THK5351 uptake in sCJD might be an off-target binding driven by its high affinities to MAO-B.These two tau ligands showed different uptake patterns in our patient with CJD. THK5351 uptake was increased in regions similar to diffusion restricted cortical areas, while flortaucipir uptake was not. Our finding is consistent with a recent study showing that CJD patients did not have any increased uptakes of flortaucipir . Our imaThe underlying pathological substrate for THK5351 uptake in CJD might be related to increased MAO-B activity. We observed that GFAP staining of the region with diffusion restriction and high THK5351 uptake showed severe astrocytosis which was reactive for MAO-B staining. Our results are in line with previous reports showing sCJD patients have increased reactive astrocytes and MAO-B activity in the brain , whereassc immunoreactivity, moderate reactivity on GFAP staining, and severe reactivity on MAO-B staining), we assume that at the time the patient underwent brain imaging, this region might have had astrocytosis with increased MAO-B activity but not spongiform changes or PrP deposition.Although THK5351 uptake regions largely overlapped with diffusion restricted areas, left frontal region showed discrepancy. The discrepancy between negative DWI and positive THK5351 uptake in the left frontal region might be explained by the difference in the underlying pathological substrate. Previous pathological studies showed that diffusion restriction on DWI correlated best with spongiform changes and PrP deposition, followed by reactive astrocytic gliosis , 15. On The limitation of this study is the 13-month delay between imaging scans and autopsy. As histopathological data were obtained at a more advanced stage than imaging data, vacuolation, PrPsc deposition, and reactive astrocytic gliosis with increased MAO-B activity were likely less severe at the time the patient underwent imaging. Therefore, DWI and THK5351 might not fully reflect the pathological findings. However, as there was no evidence of neuritic plaques or NFT even at the advanced stage, we conclude that THK5351 uptake in this sCJD patient represents off-target binding.In conclusion, our imaging-pathological correlation study of THK5351 and flortaucipir suggested that flortaucipir is more specific to PHF tau, and increased THK5351 uptake in sCJD might be an off-target binding driven by its high affinities to MAO-B.Additional file 1:Supplementary Methods. Detailed methods for PET acquisition and analysis. (DOCX 15 kb)


Indeed, HIV-induced loss of NK cell-mediated control of lytic EBV infection has been specifically shown to cause lymphoma and also increases replication of CMV. In this review, we will discuss current understanding of NK cell modulation of HIV disease, reciprocal exhaustion of NK cells, and how this may impact increased cancer incidences and prospects for NK cell-targeted immunotherapies. Finally, we will review the most recent evidence supporting adaptive functions of NK cells and highlight the potential of adaptive NK cells for cancer immunotherapy.Despite efficient suppression of plasma viremia in people living with HIV (PLWH) on cART, evidence of HIV-induced immunosuppression remains, and normally benign and opportunistic pathogens become major sources of co-morbidities, including virus-induced cancers. In fact, cancer remains a primary cause of death even in virally suppressed PLWH. Natural killer (NK) cells provide rapid early responses to HIV infection, contribute substantially to disease modulation and vaccine protection, and are also major therapeutic targets for cancer immunotherapy. However, much like other lymphocyte populations, recent burgeoning evidence suggests that in chronic conditions like HIV, NK cells can become functionally exhausted with impaired cytotoxic function, altered cytokine production and impaired antibody-dependent cell-mediated cytotoxicity. Recent work suggests functional anergy is likely due to low-level ongoing virus replication, increased inflammatory cytokines, or increased presence of MHC While efficient suppression of plasma viremia by combination antiretroviral therapy (cART) has substantially decreased mortality of people living with HIV (PLWH), burgeoning evidence suggests a higher occurrence of a vast range of comorbidities linked to long-term treatment and aging among PLWH, including cancers. The incidence of AIDS-defining cancers such as Kaposi Sarcoma, Non-Hodgkin lymphoma, and cervical cancer, has substantially decreased with access to cART. However, cART-treated PLWH still have a higher susceptibility to non-AIDS defining cancers (NADCs) compared to the general population, and NADCs currently represent a major cause of mortality among PLWH , 2. In pnegCD56pos lymphocytes  cells are large granular leukocytes that play a central role in the control of viral infections and neoplasms. Human NK cells are defined as CD3phocytes . CD56briunctions , while Ceceptors . In addiNK cells –12.bright and CD56dim NK cell subpopulations can be found in peripheral tissues. Tissue-resident NK cells differ from circulating NK cells and are found not only in secondary lymphoid organs but also in many peripheral tissues including the uterus, lung, and liver where they represent up to 50% of lymphocytes  exocytosis of cytotoxic granules containing perforin and granzyme that results in cell lysis, (ii) signaling through Fas ligand or TRAIL death receptors which induces apoptosis, (iii) release of cytokines with potent anti-viral and anti-tumor activities, and (iv) antibody-dependent cellular cytotoxicity (ADCC), triggered through binding of the FcγRIIIA receptor (CD16) on NK cells by the constant (Fc) domain of IgG antibodies. NK cells also play major roles in tuning and controlling adaptive immune responses .Unlike other lymphocytes, NK cells lack antigen-specific receptors but lyse target cells following the integration of inhibitory and activating signals. These signals are generated by an arsenal of germline encoded cell surface molecules, with effector functions taking place when activating signals overcome inhibitory ones . The majThe classical HLA-Ia group includes the highly polymorphic and ubiquitously expressed HLA-A, -B and -C antigens. Non-classical HLA-Ib antigens comprise HLA-E, -F, and -G molecules which are expressed in a tissue-specific manner, display low genetic diversity, and limited peptide repertoire . While tdim and CD56bright NK cells, other major NK cell receptors are differentially expressed on distinct subsets of NK cells -related receptors such as 2B4 –37 that in vitro as well as their ability to prevent growth and metastasis of certain tumors in vivo, principally hematological cancers, has been clearly established  ligand in patient sera . MICA isn tumors , 100. ULn tumors , yet levn tumors .Tumor progression has been associated with lower levels of NKG2D (as well as NKp30 and NKp46) expression on NK cells from patients with cervical cancer , and defFunction of additional NK cell receptors is modulated by HIV infection and play an important role in NK cell responses to cancerous cells, including the activating receptor DNAM-1 that is expressed on the majority of peripheral blood NK cells –115. Thedim NK cells, respectively , MDS (phase II NCT02599649), lymphoma (phase II NCT01592370), and CLL (phase I NCT02481297). However, long-term use of inhibitory KIR blocking agents might lead to desensitization of NK cells . Finallypos T cells. In particular, clinical studies assessing the blockade of PD-1 or its ligand PD-L1 reported potent therapeutic efficacy against several cancers such as melanoma and non-small cell lung cancer. Selective PD-1 expression on CD56dimCD57pos mature NK cells in some but not all healthy individuals has been reported  . irAEs ain vivo proliferation and survival of NK cells. To overcome this issue, TriKEs were manufactured to engage the IL-15 receptor and are evaluated in different tumor pathologies . Alternatively, CAR expression in adaptive NK cell subsets discussed in the next section may overcome expansion and persistence issues while simultaneously boosting anti-tumor activity. Finally, implementation of CAR-based strategies optimized for NK cells is warranted. For instance, induced pluripotent stem cell (iPSC)-derived NK cells transduced with novel CAR constructs that include NK cell-specific signaling domains instead of CD3ζ signaling-based domains are being evaluated and may significantly enhance their potency (A new tool for immunotherapy is chimeric antigen receptor (CAR)-engineered NK cells. CAR are artificial receptors composed of an extracellular antibody-derived tumor antigen binding domain as well as transmembrane and intracellular domains for activating signal transduction . Thus fa potency . Importa potency .While NK cells are classically viewed as non-specific effector cells of the innate immune system, a vast amount of independent studies has demonstrated that subsets of murine, non-human primate and human NK cells are capable of adaptive immune functions, including antigen-dependent expansion and long-lived immunological memory , 168. Adpos NK cell subset is driven by the HCMV UL40 peptide presented by HLA-E, the ligand for NKG2C  that has more recently also been characterized in HCMV-seropositive subjects , demethylation of the conserved upstream non-coding enhancer region of the IFN-γ gene, recruitment of anergic unlicensed NK cells, enhanced antibody-mediated functions and release from KIR-mediated inhibition  NK cells that display enhanced effector functions after a short pre-activation with a combination of IL-12, IL-15, and IL-18 followed by a prolonged rest period . Re-stimNK cells . Potent hibition , 203. Thhibition , 199.in vivo and exert robust responses against leukemia targets, leading to remission in a subset of AML patients (The long-lived properties of CIML NK cells have tremendous potential to be exploited for cancer immunotherapy, and preliminary results from a first-in-human phase 1 clinical trial have shown that NK cells pre-activated with IL-12, IL-15, and IL-18 can expand patients . A bettepatients .Strikingly, burgeoning evidence also supports the existence of true antigen-specific memory NK cells in humans , 205, inex vivo, are long-lived and persist in vivo, are less sensitive to regulatory T cells-mediated suppression (Overall, the immunotherapeutic potential of adaptive NK cells is expected to exceed that of conventional NK cells as they may overcome some of the major limitations faced in NK cell-based cancer therapies that have been evaluated so far in pre-clinical or clinical studies. For instance, adaptive NK cells can be expanded pression  or myelopression  and can pression  or antigpression . Importapression  and confpression , 192. ThOL and SJ contributed to writing of specific sections. RR and SJ edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium knowlesi (Pk) and Plasmodium vivax (Pv) attach to human Duffy antigen receptor for chemokines (DARC) via their Duffy binding-like domains (DBLs) for invasion. Here we provide a novel, testable and overarching interaction model that rationalizes even contradictory pieces of evidence that have so far existed in the literature on Pk/Pv-DBL/DARC binding determinants. We further address the conundrum of how parasite-encoded Pk/Pv-DBLs recognize human DARC and collate evidence for two distinct DARC integration sites on Pk/Pv-DBLs.Malaria parasite erythrocytic stages comprise of repeated bursts of parasites via cyclical invasion of host erythrocytes using dedicated receptor-ligand interactions. A family of erythrocyte-binding proteins from  Plasmodium falciparum (Pf) erythrocyte membrane protein 1  of DBLs  and Plasmodium vivax (Pv) contain only single copy of DBL each. There are multiple EBPs present in the malaria parasite Pk , and it is the Pkα-DBL and Pv-DBL that mediate Duffy antigen receptor for chemokines (DARC)-dependent invasion of erythrocytes in humans  are present on merozoite surface and are responsible for binding to the DARC receptor on reticulocytes  by 12-cysteine residues that are disulfide linked -DBL interaction have been previously described -DBL binding residues have been mapped to an N-terminal region of DARC -DBL/DARC binding -DBL with DARC, but contributed to IL8 association with DARC  and non-polars  later identified another site for DARC binding, hereafter labeled Site 2 . The Pv-DBL/DARC engagement was further proposed to lead to dimerization of Pv-DBL  were found in proximity to Site 2  and EPPIC (http://www.eppic-web.org/), two software that analyze the oligomeric states of biomolecular structures deposited in the public database PDB . However, we do not preclude the oligomerization of Pv-DBLs when bound to DARC in vivo, and/or in the context of full-length proteins of each when they interact on their respective cell surfaces. Yet from the available structural data in PDB, it can be construed that the Pv/Pk-DBL monomer contains most vital structural elements required for engagement with two sites on sulfated DARC. Indications of dimeric states gathered from in solution studies like SAXS, ITC and analytical ultracentrifugation do not necessarily reveal the exact binding interfaces in a biomolecular complex. The structural resolution of Pk/Pv-EBPs bound to fully sulfated DARC will eventually reveal the modes of complex formation between these receptor ligand pairs.It has been also proposed that Pv-DBL (PDB: 3RRC) has two bound phosphates near/at Site 2, where later DARC peptide residues (19–30) were also found . During process of routine structure re-refinement of deposited crystal structures, as recommended  it is also an unambiguous monomer—even when bound to a SO4 in Site 1 (PDB ID: 5X6N and kindly assess via PISA/EPPIC). The likely irrelevant lone detergent binding site in Pk-DBL (PDB: 5X6N) is distal from both Sites 1 and 2. The presence of bound sulfate at Site 1 in Pk-DBL is clearly a striking observation, and it immediately opens the possibility of reassessing and reinterpreting the engagement rules for DARC recognition and binding by Pk/Pv-DBLs.The Pk-DBL-sulfate complex  between α6 and α7 is highly conserved in Pk/Pv-DBLs , but is ordered in sulfate bound crystal structure of Pk-DBL (5X6N).Therefore, based on (a)  Figures . We envis Figure . The loos Figure . It is nPk/Pv-DBLs offers a more complete binding footprint of DARC , or mostly (2/3) conserved for Site 2  are recognized by the Pv/Pk-DBLs , but not in other regions of Pv-DBL, including the proposed dimer interface site (Site 2), abrogated DARC binding  identified earlier  that underpin this complex. Our analyses integrate new evidence for two distinct DARC binding sites on Pk/Pv-DBLs  that together likely engage the extracellular domain of DARC via sulfated Tyr41 and Tyr30, respectively (Figures Pk/Pv-DBL-DARC interactions as a possible avenue to prevent invasion of Pk/Pv malaria parasites into human erythrocytes.Finally, the indicated structural framework model of  Figures . Our anaMY, JC-G, AJ, and SG analyzed the data and prepared figures. MY, JC-G, and AS wrote and finalized the manuscript. MY and AS designed the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cisplatin, carboplatin, and oxaliplatin are Pt-based drugs used in the chemotherapeutic eradication of cancer cells. Although most cancer patient cells initially respond well to the treatment, the clinical effectiveness declines over time as the cancer cells develop resistance to the drugs. The Pt-based drugs are accumulated via membrane-bound transporters, translocated to the nucleus, where they trigger various intracellular cell death programs through DNA interaction. Here we illustrate how resistance to Pt-based drugs, acquired through limitation in the activity/subcellular localization of canonical drug transporters, might be circumvented by the facilitated uptake of Pt-based drug complexes via nanocarriers/endocytosis or lipophilic drugs by diffusion. In earlPEG-NH2) A and subthe mice .Targeting the transferrin receptor underlines the therapeutic potential of specific ligand conjugations, as demonstrated with doxorubicin-loaded lipid-coated transferrin-conjugated nanoparticles that inhibit more efficient lung tumor growth compared to non-targeted nanoparticles  and with50 values for cisplatin, CIS-liposomes, and RGD-CIS liposomes were determined at 15, 10, and 2 µM, respectively, indicating that cisplatin-loaded RGD-CIS-liposomes enhance the therapeutic effect at low concentrations, presumably with fewer systemic side effects ) (S- and R-[Pt(DABN)Cl2] were investigated for the first time by Bombard et al., who demonstrated that the two enantiomers are able to overcome cisplatin resistance in human ovarian A2780 and colorectal HCT116 cell lines [R-isomer [S-[Pt(DABN)Cl2] nor R-[Pt(DABN)Cl2]  were determined to be more active compared to cisplatin in both GUMBUS and CDDPrGB cells  turned out to be significantly larger compared to cisplatin but insensitive to hypoosmotic exposure, which is known to activate the volume-sensitive, LRRC8A/D-dependent VRACs and facilitate cisplatin accumulation (The anti-cancer activity of two enantiomeric BN)Cl2]) A in cispll lines . FurtherR-isomer . Using rR-isomer . As the R-isomer . SimilarR-isomer , later wmulation B. The ceThe design of new Pt-based drug candidates is a challenging task. Several pharmacokinetic parameters have to be taken into consideration, e.g., renal clearance, solubility (hydrophilic vs. lipophilic), metabolic half-life, bioavailability, protein binding, and possible side effects. These are generally known as ADME-Tox parameters . The next challenge is to develop solid pre-clinical model systems that mimic the tumor microenvironment to test the relevance of information obtained with 2D cultures of wild-type/drug-resistant cell lines.Facing the unwanted development of drug resistance, new drugs exploiting various facilitating uptake mechanisms are being developed and evaluated for their putative anti-tumor activities. As resistance to cisplatin often involves reduced expression/activity of Cu-transporters and/or the volume sensitive osmolyte transporters (VRACs), the emphasis has been on new Pt-based, chemotherapeutic nanocarriers  conjugated with specific ligands  to specifically target tumor cells overexpressing the appropriate receptors. Unfortunately, the antitumor efficiency of nanocarriers relies on receptor-mediated endocytosis, intracellular lysosomal degradation, and release of the drug in an active form, which might limit their therapeutic use. The lipophilic Pt-based agents may be suitable for oral administration, as they are easily absorbed through the intestinal mucosa cells by passive diffusion. However, these compounds might also have a tendency to accumulate in adipose tissue, which limits their excretion. Water-soluble compounds, in contrast, may not be suitable for oral administration, as these compounds mostly rely on the presence of a drug transporter or channel in the plasma membrane of the intestinal mucosa cells. Moreover, the bioavailability of these compounds may be lower due to high renal clearance and detoxification.Designing Pt-based conjugates or dual-drug co-delivery nanocarriers with a balanced ratio of drugs that are capable of inducing cell death through multiple intracellular mechanisms and at the same time selectively targeting rapidly dividing cancer cells (through specific biomarker recognition and targeting) may be a strategy to overcome the development of chemotherapeutic resistance and minimize systemic toxicity. However, some caution should be employed as the resistance mechanisms seen in experiments on resistant cell lines might have questionable relevance to the multifactorial resistance seen in solid tumors and the gain that EPR provides might be outweighed by the costs of lower tumor penetration by large entities.


Mycobacterium tuberculosis (TB) – and Tuberculosis-Associated Ocular Inflammation (TB-AOI) represent uncommon manifestations of TB. There is limited data and a lack of diagnostic and treatment standards for these conditions.Erythema induratum of Bazin (EIB) – nodular vasculitis associated with Eleven-year retrospective review of EIB and TB-AOI cases managed in a provincial TB program with prospective phone-based follow-up of anti-tubercular therapy (ATT) recipients. Presumptive TB-AOI and EIB diagnoses were determined by ophthalmologist or dermatologist assessments correlated with positive tuberculin skin test and/or QuantiFERON-TB Gold, along with pathologic criteria in EIB cases.Of 21 EIB and 20 TB-AOI cases that received ATT, 13 and 11, respectively, were reached for follow-up. The majority of EIB and TB-AOI cases were female and immigrated from TB high-burden countries. Median durations of pre-diagnosis symptoms were 2 and 0.8 years (IQR 2.5 & 1.1) for EIB and TB-AOI cases, respectively. Overall, 14 different ATT regimens were used for a median duration of 6 months (range 5–9). ATT related adverse events resulting in treatment discontinuation occurred in 14% of EIB and 10% of TB-AOI cases. On last follow-up, 76% of EIB and 42% of TB-AOI had improvement or resolution of disease.EIB and TB-AOI were uncommon presentations receiving variable therapy. While treatment response was modest for EIB cases, TB-AOI cases had sub-optimal treatment outcomes. The unique diagnostic and management challenges presented by these conditions in TB low-incidence settings highlight a need for improved treatment candidate selection, therapy standardization, and cross-specialty medical collaboration.The online version of this article (10.1186/s12879-019-3737-5) contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis complex. When believed to be related to a hypersensitivity reaction to TB antigens such conditions have been referred to as tuberculids, and more recently tuberculosis-associated ocular inflammation (TB-AOI) when involving the eye . Further specific treatment effectiveness statistical comparisons were limited by the high variability in ATT regimens used and small sample size.per degree of base-line TST induration (≥15 mm versus <15 mm) for either EIB or TB-AOI cases. Comparing outcomes of those who completed ATT treatment regimens containing three or more anti-TB drugs for a total of six or more months  with those that did not, outcomes were not significantly different for either EIB or TB-AOI cases  predominate across case series , 20–22. The non-specific nature of EIB and TB-AOI clinical syndromes and a frequent lack of confirmatory diagnostics make treatment candidate selection for these conditions particularly challenging. Whereas tissue sampling for histopathology and microbiology evaluation may aid diagnostic differentiation in cases of suspected EIB , the lowTuberculosis molecular diagnostics (e.g. nested polymerase chain reaction (PCR)) appear to have an emerging role in EIB and TB-AOI diagnosis. The reported sensitivity of ocular fluid PCR for TB has varied considerably across cases series in TB high-incidence settings, ranging from 31 to 47% in a pair of studies in India and Singapore , 24 to aIn the absence of confirmatory molecular diagnostics, histopathology in EIB may significantly aid treatment candidate selection. Lobular panniculitis with vasculitis - some authors consider veins and venules to be predominantly involved , 9 – and18F-FDG-PET/CT) to identify extra-ocular sites of infection for tissue sampling [In the case of TB-AOI, low culture yield of ocular aspirates and potential risks associated with ocular tissue biopsy place diagnostic emphasis on ophthalmological examinations. Findings of choroidal granulomas, occlusive retinal vasculitis, and multifocal serpiginoid choroiditis have been proposed as specific to TB-AOI in high TB incidence settings ; howeversampling . Evaluatsampling . The Colsampling .While variations in case definitions  and outcome measurements  limit cross-series ATT comparisons. Acknowledging these limitations, a trend of positive clinical response and relapse reduction is apparent with extended duration triple-drug ATT for both EIB and TB-AOI.Cases-series evaluating EIB treatments have shown higher non-response and relapse rates among cases receiving mono- or dual- drug ATT compared with those receiving triple-drug ATT for at least 6 months , 21. SimWhether EIB and TB-AOI represent immune-mediated response to LTBI or are manifestations of subclinical TB disease continues to be an area of scientific debate with implications for the selection of appropriate treatment , 10. DesThe medical records used for the retrospective component of this study did not contain detailed ophthalmologic exam findings from baseline and follow-up. While the majority of TB-AOI cases were designated as ‘uveitis’ (90%), further information on location, extent and nature of ocular inflammation could not be reliably captured. Previous studies have shown variable ATT response between predominantly anterior, posterior or panuveitis cases of TB-AOI , 12, 15;As highlighted by limitations of the present study and the outlined challenges in treatment candidate selection, collaboration between TB specialist and ophthalmologist for TB-AOI or dermato-pathologists for EIB is needed to optimize care for these conditions. This may be particularly true in low incidence setting where non-tuberculosis providers may be unfamiliar or uncomfortable managing these conditions. Supporting this need for collaboration, a recent report from the American Uveitis Survey found that ophthalmologists in TB low-incidence countries were much more likely to defer TB-AOI treatment decisions to an infectious disease or TB specialist compared with those in high TB high-incidence countries (76 versus 34%) . While tWhile erythema induratum of Bazin and TB-AOI represent uncommon manifestations of TB they pose unique management challenges, particularly in TB low-incidence settings. As highlighted by our study and review of current research in this area, improvements in treatment candidate selection and therapy standardization are needed to establish the optimal management of these conditions. Cross-specialty collaborative management of these conditions along with prospective multi-site research collaborations are needed to ensure these uncommon conditions are better understood and effectively managed as part of TB elimination efforts.Additional file 1:Phone Interview. Standardized script used for follow-up participant phone interviews. (DOCX 15 kb)Additional file 2:. Tabular summary of treatment details, clinical outcome, and phone-interview patient reported outcome. (DOCX 18 kb)Erythema induratum of Bazin treatment and outcomesAdditional file 3:Tuberculosis-Associated Ocular Inflammation treatment and outcomes. Tabular summary of treatment details, clinical outcome, and phone-interview patient reported outcome. (DOCX 18 kb)


On direct testing, the next-generation of targeted AAVP vectors proved superior to the original prototype in vitro and in mouse models of solid tumors, by avoiding degradation in pre- and postinternalization settings: An important methodological advance. We propose this platform should be considered for clinical applications.Over the past decade, we have used adeno-associated virus phage (AAVP) vectors for discovery, preclinical imaging, and translation therapy. However, hurdles remained with AAVP-mediated transgene delivery: both preinternalization  and postinternalization . As a proof-of-concept, we designed, developed, and validated a next-generation of targeted AAVP vectors that mitigate these obstacles to efficient gene delivery through the  Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage’s minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells. Bacteriophage (phage) are viruses that infect and replicate only within 1 or another bacterial hosts. A major advantage of ligand peptide-targeted phage-based vectors—as opposed to mammalian viruses—for gene delivery to human diseases is that phages internalize into nonbacterial cells only if they are genetically modified to do so . Howevercis form, such as inverted terminal repeats (ITR) from the human adeno-associated virus (AAV), into the phage genome background  and recombinant. Other phage-based constructs have been designed to exploit the functional display of ligand peptides on the pVIII major coat proteins for various purposes, including crossing cellular membranes –23. The 24Escherichia coli K91 host bacteria. We found that the multifunctional AAVP displaying SBP (RGD4C-SBP-AAVP) bound to immobilized streptavidin as determined by the greater number of bacterial transducing units (TU). In contrast, corresponding negative control constructs  did not show any detectable binding above background  under normal conditions or following incubation with fibrinogen for 60 min  compared with cells transduced with RGD4C-AAVP-Luc  resulted in substantially more gene expression compared with the other constructs and RGD4C-AAVP-Luc, which lacks any modification to pVIII. . Furthermore, in these experiments, the multifunctional RGD4C-H5WYG-AAVP-Luc proved superior in targeted gene delivery compared with RGD4C-AAVP-Luc for all GC per cell doses, in both M21 and 9L tumor cells . Of note, no gene expression was detected in M21 and 9L cells given nontargeted control fd-H5WYG-AAVP-Luc, confirming that targeted transduction of cancer cells by AAVP is markedly improved with the multifunctional RGD4C-H5WYG-AAVP-Luc.We performed additional experiments to evaluate transgene expression following mammalian cell transduction with various genomic copies (GC) of the multifunctional AAVP carrying the − ions to prevent the build-up of a charge gradient. This influx of both protons and Cl− ions increases the osmolarity of the endosomes that leads to osmotic swelling, subsequent destabilization, and release of their contents into the cytoplasm. To assess the buffering capacity of the RGD4C-H5WYG-AAVP, we performed acid–base titrations of RGD4C-H5WYG-AAVP, RGD4C-AAVP lacking the H5WYG peptide on rpVIII, or aqueous vehicle as controls. We found that the RGD4C-H5WYG-AAVP had a markedly higher buffering capacity compared with RGD4C-AAVP and required additional HCl to lower the pH from 7.0 to 4.0 , we used vectors carrying the luciferase reporter gene and assessed gene expression by bioluminescence imaging (BLI)  , 10. We AAVP-Luc A. For exring M21 B and 9L ring M21 C, respecstration D, consisver time D. No tumIn this study, we designed, generated, and evaluated a next-generation of targeted AAVP constructs with enhanced abilities as gene delivery agents to cancer. To date, development of most multifunctional vectors still relies on synthetic conjugates introduced into genetic or molecular constructs . They tyHere we explored how the capsid proteins of filamentous M13 phage, especially the multithousand copies of the major coat protein (pVIII) and the much smaller number of minor pIII protein copies on externally projecting filaments at one end of the phage particle, could be reengineered to display a wide range of functional peptide motifs with desirable attributes. Furthermore, incorporating ITR-flanked transgene expression cassettes from AAV vectors enables our multifunctional AAVP to transduce cancer cells in a targeted and specific manner . Thus, iMycobacterium abscessus infection was reported by using a 3-phage mixture , then by the Home Office.Statistical analyses were done with Student’s SI Appendix, SI Materials and Methods.Additional methods are detailed in the Supplementary File


Protein ADP-ribosylation is essential for the regulation of several cellular pathways, enabling dynamic responses to diverse pathophysiological conditions. It is modulated through a dynamic interplay between ADP-ribose readers, writers and erasers. While ADP-ribose synthesis has been studied and reviewed extensively, ADP-ribose processing by erasing enzymes has received comparably less attention. However, major progress in the mass spectrometric identification of ADP-ribosylated residues and the biochemical characterization of ADP-ribose erasers has substantially expanded our knowledge of ADP-ribosylation dynamics. Herein, we describe recent insights into the biology of ADP-ribose erasers and discuss the intricately orchestrated cellular processes to switch off ADP-ribose-dependent mechanisms. ADP-ribose erasing enzymes are increasingly recognized as critical regulators of protein ADP-ribosylation dynamics in living systems. Here, the authors review recent advances in the discovery and characterization of ADP-ribose erasers and discuss their role within the cellular ADP-ribosylation machinery. Similar to other transient biological processes, the ADP-ribosylation turnover relies on synthesis and degradation mechanisms3. The enzymes that perform these functions can essentially be described as writers and erasers, a nomenclature borrowed from the classification of proteins involved in epigenetic regulation. ADP-ribose writers are collectively referred to as ADP-ribose transferases (ARTs), a family of proteins with mono- or poly(ADP-ribose) transferase activities. These enzymes, especially the promising drug target poly(ADP-ribose) polymerase-1 (PARP-1), have been intensely studied by the ADP-ribosylation community for many years. More recently, attention has shifted towards the biological roles of ADP-ribose erasers, stimulated by the identification of a variety of ADP-ribose degrading enzymes with different substrate specificities. These recent findings have profoundly changed the prevailing view that ADP-ribose erasing depends almost solely on poly(ADP-ribose) glycohydrolase (PARG) activity.Reversible post-translational modifications (PTMs) contribute to the dynamic regulation of the proteome through a diversified repertoire of functions. Protein ADP-ribosylation has emerged as a complex, dynamic, and reversible PTM system within which fundamental components work antagonistically to fine tune and tightly regulate protein behavior+ through the cleavage of the nicotinamide-ribosyl bond. Therefore, ADP-ribosylation reactions generally depend on NADase activity. A fundamental distinction exists between mono-ADP-ribosylation (MARylation), i.e., the transfer of a single ADP-ribose monomer, and poly(ADP-ribosylation) (PARylation), which involves the biosynthesis of elongated ADP-ribose polymers . MARylation can impact protein activity, stability, substrate specificity, folding, or localization. For instance, substrates of the bacterial MAR transferases can undergo substantial structural rearrangements that profoundly modify host cell physiology and promote cellular intoxication5. The functional divergence between MARylating and PARylating enzymes is consistent with a biological system that involves multiple layers of antagonizing activities. This concept is supported by a rapidly expanding repertoire of ADP-ribose-degrading enzymes, suggesting that MAR and PAR modifications are continuously transferred to, and removed from, substrates by an antagonizing set of enzymes.ADP-ribosylation—in its strictest sense—refers to the enzymatic addition of an ADP-ribose molecule to a target substrate. The transferrable ADP-ribosyl units are typically derived from NADers Fig. . PAR polThis review will first focus on PARG and the more recently characterized enzymes that can reverse ADP-ribosylation. Subsequently, we will discuss the biochemical methods used to detect ADP-ribosylation turnover, and expand on the regulation of ADP-ribosylation through combinatorial selective erasing mechanisms. We will conclude by discussing the therapeutic target potential of ADP-ribose erasers, focusing on the use of PARG inhibitors in synthetic lethal approaches against cancer.6. This view is supported by the identification of a growing number of proteins implicated in writing, reading, and erasing the ADP-ribosylation modifications. Although a synthesis and degradation duality is inherent to transient PTMs, specialized erasers might occupy different catalytic niches to provide a functional and temporal reversibility of the reaction and for the recycling of ADP-ribosylated substrates. The inability of PARG—the main dePARylating enzyme—to remove MARylation marks8, and its limited processivity on short PAR polymers, leaves room for the involvement of other erasers , only PARylation occurs in conjunction with a substantial decrease of intracellular NAD+ concentrations when extensive DNA damage is encountered. Globally, PARylation processes account for a large proportion of the ART activity in cells. Therefore, dePARylation can be viewed as the predominant erasing activity.Although a role of MARylation in response to genotoxic stress has become better established recently  connects the N-terminal region to the catalytic domain through a hinge region11 and contains a regulatory segment15. Domain C (exons 9–14) contains the catalytic active site and the PARG signature motif16. Domains C and the C-terminal domain D (exons 15–18) form the PARG macrodomain17. A number of nuclear export signals (NES) and nuclear localization signals (NLS) are distributed throughout the PARG sequence.PARG is the major dePARylating enzyme, and is primarily responsible for hydrolyzing the glycosidic linkages between ADP-ribose units of PAR polymers to generate free ADP-ribose monomers. Only a single PARG splice variants with different localizations enables functional specialization18. In human cells, major isoforms include a full length 111 kDa PARG enzyme and splice variants that generate proteins of 102 and 99 kDa . The prevention of RPA loading eventually leads to increasing areas of uncovered ssDNA, which then transform into DNA double-strand breaks (DSBs), resulting in the formation of more PAR. Ultimately, this amplification loop promotes apoptosis and/or necrotic cell death in proliferating cell populations. These observations are in agreement with the finding that PARG localizes to replication foci throughout S-phase and interacts with the replication protein PCNA30. Furthermore, they complement an earlier report that PARG-deficient cells treated with DNA alkylating agents have an increase in S-phase arrest together with high levels of the DSB marker γH2AX31. Correspondingly, the Lopes group showed that PARG inactivation affects the progression of all replication forks and alters the molecular architecture of a significant fraction of replication intermediates32. These results provided mechanistic insight into the essential role of PARG in cell growth and development, in line with the observed embryonic lethality of PARG−/− mice33.The importance of the catalytic activity of PARG became clear with the observation that 34. Parthanatos is triggered by the release of the apoptosis-inducing factor (AIF) from the mitochondria to the nucleus36. Once translocated to the chromatin environment, AIF leads to large-scale DNA fragmentation and chromatin condensation, which is followed by cell death37. Depletion of PARG has been shown to be protective against oxidative stress-induced parthanatos by preventing the release of AIF from the mitochondria38.Another important role of PARG during the DNA damage response is to maintain stable levels of PAR and to recycle highly automodified PARP-1. The stabilization of PAR levels is crucial for protecting the cell against parthanatos, a caspase-independent PAR-mediated type of cell death40. Overall, these examples show that the dynamic equilibria established between PARP-1 and PARG activities, and therefore PAR levels, are key for controlling cell fate, suggesting that PAR erasers are as important as PAR writers for cellular homeostasis.Lastly, PARG has also been implicated in telomere maintenance. PARG is capable of negatively regulating the access to telomeric DNA by reversing ADP-ribosylation of the telomeric-specific protein TRF1, contributing to the regulation of telomere repair and replication41. Bacterial MAR transferases (MARTs) have related genes in humans whose extracellular expression makes them irrelevant or inoperative with respect to intracellular ADP-ribose-mediated pathways42. In human cells, intracellular protein MARylation is performed by members of the ADP-ribosyl transferases diphtheria toxin-like proteins (ARTDs). Formerly classified as PARPs43, the 17 members of the ARTD family in human were renamed according to a systematic nomenclature that better reflects their structural features and catalytic properties44. There are currently 11 members of the human ARTD family characterized as MARTs, typically renamed after the type of ADP-ribose molecule  they transfer onto themselves or target substrates46.ADP-ribose conjugation was first described as a PTM catalyzed by bacterial ADP-ribosylating exotoxins (bAREs)47. While ARH2 substrates are yet to be discovered, ARH1 is a highly active ADP-ribosyl-arginine hydrolase48 and ARH3 is an ADP-ribosyl-serine hydrolase49  family consists of three related proteins56 This apparent discrepancy will be discussed later on in this article.While ARHs only erase arginine- and serine-MARylation and macrodomain-containing enzymes specifically target aspartate- and glutamate-MARylation (see next section), ARTDs have been shown to mediate ADP-ribosylation on a wide range of amino-acid residues57. It can be found as a stand-alone module or integrated into multi-domain proteins. The macrodomain was the first characterized ADP-ribose-binding module. It can bind terminal ADP-ribose structures with nanomolar affinity58. There is functional diversity related to structural variation in the macrodomain protein family. A subgroup of macrodomains lacks the ability to bind ADP-ribose while others acquired glycosidic activity involved in ADP-ribosylation reversal4. There are ten human macrodomain-coding genes: the histone H2A variants Macro H2A.1 and Macro H2A.2; MacroD1, D2, and D3; TARG1; the chromodomain-helicase-DNA-binding protein 1-like (CHD1L) and the macrodomain-containing ARTDs 7-8-9 (formerly named PARPs 15-14-9). Among these, MacroD1, MacroD2, and TARG1 were classified as ADP-ribose erasers because of their ADP-ribose hydrolase activities. MacroD1 and MacroD2 cleave the chemical link between MAR and an acceptor protein while TARG1 presents the unique capability of cleaving both MARylated and PARylated side chains of aspartate and glutamate residues59 . Current experimental data suggest that ester-type ADP-ribose bonds in protein substrates are specific targets of the macrodomain erasers. This activity could play a regulatory role in vivo as MacroD2, for example, has been implicated in the recycling of automodified PARP-163. The removal of the autoinhibitory MAR moieties from PARP-1 by MacroD2 has been suggested to explain the accumulation of MARylated PARP-1 in the context of MacroD2 gene deletion in human colorectal cancer cells64. The underlying MacroD2-dependent PARP-1 recycling model proposed by Sakthianandeswaren et al. involves a biphasic erasing of PARP-1 automodification, which implicates PARG as the primary PAR trimming enzyme responsible for the generation of MAR adducts that can subsequently be targeted by MacroD264.Similarly to TARG1, the mono-ADP-ribose hydrolase activities of MacroD1 and MacroD2 are also selectively directed toward ester bonds established by ADP-ribosylated aspartate and glutamate residues, although with different catalytic modes  superfamily members NUDT9 and NUDT16 as well as ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1)68. These erasers target the phosphodiester bound in ADP-ribose moieties. Therefore, their activity is independent of the type of ADP-ribose linkage established with the substrate protein. However, these enzymes should be classified as partial erasers since they leave a phosphoribose remnant attached to the target protein 68.The extracellular ENPP1 phosphodiesterase, which lacks a NUDIX and a macrodomain, is yet to be characterized regarding its involvement in ADP-ribose processing. ENPP1 shows considerable phosphodiesterase activity in vitro against MAR and PAR, exceeding that observed for NUDT16 in a cell-free system75. Based on this observation, a thin-layer chromatography (TLC)-based strategy coupled to a radiolabed PAR substrate was developed to monitor ADP-ribose accumulation rather than substrate disappearance75. This TLC method has been used successfully to measure PARG activity in cell extracts and tissues76, characterize site-directed mutants77 and to evaluate the inhibitory strength of small molecules79. Later, the conversion of the reaction product, the monomeric ADP-ribose, into a quantifiable fluorophore has been reported as a nonradiometric and high-throughput assay for PARG activity80.The emergence of a variety of new players that modulate ADP-ribose catabolism underscore the urgent need for methods capable of rapidly measuring erasing activities. Historically, most assays were developed to measure the disappearance of PAR as a consequence of PARG glycosidic activity. Usually based on residual PAR precipitation assays, these methods give rise to inconsistencies when monitoring PARG activity75. However, no significant additional PAR erasing activity has been detected in most cell extracts using this approach, contributing to the long held belief that PARG was solely responsible for mediating PAR degradation in cells.TLC assays are inadequate to demonstrate the contribution of individual PARG isoforms or additional PAR-degrading enzymes to global PAR erasing activity in cells. In this respect, PAR zymograms were developed to detect alternative catabolic activity against PAR in complex samples. Zymograms are essentially composed of radiolabeled automodified PARP-1 co-polymerized with a polyacrylamide gel. Following renaturation, digested regions can be visualized on the autoradiogram. Although protein renaturation and in-gel activity constraints the applicability of this strategy, zymography proved to be an effective and sensitive method to detect PAR hydrolysis by PARG24 and by HPLC81. Despite being experimentally challenging, HPLC analysis of PARG degradation products following digestion with snake venom phosphodiesterase (svPDE) remains the method of choice to determine the relative contribution of both PARG glycosidic activities to PAR erasing. In this assay, the exoglycosidic activity allows PARG to attack PAR polymers at the protein-distal chain end to release ADP-ribose units, which are subsequently converted to AMP by cleaving the phophodiester bond with snake venom phosphodiesterase. The endoglycosidic actvity of PARG generates additional chain termini that release supplementary AMP upon double digestion with svPDE, which can be measured to estimate the relative endo/exo activities23.Consistent measurements of PARG activity under standardized conditions are hindered by the absence of a well-defined substrate . Additionally, none of the above-mentioned methods is sufficiently accurate to discriminate between the exo- and endoglycosidic activities of PARG. A number of assays have been designed to specifically monitor the endoglycosidic activity of PARG in protein-bound and protein-free polymer populations, but the most widely used methods are based on the analysis of PAR reaction products on high-resolution DNA sequencing gels61. In this approach, PAR termini are protected with an inactive bacterial PARGE115Q mutant that blocks exoglycosidic cleavage. When human PARG is added to the blocked PAR substrate, only endoglycosidic cleavage can occur. The accumulation of PAR fragments (ADP-ribose oligomers) is detected by LC-MS in the form of specific mass-to-charge ratios and correlated to the endoglycosidic activity of PARG61. Although exo- and endoglycosidic mechanisms are essential for efficient dePARylation, it is common to reflect PARG activity in a single value that integrates both activities based on commercially available chemiluminescence- and colorimetric-based detection systems. These assays are suitable for high-throughput screening of PARG inhibitors in addition to antibody-based detection methods82. However, immunological detection of PAR is prone to underestimating the presence of residual ADP-ribose oligomers for which the antibodies generally possess low affinity83. The recent development of antibody-like MAR- and PAR-binding reagents should prove beneficial to the evaluation of PARG inhibition in cells84.A more recently developed alternative to measure PARG endoglycosidic activity is based on the detection of ADP-ribose oligomers by LC-MSE988Q mutant as a substrate for MARylation erasers. This PARP-1 mutant is significantly more active than other MARTs and thus represents a robust approach to generate a MARylated substrate. Bona fide MARTs such as PARP-10/ARTD10 also have been used as MARylated substrates85. Furthermore, the oligo(ADP-ribosyl)ated PARP of H. aurantiacus was employed as an intermediate length substrate for ADP-ribose erasing assays66. Reaction products are generally resolved by SDS-PAGE or TLC. These approaches provide valuable substrate models for ADP-ribosylation erasing studies but may not reflect the diversity and wide range of ADP-ribose polymer species, which could explain the persisting confusion regarding the linkage selectivity of MAR hydrolases. While some MARTs such as PARP-10/ARTD10 appear to be MARylated exclusively on acidic residues85, it is less clear which types of ADP-ribose−protein linkages exist in other MARTs and PARP-1E988Q. A panel of linkage-specific substrates would be necessary to assess the diversity of MARylation reversal. For example, ADP-ribosylated actin by the arginine-specific ADP-ribosyltransferase CDTa provides a defined substrate for arginine-mediated ADP-ribosylation studies while the threonine-specific transferase TccC3 mono-ADP-ribosylates threonine residues of the same substrate85.Given the increasingly important role of MAR erasers, a number of approaches have also been developed to facilitate the detection of MAR hydrolase activities. One of the most effective approaches is to use the auto-MARylated PARP-186. Trans-ADP-ribosylation of synthetic peptides with PARP-1E988Q has been demonstrated by MS analysis but the actual yield of peptide MARylation is probably too low for subsequent biochemical analysis, even after affinity purification87. Alternatively, peptide microarrays containing several ADP-ribosylated residues in a variety of sequence contexts may allow profiling of the recognition and processing specificity of MARylation erasers.It should be kept in mind that the amino-acid sequence surrounding the ADP-ribosylation site is unknown in these substrates and might influence the recognition by the eraser. Similarly, MARylated substrates, such as histones, may carry additional PTM decorations that could also tune the binding affinity of the erasing enzyme. The development of synthetic peptides with site-specific ADP-ribosylations will be particularly useful for dissecting the substrate specificity of ADP-ribose erasers92.An intrinsic characteristic of ADP-ribosylation is the molecular heterogeneity and complexity of the reaction product transferred to target substrates. Therefore, ADP-ribosylation needs to be viewed in a length- and site-dependent manner. The site-specific length of PAR polymers is difficult to test experimentally and further studies are needed to characterize the length diversity of PARylated substrates. More progress has been made with regard to determining ADP-ribosylation sites within proteins. The site-specific localization of ADP-ribosylation modifications could be mapped in a system-wide manner in several recent MS-based proteomics studies. Notably, these methods are significantly more challenging and difficult to implement than standard MS-based approaches that only aim for protein identification93. The biological significance of differential ADP-ribosylation site usage is unknown but ADP-ribose−protein linkages appear to be processed by erasers with rigid selectivity  may be targeted by ADP-ribosylation under physiological conditionsty Table . For ins95. Furthermore, PAR polymer populations with different half-lives, depending on their length and complexity, have been reported96. This suggests that recognition and processing of multi-site and multi-structural ADP-ribosylation involves complex coordination of the erasers. However, current atlases of ADP-ribosylation signatures, notwithstanding their importance, from human cancer cells provide little information regarding the occupancy rate of different ADP-ribosylation modifications. At the moment, it is unclear how multiple ADP-ribosylation linkages can be read and transformed into meaningful signalling information.The different susceptibility of each type of ADP-ribosylation to degradation by the erasers suggests that the stability of ADP-ribosylation in cells may vary depending on the type of linkages. For example, the absence of a specific enzyme to erase ketoamine-linked ADP-ribose from lysine residues has been hypothesized to be involved in the long-term maintenance of histone epigenetic marks100, and the ADP-ribose ester bonds of MARylated, phosphorylated double-stranded DNA can be hydrolyzed by MacroD1101. PARP-3-mediated MARylation of DNA can also be erased by MacroD2, TARG1, PARG, and ARH399. For DNA MARylation reversal, MacroD1, MacroD2, and TARG1 target the same type of ester bonds  as a regulator of both histone ADP-ribosylation and PARP-1 automodification. This finding demonstrated that a PARP-1-interacting factor can modulate PARP-1’s PARylation activity, switching it to O-linked ADP-ribosylation117. HPF1 accumulates at DNA lesions in a PARP-1-dependent but PAR-independent manner105. Considering that PARP-1 relocation to DNA damage sites precedes HPF1 recruitment, PARP-1 could be involved in the glutamate and aspartate ADP-ribosylation of the nucleosomal surface118 before switching its catalytic activity toward serine residues as HPF1 accumulates locally. This may indicate a regulatory mechanism with several overlapping waves of linkage-specific ADP-ribosylation  and erasers adds an additional layer of complexity as PAR readers might influence the kinetics of degradation of polymers and MAR production.Non-covalent interactions are thought to play an important regulatory role in ADP-ribose catabolism. For example, a marked inhibition of PARG activity is observed when free PAR is associated with histones or nuclear matrix proteins, most likely through protection of ADP-ribose polymers from PARG-mediated degradation130. Following a rapid relocation of MacroD2 in a PARP-1-dependent initial phase, a second slower phase is observed, presumably as a consequence of the accumulation of MARylated species generated through PARG activity. This two-step mechanism reveals that ADP-ribosylation reversal provides a temporal ordering to orchestrate MAR/PAR-regulated pathways. More generally, the different states of ADP-ribosylation and the proteins responding to them may help to sequence and coordinate related reactions and eventually decide cell fate  were the first class of compounds used to inhibit PARG. However, the influence of DNA intercalators on PARG activity is primarily indirect by restricting access of substrates rather than through direct interaction with PARG. Later, naturally occurring polyphenolic compounds such as the tannins were found to inhibit PARG activity133. In particular, Gallotannin134 was shown to inhibit PARG and to be synthetic lethal to BRCA2-deficient tumors135. However, the utility of this compound was subsequently called into question as it exhibits nonspecific effects and is essentially cell membrane impermeable79.Fueled by the success of PARP inhibition (PARPi) as a therapeutic strategy for the treatment of many cancers, the field is now exploring the therapeutic potential of PARG inhibition (PARGi). Probably inspired by the postulated nucleic acid-like helical conformation of PAR137. Photoincorporation studies with analogues of ADP-HPD showed that the high molecular weight and branched PAR bind PARG at a different sites than short, linear polymers and ADP-HPD138. This is consistent with the identification of a secondary substrate binding site on PARG, hypothesized to be involved in its processive behavior77. These results indicate that small-molecule inhibitors of PARG might have different effects on PAR processing by modulating its ratio of exo/endoglycosidic activities.Despite its lack of cell permeability, one of the most widely used and best characterized PARG inhibitors is adenosine diphosphate hydroxymethyl pyrrolidinediol (ADP-HPD)78. Rhodamine-based PARG inhibitors (RBPIs) proved to be potent and selective PARG inhibitors since they do not inhibit ARH3 as does ADP-HPD78. However, these compounds exhibited only low micromolar inhibitory activity against PARG. Other non-tannin inhibitors such as the GPI 1552 were reported to protect against neuronal damage139 and potentiate temolozomide anti-metastatic activity in brain tumours140. A careful reexamination of the actual evidence for PARG inhibition leads to the conclusion that GPI 1552 was inadequate for a pharmacological evaluation of PARG79.To overcome cellular permeability issues with PARG inhibitors, a new generation of compounds was developed by Hergenrother and colleagues141, Bryant and colleagues demonstrated that PARGi treatment selectively kills BRCA1-, BRCA2-, PALB2-, FAM175A/ABRAXAS-, and BARD1-depleted cells in the absence of any exogenous DNA damaging agents. The underlying mechanism for this synthetic lethality is that PARGi provokes replication forks stalling and a reduction of DNA double-strand break repair via homologous recombination (HR). An alternative explanation is that inhibition of PARG might cause irreversible PAR association with several proteins needed to complete HR142. Importantly, PARGi does not phenocopy PARPi. PARGi induces a rapid increase in IR-induced activation of DNA-PK and impairs normal mitotic progression. This suggests that PARG has different effects on activation of DNA damage repair pathways following ionizing radiation, consistent with the notion that blocking PAR removal has a different consequence to inhibiting PAR addition143.More recently, using the cell permeable PARGi PDD00017273144. ES cells derived from knock-out PARG mice showed enhanced sensitivity towards γ-irradiation and other forms of ionizing radiation145. More recently it was shown that PARG suppression potentiates the toxicity of radiation therapy in BRCA-deficient cells142 and that PDD00017273 radiosensitizes MCF-7 cells143.Genetic studies have suggested PARG inhibitors as chemosensitizing agents. PARG-deficient cells display centrosome amplification and accumulate aberrant mitotic figures, which induced either polyploidy or cell death by mitotic catastrophe146. In pancreatic ductal adenocarcinomas (PDA) cells, genetic deletion of HuR enhances PARPi sensitivity. In this context, the PARPi-induced toxicity is attributable to downregulation of PARG expression. The inhibition of HuR can also re-sensitize PDA cells to PARPi, suggesting that the loss of PARG activity could enhance the clinical effectiveness of PARP inhibitors146. In contrast, Gogola and colleagues have shown that PARPi-resistance can be mediated by PARG downregulation147. The loss of PARG activity in BRCA2-deficient tumours treated with potent PARP-1 inhibitors is sufficient to restore PAR formation and rescue PARP-1 downstream signaling. PARG depletion indeed occurs in triple-negative breast cancers and serous ovarian cancers. When treatment-naive TNBC biopsies from women eligible for PARPi treatment were analyzed for PARG expression, lack of PARG occurred in some areas of the tumours147. This suggests that these tumour sections can become de facto resistant to PARPi treatment. Further studies are needed to clarify the role of PARG and the accumulation of PAR polymers that survived erasing in a dynamic system that has undergone profound alterations. Differences in genetic backgrounds can certainly account for contradictory results  but the nature of PAR itself (either free or protein-bound) might also be an important and significant clue to interpret these results. Although there is a bright future for PARG inhibitors, so far they are only effective at relatively high doses in contrast to PARP-1 inhibitors characterized with half maximal inhibitory concentration in the low nanomolar range. In addition, the fact that PARG activity can be regulated at multiple levels with respect to PAR length and branching patterns poses a particular challenge.PARG protein expression can be regulated by the stabilization of its mRNA by the RNA-binding protein HuRIn this review, we have provided an overview of the dynamic and reversible processes that regulate ADP-ribosylation. Determining the contribution of each regulator in this delicate equilibrium represents a daunting challenge. Experimental dissection of these processes are complicated by the heterogeneous nature of ADP-ribosylation, which needs to be addressed by the development of specialized analytical methods.Substantial progress has been made to understand the mechanisms that contribute to ADP-ribosylation reversal, yet several obstacles need to be overcome: (1) Sensitive and reproducible methods to monitor physiological MAR and PAR levels in cells are difficult to implement; (2) better methods are needed to evaluate site-specific PAR chain length distribution in ADP-ribosylated substrates; (3) measuring kinetics and performance of erasers are precluded by the lack of standardized and defined substrates; (4) the ADP-ribosylation conjugation chemistry and linkage selectivity of erasers need to be further clarified; (5) enzyme-specific targets and interactors, especially for the MARTs and ARHs, remain largely unknown; (6) the modulatory effect of many ADP-ribose readers, which have multiple binding sites for the same ligand is largely unresolved; (7) the biological relevance of site-specific ADP-ribosylation events is often difficult to determine.Despite these obstacles and intricacies, the machinery responsible for the processing of ADP-ribose is now beginning to be revealed. A more detailed understanding of the interplay between ADP-ribosylation writers, readers and erasers, at a molecular level, will be required to correlate these dynamics with cellular responses and translate them into clinical applications.


Breast cancer (BC) is a global public health burden, constituting the highest cancer incidence in women worldwide. Connexin43 (Cx43) is a member of a family of transmembrane proteins responsible in part for intercellular communication between adjacent breast epithelial cells, via gap junctions. Cx43 plays key role in mammary gland development and differentiation and its spatio-temporal perturbation contributes to tumorigenesis. Thus, Cx43 acts as a breast tumor-suppressor. Signaling pathways and phenotypes downstream of Cx43 mRNA loss/mis-localization in breast cells have been well-studied. However, axes parallel to Cx43 loss are less understood. microRNAs (miRNAs) are small endogenous non-coding RNAs that repress translation and circularRNAs (circRNAs) are a class of endogenous RNAs that originate from RNA splicing and act as miRNA “sponges”. CircRNAs and miRNAs are dysregulated in cancers and are highly abundant and stable in the circulation. Thus, they present as attractive liquid biopsy cancer biomarkers. Here, an axis for Cx43 mRNA-circRNAs-miRNAs interactions along BC initiation  is proposed to potentially serve as a signature biomarker toward BC early-onset detection and prevention. BC registers the highest incidence and mortality rates in females and is the second most commonly diagnosed cancer (after lung cancer) . IncidenMammary gland development requires well-orchestrated cell-cell and cell-ECM communication by gap junctions and systemic signals. Connexins (Cxs) are a family of transmembrane proteins. They are responsible for establishing gap junction intercellular communication (GJIC), capable of linking cytoplasm of two neighboring cells, allowing intercellular exchange of ions, second messengers, and metabolites –13. EachRecently, we revealed an apicolateral distribution/localization of Cx43 in luminal human breast epithelium, and that loss of Cx43 expression contributes to breast tumorigenesis by disrupting apical polarity and promoting cell multi-layering, a hallmark of tumor initiation . FurtherI130T/+) exhibited delay in ductal elongation and atrophied glands pre-puberty (Cx32/+) heterozygous knock-in pups, due to perturbation in milk ejection . Silencing Cx43 activates signaling pathways that promote invasion in non-tumorigenic breast epithelium . Thus, these circRNAs are important promoters of carcinogenesis and may be useful biomarkers for BC . Nair etmiR-1271 , 81. TheAlthough there exists a correlation between obesity and loss of Cx43 apical distribution and cell multi-layering in breast epithelial tissues in an inflammatory micro-environment , 22, no Cx43 acts as a tumor suppressor, its loss/mis-localization is an important player in breast tumor initiation , plays rin situ (LCIS), a benign precursor lesion, to invasive breast lobular carcinoma (ILC) (To predict human circRNA isoforms that originate from linear Cx43 (GJA1) transcript, CircularRNA Interactome was used and three Cx43-derived circRNA isoforms  along with their sponged miRNAs were identified  Supplem. We propma (ILC) , 19. OveIn this review, we propose a possible biomarker signature axis of Cx43 mRNA-circRNAs-miRNAs in BC early-onset detection and prevention. We highlighted potential regulatory roles that Cx43-derived circulating circRNAs and their sponged miRNAs may play, which almost parallels the differential roles Cx43 plays along breast tumorigenesis. The Cx43 mRNA- “initiation circRNAs”-miRNAs axis is denoted by three “initiation circRNAs” and a panel of their sponged miRNAs (identified to date in the literature), miR-182, miR-375, miR-203, miR-520g, and miR-520h. This axis, when dysregulated in breast tissues, recapitulates phenotypes due to loss of Cx43 mRNA, associated with loss epithelial polarity and cell-multilayering during initiation stages of tumorigenesis  16–19).–19.16–19However, circRNAs and miRNAs present with few caveats that should be addressed. Interestingly, the proposed Cx43-derived circRNAs may circumvent them. First, miRNAs and circRNAs are highly expressed in circulating blood cells and their increased levels in blood might be due to high number of blood cells. Future studies thus should focus on defining actual abundance of circRNAs in different sub-populations of blood cells, characterize their mode of transportation in serum and plasma and devise markers that predict their origin . Cx43, hin silico analysis. RT and MA mentored NN throughout the writing process and critically revised all the drafts and approved the final version for submission.NN and RT assembled the relevant literature and proposed the axes. NN performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mice were treated with either vehicle (PBS), 5-Fluorouracil (5-FU), Oxaliplatin, or combination (FOLFOX). Antigen-specific cytotoxic T cell (tet+Tc) were detected using Db-CEA-tetramer obtained from the NIH-tetramer core facility. Flow cytometry was performed for phenotypic analysis and tetramer positivity. Tumor growth was measured using standard caliper measurements. Statistical analysis was performed using t-test for continuous variables and ANOVA was used when comparing multiple groups. Statistical analysis was performed using SPSS. All arms were completed with n=3–7. RESULTS/ANTICIPATED RESULTS: To determine how systemic treatment with chemotherapy affects cytotoxic T cell development (Tc), we established that we could detect antigen-specific Tc (tet+Tc) in the spleen, tumor, and draining lymph nodes of tumor-bearing mice. After establishing that the system worked appropriately, tumor-bearing mice were treated with different chemotherapy regimens and tumor growth was monitored. As expected, the combination of FOLFOX was significantly better than either drug individually . FOLFOX therapy also showed a significant (p<0.05) increase in the number of tumor-associated tet+Tc, and tet+Tc expressing phenotypic markers of effector (Te) and resident memory (Trm) subsets. Tumor-associated tet+Tc highly expressed PD-1 (>50%); however, this was not significantly different between treatment or vehicle arms. Since 5-FU, one component of FOLFOX has previously shown a selective reduction of myeloid-derived suppressor cells, we also investigated the myeloid compartment. There were no significant differences in conventional or plasmacytoid dendritic cells, myeloid-derived suppressor cells, or tumor-associated macrophages. DISCUSSION/SIGNIFICANCE OF IMPACT: The future of cancer care involves multi-modality care tailored to patients. To more effectively combine therapy it is critical that we understand how currently utilized therapy works. In this study, we show that the primary chemotherapy regimen utilized in colorectal cancer increases tumor-associated antigen-specific cytotoxic T cells and the majority of these cells are PD-1 positive. This suggests that FOLFOX may work in concert with immune-based therapy when selected appropriately. Further study is warranted to determine optimal combination therapy and ways to maximize anti-tumor immunity in order to improve the treatment of patients with this deadly disease.OBJECTIVES/SPECIFIC AIMS: Despite significant advances in screening and treatment, colorectal cancer is the second leading cancer killer in the United States today. Some of the most promising recent developments in cancer therapy have come from immune-based therapy. Immune-based therapy, however, has shown limited utility in patients with colorectal cancer. Studies have previously shown that certain chemotherapy regimens may be more effective in combination with immune-based therapy due to induction of inflammation in the tumor microenvironment. In this study, we sought to determine how standard chemotherapy (FOLFOX) affects the generation of antigen-specific anti-tumor immunity in colorectal cancer. METHODS/STUDY POPULATION: To determine the how antigen-specific immunity and T cell responses are affected by FOLFOX, we utilized a model antigen expressing murine colon cancer cell line syngeneic to C57BL/6 (MC38-CEA). Treatment was initiated when tumor size reached 50 mm


A 57-year-old woman was treated by cholecystectomy for gallbladder lithiasis. Coelioscopic surgery was uneventful and the patient was discharged the next day, but she came back to the hospital three days after surgery, with bilious fluid extruding from the right surgical orifice. Abdominal computed tomography (CT) showed a right subcutaneous fluid collection with a small biloma at the site of cholecystectomy (not shown). For direct identification of the biliary breach, Magnetic Resonance Imaging (MRI) with hepatobiliary-specific contrast agent (Gadoxetic acid – Primovist®) was performed. Only the hepatobiliary phase (i.e. one hour after intravenous injection of the MRI contrast at bedside) was performed using tridimensional fat-saturated T1-weighted imaging. Opacification of the biliary ducts was excellent and opacified bile was depicted in the peritoneum. Thick reformations (based on 1.5 mm thickness FS T1 acquisition) located the biliary leak at the proximal portion of the common biliary duct Figure . The MRIBiliary leak is a rare post-surgical or post-traumatic event. The presence of a collection can be identified by cross-sectional imaging. Bile content versus fluid collection of other nature is less easy to predicate. Identification of the exact location of the biliary breach is of great importance and often requires invasive retrograde cholangiography. MRI is the most reliable non-invasive technique for biliary imaging. Although conventional T2-weighted cholangiography provides anatomic information, the exact site of a bile breach is not usually identifiable. Delayed contrast-enhanced MRI with hepatobiliary-specific contrast agent adds the ability to evaluate the site of bile leak. Gadoxetic acid (Gd-EOB-DTPA), Primovist®, is a hepatobiliary-specific contrast agent taken up by functioning hepatocytes with nearly 70% biliary excretion. Because of its paramagnetic properties, this agent causes T1-shortening of the liver and biliary tree on delayed T1-weighted images. In addition, fluid collections, ascites, and fluid-containing structures that can obscure biliary tree depiction on conventional T2-weighted MRCP are inherently erased on T1-weighted sequences [MRI with hepatobiliary-specific contrast agent is a promising substitute to invasive cholangiography when a biliary leak is suspected. It allows definite demonstration of ongoing biliary leak, precise breach identification and ideal treatment planning.