Wound healing is the physiologic response to a disruption in normal skin architecture and requires both spatial and temporal coordination of multiple cell types and cytokines. This complex process is prone to dysregulation secondary to local and systemic factors such as ischemia and diabetes that frequently lead to chronic wounds. Chronic wounds such as diabetic foot ulcers are epidemic with great cost to the healthcare system as they heal poorly and recur frequently, creating an urgent need for new and advanced therapies. Stem cell therapy is emerging as a potential treatment for chronic wounds, and adult-derived stem cells are currently employed in several commercially available products; however, stem cell therapy is limited by the need for invasive harvesting techniques, immunogenicity, and limited cell survival in vivo. Induced pluripotent stem cells (iPSC) are an exciting cell type with enhanced therapeutic and translational potential. iPSC are derived from adult cells by in vitro induction of pluripotency, obviating the ethical dilemmas surrounding the use of embryonic stem cells; they are harvested non-invasively and can be transplanted autologously, reducing immune rejection; and iPSC are the only cell type capable of being differentiated into all of the cell types in healthy skin. This review focuses on the use of iPSC in animal models of wound healing including limb ischemia, as well as their limitations and methods aimed at improving iPSC safety profile in an effort to hasten translation to human studies. Wound healing is a complex physiological response to the disruption in the normal architecture of the protective skin barrier. It involves the spatial and temporal coordination of various cell types and cytokines and is divided into three distinct phases: inflammation, proliferation, and remodeling , 2. ThisImmediately post-wounding, hemostasis is achieved by platelet aggregation and initiation of the coagulation cascade. Chemotactic signals including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are released and attract macrophages, neutrophils, and fibroblasts to the wound bed. Neutrophils and macrophages clear the wound of any bacteria and debris and elaborate further cytokines including interleukin 1 (IL-1), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha (TNF-α), all of which promote cellular recruitment and proliferation. A provisional matrix composed of fibronectin and hyaluronic acid (HA) is synthesized and secreted, upon which epithelialization occurs with the aid of stem cells from hair follicles and adjacent epidermis. Eventually, fibroblasts convert fibronectin-based microfibrils into collagen-enriched fibers. During these events, angiogenesis results in re-vascularization of the wound –6.Chronic cutaneous wounds, such as those seen in diabetic foot ulcers (DFU) and pressure ulcers, contribute significantly to patient morbidity and mortality. They affect over six million Americans annually and cost upwards of $25 billion . Nearly The gold standard of chronic wound management involves careful diagnosis of etiology, control of infection, optimization of vascular inflow to reduce ischemia, debridement of nonviable tissue, and offloading of pressure. Despite optimal care, only 50% of DFU heal within 12–20 weeks and 50% recur within 18 months. As such, novel and improved methods of wound healing are urgently needed .Stem cell therapy has emerged as an exciting potential therapy for wound healing. When transplanted into a wound, stem cells act in a direct and paracrine manner to promote cell recruitment, immunomodulation, extracellular matrix remodeling, and angiogenesis by secretion of cytokines and growth factors –19. AdulInduced pluripotent stem cells (iPSC) represent a groundbreaking innovation for adult-derived stem cells that carry enhanced therapeutic and translational potential. First developed in 2006, iPSC are pluripotent stem cells derived from adult somatic cells. They are reprogrammed into a pluripotent state in vitro by induced expression of four transcription factors including Oct4/Sox2/c-Myc/KLF4 or Oct4/Sox2/NANOG/LIN28 –32. AlthSimilar to embryonic stem cells, iPSC are pluripotent, have the potential for self-renewal, and can differentiate into any adult cell type. iPSC have certain advantages over other stem cell types in models of regenerative medicine and wound healing. Because they are derived from adult somatic cells, and not embryos, iPSC are not associated with the ethical dilemmas surrounding the use of embryonic stem cells. They are easily harvested from cutaneous sources such as skin fibroblasts, obviating the need for invasive harvesting procedures such as bone marrow or adipose tissue biopsies. iPSC are pluripotent and can therefore be differentiated into any adult cell type, enhancing their potential in models of various disease processes. Since iPSC can be derived in principle from any adult tissue including skin, the potential pool of source cells is many orders of magnitude greater than other stem cell types. Lastly, iPSC can be transplanted in an autologous fashion to avoid immunogenicity, enhancing their in vivo survival , 37.iPSC are currently being evaluated in pre-clinical studies of three-dimensional (3D) organ printing, wound healing, and angiogenesis . AlthougBecause they can differentiate into descendants of all three germ layers, iPSC-derived terminally differentiated cells have the potential to enhance each of the phases of diabetic wound healing through their paracrine and direct cellular effects Table  38]. Du. Du38]. The following sections review the use of iPSC in animal preclinical studies of non-ischemic and ischemic wound healing and the limitations that need to be overcome prior to clinical use. Major findings of studies relating to cutaneous wound healing in a murine model are outlined in Table Angiogenesis is a vital component of wound healing, as it reestablishes perfusion to injured tissues and delivers key nutrients. Unfortunately, angiogenesis is diminished in the hypoxic environment of chronic wounds. Additionally, diabetic patients have reduced homing potential of endothelial progenitor cells to sites of injury, further suppressing their angiogenic potential. Endothelial cells are critical for vessel formation and upregulation of VEGF expression. As such, delivery of human-induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) holds great promise for accelerating diabetic wound healing , 9.Several mechanisms by which hiPSC-EC improve wound healing have been identified thus far. Increases in wound perfusion and vessel density may be seen within the first 4 days following treatment in hiPSC-EC-treated wounds in a murine model . hiPSC-EThe optimal delivery platform for iPSC and enhancement of their in vivo survival in chronic wounds is currently under investigation, with several biomimetic materials showing promise , 49. TheOne study showed there was no significant difference between healing in wounds treated with hiPSC derived from healthy versus type I diabetic donors in terms of both healing rate and time to reach maximum rate . AlthougAs with many stem cell approaches, the low rate of in vivo cell survival has been a major limitation in wound healing. The in vivo lifetime of hiPSC-EC increased by culturing them on electrospun polycaprolactone (PCL)/gelatin scaffolds; this mode of cell delivery also increased blood perfusion and arteriole density in the tissue surrounding hiPSC-EC-seeded scaffolds compared to controls . SimilarTogether, these studies confirm that hiPSC-EC not only accelerate wound healing via increased angiogenesis, but have potential to do so to a greater extent than primary cells. Although equivocal in animal-derived iPSC, some studies examining hiPSC derived from diabetic humans suggest that they are not inferior to those from healthy donors, potentially allowing diabetic patients to undergo autologous transplant of their own cells with equal regenerative potential. This finding broadens the scope of hiPSC translational potential. Although the optimal delivery vehicle and hiPSC niche are yet to be elucidated, early studies show promising results at increasing cell survival in vivo, while advances in hiPSC-EC differentiation are becoming more efficient and rapid , 52.During wound healing, fibroblasts are responsible for the production of collagen, fibronectin, and proteoglycans, which form the extracellular matrix on which re-epithelialization and healing occur . FibroblKashpur et al.  found thIn vivo, self-assembled 3D extracellular matrix tissues from hiPSC-derived fibroblasts improved re-epithelialization in a diabetic mouse model, when applied topically . TissuesMSC are multipotent stem cells that promote cutaneous healing by homing to wounds and differentiating into myogenic, chondrogenic, osteogenic, and adipogenic derivatives. MSC also produce growth hormones that drive angiogenesis and re-epithelialization, while mobilizing the resident stem cell niche and contributing to favorable immunomodulation . HoweverNakayama et al.  successfAlthough pluripotent iPSC hold tremendous promise in wound healing and regenerative medicine, their use in clinical trials is currently limited due to their teratogenic potential. Although teratoma formation after topical transplantation has not yet been studied, subcutaneous injection of differentiated iPSC led to teratoma formation . Thus, hExtracellular vesicles released from hiPSC-MSC facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis , 60. SpeThe primary hurdle to overcome before clinical application of hiPSC-derived cells is the elimination of their tumorigenic potential. The aforementioned studies were able to harness the therapeutic potential of hiPSC without obvious negative side effects, a major step in furthering these cells’ translational potential.In addition to their therapeutic potential in cutaneous wound healing, iPSC are currently being investigated in multiple disease models of ischemia, including peripheral arterial disease, myocardial infarction, osteonecrosis, and retinopathy. Consistent with findings from cutaneous wound healing studies, the primary driver of iPSC healing in the majority of these models is their pro-angiogenic potential . AlthougiPSC ameliorate ischemia in models of peripheral arterial disease by reducing inflammation, promoting angiogenesis, and reconstituting viable cellular populations such as endothelial cells. In a nude mouse hind limb ischemia model, hiPSC-EC in conjunction with VEGF reduce inflammation and promote muscular regeneration . hiPSC-EIn a similar model, when compared to bone marrow-derived MSC, hiPSC-MSC reduced muscle fibrosis, improved ambulatory impairment and tissue loss, and enhanced perfusion . hiPSC-MThe majority of studies employing iPSC in models of peripheral arterial disease have delivered cells or extracellular vesicles via intramuscular (IM) injection with cells suspended in buffer or culture medium , 63–69. Despite their great promise, the use of pluripotent stem cells whether induced or embryonic is limited by their tumorigenic potential –73. BecaNumerous strategies are currently under investigation aimed at eliminating iPSC teratogenic potential, in addition to the use of extracellular vesicles mentioned above. Cellular differentiation prior to cell transplantation is one approach to decreasing the risk of teratoma formation. For example, cellular-based therapies, such as those used in some wound healing studies, use terminally differentiated cells. This strategy, however, does not eliminate the risk of inadvertent transplantation of residual undifferentiated cells. In fact, multiple studies using neural and chondrogenic derivatives have shown persistent teratoma formation , 77, 78.Several methods are currently employed for generating iPSC, with the oncogenic safety profile of some superior to others Table . RetroviOther modes of eliminating iPSC oncogenic potential are being investigated. Cells are now successfully reprogrammed without the use of c-Myc, the most oncogenic Yamanaka factor . Small miPSC are an innovative and exciting new cell type with potential to revolutionize the fields of regenerative medicine, inherited genetic disease, and drug therapy. hiPSC hold great promise to accelerate chronic wound healing, with increased healing and reperfusion following wounding or ischemia in rodent preclinical models. Although much has been learned about iPSC generation and optimization in the short time since their development, their safety profile, particularly in relation to tumorigenic potential, remains to be understood in sufficient detail to allow clinical translation. In addition, hiPSC can be derived from patients with chronic diseases and reprogrammed into cells functionally resembling those derived from healthy individuals. However, iPSC-EC derived from obesity-induced diabetic mice showed decreased healing and angiogenic capacity, and thus, further studies will be required to understand the differences between hiPSC derived from diseased and healthy donors.Major hurdles must be overcome before iPSC use in humans becomes possible. Methods for generating hiPSC with non-integrative technology, preferably by protein and small particle transfer, must become more efficient and speedier. Standardized protocols for teratoma formation assays as proof of pluripotency must be developed in order to derive meaningful conclusions about cell lines. The optimal platform for delivery of hiPSC into a wound, including the ideal niche to prolong cell survival and intracellular signaling, needs to be determined. New methods for eliminating undifferentiated cells capable of tumorigenic potential prior to cellular transplant are necessary. Although studies have examined the tumorigenic potential of cells injected directly into murine tissues, no study to date has examined this potential after cutaneous application of hiPSC. Thus, further animal studies to evaluate the safety and efficacy of iPSC are crucial prior to their translation into humans, including large animal models that more closely mimic human skin . Lastly,


Computational machine learning, especially self-enhancing algorithms, prove remarkable effectiveness in applications, including cardiovascular medicine. This review summarizes and cross-compares the current machine learning algorithms applied to electrocardiogram interpretation. In practice, continuous real-time monitoring of electrocardiograms is still difficult to realize. Furthermore, automated ECG interpretation by implementing specific artificial intelligence algorithms is even more challenging. By collecting large datasets from one individual, computational approaches can assure an efficient personalized treatment strategy, such as a correct prediction on patient-specific disease progression, therapeutic success rate and limitations of certain interventions, thus reducing the hospitalization costs and physicians’ workload. Clearly such aims can be achieved by a perfect symbiosis of a multidisciplinary team involving clinicians, researchers and computer scientists. Summarizing, continuous cross-examination between machine intelligence and human intelligence is a combination of precision, rationale and high-throughput scientific engine integrated into a challenging framework of big data science. IntroductionBasic aspects of machine learningBasic aspects of ECG-machine learningDetection of ECG components and disease-specific abnormalities in ECGECG prediction of cardiovascular events and detection of unknown patternsFuture considerationsConclusionTable 1), which are improved with experience, to give remarkable effectiveness in applications, including cardiovascular medicine.As an area of computational science, the machine learning subfield has at its core self-enhancing algorithms  has been not yet established, and future efforts should be directed to this field7.From all diagnostic methods in cardiovascular diseases, ECG is the most accessible and, therefore, an area of intense research. Despite of different pattern recognition algorithms applied in ECG signals recognition in the past decades, a valuable application . Detection of the components on the electrocardiogram requires the application of a classifying method, which acts in a discriminative manner based on the input features.The electrical activity of the heart on ECG recording is represented by a time-series of waves , intervals , segments   supervised or (2) unsupervised learning methodSupervised methods are taught with annotated examples from which the algorithm learns to recognize or predict patterns on unlabeled examples, whereas unsupervised methods learn to spot patterns in a data set without labels. A typical application of algorithms trained via supervised learning techniques is the classification of data in known categories. The data must be annotated by a human supervisor or through automatic collection54. Illustrative examples of this approach are found in fields ranging from handwritten digit recognition55,56 to the classification of cancerous cells57.29. Another supervised learning method used in the diagnosis of cardiac arrhythmia is Probabilistic Neural Network implemented by Gutiérrez-Gnecchi et al4. This platform was intended for on-line, real-time ambulatory operation and can classify eight heartbeat conditions. The results derived from confusion matrix tests yielded an overall on-line classiﬁcation accuracy of 92.746%.A supervised and inductive learning algorithm, VFI5, was used successfully in the diagnosis of cardiac arrhythmia from standard 12 lead ECG recordings, unsupervised learning attempts the extraction of the most significant features of a non-annotated set of data: it is in fact a technique often used for denoising purposes, to extract not explicit correlations from distributions, to cluster data into groups sharing similar characteristics54,58.On the other hand59, the clustering of ECG beats in holter records60, the preprocessing of color-Doppler data61, the prediction of patients' future from electronic health records62.Examples of tasks involving unsupervised learning models are: the extraction of fetal QRS complex from the maternal ECG1 on the arrhythmia dataset provided by MIT-HIB. The authors claim that their proposed work is also suitable for mobile health monitoring and well-suited for wireless body area network (WBAN) environment.Fuzzy C-Means (FCM) clustering algorithm improved with the attribute selection model based on Mahalanobis-Taguchi System (MTS) is an unsupervised learning method. It was implemented by Nur Al Hasan Haldar et al.40,54: quite often these two different methods overlap and are combined to perform classifications and regression tasks in big datasets, which require complex preliminary analysis in order to be successfully processed63.It is important to note that there is not a strict distinction between supervised and unsupervised learning algorithmsSemi-supervised learning is in fact another approach exploiting the data decomposition and features extraction capabilities of unsupervised learning algorithms together with the high accuracies provided by the presence of a supervisor during the training process. Among the most used, Artificial Neural Networks allow the analysis of cardiovascular issues using both supervised and unsupervised learning methods.64 use both unsupervised and supervised learning algorithms in order to achieve outstanding performances (Table 1).Recent works in ECG classificationAs much as it would be desired in artificial intelligence, there is no general approach that can unrestrictedly handle any task. The selection of specific machine learning algorithms is dependent on the individual practical problem to be solved. More importantly, it is advisable to thoroughly explore and understand the tasks’ critical details. An appropriately selected method does not lead to success unless matched perfectly with the prior field-specific knowledge.For example, consider morphological feature extraction step from an ECG trace. Some heart conditions can drastically change ECG components, thereby feature extraction step should respect all potential morphological variations.Figure 2 [A]). While some algorithms can filter QRS complex65, concerns will be raised whether high peaked T waves  can be discriminated from QRS complex when extracting all ECG components. For instance M. Vijayavanan et al. used an algorithm to calculate the heart rate based on that R wave within the QRS complex invariably which has the highest amplitude66. It can be anticipated that such a method can easily misinterpret, since there are examples when T waves have the highest amplitude .When looking at the ECG, the first wave which follows QRS complex is called T wave. T waves can present as a positive deflection, inverted (negative deflection), biphasic wave  or even become flattened during certain dynamic heart conditions (e.g. ischemia) , auricular fibrillation (AF), premature atrial contraction (PAC), left bundle branch block (LBBB), right bundle branch block (RBBB), premature ventricular contraction (PVC), sinoauricular heart block (SHB) and supraventricular tachycardia (SVT) is implemented on a platform intended for on-line, real-time ambulatory operation. The algorithm uses a wavelet transform process based on quadratic wavelets for identifying individual ECG waves67. Meanwhile machine learning-based methods for variable selection and further prediction of cardiac events showed promising results67.Extensive clinical evidence provided by scientists is the most important instrument for identifying health risks. Using empirical data and autonomic learning, we can expand our horizons and nourish our fundamental knowledge with relevant hypotheses. Disease prevention is an action of developing early detection strategies, which aim to protect and improve global health. The exploration of unknown patterns and its assembly into prediction systems for cardiovascular events are essential sources for innovation. For example, there are reports of low sensitivity of the vital signs as predictors of clinical outcome in critically ill emergency department patients8 used a rough sets (RS) and a Quantum Neural Network (QNN) to recognize ECG signals, classify and obtain fast and realistic forecast of cardiological events. In one of the most encouraging recently work, Stephen F. Weng et al.21 predicted first cardiovascular event over 10-years with four machine-learning algorithms: random forest, logistic regression, gradient boosting an neural networks compared to an established algorithm (American College of Cardiology guidelines). In the study, they used routine clinical data of 378,256 patients from UK family practices, free from cardiovascular disease at outset. Neural networks was the highest achieving algorithm, which predicted 4,998/7,404 cases  and 53,458/75,585 non-cases (specificity 70.7%), therefore correctly predicting 355 more patients (+7.6%) who developed cardiovascular disease compared to the established algorithm.In concrete examples, X. Tang and L. ShuPersonalized medicine is about applying the appropriate treatment and avoiding unnecessary interventions, and consequently providing accurate and cost-efficient care. Through grouping patients based on their genetic background, past medical history, current health state, it is sought to predict the optimal medical decision. Moreover, through the collection of large data sets from single individuals, predictions on patient-specific disease progression, therapeutic success rate and limitations of certain interventions can be made.67. In this study, they have used Sotalol, which can cause Torsades de pointes, and by training algorithms to recognize early features, automated prediction in the early stages of disease development can be spotted and subsequent alarms activated. Indeed, subtle drug-induced ECG changes are difficult to recognize and machine aid in healthcare is a justifiable research direction to improve clinical decision. Computational approaches are suitable for assessing the heterogeneity of the diseases’ natural history. Extraction of the essential disease-features is the foundation of personalized and precision medicine.In light of this, data analysis is crucial; thus, the role of artificial intelligence is irreplaceable. For example, Badilini et al. trained a model which is able to detect drug-specific changes of the electrocardiogram, based on the wave’s morphology4.The results suggest that the method and prototype presented may be suitable for implementation on wearable sensing applications auxiliary for on-line, real-time diagnosisThe world-wide burden of chronic diseases is forcing healthcare providers to implement new strategies for accurate patient monitoring, meanwhile reducing hospitalization costs and physicians’ workload. Under these circumstances, technological advancements are making their print on providing medical services.67.Recent meta-analysis showed a near fifty percent drop for the time-to-treatment of the acute myocardial infarction when using pre-hospital triage with telemedicine67. In addition, to obtain more advantages of monitoring devices, these devices are compatible for integrations into a platform for automated development of databases. Eventually, stored data can be computationally embedded into a cycle of self-teaching algorithms and serve as a source of accessible raw material for research purposes.In the field of telecardiology, efforts are made towards automated real-time ECG diagnosis with wearable devicesFinally, a reasonable future trend is towards a 12-lead interpretation with real-time diagnosis and continuous algorithm improvement.Clearly, artificial intelligence application for biomedical signal processing cannot be tackled if there is a lack of perfect symbiosis of a multidisciplinary team involving clinicians, researchers and computer scientists.Summarizing, continuous cross-examination between machine intelligence and human intelligence is a combination of precision, rationale and high-throughput scientific engine integrated into a challenging framework of big data scienceAutonomic learning using empirical data can expand our horizons and nourish background knowledge with relevant hypotheses.Computational approaches are suitable to assess the heterogeneity of diseasesExtraction of the essential disease features by computational algorithms is the foundation of personalized and precision medicine.


Unimproved water, sanitation, and hygiene (WASH) behaviors are key drivers of infectious disease transmission and influencers of mental well-being. While WASH is seen as a critical enabler of health, important knowledge gaps related to the content and delivery of effective, holistic WASH programming exist. Corresponding impacts of WASH on mental well-being are also underexplored. There is a need for more robust implementation research that yields information regarding whether and how community-based, demand-side interventions facilitate progressive and sustained adoption of improved sanitation and hygiene behaviors and downstream health impacts. The purpose of this protocol is to detail the rationale and design of a cluster-randomized trial evaluating the impact of a demand-side sanitation and hygiene intervention on sustained behavior change and mental well-being in rural and peri-urban Amhara, Ethiopia.Andilaye. We randomly selected and assigned 50 sub-districts (kebeles) from three purposively selected districts (woredas); half to receive the Andilaye intervention, and half the standard of care sanitation and hygiene programming . During baseline, midline, and endline, we will collect data on an array of behavioral factors, potential moderators , and our primary study outcomes: sanitation and hygiene behaviors and mental well-being. We will perform a process evaluation to assess intervention fidelity and related attributes.Together with partners, we developed a theoretically-informed, evidence-based behavioral intervention called Andilaye, a scalable intervention designed to address these issues and complement existing service delivery within Ethiopia’s Health Extension Program. Evidence from this trial may help address knowledge gaps related to scalable alternatives to CLTSH and inform sanitation and hygiene programming and policy in Ethiopia and beyond.While CLTSH has fostered sanitation and hygiene improvements in Ethiopia, evidence of behavioral slippage, or regression to unimproved practices in communities previously declared open defecation free exists. Other limitations of CLTSH, such as its focus on disgust, poor triggering, and over-saturation of Health Extension Workers have been documented. We employed rigorous formative research and practically applied social and behavioral theory to develop NCT03075436) on March 9, 2017.This trial was registered with clinicaltrials.gov (The online version of this article (10.1186/s12889-019-7040-6) contains supplementary material, which is available to authorized users. Inadequate water, sanitation, and hygiene (WASH) are key drivers of infectious disease transmission and result in adverse mental and social well-being –4. DiarrMany WASH programs focus only on measures of infectious diseases or growth of young children to assess programmatic impact and public health relevance. This narrow focus is at odds with the World Health Organization (WHO)‘s definition of health as “a state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity” . Poor WAWhile WASH-related research generally focuses on the individual, communities are often a more critical point of investigation. Due to the dynamics of infectious disease transmission, the effects of WASH interventions often depend on community level coverage and uptake . FinanciDespite steady reductions in open defecation since 2000, Ethiopians have some of the poorest access to basic water and sanitation, and one of the highest levels of access inequity . The EthAndilaye Trial - Amharic for “togetherness/integration” - is to use behavioral theory and evidence from formative research to inform the design of a novel holistic community-based WASH intervention and evaluate its impact on sustained behavior change and mental well-being. The study’s primary research question is: To what extent can Andilaye’s enhanced, demand-side sanitation and hygiene intervention impact sustained behavior change and mental well-being, above and beyond CLTSH? We hypothesize that individuals in communities randomized to receive the Andilaye intervention are more likely to sustainably adopt improved sanitation and hygiene behaviors and demonstrate gains in mental well-being compared to individuals in communities randomized to the standard of care CLTSH programming.The objective of this study protocol is to provide a rationale for and details related to the design of our two-arm, parallel group cluster-randomized trail (CRT) evaluating the impact of an intervention designed to specifically address documented limitations of the CLTSH approach. The aim of the Andilaye Trial will be used to: (1) identify ways in which WASH-related behavior change components can be mainstreamed into the FMoH’s HEP; (2) determine the effectiveness of an enhanced holistic demand-side sanitation and hygiene intervention that promotes NTD-preventive improved WASH behaviors; (3) investigate whether changes in personal hygiene and sanitation behaviors are sustained, and which factors contribute to this; (4) document the cost-effectiveness of integrated, holistic WASH behavior change promotion; and (5) assess whether collective efficacy and water and sanitation insecurity modify intervention effectiveness.This study is designed to generate evidence to address knowledge gaps related to demand-side sanitation and hygiene programming and examine less studied, yet critical, inter-personal factors related to sustained behavioral adoption and downstream health impacts. Data from the kebeles from three woredas in West Gojjam and South Gondar Zones, Amhara National Regional State, Ethiopia. As indicated in further detail below, we randomly selected and assigned clusters; half to receive the Andilaye intervention, half to receive the current standard of care sanitation and hygiene programming . The intervention arm will receive Andilaye, a demand-side sanitation and hygiene intervention informed by social and behavioral theory and empirical evidence, particularly evidence generated during Andilaye’s formative research phase. The Andilaye intervention promotes selected, improved WASH behaviors and constituent practices deemed locally appropriate for inclusion in a demand-side intervention. The intervention focuses on positive, community-oriented motivators of behavioral change, promotes achievable incremental improvements, and incorporates strategies that facilitate behavioral maintenance . We designed the Andilaye intervention to integrate behaviors that could contribute to improvements in mental and social well-being and the control of infectious diseases, including trachoma and soil-transmitted helminthiasis. The control arm  will receive FMoH’s existing CLTSH programming, and no attempt will be made to modify the government’s roll-out of these interventions or the Health Extension Services Package. The study team will work with government partners to minimize contamination from other WASH interventions in our study sites, to the greatest extent possible.Emory University and its consortium partners are conducting a two-year impact evaluation, designed as an ex-ante two-arm, parallel CRT. Clusters are defined as rural or peri-urban Andilaye intervention, as implemented; the extent to which participant engagement and dose response are associated with the dose delivered; and the social, political, and economic factors that may influence intervention implementation. We will take a mixed methodological approach to collect process data over the Andilaye study period. We will collect quantitative data via structured household-level surveys, activities observations, post-training assessments, and systematic audits of records. We will collect qualitative data via semi-structured interviews and informal discussions with key informants  and participant observations during relevant trainings and intervention activities. These data will allow us to better interpret our impact evaluation data and determine how and why change did or did not occur .We will conduct a process evaluation alongside our impact evaluation to assess intervention implementation fidelity, participation and dose response, and contextual factors. The purpose of the process evaluation is to determine the quality and integrity of the Andilaye Trial consists of three major phases: (1) formative research and intervention design, (2) intervention implementation and process evaluation, and (3) impact evaluation  model [Theory of Triadic Influence [Risks, Attitudes, Norms, Abilities, and Self-Regulation (RANAS) approach [Kebeles in which formative research activities were conducted were similar to kebeles eligible for the Andilaye Trial; however, in most circumstances, the kebeles were not deemed eligible for inclusion in the study given their involvement in the formative research. Prior to finalizing the design of the content and implementation strategies employed by the Andilaye intervention, we conducted a month-long behavioral trial  in formative research communities to provide an opportunity for targeted change agents and intervention recipients to provide their feedback regarding intervention functions . The finalized Andilaye intervention addresses issues related to the over-extension of HEWs and over-saturation of messaging via the HEP’s Health Extension Services Package through the engagement of WDALs and additional community change agents as mechanisms for intervention delivery. The intervention leverages recent work conducted by the World Bank, and incorporates feedback from relevant stakeholders, including FMoH, ARHB, Zonal Health Departments, Woreda Health Offices, other non-governmental organizations, and individuals such as formative research community members.We employed a rigorous intervention design approach that leveraged social and behavioral theory, formative research results, intervention mapping , and inpB) model , the Thenfluence , and theapproach . KebelesAndilaye’s intervention motto, “Together we can be a strong, caring, healthy community”, and related intervention components offer aspirational messages that emphasize the need for collective action to make positive change in the community and use verbal persuasion to enhance collective efficacy perceptions. The intervention focuses on three behavioral themes, informed by formative research: (1) sanitation, (2) personal hygiene, and (3) household environmental sanitation. Within these themes are 11 constituent practices targeted by the intervention; these practices were identified through formative research and the intervention design process as ones that could be targeted using demand-side approaches, and were seen as achievable, per stakeholder feedback  comprise the intervention’s primary target audience, we designed the intervention to address inter-personal behavioral factors and promote behavior change among all household members and the community at large. The logic model presented in Additional file Andilaye intervention commences with district-level capacity building activities, such as action planning and training of trainers and intervention activity facilitators. Further, district-level refresher trainings and adaptive management activities are conducted to reinforce previously acquired knowledge and skills, address trainer/facilitator turnover, and review successes and address challenges faced in implementing group and household level activities. Community-level activities include the ‘Whole System in the Room’ [Prior to activity roll out in the community, the he Room’   sanitation, (2) personal hygiene, and (3) household environmental sanitation, more specifically, the 11 constituent practices identified above; andMental well-being, as measured through standardized mental well-being scale scores generated via the administration of the WHO-5  and HopkPrimary outcomes of interest include:Short-term behavioral outcomes, measured as the proportion of households with improved (private or shared) latrines and washing facilities that are functional and available for use; proportion of households using functional latrines and washing facilities; proportion of households with all members exclusively using a latrine for defecation; proportion of households disposing of child feces in an improved latrine; proportion of households with all children in the household with a clean face and hands;Intermediate behavioral antecedents, measured as the proportion of households with improved knowledge regarding the implications of improved WASH practices ; proportion of households that indicate positive attitudes, perceptions toward improved sanitation and good hygiene practices; change in normative expectations related to open defecation, exclusive latrine use for defecation, and personal hygiene practices;Diarrhea period prevalence, as measured through caregiver self-report;Sanitation insecurity, as measured through changes in sanitation insecurity scale scores ;Collective efficacy measures, as measured through changes in collective efficacy scale scores ; andWater insecurity, as measured through changes in water insecurity scale scores .Secondary outcomes of interest include:kebele study cluster, or 1500 households in total .We powered this study on mental well-being outcomes, as measure by the HSCL , which rFollowing baseline, we conducted an ex-post power calculation on our main outcome. Given the prevalence of poor well-being , those criteria were dropped from inclusion requirements prior to baseline data collection. As such, any kebele meeting the previously stated eligibility criteria was eligible, regardless of sanitation coverage and utilization or previous CLTSH triggering or ODF verification status.Rural and peri-urban kebeles within the sampling frame. The primary sampling unit for this study was the kebele. The secondary sampling unit for this study was the household; specifically, any household residing in a targeted, sentinel village (gott) within a randomly selected study kebele. While we randomly selected eligible study clusters , we purposively selected gott(s), from which we randomly selected study households. We utilized a ‘fried egg’ [gotts that were either situated in or near the center of the kebele (if there were centric gotts) or were not adjacent to any other study kebele (in the event there are no centric gotts). This approach minimized spill-over of intervention effects and other externalities associated with the research between intervention and control clusters, especially those adjacent to each other. The number of targeted gotts depended only on the number of eligible households identified in gott census books. Study household inclusion criteria for the Andilaye Trial included any household randomly selected from the gott census book residing in the target gott(s) that: (1) had at least one child aged 1–9 years at baseline  and consented to allowing study staff to observe the children, specifically their faces and hands, and (2) provided consent to participate in the study, with at least one adult household member consenting to serve as the primary survey respondent.We employed a structured sampling strategy to randomly select eligible ied egg’  approachkebele sampling frame, we employed a random number generator to identify 50 eligible kebeles clusters from across the three woredas targeted for our study. Given each of the three woredas vary with regard to their hydrogeological conditions and the size and number of kebeles, we used a stratified selection approach (at the woreda level). Of the 50 clusters, 22 were selected from Farta, 12 from Fogera, and 16 from Bahir Dar Zuria. Proportionally, these selected kebeles represented 51 (22/43), 38 (12/32), and 50 (16/32) percent of all kebeles in Farta, Fogera, and Bahir Dar Zuria, respectively. An even number of clusters were selected from each woreda to ensure an equivalent sample size between the intervention and control clusters selected from each woreda. Fogera had a slightly lower proportion of selected kebeles due to accessibility concerns given the frequency of floods in the low-land, marshy areas close to Lake Tana  of study clusters have been triggered with CLTSH but have not yet been certified ODF, and the remaining 8% (4 of 50) have not been triggered with CLTSH, as of the commencement of this study, though they are slated for triggering.From the Recruitment took place within the home compounds during baseline data collection. Enumerators contacted adult members of the household; they explained the purpose of the visit, the purpose of the study, and asked the respondent if s/he would be willing to consent to participate in the study using a structured consent form . Within each stratum (woreda), researchers from Emory University in Atlanta, USA used a computer-based random number generator to generate a random number between zero and one for each kebele cluster, and then placed the clusters in ascending order by their randomly generated numbers. We then partitioned the communities within each woreda into two equal sizes, assigning the first half of kebeles to the intervention arm and the second half to the control arm. We used replacement re-randomization [a prior criterion. Results from our equivalence analyses indicated balance in the number of previously triggered and ODF certified kebeles, between study arms, with 20 triggered and ODF kebeles in the intervention arm  and 19 in the control arm . Figure Following baseline data collection, we used a stratified random design to assign study mization  to securAndilaye intervention is being implemented in collaboration with the Woreda Health Offices, HEWs, WDALs, and other community change agents in the 25 kebeles randomly selected to the Andilaye intervention arm. While allocation occurred at the kebele level, intervention activities and data collection occur in one to two sentinel gotts per kebele, purposively selected to minimize spillover. The counterfactual, or control arm, includes households from gotts within randomly selected kebeles receiving the FMoH’s standard of care CLTSH programming. As this study is operating in an area where CLTSH is being rolled out nationally, we are not interfering with established CLTSH roll-out and implementation protocols. While we cannot be sure that our control communities will not receive further CLTSH interventions during the course of the trial, any such further implementation of the current CLTSH interventions would only bias effect estimates toward the null.The Survey instruments administered at baseline and follow-up data collection rounds (midline and endline) consist of several modules aimed at collecting data on key outcome indicators through reports from respondents and other household members  to improve accuracy of data entry and enable immediate review of results. Data is stored securely using the freely available Open Data Kit (http://opendatakit.org/). Logic, range, and consistency checks were incorporated into the electronic data collection file to further improve data quality and minimize data entry errors. To ensure data quality, the supervisory team, comprised of faculty and staff from Emory University and Emory Ethiopia , will coordinate and supervise data collection along with field supervisors. Field supervisors independently assess all objective measures  at 10% of households in each study kebele. Supervisor and enumerator data captured from this sub-set of households will be compared to determine inter-rater reliability of related metrics.The Andilaye intervention clusters, which may signal treatment allocation.All enumerators and field supervisors are external evaluators who are hired for discrete data collection activities . Study staff will not disclose study cluster treatment allocation to field supervisors or enumerators. However, given the nature of the intervention, they may observe intervention materials in kebele, or (3) no child aged 1–10 years (at midline) or aged 1–11 years (at endline) resides in the household any longer. Households with no eligible respondent available after three attempts during midline data collection will continue to be enrolled in the study, and will be visited for subsequent quarterly monitoring and endline data collection. A sub-set of key data points will be collected during quarterly monitoring, however, we will seek to minimize reactivity by only engaging half of all households in each cluster per quarter, such that we visit each household for monitoring only once per annum.We will measure and track individual, household, and community-level changes in key outcomes over multiple time points . Households enrolled in the study are lost to follow-up if: (1) consent to participate in our follow-up survey was refused, (2) the entire household moved out of the study Our baseline analyses will establish reference measures for follow-up analyses. We conducted bivariate assessments of balance between study arms at baseline Table . As prevkebele clusters randomly selected for inclusion in the Andilaye Trial have been triggered with CLTSH, and certified ODF, provide strong evidence that behavioral slippage is, indeed, an issue that needs to be addressed in Amhara and perhaps elsewhere in Ethiopia.At baseline, sanitation and hygiene conditions were found to be generally poor Table . Only 40Our key health outcome, mental well-being, was shown to be poor, per the WHO-5 scale, amongst approximately one-third of respondents at baseline Table . The basworeda indicator variable [kebeles.The primary method of analysis for all primary and secondary outcomes will follow an “intention-to-treat” analysis, which compares the intervention arm to the control arm, without regard to intervention fidelity or compliance. For binary outcomes, such as our targeted WASH behaviors, we will preferentially use log-linear binomial regression models. For continuous outcomes, such as the mental well-being scale scores, we will use linear regression models. All models will include an intervention variable as a fixed effect, and account for the stratified design through the inclusion of the variable , and incThe majority of our primary and secondary outcomes . We have performed such analyses in other studies [Sub-optimal adherence, due perhaps to low uptake of interventions by participants or to poor implementation of the interventions, can lead to trial results that do not reflect the true efficacy of WASH. The intention-to treat analyses show a valid and unbiased causal effect for the effectiveness of a specific WASH program/intervention, but do not show the efficacy of WASH under ideal circumstances. If fidelity and adherence are heterogeneous, we will supplement our primary intention-to-treat analyses with an analysis that attempts to assess the impact of adherence to the  studies , 55.Andilaye, a demand-side sanitation and hygiene intervention, on sustained adoption of improved sanitation, personal hygiene, and household environmental sanitation behaviors and mental well-being above and beyond current CLTSH interventions. As outlined below, there are several documented limitations of CLTSH in Ethiopia, and WASH programming more broadly, that the Andilaye intervention was designed to address.CLTSH, the key approach to improving sanitation coverage and utilization in Ethiopia, has facilitated considerable changes in coverage of basic sanitation. However, some of these gains have not been sustained, and incremental improvements to improve sanitation  have not been widely promoted or achieved .WASH programs, more broadly, have focused on catalyzing initial behavior change, and have placed little, if any emphasis on the habituation of improved behaviors and behavioral maintenance. Such approaches have fostered behavioral slippage, or regression back to unimproved behaviors and practices, and poor sustainability of behavioral outcomes and potential health impacts .CLTSH largely focuses on leveraging shame to change norms around open defecation, but these negative affective motivators may not be the most appropriate or effective drivers of change, and may actually erode mental well-being. As indicated by our formative research, focus on negative affective motivators, poor facilitation of initial triggering, and a lack of post-triggering follow-up has left many communities with negative impressions of CLTSH initiatives.Behaviors and facilities promoted by existing programs are aspirational but require considerable effort and/or capital investment to achieve. A focus on small, incremental improvements in WASH practices and facilities may be viewed as more achievable by program participants, particularly in low resource settings, and as such, may garner greater success.HEWs charged with implementing CLTSH have many responsibilities, few tools, and little capacity to continually reinforce messages . AlthougSiloed approaches within the health and development sectors, namely WASH and those vertical programs involved in the control and elimination of NTDs, prevent the integration and harmonization of NTD and WASH behavior change initiatives . For insWhile the focus on diarrheal disease prevention and growth faltering have driven investments in WASH, recent evidence suggests that, in sub-Saharan Africa, basic improvements may not be enough to impact these health outcomes , 59. HowThis study will evaluate the impact of Andilaye intervention reflects a potential alternative to CLSTH, and evidence generated by the Andilaye Trial may serve to address several knowledge gaps related to integrated and holistic community-based, demand-side sanitation and hygiene programming in Ethiopia and beyond. Few community-based WASH interventions have incorporated specific strategies to enhance behavioral maintenance capacity, and limited evidence exists regarding the influence of various inter-personal factors on sustained behavioral change. Evidence generated by this CRT may also indicate how  interventions can bring about sustained improvements in sanitation, hygiene, and less studied health outcomes, such as mental well-being.The Andilaye intervention. Our mixed methods process evaluation will allow us to assess intervention fidelity and adherence, and document any potential bottle-necks that may threaten the intended roll-out of intervention activities. Evidence from the process evaluation will help us interpret the results from the Andilaye Trial. Policy-relevant findings and related recommendations regarding the feasibility and effectiveness of the Andilaye intervention will be shared broadly when endline data have been collected and analyzed.Limitations of this CRT include reliance on local government partners to implement and supervise the implementation of the Additional file 1:Figure S1. Depicts the Andilaye logic model. (DOCX 250 kb)Additional file 2:Table S1. Summary of Andilaye activities. (DOCX 14 kb)Additional file 3:Table S2. Sample size calculations. (DOCX 13 kb)Additional file 4:Andilaye Trial consent form. (DOC 65 kb)Additional file 5:Table S3. Key Andilaye Trial indicators used to assess behavior and health outcomes. (DOCX 14 kb)Additional file 6:Table S4. Sub-set of Andilaye survey prompts and answer choices. (DOCX 51 kb)


This review highlights recent advances in the understanding of the relevance of altered lipid metabolic pathways contributing to the poor prognosis of high grade serous ovarian cancer, as they relate to cancer metastasis and cancer stemness. Increased lipid uptake regulated by the receptor CD36 and the transport protein FABP4 has been implicated in ovarian cancer metastasis. The symbiotic relationship between ovarian cancer cells and adipocytes was shown to be important for sustaining widespread peritoneal and omental metastasis. Increased lipogenesis dependent on the fatty acid desaturase SCD1 was detected in ovarian cancer stem cells. Furthermore, response to therapy, specifically to platinum, was linked to increased fatty acid biogenesis, while the survival of drug tolerant cells was shown to depend on lipid peroxidation. These recent findings suggest that lipids are necessary elements supporting oncogenic signaling and the energetic needs of rapidly proliferating cancer cells. New strategies targeting key enzymes involved in lipid uptake or utilization in cancer cells have been shown to exert anti-tumor effects and are being developed as cancer interventions in combination with chemotherapy or immunotherapy. Epithelial ovarian cancer (OC), an aggressive tumor with origins in the fallopian tube epithelium , is charMany mechanisms have been implicated in development of acquired platinum resistance, including export pathways, epigenetic modifications, and alterations in DNA damage response. More recently, it has been hypothesized that a key phenomenon implicated in disease recurrence in OC and other solid tumors is the persistence of cancer stem cells (CSCs), which are quiescent and therefore can escape the effects of cytotoxic therapy, survive and under the stimulation of certain factors in the peritoneal environment, eventually become reactivated and give rise to recurrent tumors, which are heterogeneous and highly treatment-resistant ,10,11. UMetabolic alterations have been associated with rapid growth of tumors, early propensity to metastasize, development of resistance to therapy, and survival of CSCs. While abnormal glycolysis and glucose metabolism are well understood in these contexts, new data related to the role of lipid metabolism in cancer are emerging. Due to the almost symbiotic relationship between OC and the fat containing cells in the omentum, alterations of the lipid metabolism in HGSOC remain of high interest both to advance the understanding of the mechanisms that fuel peritoneal dissemination, and to identify potential new targets for therapeutic interventions. Here, we review recent advances in the field related to lipid metabolic pathways altered in cancer of specific relevance to HGSOC.Lipids are hydrophobic biomolecules which include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids, and prenol lipids . Three mLipids are imported into cells through a variety of fatty acids transporters present on the plasma membrane ,15. ThesLipogenesis is the pathway by which lipids are generated in the cell from other metabolites. This pathway is typically activated when exogenous lipids are unavailable for import, such as during starvation or energy shortage. In these situations, the cell uses alternative metabolic units to build fatty acids. For example, cytosolic citrate resulting from glutamine metabolism and citric acid cycle downstream of glucose metabolism is cleaved by ATP-citrate lyase into acetyl-CoA . Acetyl-2 and NADH as small and mobile energy storage units that participate in different biochemical processes including the generation of ATP via the electron transport chain [Fatty acids represent an important source of fuel in the cell, which is released through oxidation, tightly regulated by several enzymes. Free fatty acids in the mitochondrial inter-membrane space are converted into acyl-CoA by ATP-dependent acyl-CoA synthetase. Carnitine palmitoyl transferase 1 tags fatty acyl-CoA with carnitine to form fatty acyl-carnitine which is transported into the mitochondrial inner membrane via carnitine-acylcarnitine translocase. Once inside the mitochondrial matrix, fatty acyl-CoA is reformed by carnitine palmitoyl transferase 2 with free CoA. The subsequent fatty acid β-oxidation strips one acetyl-CoA group off fatty acyl-CoA at a time, generating FADHrt chain . Through+ oral carcinoma cells could initiate tumor metastasis more effectively than CD36− cells, while retaining the same ability to generate tumors at the primary site. Interestingly, tumor metastasis was augmented by a fat rich diet, in a CD36-dependent manner. Strategies targeting CD36 by using neutralizing antibodies decreased the metastatic potential [Cancer cells utilize various strategies to boost lipid uptake in order to fulfil their high energetic needs for cell growth and altered oncogenic signaling. Alterations in lipid uptake were described in different cancer types, including HGSOC. For example, breast cancer cells were shown to import exogenous oleic acid through upregulated CD36 and this mechanism alleviated the apoptotic effects induced by SCD1 inhibition . The verotential , pointinThe role of lipid uptake has also been studied in ovarian cancer. Recognizing that the preferred site of metastasis in ovarian cancer is the omentum, a fat rich organ, Lengyel’s group characterized the symbiotic relationship between adipocytes and ovarian cancer cells . Direct A more recent report focusing on lipid uptake demonstrated the significance of the CD36 transporter mediated fatty acid transport into ovarian cancer cells . The autWhile the excessive anaerobic metabolism of glucose, i.e., the Warburg effect ,37 in caAdditionally, fatty acid desaturation, a critical step in the process of lipogenesis, is essential for the maintenance of membrane fluidity, inter- and intra-cellular signaling, and provision of lipids for energy generation through oxidation . NotablyRecently, our group developed a new imaging approach based on Raman spectroscopy that allowed us to probe the different lipid species in rare cell populations, such as stem cells ,51. By uOur current understanding of fatty acid desaturation supports that SCD1 is the sole enzyme responsible for converting saturated fatty acid to monounsaturated fatty acid. However, very recently, Vriens et al. discovered an alternative fatty acid desaturation pathway, which is operative in multiple cancer types, including lung, breast, prostate, and liver cancer . In thosFurthermore, while lipid unsaturation is critical to the survival of cancer stem cells, it has also been linked to normal stem cell physiology. FASN was found to be upregulated in neural progenitors, where suppression of its activity affected normal neurogenesis . EmbryonAbnormalities in lipid metabolism have been linked to several cancer phenotypes, including metastatic potential, cancer stemness and resistance to chemotherapy. Specific alterations and the cellular mechanisms engaged in these contexts are reviewed bellow.Advanced stage ovarian cancer is typically characterized by omental or peritoneal metastasis, which leads to the typical complications of the disease, ascites and bowel obstruction. The omentum is mainly comprised of adipocytes which serve as a chemical attractant for ovarian cancer cells. Nieman et al. have identified that the interleukins, IL-6, IL-8, monocyte chemoattractant protein-1, and tissue inhibitor of metalloproteinases-1 are released by the omentum to promote dissemination of ovarian cancer cells . SpecifiAside from metastatic ovarian cancer, altered lipid metabolism was also observed as a regulator of tumor metastasis to lymph nodes in melanoma by Lee et al. . The autCancer stem cells represent a small population residing within heterogeneous tumors and have been reported to mediate ovarian cancer initiation ,63,64. TA recent report from our group utilized stimulated Raman Scattering Microscopy to study ovarian cancer stem cells and found that these cells harbor higher level of unsaturated fatty acids and increased levels of the SCD1 desaturase mRNA as compared to non-stem cancer cells . SuppresIn order to identify the molecular mechanisms linking unsaturated fatty acids and ovarian cancer cell stemness, ovarian cancer stem cells treated with vehicle and an SCD1 inhibitor were probed by using a pathway-specific quantitative RT-PCR array. Transcriptional activity of NF-κB was found to be compromised in ovarian cancer stem cells upon suppression of SCD1 desaturase activity. Furthermore, the NF-κB signaling pathway was shown to directly regulate the transcription of SCD1, through a positive feedback loop. It is still not clear whether the pathway is activated directly or indirectly by the increased amounts of unsaturated fatty acids in this context.These observations were corroborated in other cancer types, such as colon and breast cancer ,72 as weBased on the significance of lipid metabolism to cancer stem cells, it was reasonable to hypothesize that similar deregulation is associated with development of resistance to chemotherapy. A report using used ovarian cancer cell lines Hey, Igrov-1, and SKOV-3 as a relative model for different levels of cisplatin-resistance, i.e., sensitive, intermediate-resistant and resistant, described the potential role of lipogenesis to this process . Pre-treThe role of fatty acid metabolism was also studied in other cancer types. For example, Wu et al. found that FASN mediated cellular responses to cisplatin treatment in breast cancer cells . The undFurthermore, recent reports have suggested that drug-tolerant cells adopt a mesenchymal state and are dependent on lipid peroxidation mediated by the lipid hydroperoxidase GPX4 . TargetiGiven the importance of lipid metabolism to tumor progression, in the past few years efforts have been channeled towards the development of small molecule inhibitors targeting different enzymes involved in this metabolic network. The compounds listed in Acetyl-CoA carboxylase is indirectly targeted by metformin and directly blocked by specific inhibitors ND-646 and ND-654. The latter are being studied in preclinical models of lung cancer and hepatoma, while metformin has been studies extensively in combination with chemotherapy in clinical trials in breast, ovarian, and endometrial cancer or as a preventive cancer agent. Multiple inhibitors are also under development for SCD1 and SREBP1; however, none has yet entered the clinical arena. Inhibitors for ATP citrate lyase are being developed as cholesterol lowering drugs, with the most advanced being ETC-1002, a first in class compound, which completed phase III studies, demonstrating efficacy in blocking cholesterol synthesis . EvaluatCPT1 is the rate limiting enzyme controlling fatty acid oxidation, which is a major source of energy for several tumor types. The best studied CPT1 inhibitor is etomoxir which has been evaluated in preclinical models of prostate, breast, and bladder cancer and leukemia . EtomoxiInterestingly, inhibition of lipid metabolism can also affect normal cells, particularly the immune system, including T cells, tumor-associated macrophages, regulatory T cells and myeloid derived stromal cells (MDSCs). An example is the fatty acid transport proteins which are upregulated in MDSCs, cells with immunosuppressive roles in tumors . By reduIn summary, emerging reports support the role of lipid metabolism in cancer stemness and metastasis. Several alterations in key molecules regulating pathways involving lipid uptake , lipid synthesis (FASN) or desaturation (SCD1), or fatty acid oxidation (CTP1) have been described in ovarian cancer and linked to peritoneal dissemination, survival of stem cells and response to chemotherapy. Development of strategies targeting these key enzymes or lipid transporters, alone or in combination with other biologics or chemotherapy, is ongoing.