The metabolism of cancer cells differs from that of their normal counterparts in a spectrum of attributes, including imbalances in diverse metabolic arms and pathways, metabolic plasticity and extent of adaptive responses, levels, and activities of metabolic enzymes and their upstream regulators and abnormal fluxes of metabolic intermediates and products. These attributes endow cancer cells with the ability to survive stressors of the tumor microenvironment and enable them to landscape and exploit the host terrain, thereby facilitating cancer progression and therapy resistance. Understanding the molecular and physiological principles of cancer metabolism is one of the key prerequisites for the development of better anticancer treatments. Therefore, various aspects of cancer metabolism were addressed at the 5th annual meeting of the International Society of Cancer Metabolism (ISCaM) in Bratislava, Slovakia, on October 17–20, 2018. The meeting presentations and discussions were traditionally focused on mechanistic, translational, and clinical characteristics of metabolism and pH control in cancer, at the level of molecular pathways, cells, tissues, and organisms. In order to reflect major healthcare challenges of the current era, ISCaM has extended its scope to metabolic disorders contributing to cancer, as well as to opportunities for their prevention, intervention, and therapeutic targeting. Research on cancer metabolism attracts an increasing interest, not only because of efforts to better understand principles of cancer progression but equally because of the need to bring new ideas and strategies for more effective anticancer therapies. Due to the enormous metabolic plasticity of cancer cells responding and adapting to signals from intracellular and extracellular milieu, this research field is very complex and rapidly evolving. To address challenges of this dynamic research area, the International Society of Cancer Metabolism (ISCaM) was founded in 2014 as a logical expansion of the International Society for Proton Dynamics in Cancer (ISPDC) that operated from 2009 to 2014. ISCaM's scope is to foster communication and collaboration between scientists engaged in research of metabolism, acidity, proton dynamics, and microenvironment in cancer  reported on new targetable aspects of cancer metabolism. He focused on mechanisms of cellular adaptation to glutamine deficiency, which is one of the key amino acids in cancer metabolism, and described a new pathway of metabolic reprogramming and regulating amino acid uptake in conditions of hypoxia-associated low glutamine.Modern society generates a stressful and obesogenic environment promoting the development of metabolic disorders that contribute to many chronic diseases, including cancer. In the initial talk of this session, Stephan Herzig  explained the role of lipid metabolism in metabolic dysfunction-related cancer. He emphasized that obesity-associated complications do not only increase tumor risk but also promote metastatic tumor recurrence, thereby imposing a major threat on affected patients. In turn, end-stage tumor diseases have a significant impact on systemic metabolism, in particular causing body wasting, i.e., cachexia. He discussed the latest findings on key events and alterations of lipid metabolism relevant for both obesity-triggered metastasis formation and cancer cachexia, including their therapeutic aspects. Jozef Ukropec  then briefly reviewed data on the metabolic and functional flexibility of the adipose tissue, which largely determines the metabolic health of humans living in the obesogenic environment. Next, he described the skeletal muscle as an endocrine organ providing a spectrum of bioactive substances to orchestrate the whole-body adaptive response to exercise in recently finalized intervention studies. He also presented the design of a currently ongoing study aimed at investigating benefits of regular exercise in testicular germ cell cancer survivors. Elisabeth Wyart  focused on cachexia, which is a multi-organ wasting syndrome characterized by irreversible skeletal muscle atrophy that dramatically increases both morbidity and mortality in various diseases. Despite its high prevalence in cancer patients, the mechanism of cancer-induced cachexia remains unclear. So far, unpublished findings obtained by investigating cachectic mice bearing CT26-colon cancer suggest striking alterations in certain regulators of metabolism. Sofia Avnet  provided a thorough overview of the complex relationships between systemic/local acidosis and insulin resistance/signaling. Insulin influences acidosis by promoting glycolysis and, in turn, acidosis lowers insulin binding to its receptor, inhibits glycolysis, inhibits the recycling of glucose transporters and reduces insulin secretion by pancreatic β-cells via transforming growth factor β. Insulin also affects ion/proton transporters and pH sensors, and resulting acidosis may enhance insulin resistance through the NF-κB-driven inflammatory pathway. Eduardo Maldonado  presented the current view on the modulation of mitochondrial metabolism by statins in cancer cells, and suggested that it is not mediated by changes in cholesterol content and does not affect glycolysis. Overall, insights on the relationships between metabolic disorders and cancer provided in this session support the importance of continuing investigations on this topic, which is highly relevant for improving the lifestyle and healthcare of human populations.min mice, a model prone to intestinal and mammary cancer due to a non-sense mutation in an allele of the adenomatous polyposis coli (APC) gene, loss of NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) resulted in histone hyperacetylation, increased the abundance of intestinal stem cells (ISCs) and enhanced the tumor-initiating potential. Due to high pyruvate dehydrogenase kinase (PDK) activity, ISCs were highly glycolytic and had low ROS production, supporting stemness. After transformation, they were proposed to be at the origin of a Warburg-phenotype lineage of cancer cells in established intestinal tumors. Relationships between cancer cell metabolism and epigenetics were further highlighted by Thomas Grunt , who proposed systems biology as the best approach to decipher and target these networks. For example, he showed that fatty acid synthase (FASN) not only controls lipogenesis, but also the cell membrane structure, hence signaling, and epigenetics. Similar to FASN, other enzymes, such as the acetyl-CoA carboxylase ACC1/ACC2 system, are at the crossroad between several metabolic and functional pathways in cancer cells. Precisely identifying these nodal points may be used for future co-targeting anticancer strategies.In this session, the first presentation by Carlos Sebastian  focused on the epigenetic control of tumor transformation in the intestinal tract. In APCin vivo. Paolo Ceppi  then explained how glucose metabolism controls the epithelial-to-mesenchymal transition (EMT), a de-differentiation process used by cancer cells to acquire invasive and resistance properties. He identified that enzymes belonging to the polyol pathway (PP), aldo-keto reductase family 1 member B1 (AKR1B1), and sorbitol dehydrogenase, promote EMT and cell growth. The expression of these enzymes was correlated with poor patient survival. Bárbara Sousa  described the control of breast cancer metabolism by the cell-cell adhesion molecule P-cadherin. She demonstrated that P-cadherin promotes glycolysis and reduces oxidative stress, suggesting an important role in anoikis resistance and metastasis. Altogether, these insights illustrated the crucial role of metabolic plasticity in cancer metastasis providing new opportunities of targeting.Metastasis remains a factor of poor prognosis for cancer patients and the main cause of death by cancer for which there is currently no specific treatment. In this session, speakers highlighted the key role of metabolic plasticity in controlling metastatic dissemination from the primary tumor to colonization of distant organs. Pierre Sonveaux  focused on monocarboxylate transporter 1 (MCT1), a proton/lactate cotransporter, that could contribute to the metastatic process and, therefore, constitutes an interesting target for metastatic prevention. He reported that MCT1 activates transcription factor NF-κB, which promotes cancer cell migration independently of MCT1 activity as a transporter. Unlike MCT1 activity inhibition, the downregulation of MCT1 expression inhibited migration, invasion and spontaneous metastasis in vitro. A major issue in assessing tumor metabolic heterogeneity is the limiting availability of tools to image metabolism in vivo. The talk of Valery Khramtsov  focused on the use of low-field electron paramagnetic resonance (EPR)-based techniques, in combination with specially designed functional paramagnetic probes, to measure various parameters such as oxygenation, pH, and inorganic phosphate availability, providing interesting observations on the correlation of specific nutrient availability with tumor aggressiveness.Tumors are known to be highly glycolytic, with lactate concentrations up to 40 mM in the tumor microenvironment. Hence, tumor metabolism is often considered as a homogeneous entity, with tumor relying only on aerobic glycolysis to survive and grow. This view is outdated as we are progressively acknowledging the impact of tumor heterogeneity on shaping metabolic fluxes. In particular, we are beginning to elucidate the network of “social” interaction between tumor cell populations, ranging from metabolic symbiosis to metabolic competition, based on the metabolic requirements of the ongoing biological activities and nutrient availability. The session dedicated to this topic provided several interesting points of discussion concerning tumor heterogeneity and metabolic crosstalks. For instance, Maria Yuneva  discussed her work on metabolic reprogramming controlled by the MYC oncogene, highlighting the metabolic flexibility of tumors and defining potential vulnerabilities. Then, Maria Shoshan  presented her data investigating the impact of metabolic flexibility on cisplatin resistance. On a similar topic, the work of Giuseppe Taurino  concerned the importance of amino acid transport and stromal cooperation in counteracting nutritional stress in asparaginase-treated acute lymphoblastic anemia, thus evidencing the importance of such metabolic crosstalk in supporting cancer fitness. Tumor metabolic heterogeneity is not only shaped by cellular and biological heterogeneity, but it is also promoted by nutrient availability due to inefficient perfusion. To model this, Angela Otto  presented her work concerning metabolic reprogramming under nutrient (glucose and glutamine) limitation in vivo the intratumoral pH by using MRI-based approaches. This innovative technology will be crucial to measure intratumoral pH in real-time, both for diagnostic and prognostic purpose, as well as for the monitoring of treatment response, like for the evaluation of the cytotoxic effect of anticancer drugs that target proton pumps/transporters. Among these drugs, one example has been proposed by Ivana Novak  in pancreatic cancer that expresses both gastric and non-gastric H+/K+- ATPases. In this tumor type, H+/K+- ATPases contribute to secretin-stimulated secretion and participate in intracellular pH regulation. Their inhibition by using either proton pump inhibitors (PPI) or potassium-competitive blockers reduced tumor growth and dissemination. Similarly, as discussed by Stine Falsig Pedersen  in luminal breast carcinoma cells, targeting the Na+/H+ exchanger NHE1 and the Na+/This session was focused on intratumoral acidosis and its effect on cancer biology. Extracellular acidosis is mainly due to an enhancement of tumor-associated glycolytic activity also in conditions of normal oxygen supply (Warburg effect), and it is now considered to be a hallmark of cancer. As described by Dario Longo , it is currently possible to directly measure in vivo processes where immune cells determine the fate of growing or regenerating tissues. In the opening talk, Massimiliano Mazzone  showed intriguing data on how macrophages and skeletal muscle satellite cells compete for glutamine in the tissue environment. He provided evidence for this competition by showing that genetic deletion and pharmacologic targeting of glutamate dehydrogenase 1 (GLUD1) in macrophages leads to the rerouting of glutamate to glutamine synthesis and secretion supported by glutaminase 1 (GLS1) upregulation, which in turn sustains satellite cell activation, proliferation and differentiation, altogether accelerating muscle regeneration and functional recovery. In the following talks, two tumor-specific examples of metabolic pathways participating in immune-cancer cell crosstalk were presented by Stefano Biffo  and Maša Ždralević . First, Stefano Biffo has shown that a unique translational control of lipid synthesis enzymes (including ACC1) via eIF6 and eIF4E, operates in both cancer cells and immune responses, providing a potential adaptive framework for tumor evolution. Maša Ždralević presented further details on the mechanism of lactate secretion by cancer cells using cellular models with LDH-A/B depletion. She showed that knockout of both enzymes leads to re-activation of OXPHOS, which is sufficient to maintain tumor growth and renders tumor cells uniquely sensitive to OXPHOS inhibition. This raises the possibility that the primary role of lactate production by cancer cells is to provide signals to the tumor environment, including immune cells, as previously suggested. The closing talk by Mikhail M. Dikov  reported exciting technological advances to assess these signals by profiling the tumor and lymph node metabolic microenvironment using EPR spectroscopy with a newly generated set of paramagnetic probes.Interactions between cancer cells and their microenvironment are now recognized as major players in tumorigenesis, tumor growth, and shaping the evolutionary path of many if not all cancers. A large fraction of tumors display infiltration by immune cells, and intense research has been initiated to deconvolute the role of the immune system in the process. These interactions are, however, not unique to tumors, and insights in cancer metabolism and immune responses can be achieved by studying other in vivo studies of pancreatic ductal adenocarcinoma (PDAC), revealed that growth-suppressive effects of CR in the form of rapamycin or IGF-1R/insulin receptor inhibition were strongly potentiated by co-treatment with chloroquine-related compounds to inhibit autophagy. There is, however, a serious lack of specific anti-autophagy treatments suitable for clinical use. Following up on this theme, Angelo de Milito  presented data on the development and efficacy of inhibitors of class III phosphatidyl-3-kinase Vps34. Vps34 inhibition would be particularly powerful in combination with treatment schemes that elicit upregulation of autophagy. The team found that cytotoxicity of receptor tyrosine kinase inhibitors Sunitinib and Erlotinib in breast cancer cell lines was potentiated by Vps34, consistent with upregulation of autophagy by these treatments. Finally, Margherita Cortini  enlightened the crucial role of lipid metabolism in determining the aggressiveness of osteosarcoma in response to extracellular acidosis, with particular regard to the sphingolipid pathway.Acquired treatment resistance is a predominant cause of treatment failure and death of cancer patients. Recent investigations showed that acquired resistance often reflects metabolic pathway rewiring. This session focused on novel treatment schemes co-targeting metabolic pathways upregulated as a consequence of treatment or tumor microenvironment conditions. Miguel Quintela-Fandino  presented results from transcriptomic, phosphoproteomic and metabolomics studies exploring the combination of anti-angiogenic treatments with inhibitors of mitochondrial respiration in mouse models of lung and breast cancers. The group showed that small molecule anti-angiogenics led to vascular normalization associated with a metabolic shift toward OXPHOS, and that combination with e.g., metformin could induce metabolic synthetic lethality. On the other hand, monoclonal antibody anti-angiogenics exacerbated blood-vessel abnormality and hypoxia. This was associated with upregulation of a tumor-immune-suppressive phenotype, rendering such treatment synergistic with e.g., anti-PD-L1 treatment. Steven Hurstings  combined a calorie restriction-mimetic diet (CR) with inhibition of autophagy, which is upregulated as a consequence of CR. Cell culture, as well as The somatic evolution of cancers leading to their invasive behaviors includes metabolic rewiring that endows cancer cells with the ability to survive and proliferate in a highly selective microenvironment of hypoxia, acidosis, and severe nutrient restriction. Over many months or years in this environment, cells emerge with more adaptable, aggressive and de-differentiated phenotypes. An important component of such phenotypes is aerobic glycolysis, known as Warburg effect. In the closing lecture, Robert J. Gillies  discussed the nature of the microenvironmental conditions and mechanisms that select for the glycolytic phenotype, highlighting work using an approach based on exposure of non-metastatic/non-glycolytic cancer cells to microenvironmental selection including acidity, hypoxia and low glucose followed by regrowth in normal conditions. Resulting clones exhibited increased rates of aerobic glycolysis and upregulation of a number of cancer-related genes. This approach explains why and how cells acquire a glycolytic phenotype in early cancers, which is a hallmark of aggressiveness and poor outcome.ISCaM aims to promote interactions between basic and clinical researchers and between young and senior scientists, which was facilitated by the international composition of the meeting (80 attendees representing 14 countries), vivid discussions in the audience and beyond, and social activities. Oral and poster communications were demonstrated in sessions focusing on emerging aspects of cancer metabolism that were co-chaired by a principal and by a young investigator. Three young investigators  received travel grants from the Local Organizers. Prizes were awarded to young investigators who delivered best scientific presentations: the first ISCaM award for best oral presentation went to Elisabeth Wyart (her topic is highlighted above) and the second ISCaM award for the best poster presentation went to Joao Santiago de Jesus  for his study of acidosis-induced lipid metabolism in cancer invasiveness. In line with ISCaM's objective to improve the outcome of cancer patients through the discovery and development of new anticancer drugs, several potential anticancer strategies related to tumor metabolism were presented.https://www.iscams.org/meeting/iscam2019-6th-annual-meeting-braga).Conclusively, ISCaM2018 provided the audience with most recent discoveries on metabolic adaptations and targets in cancer. The follow up discussions will be held at the 6th annual ISCaM2019 meeting in Braga, Portugal, 19–21 October 2019, entitled “Cancer Metabolic Rewiring: Mapping the Road to Clinical Translations” .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Survivors of stroke often experience significant disability and impaired quality of life. The recovery of motor or cognitive function requires long periods. Neuroimaging could measure changes in the brain and monitor recovery process in order to offer timely treatment and assess the effects of therapy. A non-invasive neuroimaging technique near-infrared spectroscopy (NIRS) with its ambulatory, portable, low-cost nature without fixation of subjects has attracted extensive attention.Methods: We conducted a comprehensive literature review in order to review the use of NIRS in stroke or post-stroke patients in July 2018. NCBI Pubmed database, EMBASE database, Cochrane Library and ScienceDirect database were searched.Results: Overall, we reviewed 66 papers. NIRS has a wide range of application, including in monitoring upper limb, lower limb recovery, motor learning, cortical function recovery, cerebral hemodynamic changes, cerebral oxygenation, as well as in therapeutic method, clinical researches, and evaluation of the risk for stroke.Conclusions: This study provides a preliminary evidence of the application of NIRS in stroke patients as a monitoring, therapeutic, and research tool. Further studies could give more emphasize on the combination of NIRS with other techniques and its utility in the prevention of stroke. Stroke, which refers to a medical condition in which insufficient brain blood supply results in cell death, is a major cause of death and disability worldwide , 2. SurvNeuroimaging has been shown to be an effective monitoring and therapeutic tool, evaluating the evolution of neural activity and stroke rehabilitation and recovery . TraditiNear-infrared spectroscopy (NIRS), introduced in 1977 by Jöbsis et al. as a monitoring tool of cerebral and myocardial oxygenation .NIRS can be divided into continuous wave NIRS (CW NIRS), time domain NIRS (TD NIRS), and frequency domain NIRS (FD NIRS). CW NIRS emits continuous wave and measures the changes in the intensity of the light that passed through the tissue, whereas TD NIRS utilizes a short pulse of laser light and measures the arrival times of photons emerging from the tissue. FD NIRS records the intensity of the detected light as well as the phase shift. These signals can then be converted to the concentration of oxygenated (oxy-Hb) and deoxygenated hemoglobin (deoxy-Hb). One of the most common used algorithm is Modified Beer–Lambert Law (MBLL). CW NIRS could not measure absolute concentrations of oxy-Hb and deoxy-Hb, because this method assumes a homogenous tissue which is not true. This does not change the results of qualitative analysis, but may lead to error in quantitative outcome. TD NIRS recording the temporal broadening of the pulse as it penetrates the investigated area allows accurate quantification of the concentrations and has better spatial resolution . Neural Stroke includes two major types: ischemic, due to lack of cerebral blood flow, and hemorrhagic, due to bleeding. During ischemic stroke cerebral blood supply is disrupted by narrowing of vessels caused by atherosclerosis or embolism. In hemorrhagic stroke caused by hypertension or ruptured aneurysm blood flow is reduced due to direct blood loss or vessel compression. In either case, a significant decline in blood supply could be observed. The reduction in cerebral oxygenation results in injury of neurovascular unit, decreased neuronal activity and accumulation of anaerobic metabolites. NIRS could monitor oxygenation signal changes, thus reflecting this pathophysiological process .NIRS is well-established as a safe and effective monitoring tool for stroke recovery, including upper limb, lower limb recovery, motor learning, cortical function recovery , cerebraTo evaluate how NIRS has been employed for surveillance and treatment of stroke patients, we conducted a systematic literature review of all published original research involving NIRS in stroke patients. The literature search, conducted in July 2018, began with an initial search of the NCBI Pubmed database using the following search terms: “ AND (stroke[MeSH Major Topic] OR “cerebrovascular disorders”[MeSH Major Topic] OR “brain infarction”[MeSH Major Topic])”. Additional spot check searches stemming from key references in identified works were also carried out to further strengthen the literature review's reliability. Only English language articles published in peer-reviewed journals were included. Then we searched EMBASE database, Cochrane Library and ScienceDirect database . All stuWe identified 14 papers –37 reporFive papers –28 reporWe found six papers –34 descrBalance problem is one of the major sequelae of stroke. Two papers , 36 focuMotor learning is essential in function recovery, including motor adaptation, and motor sequence learning. Motor adaptation refers to the ability to compensate for environmental changes such as external force. Motor sequence learning is defined as learning of sequential motor actions, which is essential for activities of daily living. Impaired motor sequence learning may be correlated with reduced rehabilitation gains for ataxic patients with stroke .The cortical functions including cortical plasticity, functional connectivity or cerebral autoregulation that could be reflected based on oxygenation levels are directly related to motor and cognitive recovery after stroke. We reviewed 10 papers –47 reporTwo studies have reported significant improvement in task performance or cortical plasticity after constraint-induced movement therapy , 41. In Post-stroke patients exhibit changes in cerebral autoregulation (CA) which could be assessed by recording spontaneous oscillations in mean arterial blood pressure and cerebral blood flow. The etiology of oscillations remained unknown, however, its significance in reflecting CA has been demonstrated. NIRS with a high time resolution is suggested as a promising tool for measuring oscillations, identifying risk and evaluating treatment strategies in patients with carotid artery disease and ischemic stroke , 43.Mori et al. investigated dual-task interference on physical and cognitive performance in stroke patients. In consistent with previous studies, during dual-task walking, PFC activation might prioritize physical demands in stroke patients, but might prioritize cognitive demands in healthy subjects . AnotherFourteen papers –61 focusNIRS could be used in intensive care unit as a neuromonitoring tool, reflecting blood flow signals , 55 as wSpontaneous activity in prefrontal cerebral oxygenations has been demonstrated to be disturbed in the elderly and in patients with cerebral infarction (CI), which could be analyzed by continuous recording based on the wavelet transform of NIRS signals, could reflect resting state functional connectivity . Recent Hemodynamic changes recorded by NIRS could also provide critical information for other techniques. A method for EEG-NIRS based assessment of neurovascular coupling (NVC) during anodal transcranial direct current stimulation (tDCS) was proposed in case series of four chronic ischemic stroke survivors . Cooper We reviewed twelve –73 paperOximetry by NIRS evaluates oxygenation changes in stroke patients  and the One of the most important predictors of clinical outcome after acute ischemic stroke is recanalization. Endovascular treatment is one of major therapies of stroke patients. NIRS may be useful to monitor oxygenation during management of acute ischemic stroke . NakamurCerebral hyperperfusion syndrome (CHS) was described in a case report of a 77-year-old man with acute cerebral infarction, which is known to be a rare but devastating complication of carotid artery revascularization. The patient underwent successful recanalization with thrombectomy by using a stent retriever for the middle cerebral artery and stent placement for the origin of the internal carotid artery. However, 12 h after treatment acute intracranial hemorrhage happened, which was considered to have been caused by CHS under the evaluation of NIRS. This case emphasized the significance of performing routine monitoring of regional cerebral oxygen saturation by NIRS to timely recognize CHS .NIRS may be more useful in combination with other techniques. Vernieri et al. combined NIRS with transcranial Doppler to investigate the effects of hypercapnia on healthy individuals and patients of ischemic stroke . The resDespite several efficient therapies, neurorehabilitation is an intricate process requiring time and numerous factors. Given this circumstance, BCI which use brain activity to control external devices may be the only solution for patients with severe function deficiency, failing to interact with the environment and sustain normal daily lives.BCI can be divided into two types, assistive BCI and rehabilitative BCI system. The assistive BCI system aims to substitute lost functions, for example, to achieve high-dimensional movement control of robotic devices. In contrast, the rehabilitative BCI system aims to facilitate the restoration of brain function by normalizing the neurophysiologic activity . The patNIRS is a relatively new measurement modality that offers a portable, low-cost, sensitive option for BCI development. The idea of using NIRS as an optical BCI has been introduced by Coyle in 2004 . Since tIn addition, NIRS was used in neurofeedback. Neurofeedback is a type of biofeedback that measures brain waves to produce a signal that can be used as feedback to modulate brain function voluntarily by the patients themselves. Real-time feedback of neural activity to the subjects may modulate plastic reorganization without external stimulation of the brain. NIRS, as a neurofeedback tool detecting blood flow signals, could improve cortical plasticity and enhance the therapeutic effect of motor imagery. In a pilot study twenty hemiplegic patients with subcortical stroke were analyzed and the results suggested that NIRS-mediated neurofeedback may enhance the efficacy of mental practice with motor imagery and augment motor recovery .Apart from applications above, NIRS has been used in evaluation of the risk for stroke, clinical studies and monitoring muscle metabolic indexes , 7.Continuously monitoring oxygenation levels by NIRS during operation could diagnose and predict the occurrence of perioperative stroke. In a study of 46 patients underwent selective antegrade cerebral perfusion of the right subclavian artery, decreased regional cerebral tissue oxygen saturation was observed. Monitoring of regional cerebral oxygenation by NIRS allows detection of clinically important cerebral desaturation, and could predict perioperative neurologic sequelae . Two stuStudies have also evaluated the risk for stroke in other diseases. We reviewed three papers. Viola et al. assessed hemodynamic changes in migraine patients. The results showed a mild vasoconstriction, which may be the possible explanation of the association between migraine and an increased risk for ischemic stroke . The spoIn addition, NIRS presents as an effective research tool in clinical studies. The concentration of oxy-Hb and deoxy-Hb obtained by NIRS could be measured, calculated and analyzed, evaluating hemodynamic, oxygenation changes and cortical function, thus widely used in assessing the effects of different therapies or the evolution of diseases , 21. FerNIRS, a non-invasive neuroimaging technique with the advantages of low price, simplicity, portability and small devices, has a wide range of utility . The exiNIRS is applied in healthy population at first and then used in stroke subjects. By monitoring of NIRS could reflect the function recovery and the therapeutic effect in patients; compared with normal people, it can be used to study the pathophysiological mechanism of disease and predict the risk, serving as an effective monitoring and research tool. In addition, it can be used as therapeutic tool combined with BCI and neurofeedback, but the current studies mainly limited in healthy people, however, the results have shown potential value, requiring further investigations in stroke patients.Despite its non-invasive, portable and ambulatory nature, its accuracy is influenced by several factors, thus limiting its further application. fNIRS detects neurovascular coupling and its accuracy may be impaired by extracerebral and systemic changes such as blood pressure and concentration of CO2 , 99. ThiConsidering the inevitable shortcomings, one promising prospect is the combination of different neuroimaging techniques, including diffusion-tensor imaging (DTI), magnetic resonance spectroscopy (MRS), ligand-based positron emission tomography (PET), single-photon emission computed tomography (SPECT), functional magnetic resonance imaging (fMRI), NIRS, and EEG. Conjugated with other techniques, the surveillance might be more comprehensive and accurate. Moreover, at present most studies have focused on its application in stroke recovery, its ability to assess the occurrence of stroke, for example, during operation and in people with higher risk, is of great significance. Further researches could emphasize NIRS in the prevention of stroke as well as stroke recovery and prognosis.NIRS could also used in combination with neurostimulation tools. Medical communities have been interested in electrical stimulation for a long time. Recently transcranial direct current stimulation (tDCS), which was firstly used in humans in 2,000 by Nitsche et al. , has shoPW contributed to the conception of the study. MY contributed significantly to manuscript preparation and wrote the manuscript. ZY, TY, and WF revised the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


International disparities in outcomes from pediatric solid tumors remain striking. Herein, we review the current literature regarding management, outcomes, and barriers to care for pediatric solid tumors in low- and middle-income countries (LMICs). In sub-Saharan Africa, Wilms Tumor represents the most commonly encountered solid tumor of childhood and has been the primary target of recent efforts to improve outcomes in low-resource settings. Aggressive and treatment-resistant tumor biology may play a role in poor outcomes within certain populations, but socioeconomic barriers remain the principal drivers of preventable mortality. Management protocols that include measures to address socioeconomic barriers have demonstrated early success in reducing abandonment of therapy. Further work is required to improve infrastructure and general pediatric care to address disparities. Disparities in outcomes from pediatric solid tumors remain striking. In high-income countries (HICs), several multi-institutional collaborations, including the International Society of Pediatric Oncology (SIOP) and the National Wilms Tumor Study Group  have succeeded in raising overall survival for Wilms Tumor (WT) to greater than 90% at five years ,2,3. In This review seeks to report available literature regarding the burden of disease, management, and outcomes from pediatric solid cancers in low-resource settings, as well as published evidence regarding barriers to care.Medline and Cochrane database searches were performed in July 2018. Search terms included “Wilms tumor, Africa”, and “pediatric tumor, Africa.” The reference lists of articles identified using these search terms were also reviewed. Studies were selected based on the relevance to burden of disease, management, and outcomes of pediatric solid tumors in LMICs.Most published literature on the burden of disease and outcomes from pediatric solid tumors in LMICs were derived from sub-Saharan Africa (SSA) where disparities are alarming. In a recent review of cancer registries in selected major urban centers in Zimbabwe, Uganda, and Kenya, WT was the most common solid tumor identified in children and third most common pediatric cancer after leukemia and lymphoma. Survival at one year was only 61%, 39%, and 66% at each respective site . Five-yeHereditary predisposition and biologic behavior of tumors may contribute to disparate outcomes across ethnic groups. Over-representation of certain Kenyan tribes among patients treated for WT suggests increased incidence within specific populations . Using aTP53 mutation and MYCN alteration [Inadequate access to and abandonment from care remain the likely principal drivers of mortality . Effortsteration . Accuratteration .Completion of therapy for WT in LMICs remains a significant challenge. A review of outcomes from eight referral centers in SSA found treatment abandonment rates ranging from 14–48% . Root caBarriers to care can be reduced through social programs. Excellent evidence exists that health insurance reduces mortality for children burdened with cancer in LMICs. For example, in Kenya, children with WT who were enrolled in the National Hospital Insurance Fund were more likely to initiate therapy, complete preoperative chemotherapy, undergo operative resection, complete post-operative chemotherapy, and finish radiation therapy . Health Two strategies for the multidisciplinary treatment of WT have been employed yielding comparable results in HICs. The COG in North America practices operative resection of renal tumors followed by adjuvant chemotherapy for certain risk groups. In Europe, the SIOP initiates preoperative chemotherapy of all renal tumors without biopsy, which potentially can downstage the tumor at surgery and reduce tumor rupture, intensity of postoperative chemotherapy, and need for radiotherapy . Both apRecent years have seen renewed efforts to standardize treatment protocols for management of WT in LMICs. The SIOP Paediatric Oncology in Developing Countries (PODC) committee published clinical guidelines for management and supportive care of WT patients in low-income settings, which outline the requirements for therapy with curative intent ,32. ThesWT represents the most common and intervenable solid tumor of childhood and so has been the target of the greatest efforts to improve outcomes in LMICs given its profound disparity with industrialized countries. Socioeconomic factors among patients and families that limit WT treatment completion are likely similar among other pediatric solid tumor patients. SIOP-PODC guidelines for management of neuroblastoma and retinoblastoma in LMICs are now also available ,35. A reRecent literature indicates that mortality rates for pediatric solid tumors in LMICs remain unacceptably high due to complex interactions between social and economic status, access to care, race and ethnicity, health literacy, and tumor biology. Collectively, late presentation with high risk for treatment abandonment, on-therapy mortality due to deficiencies in supportive care, and aggressive and treatment-resistant biology remain the biggest factors driving mortality in poor settings. Despite persistent challenges, there are great gains to be made in this field. When working with a young population of patients, many of whom have eminently treatable disease, the impact of systemic improvements in care in terms of disability-adjusted life years and quality-adjusted life years may be profound .Since socioeconomic barriers are major obstacles to successfully completing therapy for pediatric solid tumors in LMICs, we are encouraged to see improved outcomes when these obstacles are specifically addressed. The success of eight SSA centers to reduce abandonment of treatment through implementation of PODC guidelines, including financial and social support for poor families, represents a key step towards reducing widespread disparities. Emphasis and assistance for families to enroll in national health insurance plans has proven benefits on survival at least from WT. However, much work remains to be completed in expanding access to this type of support and providing scalable, sustained, multi-disciplinary prevention of treatment abandonment.To overcome this burden and major cancer health disparity in LMIC settings, our group has emphasized the professional training of native, in-country pediatric surgeons, anesthesiologists and anesthetists, oncologists, intensivists, radiologists, and pathologists. Indeed, we have begun to witness a growing number of these health care providers assume increasing responsibility and independence in the care of the often-complex pediatric cancer patients See . MoreoveAttempts to clarify population-specific tumor biology have revealed unique molecular profiles that, with additional work, could yield novel druggable targets and optimized, more precise care schemes in low-resource settings. Recent research suggests that population-specific genetic variations may increase risk for adverse drug reactions to commonly-prescribed antineoplastic agents, and contribute to difficulty completing therapy in particular settings . ProgramLimitations of this review include the heterogeneous nature of populations within the referenced studies. It is important to note the vast diversity in socioeconomic and medical environments included within the umbrella terms of “LMICs” or “low-resource settings.” Much of the available literature stems from referral centers in urban settings in SSA. The full extent of geographic obstacles to care felt by rural populations may be poorly represented. This review focuses on WT as a pediatric solid tumor that has been proven in HICs and even in certain LMIC settings to be highly intervenable. The available evidence suggests that overall outcomes for other solid tumors of childhood in LMICs remain dismal.Efforts to improve oncologic outcomes in LMICs may be hampered by competing health priorities. Prior authors have noted the need for interventions that address oncologic disparities while strengthening infrastructure for general pediatric care ,45. As o


Iron dyshomeostasis can cause neuronal damage to iron-sensitive brain regions. Neurodegeneration with brain iron accumulation reflects a group of disorders caused by iron overload in the basal ganglia. High iron levels and iron related pathogenic triggers have also been implicated in sporadic neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple system atrophy (MSA). Iron-induced dyshomeostasis within vulnerable brain regions is still insufficiently understood. Here, we summarize the modes of action by which iron might act as primary or secondary disease trigger in neurodegenerative disorders. In addition, available treatment options targeting brain iron dysregulation and the use of iron as biomarker in prodromal stages are critically discussed to address the question of cause or consequence. Iron is involved in an abundant number of cellular processes in the brain including mitochondrial respiration, myelin synthesis, DNA synthesis, oxygen transportation, neurotransmitter synthesis and cellular metabolism . In the Within the cell, iron mediates essential functions due to its capability to participate in reaction of electron transfer thereby switching between two states: ferrous (II) and ferric (III) iron. This mechanism represents a double-edge sword because distinct levels of reactive oxygen species (ROS) are produced during these reactions enabling calcium-mediated basal synaptic transmission and long-term potentiation , but on Iron levels in the brain and body increase sharply up to 30 years of age due to a metabolic need during the growth process and remain stable during adulthood . In the Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies, amyotrophic lateral sclerosis, Huntington’s disease (HD), frontotemporal dementia, corticobasal degeneration, and progressive supranuclear palsy (PSP) are primarily characterized by the deposition of insoluble protein aggregates which colocalize with iron , suggestIn this review we aim to outline the potential links between pathophysiological mechanisms and the role of iron in neurodegeneration.Both excess and deficiency of iron lead to impaired brain function, thus tight regulation of iron metabolism is critical.in vitro  barrier , or possin vitro . DMT1, ain vitro . How iroin vitro . Within in vitro  and tranin vitro .Iron, prone to contribute to oxidative stress, can be (i) stored within ferritin , (ii) imIron accumulates as a function of the aging brain and thereby the levels of labile, potentially harmful iron increase . Iron acThe underlying mechanisms of age and disease related region and cell specific differences in iron levels have not been fully elucidated yet, but the distinct iron richness of the basal ganglia may explain its increased vulnerability to neurodegeneration . In neurAstro- and microglia exhibit increased iron deposition and ferritin concentrations throughout their lifespan. In contrast, oligodendrocytes that constitute the main iron reservoir in the CNS  do not aIron accumulation as a hallmark feature has been confirmed in many neurodegenerative diseases. Mitochondria provide neurons and glia with energy in the form of ATP via oxidative phosphorylation of glucose. Functional disruption of this pathway is likely to contribute to neurodegeneration. In PD, environmental toxins that disrupt mitochondrial complex I function like rotenone induce parkinsonism . Also, gNeurodegeneration with brain iron accumulation comprises a number of pediatric and adult neurological diseases characterized by parkinsonism and dementia, and excessive iron accumulation in the basal ganglia among other regions . DistincDrosophila model of Friedreich’s ataxia, upregulation of mitoferrin associated with frataxin deficiency is observed. Interestingly, targeted downregulation of mitoferrin ameliorates the phenotype while worsening overall life span, indicating that due to its physiological function dysregulation of mitoferrin has detrimental consequences  forms a reversible interaction through threonine residues, Fe (III) induces an irreversible conformational change, both of which promote aggregation of tau . HyperphAutophagic-lysosomal dysfunction is a hallmark feature of neurodegenerative proteinopathies, either due to mutations directly affecting the autophagy-lysosome machinery, or secondary to aggregation of misfolded proteins. Iron dyshomeostasis may occur as a consequence of inadequate ferritinophagy, a term coined to describe the lysosomal degradation of ferritin molecules mediated via nuclear receptor co-activator 4 (NCOA4) . As bothMicroglia are in a constant cross-talk with neurons and are capable of sensing neuronal dyshomeostasis. Microglial activation is a healthy reaction against disease and injury, accompanied by the release of pro- and anti-inflammatory factors, ROS and recruitment molecules . ChronicInterestingly, microglia turn out to be far more efficient in sequestering iron compared to other brain cell types, as levels of ferritin induced by iron exposure in an organotypic slice culture model are highest in microglia and oligodendrocytes, whereas ferritin is rarely detected in astrocytes and, surprisingly, never in neurons .Exposing cultured neurons to ferric iron reveals accumulation of iron, however microglia accumulates eightfold more iron than neurons and 4.7-fold more than cultured astrocytes after 24 h . ActivatIncreased iron uptake in substantia nigra in PD patients has been linked with altered expression of lactoferrin receptor on vulnerable dopaminergic neurons . In addiin vitro studies in cultured astrocytes show that cultured astrocytes are surprisingly resistant to ferrous iron compared to neurons and oligodendrocytes, even though intracellular iron burden is comparable  in mice, which results in iron accumulation of ventral motor neurons. Intriguingly, an increase in the enzymatic activity of TNF-α – converting enzyme and subsequent enhanced secretion of soluble TNF-α is observed triggered by iron whereas chelation with deferoxamine mesylate delays disease onset and prolongs overall lifespan .Ferroptosis represents a recently discovered form of cell death independent of the caspase pathway and involves iron dysregulation, lipid peroxidation and inflammation as major hallmarks . CentralKey features of ferroptosis including lipid peroxidation and reduced glutathione levels have been detected previously in the substantia nigra in PD  and coloTherefore, targeting ferroptosis in neurodegeneration may represent an attractive target for disease modification. Potential candidates include vitamin E analogs, liproxstatins and ferrostatins, deuterated lipids and ACSL4 (Acyl-CoA Synthetase Long Chain Family Member 4) inhibitors .Based on the literature reviewed to this point it is certain that iron contributes substantially to neurodegeneration, however so far, no compelling evidence was provided to attribute a causal role to iron, which would imply (1) detection at an early stage if not as first sign, and (2) evidence for disease modification if targeted appropriately. We aim to address both issues based on current knowledge and progress made to this point.In the past few years numerous targets for treatment of iron dysregulation have been identified including reduction of iron burden but also inhibition of downstream effects. However, the numerous physiological functions of iron and defects resulting from aggressive iron depletion need to be considered. Therefore, the following features of treatment of brain iron dyshomeostasis could be taken into account: ability to pass BBB and consecutive cell membranes; chelation of excess iron with moderate affinity to avoid depletion of transferrin and iron-associated proteins; prevention of side effects due to iron removal in areas lacking iron overload . Here, wDeferiprone is an iron chelator currently in use for systemic iron overload conditions like thalassemia major, but its benefit in neurodegenerative conditions lacking systemic iron dysregulation has been shown in a small trial in 9 patients with Friedreich’s ataxia, who received deferiprone for 6 months. Treatment results in decreased iron load in dentate nuclei as visualized by MRI, an area implicated in neurodegeneration. Moreover, motor skills and signs of neuropathy are improved especially in the younger patients, and no severe side effects are reported . DeferipInterestingly however, in another phase II trial on deferiprone 22 early-disease patients  reveal nLactoferrin (LF) is an iron-binding glycoprotein with 60% amino acid sequence homology to transferrin . However42, phosphorylated tau, interleukin-6 and caspase-3 as well as improved cognitive function (There is evidence of a protective function of lactoferrin as exogenous application results in reduced Aβ deposition and improvement of cognitive decline in AD mice via enhanced non-amyloidogenic processing of APP and α -secretase expression and activity through ERK/1/2-CREB and HIF-1α pathways . In MPTPfunction , possibl42 and improved cognition by two measures of executive function (Metal chaperone PBT2 is a derivate of clioquinol, an iron, copper and zinc chelator. In a phase II trial for AD clioquinol improved cognition and decreased Aβ plasma levels , howeverfunction .Another promising target in the self-perpetuating cycle of iron and disease-associated protein accumulation represents the inhibition of the IRE in the 5′-UTR of α-synuclein and APP . Two com42 is observed (Posiphen potently inhibits translation of APP and α-synuclein in preclinical models . In a phobserved .in vitro and in vivo evidence supports the effects of paroxetine as chemical IRE modulator on APP expression (Besides, paroxetine is widely used in the treatment of depression acting through selective inhibition of serotonine reuptake and may represent a suitable candidate for drug repurposing in AD. Preclinical pression , and itsOne of the major drawbacks in therapeutic research of neurodegenerative diseases is a lack of biomarkers for earlier diagnosis, providing a chance for successful disease-modifying treatments. Advances in MRI technology to facilitate more accurate and specific detection of iron may help to determine the biomarker status of iron accumulation at preclinical stages of neurodegeneration. For an extensive review on the use of MRI for brain iron detection we refer to excellent reviews .∗ map, a sign for low neuromelanin reflecting the loss of dopaminergic neurons (∗ parameter value, indicating alterations in iron burden, was observed. In this case, iron measurement by MRI may rather be a marker of disease progression (In premotor PD patients, MRI scans revealed increased echogenicity in substantia nigra, probably indicative of labile iron . De Marz neurons , was det neurons . In a co neurons . In Huntgression . In genegression . Direct gression . Taken tExperimental data show that many neurodegenerative diseases represent conditions of multifactorial cellular dysfunction associated with iron dyshomeostasis present already at early disease stages, yet preclinical models may not be able to answer what comes first in sporadic neurodegenerative diseases. Intriguingly however, iron substantially contributes to and drives many aspects of neurodegeneration, plus, genetic modification of iron metabolism resulted in rescue of neurodegeneration in different animal models, indicating a causal role of iron in neurodegenerative disorders. Advances in MRI technology to facilitate more accurate and specific detection of iron hold great promise to implement iron as a biomarker for preclinical stages of neurodegeneration, probably a crucial treatment window for upcoming therapies. Moreover, CSF levels of iron related proteins in sporadic neurodegenerative diseases may aid in early diagnosis in the future, like CSF ferritin in AD , 2017. CAN wrote the manuscript. CK and GW edited and revised the manuscript.CK and GW are employed by the Medical University of Innsbruck, Austria. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Preclinical studies suggest cannabinoids affect functioning of the hypothalamic-pituitary-adrenal (HPA) axis, but little is known about the effects of marijuana (MJ) use on HPA axis functioning in humans. Since previous work indicates substances of abuse may dysregulate the HPA axis, it is critical to understand how MJ use affects HPA axis activity. Here, we review studies that (a) examined the effects of acute MJ administration on HPA axis functioning, (b) investigated the impact of stress on HPA axis functioning in MJ users, (c) examined the effect of chronic MJ use on basal cortisol levels, and (d) studied the relationship between MJ use and the cortisol awakening response (CAR). Findings indicate acute MJ administration typically raises cortisol levels, but this increase is blunted in MJ-dependent users relative to controls. Frequent MJ users have blunted adrenocorticotropic hormone and cortisol reactivity in response to acute stress. These findings suggest HPA axis activity may be dysregulated by heavy MJ use. Alternatively, dysregulation of the HPA axis may be a risk marker for heavy MJ use. There is mixed evidence for how MJ use affects basal cortisol levels and the CAR. Future studies should consider MJ use characteristics, method of hormone collection, time when samples are collected, and environmental factors that may influence HPA axis activity in MJ users. By examining existing studies we provide one of the first reviews aimed at synthesizing the literature on HPA axis functioning in MJ users. Marijuana (MJ) is the most commonly used illicit substance worldwide, with ~147 million past year users . These iSeveral studies indicate that the hypothalamic-pituitary-adrenal (HPA) axis, the major neuroendocrine system that responds to stress , is dysrThe purpose of this review is to provide a descriptive overview of prior research on the effects of acute MJ administration on HPA axis activity, the impact of stress on HPA axis functioning in MJ users, and the role of chronic MJ use on basal cortisol levels and the cortisol awakening response (CAR). Additionally, we compare methodological differences among studies that may have contributed to discrepant findings, and comment on future directions for advancing research in this field. Articles for this mini review were included based on combinations of keywords searched on Pubmed, including “marijuana”, “HPA axis”, “cortisol”, “tetrahydrocannabinol (THC)”, and “endocannabinoid.” Titles and abstracts were reviewed for relevance to the topic on human MJ users. Additional articles were found through citations included within the manuscripts found using the keyword search.Several studies have investigated the effects of acute MJ administration on HPA axis response by examining ACTH and/or cortisol levels in a laboratory setting Table . Cone etIn other studies, cortisol levels were compared between abstinent MJ-dependent individuals and non-users following intravenous THC administration. MJ-dependent individuals exhibited a blunted cortisol increase after THC administration compared to non-users , 14. PreSome studies have found no significant effect of acute MJ administration on cortisol levels. In a study conducted by Benowitz et al. , the effA number of studies in MJ users have examined the effects of acute stressors on HPA axis activity Table . A studyN = 591) of adolescent MJ users who had lifetime or repeated MJ use  and psychosocial stress (solving math problems). The acute stress manipulation resulted in blunted cortisol response in the daily MJ users compared to healthy controls , 26. Ford MJ use . The autd MJ use . HoweverOther types of stress, such as previous trauma exposure, which may influence cortisol response in MJ users, was examined in a study of non-treatment seeking daily MJ users . Daily MTaken together, the findings to date suggest that stress exposure in adult heavy MJ users , 25, 26,Studies have measured cortisol levels in frequent MJ users and non-users to determine whether the groups differ in basal cortisol levels, and findings suggest MJ has either no effect or increases basal cortisol Table . Block eContrary to the findings above, an fMRI study examining psychomotor function found that chronic MJ users had higher levels of salivary cortisol compared with controls and greater superior frontal gyrus (SFG) but reduced visuomotor activity relative to controls . The autCortisol levels exhibit diurnal variation, such that levels rise during the morning hours, peak 30 min after awakening, and are lowest in the evening. This increase of cortisol in the morning, known as the CAR is believed to be a reliable marker for individual differences in HPA axis activity . StudiesThe purpose of the current mini review is to highlight and integrate the existing, albeit limited literature on the effects of MJ use and stress on HPA axis functioning in adult MJ users and youth at risk for heavy MJ use. Understanding these findings comes at an important time when MJ decriminalization and legalization has made MJ increasingly available, while the perceived risk of MJ use has declined . PreclinAll authors contributed to the article search for this review. AC and JD-B wrote the Introduction, SL wrote the section on acute THC and MJ effects on HPA axis functioning, and AC wrote the sections on stress, basal cortisol, cortisol awakening response, and conclusions and future directions. AC and SL created the table. AC edited and finalized 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.


To achieve this, it is imperative that accurate hormone concentrations be utilized when modeling progestin-mediated outcomes, as the down-stream effects are dose-dependent. The local concentrations of progestins to which the lower female genital tract tissues are exposed after initiation of LARCs are unknown, but they likely differ from peripheral concentrations, dependent upon the progestin type and delivery method. Here, we measured in vivo endocervical and plasma concentrations of (1) systemically-delivered depo MPA (DMPA), (2) levonorgestrel (LNG) delivered via intrauterine system (IUS) and (3) etonogestrel (ETG) delivered via vaginal ring in women who recently initiated contraception treatment. Levels of ETG and LNG in cervical secretions were 100–200 fold higher than plasma levels. In contrast, measurable MPA levels were approximately 10-fold higher in plasma compared to cervical secretions. These results will inform the design of accurate in vitro studies on the influence of progestins on epithelial cells, tissue explants, and peripheral blood cells, to be able to better predict in vivo outcomes. Subsequent observations will aid in determining how MPA might influence HIV acquisition and may facilitate identification of optimal progestin-containing LARC alternatives for women at high risk for HIV infection.Progestin-only long-acting reversible contraceptives (LARCs) are increasingly popular among women seeking contraception; however, recent epidemiological studies suggest that systemically administered medroxyprogesterone acetate (MPA) may increase HIV acquisition. In order to determine the exact mechanisms underlying increases in transmission specific to MPA use and to test safer, alternative contraceptive progestin types and delivery methods,  Long-acting reversible contraception (LARC) options, particularly injectables and intrauterine devices, are becoming increasingly popular in developed and under-resourced countries due to their discrete nature, high efficacy, and low user-failure rates . Howeverin vitro studies are required using cells and/or tissues from the female genital tract (FGT) where heterosexual transmission of HIV occurs. There are several factors that complicate accurate modeling of hormonal influences on the FGT during HIV transmission and the translation of in vitro observations to in vivo outcomes. For instance, several types of progestins with disparate receptor binding capacities . orldwide –54. The orldwide  and DMPAorldwide . TherefoIn vitro and in vivo comparisons of the different progestin-containing contraceptive options and their influence on sexual transmission of HIV are plagued by many issues. These include the variety of progestin subtypes included in female contraceptives and their respective hormone receptor binding profiles, differences in the dosages and routes of progestin administration, and the use of formulations that are combined with contraceptive estrogens. Particularly striking among these factors is the remarkable paucity of information on local FGT levels of exogenous hormones that could allow accurate in vitro modeling. To date, numerous in vitro studies have been performed using serum levels of progestins as clinical surrogates. Given that some progestins are delivered systemically  while others are delivered into the lumen of the FGT , it is likely that serum levels do not reflect the local progestin concentrations to which epithelial cells and underlying tissues are exposed for some or all methods. This is a particularly important factor to consider for in vitro modeling because the contraceptive and non-contraceptive effects of each progestin are concentration dependent [in vivo. Thus, the purpose of this study was to determine the levels of several commonly used contraceptive progestins in cervical secretions and compare these to plasma levels in order to establish proper in vitro modeling conditions for future investigations.ependent , 24, andWe focused on the cervix in this study because non-human primate studies indicate the endocervix as a major site of SIV transmission . This isin vivo. We observed that levels of locally delivered progestins, LNG delivered via an IUS and ETG via a vaginal ring, were approximately 100–200 times higher in the local cervical secretions when compared to the periphery. Levels of MPA after delivery by intramuscular injection, however, were >10 fold higher in the plasma compared to cervical secretions. Interestingly, approximately 50% of samples collected from women treated with DMPA had unmeasurable levels of MPA in their cervical secretions. This is not unexpected, since MPA is delivered systemically, so epithelial cells and other tissues are exposed to this hormone basolaterally via the bloodstream rather than apically within the FGT as with contraceptive progestins delivered via a locally-placed device. In the latter scenario, the measurement of locally-delivered progestin concentrations in samples obtained from the FGT lumen, where delivery occurs, is feasible, reproducible and more physiologically relevant to HIV transmission across these epithelial barriers than peripheral measurements. While significantly more variability is encountered when measuring systemically-delivered progestin levels in luminal samples, these measurements also provide improved physiologic application and enable more informed modeling of in vitro investigations. In short, it is important to utilize progestin concentrations in a polarized manner when modeling progestin effects on epithelial cells or tissue explants in vitro. Specifically, the working concentrations utilized in vitro should reflect median hormone levels for both peripheral and local concentrations for MPA, 10-8M MPA at the basolateral epithelial surface and 10-10M MPA at the apical surface of polarized in vitro models. For investigating the effects of locally delivered progestins on epithelial cells or tissue explants, the basolateral surface should be exposed to median peripheral concentrations, e.g. 10−9 M ETG, 10-10M LNG, and the apical epithelial surface exposed to median luminal concentrations at, e.g. 10-7M ETG, 10-8M LNG. Similarly, when performing experiments investigating the influence of progestins on peripheral blood cells, peripheral progestin concentrations, e.g. 10-8M MPA, 10−9 M ETG, 10-10M LNG, should be replicated, as these represent the approximate median hormone levels to which circulating leukocytes are exposed. These findings provide context for previously published in vitro progestin studies and their observations. For instance, by using a range of exposure levels, Sampah et al. demonstrated an increase in HIV infection of MPA exposed target cells using concentrations representative of those found in blood (approximately 10-9M) as well as the cervical environment (approximately 10-10M). This suggests that MPA may influence infection and replication of virus in the local tissues as well as the periphery [We then used this methodology on ophthalmic sponge and plasma samples collected pre- and post- exposure to several progestin-containing LARCs eriphery .in vitro and in vivo progestin exposure study designs. Our optimized methodologies can be utilized by others to elute steroid hormones from ophthalmic sponges for further investigation of the local genital tract milieu in women who have initiated progestin treatments. Furthermore, the progestin levels reported here for local secretions and plasma inform the community on study design for epithelial, tissue explant, or peripheral blood cultures in the context of synthetic progestins that depend upon both the type of progestin and delivery method. Such study design parameters are essential, as the immunological and contraceptive effects of progestins are concentration dependent and uninformed design could yield results that are not applicable to in vivo outcomes. Our results should help to uncover specific, concentration-dependent, progestin-mediated mechanisms by which systemic and local exposure to DMPA increases HIV infection and/or replication in target cells. Moreover, these results will facilitate appropriate in vitro testing of alternative contraceptive progestin replacement options and thereby guide physicians in making safe contraception recommendations for women at high risk for HIV acquisition.Overall, the findings in this study provide a valuable guide for S1 File(XLSX)Click here for additional data file.


PD-1), and interleukin-12p40 (IL-12B). A KIR haplotype (2DS4+) is proposed to be protective for CMV activation among hematopoietic stem cell transplant patients. Polymorphisms in the interferon lambda 3/4 (IFNL3/4) region are shown to influence susceptibility to CMV replication among solid organ transplant patients. Interestingly, the IFNL3/4 region is also associated with AIDS-related CMV retinitis susceptibility in HIV-infected patients. Likewise, interleukin-10 receptor 1 (IL-10R1) variants are shown to influence CMV retinitis development in patients with AIDS. Results from genome-wide association studies suggest a possible role for microtubule network and retinol metabolism in anti-CMV antibody response. Nevertheless, further genetic epidemiological studies with large cohorts, functional studies on the numerous HCMV genes, and immune response to chronic and latent states of infection that contribute to HCMV persistence are clearly necessary to elucidate the genetic mechanisms of CMV infection, reactivation, and pathogenesis.Human cytomegalovirus (HCMV) is a ubiquitous herpes virus (human herpes virus 5) with the highest morbidity and mortality rates compared to other herpes viruses. Risk groups include very young, elderly, transplant recipient, and immunocompromised individuals. HCMV may cause retinitis, encephalitis, hepatitis, esophagitis, colitis, pneumonia, neonatal infection sequelae, inflammatory, and age-related diseases. With an arsenal of genes in its large genome dedicated to host immune evasion, HCMV can block intrinsic cellular defenses and interfere with cellular immune responses. HCMV also encodes chemokines, chemokine receptors, and cytokines. Therefore, genes involved in human viral defense mechanisms and those encoding proteins targeted by the CMV proteins are candidates for host control of CMV infection and reactivation. Although still few in number, host genetic studies are producing valuable insights into biological processes involved in HCMV pathogenesis and HCMV-related diseases. For example, genetic variants in the immunoglobulin GM light chain can influence the antibody responsiveness to CMV glycoprotein B and modify risk of HCMV-related diseases. Moreover, CMV infection following organ transplantation has been associated with variants in genes encoding toll-like receptors (TLRs), programmed death-1 ( Human cytomegalovirus (HCMV), also called human herpes virus 5 (HHV5), is a beta herpesvirus that belongs to the Herpesviridae family . HCMV exHCMV, with a double-stranded linear DNA genome ranging between 196 and 241 kbp (thousand base pairs), has the largest genome among the betaherpesviruses. The genome can encode over 160 gene products, a number much higher than other betaherpesviruses . Only a The recognition of CMV as a medically important virus goes back to early 1930s when cytomegalic inclusion disease, a severe form of congenital CMV disease with an owl’s eye appearance of inclusion bodies in cells from multiple organs of the infants, was observed. By 1970s, the pathogenic organ disease and HCMV link was well established, and HCMV-like viruses were isolated from other mammals. Due to the high social and medical cost of congenital CMV disease , vaccine development is a high public health priority . HCMV coMost reviews in CMV literature focus on the viral and immune response aspects of the pathogenesis. However, host genetics of viral infection and pathogenesis can identify biological pathways that may lead to novel therapeutics. This review takes a different approach and aims to cover the current cumulative state of the knowledge in the host genetics of CMV pathogenesis in different risk groups. Different phenotypic outcomes of HCMV susceptibility are presented in the following sections. The details of genetic associations such as cohorts, odds ratios, P-values, and sample size are presented in HCMV immune human sera contain neutralizing antibodies against principal CMV envelope proteins (such as gB), tegument phosphoprotein pp150 (UL32), and nonstructural DNA binding phosphoprotein pp52 (UL44) . HumoralRL13 gene) that can bind to the anti-HCMV IgG antibody, thus reducing the number of free anti-HCMV antibodies in circulation, and giving survival advantage to the virus. The GM 3 allele has higher affinity to HCMV FcgR-like protein (through bipolar bridging) compared to the GM 17 allele, leaving lower concentration of free anti-HCMV gB antibodies circulating in the system. They also drew attention to B-cell-mediated antigen processing/presentation pathway as an alternative mechanism underlying GM allotypes’ differential responsiveness to HCMV gB. One of the strategies that HCMV has evolved for evading host immunosurveillance involves generating proteins with similar functional properties to the Fcγ receptor for IgG (FcγR). FcγR interferes with the anti-HCMV IgG antibody’s binding to the virus, thus giving survival advantage to HCMV against antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and antibody-dependent complement-dependent cytotoxicity  GM (gamma marker) creates several alleles ,  with different binding affinities to antigens such as HCMV glycoprotein B (gB). The effect of genetic variation in humoral immunity on susceptibility to HCMV disease has been suggested by the studies that focus on association between immunoglobulin (Ig) GM allotype variation and HCMV antibody response. Pandey et al. reported a significant effect of immunoglobulin GM genotypes on antibody responsiveness to HCMV glycoprotein B (gB) . The stutoxicity .A major issue in genetic epidemiology studies of antibody variation is that most of the genetic variation is ethnic and population specific. For example, GM 3 is rare among people of African descent. Moreover, the genetic background influencing overall immune response will be different between races and populations. Therefore replication and translation of results from one study to another is not always possible. Ethnic and population-specific genetic variation can also lead to hidden population stratification even in the same country, which is major factor confounding the genetic association results. Given the complex genetic nature of humoral immune response to HCMV, much larger studies with more balanced case and control groups are needed to test the GM associations with HCMV response.HCMV is not considered an oncogenic virus; however, HCMV viral DNA, RNA, and protein have been frequently found in neoplastic tissues including gliomas, breast cancer, and neuroblastoma . MoreoveSignificant association of rare GM genotypes with neuroblastoma, a rare extracranial solid tumor, has been reported , but theIndependent cohort and functional studies make a case for significant influence of host genetic variation in humoral immunity on response to HCMV disease Figure 1HCMV is a common opportunistic infection among immunocompromised individuals. Individuals are maximally immunocompromised due to use of immunosuppressants during solid organ or HSCT procedures and thus are prone to HCMV reactivation (of the latent virus), primary infection, and reinfection. HCMV infections can cause severe morbidity and transplant failure, which frequently results in extended hospital stay and substantially higher cost of care . TransplCoordinated innate and adaptive immune response is crucial for control of HCMV infection in immunocompromised transplant recipients. Whereas innate interferon (IFN) and natural killer (NK) cell responses are important in immediate control of CMV infection, adaptive T cell immune responses are important in both active infection and reactivation control phases . To reduA family of transmembrane proteins, the Toll-like receptors (TLRs), are part of the innate immune system and play crucial roles in the activation of the immune system by regulating the production of antiviral peptides and inflammatory cytokines against viral replication. The detection of CMV envelope glycoproteins B (gB) and H (gH) by TLR2 leads to nuclear factor-kB (NF-kB) activation and cytokine secretion against CMV . ClinicaGenetic variants of MICA (major histocompatibility complex class I chain-related protein A) and its activating receptor NKG2D  receptor may be associated with HCMV disease risk among kidney transplant patients. A candidate gene association study identified a regulatory MICA variant (rs2596538) in the kidney donors that can be a protective prognostic determinant for CMV disease. This functional variant was able to predict the development of CMV infection and disease during the first year after kidney transplantation . MembranCytokines, signaling molecules of the immune system, regulate pro-inflammatory and anti-inflammatory responses, and play important roles in antiviral response. Cytokines also play a role in HCMV infection, reactivation, and disease . StudiesType III interferon, also called interferon lambda (INFL3 or formerly IL-28B), has gained much attention as an important viral response element in recent years . In a coFOXP3 , up to 40% of adults with AIDS developed CMV disease . AlthougCCR5 and stromal derived factor (SDF-1)] genetic variants have been observed to influence retinitis progression  cohort Table 1.gression . In a diple size . In a lapatients , was alspatients .Although subject to complex confounding factors and high false discovery rates, host candidate gene studies of immunocompromised groups cumulatively indicate possible effects of innate and adaptive immune gene variants on CMV disease Figure 1Vertical transmission of HCMV from mother to fetus or newborn is common and plays an important role in maintaining infection in the population . PrenataHost candidate genetic studies of congenital HCMV infection mainly have focused on innate immune system, such as TLRs and Mannan-binding lectins, and cytokine genes Figure 1MBL2 were reported to be associated with lower serum MBL concentrations (Another important player in the innate immune system is the Mannan-binding lectin (MBL), a pattern recognition molecule and a first line defense antimicrobial factor . Mutatiotrations . In a Potrations . Howevertrations .HCMV infection during pregnancy can affect the cytokine profile within a HCMV-infected placenta and shift the cytokine expression toward a proinflammatory state with implications for adverse pregnancy outcomes . As hostDeciphering the contribution of hot genetics to HCMV vertical transmission and related disease outcomes may be the hardest of all HCMV-related disease studies. Firstly, the susceptibility of the pregnant mother to HCMV needs to be considered, where the immune response will be modified due to pregnancy further complicating the interaction between the host and HCMV. Secondly, if the mother cannot clear the infection, and HCMV finds its way to fetus, then the immune response by the infant, which is rather immature and still developing, will be involved with a genetic make-up different than that of the mother. Aforementioned reports should be considered as early attempts of a rather challenging research agenda. High-throughput genetic and immune profiling methods with much larger cohorts are necessary to understand the genetic and non-genetic factors involved in HCMV vertical transmission.Genome-wide association studies (GWASs) have made significant contributions for discovering genetic factors underlying complex phenotypes and diseases. Unlike traditional hypothesis-driven candidate gene studies, where only a few candidate genes are targeted, in GWAS all human genes become potential candidates for the phenotype of interest. Therefore, GWAS approach can discover genes and their variants that may look irrelevant to the phenotype of interest, which in return can lead to discovery of novel biological pathways involved in development of this phenotype.DHRS4, near the associated SNP, was proposed to be a candidate for further evaluation. These two studies show that GWAS approach can be productive in HCMV field; however, one also needs to consider the fact that there was no overlap of identified genes between the two GWASs, although the phenotypes were similar. Curiously, none of the candidate innate and adaptive immune genes examined so far were top hits in these GWASs.The first GWAS was conducted to identify genetic polymorphisms associated with the susceptibility to HCMV and strength of anti-HCMV immunoglobulin G (IgG) response to CMV infection . The stuClinical management of CMV infection is particularly challenging due to the arsenal of host immune evasion strategies encoded by its large genome and its complex interactions with its human host. As we show in this review, with a few exceptions, most of the genetic loci identified to date have not been replicated or validated in sufficiently powered cohort studies, suggesting that only a small fraction of variance in host response is likely due to genetic variation. To address the role of host genetic variation in immune response to HCMV and CMV disease, large prospective studies and genome-wide approaches are required to securely identify causal variants involved in immune response and pathophysiological mechanisms leading to CMV disease.ES designed the study, conducted literature research, and wrote and edited the manuscript. PA and CW wrote and edited the manuscript.This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN26120080001E. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.PA and CW are employees of Leidos Biomedical Research, Inc., and declare no competing interest.The remaining authors declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While primarily unresectable locally advanced pancreatic cancer (LAPC) used to be an indication for palliative therapy, a strategy of neoadjuvant therapy (NAT) and conversion surgery is being increasingly used after more effective chemotherapy regimens have become available for pancreatic ductal adenocarcinoma. While high-level evidence from prospective studies is still sparse, several large retrospective studies have recently reported their experience with NAT and conversion surgery for LAPC. This review aims to provide a current overview about different NAT regimens, conversion rates, survival outcomes and determinants of post-resection outcomes, as well as surgical strategies in the context of conversion surgery after NAT. FOLFIRINOX is the predominant regimen used and associated with the highest reported conversion rates. Conversion rates considerably vary between less than 5% and more than half of the study population with heterogeneous long-term outcomes, owing to a lack of intention-to-treat analyses in most studies and a high heterogeneity in resectability criteria, treatment strategies, and reporting among studies. Since radiological criteria of local resectability are no longer applicable after NAT, patients without progressive disease should undergo surgical exploration. Surgery after NAT has to be aimed at local radicality around the peripancreatic vessels and should be performed in expert centers. Future studies in this rapidly evolving field need to be prospective, analyze intention-to-treat populations, report stringent and objective inclusion criteria and criteria for resection. Innovative regimens for NAT in combination with a radical surgical approach hold high promise for patients with LAPC in the future. Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related deaths and will become the second cause by 2030 ,2,3. ThiIn the last decade, a strategy of neoadjuvant therapy (NAT) followed by conversion surgery has been tested and is increasingly used in the treatment of primarily unresectable LAPC . This stHere, we aim to provide an overview about current concepts of NAT and conversion surgery in advanced pancreatic cancer including different neoadjuvant regimens, patient selection for surgery, surgical techniques, postoperative management, and outcomes.While LAPC was previously considered an incurable disease stage and indication for palliative therapy along with metastatic PDAC, it is now increasingly recognized as a potentially curable disease. Several guidelines for LAPC have been published, although the evidence on its best treatment remains limited ,31. In cIn general, NAT is performed for almost 6 months, which equals six to eight cycles of chemotherapy and can be further extended in some patients, as demonstrated in the palliative situation . In a muDuring NAT, the performance status, toxicity, and tumor response should be closely monitored. Cross-sectional imaging is usually performed every 2–3 months together with assessment of CA 19-9 levels as the current gold-standard biomarker for therapy monitoring . Novel bLocal disease response in cross-sectional imaging is not necessary to qualify for conversion surgery, as radiological restaging can be misleading after NAT in PDAC ,51,52. TConversion surgery after NAT has been performed and reported in small observational studies for many years. In 2010, a systematic review reported response and resectability rates in NAT patients . The larThe introduction of the FORFIRINOX regimen considerably advanced the treatment of LAPC and of conversion surgery. A patient-level meta-analysis investigating the outcomes in LAPC patients receiving first-line therapy with FOLFIRINOX included 315 patients from 11 observational studies . The medSeveral larger observational studies on LAPC patients were published more recently . The HeiThe overview provided in The majority of the literature on conversion surgery examines resections after NAT for localized PDAC. However, there is some sparse data on conversion surgery after chemotherapy for metastatic PDAC, as shown in Taken together, these studies mostly report favorable results of conversion surgery in highly selected “super-responders” to long-term chemotherapy. As in all studies on NAT, it remains unclear to what extent favorable survival outcomes are based on individual patient selection and/or surgical resection. Based on those reports with larger stage IV cohorts, conversion rates in an ITT population remain below 5% for this particular subgroup. With the increasing use of more effective chemotherapy regimens, more data on conversion surgery in metastatic patients will become available and will need to be carefully examined.In recent decades, surgical procedures for resectable PDAC were continuously refined and, in combination with more effective adjuvant chemotherapy regimens, markedly improved survival outcomes . SurgicaBody and tail cancers infiltrating the celiac trunk represent a special subgroup of LAPC, because in these tumors, a distal pancreatectomy with celiac axis resection (DP-CAR) without arterial reconstruction can be considered ,81. WhilTaken together, the surgical strategy during conversion surgery after NAT for LAPC should include an artery-first approach for assessment of resectability early during exploration, in order to avoid R2 resections, and to achieve local radicality . The plane of dissection should be directly at the vascular walls. Surgeons performing such conversion surgery should be well prepared to perform vascular resections. While venous resections are still necessary to achieve local radicality, arterial resections might be avoided. In patients with tumors still infiltrating an artery after NAT, the role of arterial resections needs to be further examined. These operations are technically demanding and should be performed in experienced centers.Several studies investigated morbidity of conversion surgery after NAT for LAPC with overall indecisive results—likely due to heterogeneous treatment algorithms . Larger Another important aspect is the prognostification of patients after NAT and conversion surgery for LAPC. Currently, there are no valid clinical tools available to identify patients at high risk for early recurrence and early death after NAT. The role of additional adjuvant therapy after NAT and conversion surgery is unknown and there is no evidence for a standard of postoperative cancer-directed treatment. However, a relevant fraction of NAT patients has early recurrence or die within 12 months after conversion surgery ,55. IdenAn important question in this context is if and how NAT impacts the prognostic relevance of clinical and pathological factors that are well-established predictors of survival after upfront resections. These classic predictors include tumor-related factors, such as pT-stage, tumor size, pN-stage, resection margin status, as well as CA19–9 levels ,91,92,93In other studies, a good biological response, indicated by the course of CA19–9 levels, was found to be independently predictive for overall survival, along with a major pathological response defined by rare or absent viable tumor cells ,45,65. PThe strategy of NAT and conversion surgery for patients with LAPC is rapidly evolving, especially since more effective combination chemotherapies have become available. This is reflected by an increasing number of studies investigating this topic with heterogenous short- and long-term outcomes. In highly heterogeneous studies, the reported surgical conversion rates for LAPC vary between 9–87%. These numbers should be interpreted with caution since ITT analyses are lacking in most studies on this topic. Even in the few studies providing an ITT analysis, resectability rates vary extremely with variations in local practices of indication for resection and use of extended resections. Furthermore, survival data are difficult to read since the anatomical inclusion criteria for borderline-resectable or LAPC subgroups differ enormously between centers and studies. Future studies should try to incorporate objective biological criteria for resectability and response evaluation, instead of relying only on radiological criteria, which are often applied in a subjective and non-standardized fashion . Of note


Cardiac computed tomography (CT) allows rapid visualization of the heart and coronary arteries with high spatial resolution. However, analysis of cardiac CT scans for manifestation of coronary artery disease is time-consuming and challenging. Machine learning (ML) approaches have the potential to address these challenges with high accuracy and consistent performance. In this mini review, we present a survey of the literature on ML-based analysis of coronary artery disease in cardiac CT. We summarize ML methods for detection and characterization of atherosclerotic plaque as well as anatomically and functionally significant coronary artery stenosis. Diagnosis and monitoring of coronary artery disease (CAD) is increasingly based on non-invasive imaging with computed tomography (CT), allowing excellent visualization of the coronary arteries with high spatial resolution. Cardiac CT exams consist of hundreds of slices and the number of cardiac CT studies has been steadily increasing . This haThis survey provides an overview of ML algorithms for detection, characterization, and quantification of CAD in cardiac CT. We searched PubMed for articles related to ML-based assessment of CAD in cardiac CT published within the last 10 years  classifiers, which look for training samples with similar feature values to a test sample, and assign the test sample to the majority class among these training samples. Linear classifier (LC) models like support vector machines (SVM) aim to find a linear combination of features to separate samples in different classes. Alternatively, samples can be separated by thresholding feature values along a single axis. This is unlikely to lead to highly accurate classifiers, but by consecutively applying thresholds, a decision tree model can be built for more accurate classification.Each sample can be described based on characteristics or deep learning. The success of deep learning in medical image analysis has been to a large extent due to the inclusion of trainable image filters in so-called convolutional neural networks (CNNs), which can be trained to extract valuable features from raw image data (ML performance can often be improved by combining predictions of multiple models. Ensembles (E) combine predictions of multiple simultaneously executed models, e.g., by averaging predictions of decision trees in a random forest (RF). In boosting (BO), models are applied consecutively and each model is trained to correct errors of its predecessors. Finally, artificial neural networks (ANNs) transform samples into targets through layers of trainable neurons, which are loosely based on biological neurons. While ANNs have been around since the 1950s, it has recently become possible to train networks that have many layers, i.e., age data . For a mage data .CT offers a non-invasive alternative to e.g., catheter-guided X-ray angiography, optical coherence tomography, and intravascular ultrasound (IVUS) for atherosclerotic plaque visualization. Characterization and quantification of plaque in CT provide insight in different stages of CAD . In thisscoring is typically performed in dedicated non-contrast-enhanced, ECG-triggered, calcium scoring CT images (CSCT). Using dedicated software, an expert identifies voxels with a density over 130 Hounsfield units (HU) in the coronary arteries. Identified CAC is then quantified according to its volume, density, or a combination of both  quantification or  of both . CAC canML-based calcium scoring methods proposed prior to the advent of deep learning have focused on identification of CAC lesions among a large set of samples, i.e., groups of connected voxels above 130 HU. Samples are described with features such as size, shape, appearance and location to distinguish CAC from other candidate lesions such as calcifications in the aorta. Location features are of particular importance, as recognized by Liu et al. , KurkureDeep learning-based methods have typically classified individual voxels instead of candidate lesions. Due to the extreme imbalance between numbers of CAC and background voxels in CT images, Wolterink et al.  proposedAutomatic CAC scoring methods have been validated in large data sets  and in oNon-calcified plaque is typically lipid-rich and vulnerable to rupture, causing acute coronary syndrome . ML-baseCoronary artery localization by means of centerline extraction is a typical preprocessing step for ML-based plaque analysis. Traditionally, many automatic centerline extraction methods have been based on minimum cost paths between proximal and distal artery points , 36. ML Coronary artery centerlines can be used to reconstruct CCTA volumes into images that allow better plaque visualization and identification. Zhao et al. , Jawaid ex-vivo CCTA scans. Features were extracted for each cross-sectional image and lesions were classified into advanced or early stage atherosclerosis using a linear classifier. Alternatively, Masuda et al. . Many centerline extraction methods also estimate the luminal radius at each centerline point, assuming a circular coronary artery profile , 38. HowLumen segmentation is often considered a preprocessing step for stenosis detection, but it has been shown that stenosis degree can also be directly determined based on image data. Zuluaga et al.  detectedCoronary stenoses are located in the arteries, but may restrict blood flow to myocardial segments. Mukhopadhyay et al.  used an The sensitivity of CCTA-based anatomical stenosis evaluation for detection of functionally significant stenosis is high when evaluated visually, but its specificity is moderate . The curCT) could provide reproducible physical measurement without the drawbacks of invasive procedures. FFRCT has traditionally been based on computational fluid dynamics (CFD) (CT.FFR estimation based on CCTA scans (FFRcs (CFD) , 62, i.ecs (CFD)  but compCT was reduced by two orders of magnitude. The diagnostic value of this method has been demonstrated thoroughly ].CT development is aided by the availability of large data sets with CFD-derived reference values. An important remaining step toward clinical application of FFRCT lies in performance evaluation specifically for subjects around the FFR threshold of 0.8, which were shown to be most challenging , the Netherlands Organisation for Health Research and Development with participation of Pie Medical Imaging (104003009). II received institutional research project by Dutch Technology Foundation cofunded by Pie Medical Imaging (12726). II and TL are cofounders and shareholders of Quantib-U BV. The remaining 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.