The reinforcement of resins with short or long fibers has multiple applications in various engineering and biomedical fields. The use of fiber reinforced composites (FRCs) in dentistry has been described in the literature from more than 40 years. In vitro studies evaluated mechanical properties such as flexural strength, fatigue resistance, fracture strength, layer thickness, bacterial adhesion, bonding characteristics with long fibers, woven fibers, and FRC posts. Also, multiple clinical applications such as replacement of missing teeth by resin-bonded adhesive fixed dental prostheses of various kinds, reinforcement elements of dentures or pontics, and direct construction of posts and cores have been investigated. In orthodontics, FRCs have been used also for active and passive orthodontic applications, such as anchorage units, en-masse movement units, and postorthodontic tooth retention. FRCs have been extensively tested in the literature, but today the advances in new technologies involving the introduction of nanofillers or new fibers along with understanding the design principles of FRC devices open new fields of research for these materials both in vitro and in vivo. The present review describes past and present applications of FRCs and introduces some future perspectives on the use of these materials. Fiber-reinforced composites (FRCs) have been studied for biomedical applications for over 40 years  and wereThe incorporation of fibers into the organic matrix provides material-specific characteristics. Fiber bundles can be discontinuous or continuous, with randomly directed or directional fibers. The strongest FRC devices are typically made of continuous unidirectional fibers . Fibers Some FRCs are hand fabricated, with a polymeric matrix added to the fibers at chairside. This approach might not produce an effective composite, because coupling between the fiber and the polymer might be inadequate and leave voids. On the other hand, partial- or full- preimpregnated FRCs are partially or fully polymerized continuous long fibers, which offer superior properties, because they combine both polymer and fibers .Reinforcement of polymers with long, continuous fibers is an effective mean for engineering materials for many applications. FRCs have been proposed in many fields in dentistry for different purposes, namely, prosthodontics, endodontics, conservative dentistry, orthodontics, periodontology, and paediatric dentistry . Previou  TITLE-ABS-KEY ( fiber AND reinforced AND composite )A broad search on Scopus Database has been conducted using the following MeSH terms: The search strategy included an initial analysis of the results in the specific Scopus sections dedicated to the different document types, thus allowing to highlight the kind of document . No exclusion criteria have been applied in order to provide a whole publications count.  ( TITLE-ABS-KEY ( fiber AND reinforced AND composite ) ) AND   ( TITLE-ABS-KEY ( fiber AND reinforced AND composite ) ) AND ( prosthodontics )  ( TITLE-ABS-KEY ( fiber AND reinforced AND composite ) ) AND ( endodontics )  ( TITLE-ABS-KEY ( fiber AND reinforced AND composite ) ) AND ( conservative AND dentistry )  ( TITLE-ABS-KEY ( fiber AND reinforced AND composite ) ) AND ( orthodontics )  ( TITLE-ABS-KEY ( fiber AND reinforced AND composite ) ) AND ( periodontology )  ( TITLE-ABS-KEY ( fiber AND reinforced AND composite ) ) AND ( paediatric AND dentistry )Furthermore, the analysis has been refined with the function “search within results,” with the following MeSH terms for each discipline considered in the investigation:The results of this research revealed that, today in the literature, more than 80.000 documents have been published on FRC materials when Scopus-indexed journals are considered. Based on the published material, it could be stated that the main subjects of investigation were engineering, materials science, physics, and chemistry. In total, 1797 studies have been reported in medical field, of which 1473 were on dental related topics. This remarkable production mainly consists of original articles (1333). Other contributions are conference papers (62), reviews (45), and book chapters (19). The main part of this type of research (1444 documents) was published in sources that require university/hospitals special access or consultation under payment, whereas only 29 documents were free access with an open access route. The research on FRC materials in dentistry seemed to start in 1975 , 27. HowAmong various dental fields, the main topic of published material was on material properties (1294) where 897 documents were on prosthodontics, 448 on endodontics, 215 on conservative dentistry, 194 on orthodontics, 164 on periodontology, and 132 on paediatric dentistry . Many stThe main application of FRCs in dentistry is related to provisional or definitive prosthodontics. By using FRCs, FDPs and veneers can be realized in a minimal invasive fashion, utilizing combinations of various kinds of adhering and retentive elements . A resinFRC FDPs could be fabricated as surface-retained, inlay-retained, or full coverage crown retained prostheses . The fabThe results of mechanical  and adheThe applications of FRCs in conservative dentistry mainly consist of direct composite restorations. The advantages of the use of FRCs over conventional filling materials are related to their biomimetic properties. In fact, the dental restorations ideally would be as minimally invasive as possible and substitute the missing hard dental tissues resembling mechanical features and properties of natural teeth . FollowiSuperior mechanical properties of FRCs could improve their bond durability with universal adhesives, even if there is little evidence comparing the bond durability of FRC to dentin with that of other composite resins . On the In endodontic clinical practice, the use of FRCs is mainly reported as root canal anchoring system. Studies evaluated both prefabricated and individualized FRC posts , 41–43. Generally, FRCs present limited radio-opacity due to the low concentration of radio-opaque elements. This shortcoming of E-glass fiber would limit its application in dentistry as sufficient radio opacity is highly desirable for dental materials. The addition of synthesized iodine containing a new methacrylate monomer HMTIB has been tested to increase the radio opacity of FRCs with the results showing that FRCs present higher radiopacity than natural tooth enamel .Finally, in the field of endodontics, FRCs showed excellent integration with other new technologies such as laser applications  and CAD/The main use of FRCs in clinical orthodontics is as fixed retention . After oA great advantage of FRC splints over conventional metallic retention is aesthetics. Fibers are barely invisible and do not affect the translucency of teeth . This aspect is important, considering the higher number of adult patients who request an orthodontic therapy. Finally, FRCs are metal-free and are indicated for adult and young patients screened by Nuclear Magnetic Resonance or in subjects allergic to metals. On the other hand, FRC splints are more rigid than conventional metallic splints, thus leading to a higher ankyloses risk of teeth involved. However, the application of FRC with a spot-bonding technique has been proposed, in order to reduce framework rigidity, thus allowing physiologic tooth movement .Clinical success of FRC resins has been reported also for space maintainer purpose . The earIn addition to stabilization uses, in orthodontics, FRCs have been proposed also for active tooth movement. Groups of two or more teeth can be splinted with FRCs and moved “en masse” with sectional mechanics .One other application of FRCs has been proposed as innovative materials for fabrication of brackets  and wirePeriodontal or posttraumatic FRC splints have been reported in clinical periodontology. Splints are used to stabilize teeth, which have become loose as a result of supporting bone loss as a consequence of periodontal disease. The main advantage of stabilization splints is the reduction of tooth mobility. . FRC perIn paediatric dentistry FRCs can be used in almost all the fields as described above: restorations, space maintainers, splints, or other frameworks . The maiThe use of FRCs has been recently reported also in oral and maxillofacial surgery. These materials can be applied in oral implantology for bone replacing and bone anchoring implants. The rationale for this application is that, although metal implants have successfully been used for decades, devices made out of metals do not meet all clinical requirements. Metal objects may interfere with some medical imaging systems, while their stiffness also differs from natural bone and may cause stress shielding and overloading of bone. Glass fibers are responsible for the load-bearing capacity of the implant, while the dissolution of bioactive glass particles supports bone bonding and provides antimicrobial properties for the implant .Moreover, FRCs materials can be used in maxillofacial discipline for orbital floor implants , craniopThe main advantages of the use of FRCs over conventional materials are mainly due to their easy manipulation and high mechanical properties especially in dynamic loading conditions. For many FRC applications, no or minimal laboratory work is needed and often frameworks can be prepared at chairside, directly in the oral cavity . The othThe main limitations of FRC clinical use are that, even though many in vitro studies have been conducted, research is still lacking regarding long-term clinical performance. The most important weakness of FRC is the interface between the fiber and the organic matrix. Intraoral hydrolysis and degradation weaken this interface and failure can occur. Maybe this might also be a reason for missing long-term results.Principal failure reasons of FRC devices are fracture and delamination but such events could be easily repaired with resin composite materials .Finally, the higher cost than unreinforced or metallic materials is a factor that has to be considered for a global evaluation of FRC employment.Future research on FRCs needs to focus on many aspects such as optimization of the design of the frameworks in FRC devices , incorpoAnother improvement is related to nanotechnology, with the production of functional structures in the range of 0.1-100 nm by various physical or chemical methods. Dental nanocomposites provided a cosmetically acceptable result with excellent mechanical properties , 20. TheFRC utilization has been proposed also in combination with Computer-Aided-Design/Computer-Aided-Machining (CAD/CAM) technologies. The interaction between the two technologies seems to be promising based on limited information .One other field where FRCs are starting to be utilized is implantology. Implant applications could benefit from certain biomechanical properties of FRCs, and the possibility of incorporating additional bioactive components into the implant structure may open new research fields .FRCs have been suggested for tissue engineering for orthopaedic scaffolds . As biocThe research options with FRC materials are open and future reports about the topic are expected to widen FRC utilization in both dental and medical fields.


Human induced pluripotent stem cells (iPSCs) have emerged as an effective platform for regenerative therapy, disease modeling, and drug discovery. iPSCs allow for the production of limitless supply of patient-specific somatic cells that enable advancement in cardiovascular precision medicine. Over the past decade, researchers have developed protocols to differentiate iPSCs to multiple cardiovascular lineages, as well as to enhance the maturity and functionality of these cells. Despite significant advances, drug therapy and discovery for cardiovascular disease have lagged behind other fields such as oncology. We speculate that this paucity of drug discovery is due to a previous lack of efficient, reproducible, and translational model systems. Notably, existing drug discovery and testing platforms rely on animal studies and clinical trials, but investigations in animal models have inherent limitations due to interspecies differences. Moreover, clinical trials are inherently flawed by assuming that all individuals with a disease will respond identically to a therapy, ignoring the genetic and epigenomic variations that define our individuality. With ever-improving differentiation and phenotyping methods, patient-specific iPSC-derived cardiovascular cells allow unprecedented opportunities to discover new drug targets and screen compounds for cardiovascular disease. Imbued with the genetic information of an individual, iPSCs will vastly improve our ability to test drugs efficiently, as well as tailor and titrate drug therapy for each patient. The groundbreaking discovery by Shinya Yamanaka and colleagues that a set of four transcription factors (Oct4/Sox2/c-Myc/Klf4) can induce reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has revolutionized the field of biomedical research, providing an accessible, versatile, and adaptable platform for precision medicine . iPSCs gDrug discovery requires years of preclinical research. After a compound is synthesized, it must be rigorously tested in preclinical studies . CurrentAlthough animal models and primary cell lines are the most common methods for establishing efficacy and safety in preclinical drug trials, there are significant problems with each approach. Animal model systems are inherently limited due to fundamental species differences in physiology, reproducibility, ethical concerns, and a poor correlation with human clinical trial data . For exaAs an alternative to animal models and primary cells, iPSC technology has triggered a paradigm shift in the drug discovery and clinical trial landscapes. iPSCs circumvent many of the problems associated with animal and primary cell models and allow for the mass production of disease- and patient-specific functional somatic cells . In thisThe generation of cardiovascular cells derived from patients allows individualized disease modeling, drug screening, and drug testing. Currently, there are two approaches to generate patient-specific pluripotent stem cells from which cardiovascular cells can be derived: 1) generation of iPSCs by ectopic overexpression of the Yamanaka transcription factors, and 2) somatic cell nuclear transfer–derived embryonic stem cells using enucleated human oocytes, a more technically challenging and ethically fraught procedure . GeneticiPSCs can give rise to a wide variety of cell types, including cardiomyocytes (CMs) and vascular cells. iPSC-derived CMs (iPSC-CMs) provide a platform to effectively study patient- and disease-specific heart disease conditions in vitro, including Long QT syndromes , hypertrOver the past decade, low-cost methods to derive CMs from iPSCs with high efficiency (>90%) have been developed , enablin+, NANOG+), to mesodermal lineage , to cardiac progenitor lineage , and finally to relatively immature beating CMs   . Severalβ inhibitor CHIR99021 is used to promote differentiation of iPSCs to mesodermal lineage, followed by a treatment with the canonical Wnt inhibitor compound IWR-1, IWR-4, XAV939, or C59 that directs differentiation of these mesodermal cells to the cardiac lineage. In these protocols, the use of serum has been eliminated and all medium components are fully defined. These differentiation methods yield robustly beating troponin T+ iPSC-CMs with a high efficiency at manageable cost  isoforms occurs during CM maturation. In the fetal heart, slow skeletal troponin I (ssTnI) expression predominates over cTnI expression, whereas in the adult heart cTnI expression rises and ssTnI levels decrease . Similarake over . Finallyake over .β-MHC/α-MHC and MLC2a/MLC2v in iPSC-CMs is complicated due to the heterogeneity of atrial and ventricular CMs present in iPSC-CMs  or dexamethasone (DEX), culture substrate variations, three-dimensional tissue engineering, electrical stimulation, and genetic approaches . Initialα-, and β-MHC expression and to enhance the electrophysiological properties of the cells, increasing maximal upstroke velocity and peak amplitude of caffeine-induced calcium transients , polyethylene glycol, and carboxylated PCL, The dynamic cellular environment of the heart tissue is not recapitulated in a two-dimensional monolayer culture of cells. To address this, several studies have used culture substrates with altered stiffness and specific patternings that more closely resemble those of the native tissue. κB (NF-κB), myocyte enhancer factor-2, serum response factor, and EGR1 should be considered as promising targets of genetic approaches to enhance iPSC-CM maturation.Genetic approaches have also been promising. To mimic the switch of TnI isoforms during CM maturation of developing heart, Bedada et al. performed exogenous adenoviral-mediated gene transfer of human cTnI to iPSC-CMs, which successfully altered the cTnI:ssTnI isoform ratio . HistoneApproaches to create three-dimensional cardiac tissue  and to sβ1 to induce vSMCs -A to induce ECs  with place vSMCs . Similarce vSMCs . Consequce vSMCs . Notablyelopment . Overexpelopment .β1 to yield a putatively pure populations of CD140b+ vSMCs without additional cell-sorting steps  encodes the inward rectifying voltage-gated potassium channel in the heart, involved in cardiac repolarization . hERG teMeta-analysis of different drugs in the same class may also reveal that all individuals respond to drugs in the same class, a phenomenon known as a class effect. In the clinic, however, some drugs are more effective than others and have a better side effect profile. All patients are unique, and their individual responses to a drug will vary.The cause of the variation may be due to the differences in the genetic code caused by single-nucleotide polymorphisms. The goal of precision therapy is to deliver the right medicine at the right time to the right individual. In the past, cohorts were selected based on demographic, biometrics, and cardiovascular risk factors or disease state. More recently, trials such as the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin  and the iPSCs offer a window into an individual’s unique response to a drug. The iPSCs retain the individual’s genetic information, and when differentiated into the appropriate tissue subtypes, can reveal unique transcriptomic, metabolomics, and proteomic profiles . Armed wOver the past century, we have witnessed astonishing progress in the treatment of cardiovascular disease. Using animal models and primary cell cultures, mechanisms of cardiovascular disease have been uncovered and drug therapies for atherosclerosis rigorously tested in clinical trials. With the development of statins, mortality and the incidence of cardiovascular events have declined significantly. Following the mantra of lowering cholesterol levels, a multitude of clinical trials, including the Pravastatin or Atorvastatin Evaluation and Infection Therapy—Thrombolysis in Myocardial Infarction 22, a randomized trial of cholesterol lowering in 4444 patients with coronary heart disease, the Scandinavian Simvastatin Survival Study (4S), and Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering, have demonstrated the tremendous power of statins to alter the trajectory of atherosclerotic disease . The curAlthough statin therapy is essential for preventing cardiovascular deaths and improving survival, the implementation is difficult and often frustrating for both patients and clinicians. Similarly, patients with atrial fibrillation are prescribed rhythm or rate control strategies based on patients’ symptoms and preferences. Using an iterative process, clinicians and patients work together to find a drug with the best risk-to-benefit ratio. More broadly, iPSCs could identify subpopulations that would respond to therapy and experience the benefits of a drug while minimizing the adverse effects. Using iPSC-derived skeletal myoblasts, high-throughput assay for screening statin-induced myopathy was developed to test for mitochondrial integrity and cytoplasmic integrity . With loPatients who meet clinical trial entrance criteria should respond appropriately to therapy. The dilemma is that many trials, despite being adequately powered, fail to show improvement or even demonstrate adverse events in the enrolled patients. Clinical trials are designed with entrance and exclusion criteria to select responders to treatment and minimize adverse side effects. For example, the Cardiac Arrhythmia Suppression Trial (CAST) was designed to show the efficacy of antiarrhythmic drugs in suppressing ventricular arrhythmias in ischemic cardiomyopathy . HoweverThere already exist several examples of iPSC-CMs in modeling drug-induced toxicity with the potential to guide clinical management. Tyrosine kinase inhibitors (TKIs) are a powerful family of cancer therapies, but, unfortunately, some patients exhibit susceptibility to cardiotoxicity and arrhythmias from TKIs. Section VI of this review.The cardiotoxic effects of a drug may be patient-specific due to inherited factors. In another study, The cost of developing a drug is approximately $ 2.6 billion, with over 80% of the funds spent in the clinical trials phase of drug development and with approximately seven of eight drugs failing to make it into the clinic . iPSC-baTraditional biomarkers require the use of biometrics, serum proteins, and imaging. In this omics era of today, future trial designs may incorporate individual profiles from genetics, transcriptomics, metabolomics, and proteomics. Using this highly tailored approach, a drug will be more efficacious in receptive populations. Clinical trials that may have shown a signal for a positive response using traditional selection criteria would be more likely to be clinically significant.Drug screening and testing using patient-specific iPSCs enable the realization of “clinical trial in a dish” to reduce the exorbitant cost and inefficiency of the current drug development pipeline in the United States and other countries. The use of disease- and patient-specific iPSCs in drug screening and testing will also reduce trial and error and optimize patient selection for clinical trials, by accurately predicting responders versus nonresponders with greater accuracy .In silico analysis predicts compounds binding to drug target receptors. The structure of a receptor must be known to allow a known ligand to facilitate the modeling of binding with the use of a compound library. Designed algorithms can predict binding to putative ligands, which must be subsequently validated in vitro or in vivo . Use of An existing drug may be repurposed for a new indication. For example, colchicine is an anti-inflammatory drug for gout, which found off-label use in other inflammatory conditions such as pericarditis and postoperative pericarditis syndrome , 2013. EDrug screening can involve creating new compounds from scratch or using existing molecules to design novel compounds with improved efficacy or side effect profile. Rational drug design can be used to synthesize a multitude of compound . The effDespite the many advantages for drug discovery, pluripotent stem cells have not yet gained traction in the pharmaceutical industry. ESC-based strategies face a significant stigma with ethical concerns. The use of iPSCs can avoid this issue and has many advantages over ESCs. iPSCs retain the genetic information of an individual and provide a unique opportunity to advance personalized medicine. In regenerative therapy, the use of iPSCs may eliminate the need for immunosuppression, which is needed for the case of ESCs. Genome editing can further augment the power of iPSCs to correct mutations causative of the disease. Although gene therapy is not yet feasible, gene editing will uncover novel mechanisms of disease and targets for small-molecule therapies, which when combined with iPSCs will advance the development of novel personal therapies.However, iPSC-based platforms are not yet high throughput, remain expensive, and require storage for the delivery of regenerative therapies in a timely manner. The cost of drug discovery with iPSC platforms could be enormous, as each person would require a blood sample or tissue sample to be reprogrammed and subsequently differentiated. This individualized approach inevitably takes time because the reprogramming and differentiating process can take up to 6 months for each individual, not counting the time needed for recruiting patients into the study. Making the drug discovery process high throughput may thus prove more difficult than expected, as disease modeling likely will add considerable complexity to the drug-screening process, and titrating for each individual may require multiple iterations.From 1953 to 2013, 14% of the drugs were withdrawn from market due to serious cardiovascular adverse events . These i2. With improved methods of detecting subtle changes in cardiac function, the incidence of DIC is now thought to be much higher, occurring in up to 65% of long-term survivors of breast cancer, even at doses as low as 228 mg/m2  and topoisomerase I mitochondrial; and 3) modulation of intracellular calcium release. iPSC-CMs from DIC patients have been used to recapitulate the phenotype and elucidate the molecular mechanisms of the drug’s adverse effects  has been attributed to mutations in sarcomere and sarcolemma-related genes, including titin, lamin A/C (LMNA), MHC, and cardiac troponins . For insHypertrophic cardiomyopathy (HCM) is an abnormal thickening of the ventricle that causes microinfarction, outflow tract obstruction, and scarring, resulting in arrhythmias . Patientβ signaling leads to defects in proliferation of CMs (Development of therapies for left ventricular noncompaction (LVNC), a condition increasingly recognized as a cause of cardiomyopathy especially in children, is also in dire need. Characterization of iPSC-CMs from LVNC patients carrying a mutation in cardiac transcription factor TBX20 has shown that abnormal activation of TGF-n of CMs . Anothern of CMs . The datn of CMs . As suchThe use of iPSCs for cardiovascular regenerative medicine provides a limitless source of patient-specific cells that can repair and/or even replace damaged organs . iPSCs aConsequently, FDA approval would likely require that iPSC-derived cell therapy fully address the issues of arrhythmogenicity, tumorigenicity, and immunogenicity . ContinuIn both primary and secondary prevention studies, statins are the cornerstone of medical therapy. By lowering cholesterol, statins have been effective in reducing cardiovascular events and improving survival. Numerous clinical trials in secondary prevention have described the dose-dependent reductions in LDL levels, correlated with improved survival . The emeβ antibody Canakinumab could further reduce cardiovascular death and mortality beyond optimal treatment with existing medical management that includes the use of statins and PCSK9 inhibitor . Marfan syndrome patient-derived iPSC-vSMCs recapitulated the clinical phenotype in vitro and confirmed the critical role of TGF-β signaling in regulation of proliferation and apoptosis of aortic vSMCs . The aorta is normally an elastic structure, but defects in collagen synthesis and connective tissue can make the vessel more susceptible to shear stress from blood ejected from the heart. Traditionally, aortopathies are managed with ic vSMCs . An indeic vSMCs . Angioteic vSMCs . Howeveric vSMCs .The progression of aortopathy to dilatation, aneurysm, and possible rupture makes regenerative therapy a welcome option to surgery, either planned or emergent, which is associated with significant morbidity and mortality. iPSC-vSMCs can thus be used to discover new biomarkers of aortopathy, which are expensive and difficult to detect without serial imaging. Using an iPSC-vSMC array, it may be possible to develop new small molecular inhibitors of central medial necrosis that is pathogenomic for aortopathy. Furthermore, to best reflect the physiologic function and region-specific blood flow hemodynamics in aortopathic conditions, it is necessary to develop a vascular surrogate system that cocultures iPSC-derived vascular, stromal, and immune cell types under atheroprone hemodynamics. Such efforts are currently under development to model changes in hemodynamics and response to vascular inflammation or drug treatments using iPSC-derived vascular cells .Cardiometabolic diseases and vasculopathies stem from a combination of factors, including environmental exposure, stress, smoking, poor diet, and microbiome, in addition to genetic predisposition, requiring additional layers of investigation to address the patient-specific disease mechanisms . Most prThe primary cause for mortality in diabetic patients is cardiovascular disease . T2D is β-cell differentiation and regenerative therapy, we refer the readers to this review (Insulin resistance is associated with a metabolic syndrome that includes hypertension and dyslipidemia. Insulin resistance is often undiagnosed because it is difficult to test, but, like T2D, it is associated with an increased risk of cardiovascular disease . In addis review .γ-secretase inhibitor DAPT. Development of such iPSC-based organoid systems holds promise in advanced patient-specific disease modeling and drug screening and testing for diabetic cardiomyopathy and vasculopathies.T2D patients may also develop heart failure independent of these complications in a condition called diabetic cardiomyopathy, which was first documented in diabetic patients with adverse myocardial structural changes in the absence of hypertension and vascular dysfunction . The exaCardiovascular drug discovery is enormously challenging due to factors such as the unavailability of cardiac cells, combinatorial etiology of cardiovascular diseases, and imperfect translation of animal studies to human subjects. Combined with the state-of-the-art tools in multiomics analysis , the advHowever, a number of challenges in the use of iPSC technology for cardiovascular disease modeling and drug screening remains to be resolved. These include the generation of adult-like and tissue-specific subtypes of iPSC-derived cardiovascular cells as well as development of methods to introduce nongenetic etiological factors, all of which are currently under intensive investigation. Because the multifaceted pathophysiology of cardiovascular diseases involves multiple cell types in a number of organ systems, in vivo model validation is necessary to delineate the comprehensive disease phenotype, which should be performed concurrently to verify the findings from the iPSC-based in vitro studies. In addition, it will be necessary to generate functional and physiologic three-dimensional cardiac organoids and to develop high-throughput platforms utilizing heart and vessel mimics to enable effective and accurate drug screening and testing.Notwithstanding these challenges, continuing advances have made the use of patient-specific iPSCs an indispensable and powerful tool in the discovery of effective therapeutics for all cardiovascular diseases, helping to realize the full potential of cardiovascular precision medicine.


Many low- and middle-income countries are moving to introduce HPV vaccine into their national immunization programs. To improve coverage, equity, and sustainability, public health officials and practitioners can use planning and implementation lessons learned, including successful school-based delivery strategies, innovative approaches to reach out-of-school girls, best practices for communication and social mobilization, and integration of services to reduce delivery cost. Policy makers, donors, and global partners should continue to consider ways to drive down costs of vaccine procurement. Many low- and middle-income countries are moving to introduce HPV vaccine into their national immunization programs. To improve coverage, equity, and sustainability, public health officials and practitioners can use planning and implementation lessons learned, including successful school-based delivery strategies, innovative approaches to reach out-of-school girls, best practices for communication and social mobilization, and integration of services to reduce delivery cost. Policy makers, donors, and global partners should continue to consider ways to drive down costs of vaccine procurement. As countries move to add primary prevention to their strategies to combat death and morbidity associated with cervical cancer, many practitioners in immunization as well as experts in non-communicable and communicable diseases will benefit from keeping up-to-date with recent developments in practice and implementation regarding human papillomavirus (HPV) vaccine delivery.,Persistent infection of cervical epithelial cells with “high-risk” carcinogenic types of HPV causes 99% of the estimated 530,000 global cases,–Three HPV vaccines are currently on the global market: a bivalent product (protecting against HPV types 16 and 18), a quadrivalent product , and a nonavalent product .The World Health Organization (WHO) recommends all countries include HPV vaccine in their national immunization schedule. WHO recommends 2 doses of HPV vaccine for girls ages 9–14 years, separated by a minimum interval of 6 months, and 3 doses of HPV vaccine for girls ages 15 years and over.We offer this commentary in the hope of focusing dialogue between and among public health practitioners and public health officials on key recent developments in the planning and implementation of HPV vaccination programs.Gavi, the Vaccine Alliance provides support for vaccine introduction and immunization programs in eligible countries; country eligibility for support from Gavi is chiefly determined by the gross national income (GNI) per capita, which determines the level of co-financing and nature of vaccine program support available.Following WHO's change in HPV vaccine policy and Gavi's shift in program support, many Gavi-eligible countries are moving rapidly to introduce HPV vaccine into their national immunization programs.Achieving high vaccination coverage through a routine immunization program among adolescent girls necessitates innovative delivery strategies and communication efforts.,18—for example, for second-dose measles vaccine at school entry,Among 45 low- and middle-income countries surveyed in 2016 after having completed HPV vaccine demonstration programs or national introduction, most (87%) used primarily a school-based delivery strategy.,,,Using a school-based vaccine delivery platform has effectively achieved high coverage for girls in school but poses an equity challenge for out-of-school youth, many of whom have poor access to health services and screening later in life.School-based vaccine delivery models have effectively achieved high coverage for girls in school but pose an equity challenge for out-of-school youth.To continue to build successful HPV vaccination programs, several types of stakeholders must be engaged in the program planning process. Regardless of how and where the vaccine is delivered, education stakeholders need to be involved in program planning and communication, as the adolescent age group is largely enrolled in primary school. Other key stakeholders include adolescent and youth service providers, community service organizations, local women's groups, family planning and reproductive health advocates, cervical cancer specialists, gynecology organizations, and HIV prevention and treatment groups. Vaccine delivery may also be a promising service for integration with other development or health services for girls, such as nutrition, economic empowerment, menstrual hygiene, and disease prevention, so stakeholders who are experts in those programs may be involved.While at least 11 countries around the world, including Australia and the United States of America, routinely vaccinate boys with HPV vaccine, achieving high coverage among girls is a more cost-effective vaccination strategy in low- and middle-income countries than a “gender-neutral” vaccination strategy that immunizes both girls and boys.The current context of most countries focusing on vaccinating girls illuminates the importance of having a clear communication and social messaging campaign in place, with a realistic and nimble crisis communication strategy that can be activated quickly if rumors emerge.Although delivering vaccines to girls nationwide requires a different scale of resource commitment than a demonstration program, a number of potentially generalizable communication lessons can be drawn from studying programs that have implemented HPV vaccination to date.,Our understanding of best practices continues to evolve, highlighted by some best-case examples from Rwanda and Bhutan. In 2011, Rwanda became the first low-income country in the world to introduce HPV vaccine into its national program, and with strong leadership from its First Lady, partnership with industry, and effective, evidence-based mobilization efforts, has consistently reported between 93% and 96% full-course coverage.Although adolescence is arguably one of the healthiest periods of the life course, investment in this population, and inquiry into which services can be successfully and cost-effectively bundled with HPV vaccination, offers significant opportunity for impact.Overall, validated and relatively sophisticated economic models predict that HPV vaccination is very cost-effective in most countries, particularly in low-income countries.Robust economic models predict that HPV vaccination is very cost-effective in most countries, particularly in low-income countries.All 3 HPV vaccine products on the global market are currently WHO-prequalified; as of August 2018, the quadrivalent and bivalent products are approved for Gavi funding support to eligible countries. Gavi provides a vaccine introduction grant as part of its initial start-up package to a country to cover operational costs and social mobilization efforts. Gavi-eligible countries can also procure the prequalified HPV vaccines for US$4.60 per dose  and US$4.50 per dose .,Looking forward, new developments may be able to reduce HPV vaccine procurement and delivery costs. The eventual market entry of vaccines manufactured by companies based in low- and middle-income countries and owned by local entities may create the same downward pressures on prices as we have seen with multiple other medicines and biologics.Other factors may also play a role in reducing expected costs of program implementation. For example, an analysis by Gavi and WHO anticipates that national programs will harness economies of scale much more effectively than small demonstration programs were able to do.National HPV vaccination programs will likely harness economies of scale much more effectively than small demonstration programs.–,,,Data on whether a 1-dose schedule confers adequate levels of protection show promise,As countries introduce and scale up HPV vaccination programs, cervical cancer screening remains important for women who do not get vaccinated as children and for women who may have been infected with a high-risk HPV type that is not included in the vaccine. As national stakeholders in cancer and chronic diseases come together with immunization programs and their advisory bodies to make policy on HPV vaccination, they have an important opportunity to also inform their national policies on cervical cancer screening and surveillance programs.


Bacteriophage O276 is a laboratory-generated hybrid that carries the immunity region of bacteriophage HK022 and all remaining genes from phage λ. Its construction was instrumental in the discovery of RNA-mediated antitermination, an intriguing alternative to the protein-mediated mechanism of transcription antitermination found in most lambdoid phages. Bacteriophage O276 is a laboratory-generated hybrid that carries the immunity region of bacteriophage HK022 and all remaining genes from phage λ. Its construction was instrumental in the discovery of RNA-mediated antitermination, an intriguing alternative to the protein-mediated mechanism of transcription antitermination found in most lambdoid phages. Most early phage genes in lambdoid phages are located downstream of transcription termination sites, and the suppression of termination, or antitermination, is required for the production of viable phage particles. Early analysis of the immunity region of lambdoid phage HK022 suggested that transcription antitermination occurs but that no phage-encoded antitermination protein is required . To confEscherichia coli, and HK022 was isolated from sewage as previously described  and PECAAN (https://pecaan.kbrinsgd.org/). Comparison with the published λ and HK022 genomes provided additional verification of the annotations. Gene functions were identified/confirmed using BLASTP  are nun, cI, cro, and cII (Genetic analysis of O276 suggested that the crossovers between the parental phages likely occurred between genes  and cII . SequencEscherichia species phage O276 is MH547045. Raw reads are available in the SRA under accession number SRR7774037.The GenBank accession number for


Patients surviving severe brain injury either recover quickly from coma or go through prolonged disorders of consciousness (DOC) such as unresponsive wakefulness syndrome (UWS) or minimally conscious state (MCS). While patients in MCS show signs of consciousness, patients in UWS only show reflexive behaviors. These patients are unable to communicate and present vigilance fluctuation, language impairments, and severe sensory-motor deficits. Once patients recover functional communication or use of objects, they are emerged from MCS (EMCS).Even though behavioral assessment remains the gold standard for diagnosis, a number of studies highlight the difficulty in making the distinction between conscious and unconscious patients based on clinical examinations. Misdiagnosis can have serious consequences on patient's management, medically and ethically .The emergence of neuroimaging and neurophysiological techniques opened new opportunities to complement bedside assessment by improving diagnosis and prognosis of patients with DOC. This Research Topic, which includes 15 articles with 100 contributors, aims to give an overview on how neuroimaging research can improve diagnosis, prognosis and management of patients with DOC, and how recent applications of neuroimaging can help understand consciousness through severe brain injuries.Vanhaudenhuyse et al. report the case of a misdiagnosis in a patient who was considered in an unresponsive wakefulness syndrome for 20 years. Repeated behavioral examinations  combined with neuroimaging techniques  showed that the patient was in fact conscious and was re-diagnosed as an incomplete locked-in syndrome. Sarà et al. reported that some locked-in patients present hallucinations, and in a subgroup of 5 patients with such symptoms, they showed a reduced cortical volume in the right parahippocampal cortex, the fusiform and lingual regions, suggesting that advanced neuroimaging might help to detect small cortical changes.Aubinet et al. show how neuroimaging can help develop more sensitive behavioral tools to assess cognition in DOC patients. They describe the behavioral and cognitive profiles of 5 patients in MCS/EMCS alongside their neuroimaging results using structural MRI and PET. They introduce a new language-based neuropsychological tool, the Cognitive Assessment by Visual Election (CAVE), and showed that the cognitive profiles of the patients were consistent with the underlying brain impairments. More specifically, the presence of residual visual, motor, and language comprehension functions was, respectively, associated with a relative preservation of occipital, motor, and temporo-angular cortex metabolism.In parallel, Bodien et al. compared the rate of covert consciousness detection by hand squeezing and tennis playing motor imagery paradigms in 10 patients with traumatic DOC and 10 healthy subjects. They found that the tennis paradigm performed better in healthy subjects, but in patients, the hand squeezing paradigm detected command following with greater accuracy. The hand squeezing paradigm may therefore be a better classifier of command following in DOC patients.Using functional MRI (fMRI) command following tasks, Haugg et al. assessed the juxtaposed relationship between the default mode network (DMN) and the fronto-parietal  networks' functional time-courses. Patients who demonstrated fMRI command following showed greater differentiation between the DMN and dorsal attention network in response to movie viewing, compared to the resting condition. This effect was similar to healthy subjects and was driven by the movie's narrative. Conversely, this pattern was not present in DOC patients who showed no fMRI-based evidence of covert awareness. Naturalistic paradigms could therefore be used to dissociate between groups of DOC patients with and without covert awareness.Several articles also aimed at exploring new ways to detect consciousness in DOC patients. To investigate the potential for internal and external awareness in DOC patients, Dell'Italia et al. present a different framework to estimate fMRI network properties based on Exponential Random Graph Models (ERGM), which overcomes current methodological limitations. Longitudinal data in one patient who sustained a severe traumatic brain injury show that throughout recovery from coma, brain graphs vary in their natural level of connectivity. Separable temporal ERGM can characterize network dynamics over time and show the specific pattern of formation and dissolution of connectivity after coma.Riganello et al. measured the complexity index of the heart rate variability using non-linear multi-scale entropies in 16 MCS and 14 UWS patients. Higher complexity index values were observed in MCS compared to UWS patients with high discriminative power using machine learning. The complexity index also correlated with brain connectivity in the central autonomic networks assessed with resting state fMRI. These findings suggest that this “heart” index can provide an indirect way to monitor brain connectivity in DOC patients.To differentiate DOC patients, Naro et al. used functional transcranial doppler to differentiate between patients in MCS and UWS by assessing cerebral blood flow velocity (CBFV) during passive movement tasks. They observed group difference changes in CBFV with the pulsatility index in 21 patients with DOC and 25 healthy controls. This rapid and easy tool may allow to identify residual top-down modulation processes from higher-order cortical areas to sensory-motor integration networks related to the peripersonal space. Another potential technique that is at its infancy in the field of DOC is the functional near-infrared spectroscopy, a non-invasive optical and portable device. Rupawala et al. review its potential application to improve the accuracy of diagnoses and to provide new ways of communication.Cavaliere et al. investigated test-retest brain connectivity variability in three DOC patients. Using graph-theory and independent component analyses, they found differences between test-retest acquisitions affecting each network and each patient in a different way. Higher functional/metabolic correlation was measured in the MCS and EMCS patients compared to the UWS patient. Performing multiple acquisitions within the same session allow to assess temporal patterns of resting-state networks and improve characterization of DOC patients.A few authors tackled to better understand brain processing in DOC patients and therefore, in turn, improve diagnostic and prognostic techniques in such population. Using an hybrid PET/MR imaging, Bareham et al., who showed that measures of EEG networks correlated with behavioral variations. EEG connectivity captured both stability and recovery of behavioral trajectories within and between patients. This highlights the feasibility of bedside EEG assessments in rehabilitation setting, which can complement clinical evaluation.Longitudinal assessments using high-density electroencephalography (EEG) and CRS-R have also been performed on a longer time scale  by Bai et al. reviewed resting state EEG studies in DOC for diagnosis, prognosis, and brain interventions. Spectrum power, coherence, and entropy were the most frequently used features while power spectrum and functional connectivity had the best performance differentiating UWS from MCS and healthy subjects. Permutation entropy in the theta frequency also had high classification accuracy for differential diagnosis. Regarding prognosis, in their systematic review and meta-analysis of brain data and outcome in DOC, Kotchoubey and Pavlov report that oscillatory EEG responses, sleep spindles, and the somatosensory cortical response N20 were the best outcome predictors for DOC, whereas the poorest prognostic effects were fMRI responses to stimulation and P300. They however conclude that no practical recommendations on prognosis indicators can be given at this stage and they suggest several considerations to improve future (prognosis) studies: each group of patients should include at least 20 patients, perform blind assessments, use a flow chart to illustrate the procedure of patient selection, include the full list of measured variables, report the time since injury and the time of outcome, and describe all positive and negative results.Gottshall et al. evaluated a patient in chronic MCS who received central thalamic deep brain stimulation (CT-DBS). After 1 year of treatment discontinuation, reduced responsiveness was observed along with the abolishment of sleep spindles, marked downregulation of slow wave sleep delta power, and the return of alpha-delta sleep. The authors discuss the mechanism of sleep modulation by daytime CT-DBS in severe brain injuries and suggest a novel mechanistic framework for alpha-delta sleep generation across pathophysiologies.The last two articles refer to therapeutic options. Cheng et al. evaluated the effect of a sensory stimulation program (3 days per week for 4 weeks) using a ABAB design (for 16 weeks) in 29 patients. Higher CRS-R total scores were obtained during the treatment in the MCS group with increased arousal and oromotor function but not in the UWS group. Three patients also underwent fMRI and a modulation of brain activity related to treatment was found in specific brain regions.In conclusion, this Research Topic offers some novelties in the field of severe brain injuries and DOC, including new techniques, methodology, diagnosis/prognosis improvements, and therapeutic options. We look forward to translate the neuroscientific evidence generated from these studies to the clinical context.OG wrote the editorial. CS and SL revised it.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.


The heart is a complex organ consisting of various cell types, each of which plays an important role in both physiological and pathophysiological conditions. The cells communicate with each other through direct cell-cell interactions and paracrine signaling, and both homotypic and heterotypic cell interactions contribute to the organized structure and proper function of the heart. Cardiomyocytes (CMs) and endothelial cells (ECs) are two of the most abundant cardiac cell types and they also play central roles in both cardiac remodeling and regeneration. The postnatal cell cycle withdrawal of CMs, which takes place within days or weeks after birth, represents the major barrier for regeneration in adult mammalian hearts, as adult CMs exhibit a very low proliferative capacity. Recent evidence highlights the importance of ECs not only as the most abundant cell type in the heart but also as key players in post-infarction remodeling and regeneration. In this MiniReview, we focus on blood vascular ECs and CMs and their roles and interactions in cardiac physiology and pathologies, with a special emphasis on cardiac regeneration. We summarize the known mediators of the bidirectional CM-EC interactions and discuss the related recent advances in the development of therapies aiming to promote heart repair and regeneration targeting these two cell types. The heart is a complex multicellular organ with specialized structures and cells to take care of their own “subprojects.” Its main function, to maintain the blood circulation, depends on its pump function, making cardiomyocytes (CMs) the central cardiac cell type in both normal and pathological conditions. Cardiomyocytes are generally divided into pacemaker cells and force-producing ventricular and atrial CMs. In order for the heart to function properly, additional cell types, such as blood and lymphatic endothelial cells (ECs), vascular smooth muscle cells (SMCs), fibroblasts, and pericytes are needed. Besides their structural role in the interior surfaces of blood vessels, vascular ECs are metabolically active, control vasomotor tone, and regulate angiogenesis , 2. FibrIn the myocardium, CMs are physically connected and communicate with each other through gap junctions, adherens junctions, and desmosomes . HoweverCardiac remodeling, i.e., remodeling of the left ventricular wall, can take place in physiological or pathological conditions . While pSince the adult mammalian heart exhibits a very limited regenerative capacity , 17, a cDuring cardiac regeneration in the zebrafish and neonatal mouse, new CMs are produced through proliferation of remaining CMs , 26, 27.Cardiomyocytes produce and secrete proteins and peptides for paracrine signaling with other cardiac cells and for endocrine signaling with peripheral tissues. It has been estimated that the heart secretes 30–60 different proteins or peptides, which are called cardiokines or cardiomyokines ; howeverThe main cardiokine regulating EC activation and proliferation is vascular endothelial growth factor (VEGF) that binds to and activates the VEGF receptor 2  in ECs . VEGFR2 The first evidence indicating that angiogenesis induces cardiac hypertrophy came from a study by Tirziu et al. , where tIt is estimated that humans have about 100,000 km of blood vessels, which most likely makes vasculature the largest “endocrine organ.” In response to physiological or pathological stimuli, ECs secrete a set of proteins and other factors, angiocrines, which act on the neighboring cell types. Recent data from other tissues provide strong evidence that ECs establish an instructive vascular niche, which stimulates organ growth and regeneration via paracrine signaling , 46, 47.A growing number of EC-derived factors acting on CMs have been identified. One of the most studied over the years is NO, which is produced by many cell types in the heart and affects both vasculature and CMs. NO induces relaxation of vascular SMCs resulting in vasodilation , and canin vitro co-culture studies, connexins have also been suggested to mediate communication between ECs and CMs; however, more evidence is needed to prove the existence and relevance of direct EC-CM contacts in vivo. Furthermore, exosomes and other extracellular vesicles can carry non-coding RNAs such as microRNAs, long non-coding RNAs and circular RNAs, which have been shown to regulate cardiac remodeling and regeneration  as well as plasmids, exhibit a good safety profile also in clinical trials. However, the clinical efficacy of cardiovascular gene therapies has been somewhat disappointing [reviewed in ]. PlasmiSome randomized clinical trials with VEGF and FGF gene therapies have improved myocardial vascularity; however, convincing evidence on functional benefits is still missing see , 64]. Cl. Cl64)].www.clinicaltrials.gov). It will be administered by epicardial injections during coronary artery bypass grafting surgery. In preclinical studies, several miRNAs have been shown to regulate EC and CM survival and proliferation and are thus expected to become potential therapeutic targets as the delivery and targeting techniques improve (RNA-based drugs include short interfering RNAs, antisense oligonucleotides, messenger RNAs and CRISPR gene-editing technology . The fir improve , 69.Stem cell therapy for cardiac repair has been under intensive investigation and produces modest, although variable, improvements in cardiac function in clinical trials –73. The in vitro (The requirement for fast neovascularization of the infarcted area  and expein vitro  suggest in vitro . There iin vitro , and patin vitro . Howeverin vitro .Furthermore, the beneficial paracrine effects of cell therapy have prompted research on extracellular vesicles that carry pro-survival signals such as non-coding RNAs, which could be used to achieve similar effects as cell therapy without the delivery of cells. Prolonged cell-free delivery of hPSC-CM-derived extracellular vesicles into infarcted rat hearts promotes cardiac recovery . FurtherThe efforts to discover regeneration-inducing pharmacological agents are still in their infancy. Considering the lack of regenerative gene expression response in both CMs and ECs , a potenDue to potential adverse effects in off-target organs, regeneration-inducing therapeutics should be specifically targeted to the heart. To avoid invasive administration, cardiac targeting with heart-homing nanoformulations has provoked interest. For example, porous silicon nanoparticles coated with ANP and loaded with a novel GATA4-targeted cardioprotective compound were shown to enrich in the ischaemic endocardium and inhibit prohypertrophic signaling after systemic administration . Althougin vitro models used in drug discovery and development. More relevant in vitro systems are needed not only for screening and lead optimization of cardiovascular drugs, but also for screening cardiovascular and other drug candidates for cardiotoxicity and proarrhythmic effects. The advances in the production of human pluripotent stem cell-derived cardiovascular cells and microfabrication provide an unprecedented possibility for large-scale drug screening in humanized multicellular cardiac cell culture models and heart-on-a-chip systems (Cardiac heterocellularity also needs to be implemented in  systems , 96.Cardiac regeneration requires an orchestrated multicellular response, the mechanisms of which are not yet fully understood . The curAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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. The reviewer EA and handling Editor declared their shared affiliation.


For example, anemia and bone marrow failure are observed across different ribosomopathies—each caused by mutations in a different ribosomal protein or assembly factor—including Diamond-Blackfan anemia (DBA), Dyskeratosis Congenita, Shwachman-Diamond Syndrome (SDS), Myelodysplastic syndrome with Chromosome 5q Deletion, and Cartilage Hair Hypoplasia. Why is hematopoietic dysfunction a common nexus for an otherwise extremely diverse and unique set of phenotypic defects of ribosomopathies? This question is as important as it is puzzling. What causes mutations in the biochemical machine found in every single cell of the body, and with an equally essential role in every tissue, to have a more profound effect on hematopoietic cells? Moreover, in addition to the congenital disorders, a number of recent reports also described somatic ribosomal mutations, primarily found in hematologic cancers such as T-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.2 Hematopoietic lineages thus appear to be a preferential target for both congenital and somatic ribosomal mutations. Several new exciting studies have shed some light on the “hema-centrism” of ribosomal defects, some of which are discussed below. Broad insights into this tissue-specificity paradox can also be appreciated in recent excellent reviews.4Eukaryotic cells contain many thousands to millions of ribosomes distributed throughout the cytoplasm and attached to the endoplasmic reticulum. Capping the central dogma of biology, these ribosomes ensure faithful conversion of genomic information captured in mRNA into functional proteins. Impaired ribosome assembly and/or function stemming from mutations in one of the hundreds known ribosome assembly factors or 1 of the 80 ribosomal proteins is the underlying cause of the congenital disorders referred to as ribosomopathies.5 Other tissues that have high protein synthesis rates in the body, including the liver, gastrointestinal tract, muscle, and skin, are, however, not classically affected in ribosomopathies. Therefore, in addition to large protein synthesis requirements, other unique features of hematopoietic cells likely contribute to their sensitivity to ribosome defects. To this end, Mills et al delineated an erythroid-specific pathway with strong implications on ribosome function.6 After reaching a termination codon, ribosomes need to be recycled back to the beginning of the message for another round of translation or to begin translation of another message. Occasionally, ribosomes become trapped on an mRNA and can be freed by ribosome rescue factors. The authors found that the levels of the canonical ribosome recycling factor ABCE1 and rescue factor PELOTA are strongly and selectively reduced in reticulocytes after terminal differentiation. Resulting inefficient ribosome recycling might synergize with a shortage of ribosomes in reticulocytes to create an additive sensitivity to an imbalance in ribosome homeostasis. Furthermore, several studies demonstrated the essential role of ribosome levels on hematopoietic differentiation. It was previously shown that DBA-associated ribosomal protein defects promote defective translation of erythroid regulators of hematopoiesis BAG1, CSDE1, and GATA1.8 In a more recent follow-up,9 DBA-associated ribosomal protein lesions were observed to cause a reduction in ribosome levels, which in turn reduced translation of transcripts that are normally translated most rapidly and efficiently—those containing short and unstructured 5′ untranslated regions (UTRs)—including GATA1. Conversely, transcripts of the regulators of other hematopoietic lineages were found to contain long and structured 5′ UTRs, offering protection from the translational alterations of DBA-mutant ribosomes. The authors propose a model in which reduction of GATA1-specific translation in ribosome-mutant DBA impairs erythroid lineage commitment, ultimately causing erythroid deficiency. Similarly, deficiency for the ribosome assembly factor SBDS in SDS causes defective translation re-initiation on C/EBPα and C/EBPβ mRNAs,10 which might explain the neutropenia in SDS as these factors are essential for neutrophil proliferation and differentiation.Ribosomopathic mutations result in haploinsufficiency of key ribosome assembly factors and core ribosomal proteins, leading to defects in ribosome assembly and reduction of the cellular ribosome pool. This leaves fewer ribosomes available for cells to produce required proteins, which can lead to proliferation defects. Additionally, disrupted ribosome assembly increases the availability of free ribosomal proteins, which can activate TP53 and further augment the hypo-proliferative phenotypes. It is tempting to speculate that lower levels of functional ribosomes affect rapidly dividing cells, such as hematopoietic progenitors, more substantially. Indeed, the highest known rates of protein synthesis occur in progenitors undergoing erythroid lineage commitment.11 Two recent papers by the group of Maria Barna lend additional support to this evolving idea by demonstrating that ribosomes can display heterogeneity both at the level of core ribosomal proteins12 and proteins interacting with ribosomes13 in murine embryonic stem cells. The authors identified populations of ribosomes with altered ribosomal protein composition, tasked for translation of specific mRNA subsets.12 Specifically, translation of classes of mRNAs containing strong secondary structures in the 5′ UTRs—such as Internal Ribosomal Entry Site elements—were found to depend on ribosomes with particular ribosomal protein composition. This study also indicates that ribosomal proteins found mutated in ribosomopathies like DBA are substoichiometric, and may demarcate ribosomes with specialized functions. This provides another example of how haploinsufficiencies of specific ribosomal proteins particularly perturb the translation of specific mRNAs, which may disproportionately result in hematopoietic dysfunction. In another approach, the authors focused on a complementary aspect of ribosomal function—interaction with extra-ribosomal factors.13 The large surface area of ribosomes, which increases along with organismal complexity, enables potential interactions with a variety of trans-acting factors. The complete identification of such factors illuminated for the first time a map of the “ribo-interactome.” This revealed several hundred ribosome-associated proteins belonging to a wide spectrum of functional categories including ribosome function and modification and, more surprisingly, cell cycle, cell redox homeostasis, and metabolism. Moreover, different subcellular pools of ribosomes displayed different ribosome-associated proteins, suggesting that ribosomal function may be trans-modulated at distinct cellular environments. This study also established a metazoan-specific post-translational modification termed ufmylation, in which UFM1 proteins are conjugated to particular ribosomal proteins. While the precise roles of this modification remain to be determined, the available knockout mouse models for the enzymes of the ufmylation cascade show defects in erythrocyte differentiation and result in embryonic lethality,14 similar to the defects arising from haploinsufficiency of some ribosomal proteins.Whereas the defects described above may provide an explanation for defective erythropoiesis (DBA) and neutropenia (SDS), potential explanations regarding the disproportionate effect of ribosomal protein mutations on other hematopoietic lineages may emerge from the new provocative concept of “specialized ribosomes.” Because of their central role in all of life, ribosomes were historically assumed to be structurally and functionally invariable in every cell of an organism. Recent studies are challenging this notion. For example, a quarter of human ribosomal proteins were found to exhibit tissue-specific expression, with primary hematopoietic cells displaying the most complex expression patterns.cis-and trans-regulation are rapidly replacing the outdated concept of “the” ribosome. While still in its infancy, this recent rise of the ribosomal repertoire might be a key to solving the tissue-specificity paradox of ribosome-mutant diseases. Evidence is accumulating that hematopoietic tissues, as well as other types of tissues, contain structurally and functionally unique ribosomes  and gets disrupted in disease states. We imagine that depletion of certain ribosome species due to decreased expression of a ribosomal protein, or introduction of new ribosome species due to missense mutations, would shift the overall balance of cellular ribosome variants, and hence the proportion of ribosomes with particular, optimized functions. Divergent function and regulation of the ribosomal molecular machine can be compared to the combustion engine: while the fundamental underlying mechanism remains the same, many subtle variations of the engine exist which are fine-tuned to a diversity of applications. The specialized engine driving translation in hematopoietic lineages might make these cells more vulnerable to faults in engine composition, activation, and maintenance.These studies highlight the sensitivity of hematopoietic cells to defects in protein production, while underscoring the increasingly more heterogeneous, dynamic, and malleable nature of ribosomes. Adaptable ribosomes whose function can be modulated by differences in ribosomal composition or mes Fig. , Model 1


Cancer is a global health problem in need of transformative treatment solutions for improved patient outcomes. Many conventional treatments prove ineffective and produce undesirable side effects because they are incapable of targeting only cancer cells within tumors and metastases post administration. There is a desperate need for targeted therapies that can maximize treatment success and minimize toxicity. Nanoparticles (NPs) with tunable physicochemical properties have potential to meet the need for high precision cancer therapies. At the forefront of nanomedicine is biomimetic nanotechnology, which hides NPs from the immune system and provides superior targeting capabilities by cloaking NPs in cell-derived membranes. Cancer cell membranes expressing “markers of self” and “self-recognition molecules” can be removed from cancer cells and wrapped around a variety of NPs, providing homotypic targeting and circumventing the challenge of synthetically replicating natural cell surfaces. Compared to unwrapped NPs, cancer cell membrane-wrapped NPs (CCNPs) provide reduced accumulation in healthy tissues and higher accumulation in tumors and metastases. The unique biointerfacing capabilities of CCNPs enable their use as targeted nanovehicles for enhanced drug delivery, localized phototherapy, intensified imaging, or more potent immunotherapy. This review summarizes the state-of-the-art in CCNP technology and provides insight to the path forward for clinical implementation. Cancer is a devastating global public health problem in desperate need of transformative solutions. It is the second leading cause of death in the United States and predicted to take 1700 lives per day in 2019 [Conventional methods to treat cancer involve the surgical resection of tumors followed or preceded by aggressive chemotherapy and localized radiotherapy ,3. HowevNanotechnology offers the opportunity to create nanovehicles that can carry either single or multiple therapeutic cargos, as well as contrast agents, to tumors for improved treatment and imaging. Nanoparticles (NPs) can be “smart designed” for enhanced drug delivery, phototherapy, vaccination, immunotherapy, and imaging ,8. AdditThe tumor microenvironment is characterized by leaky tumor vasculature and poor lymphatic drainage ,8,9. AllWhile ligand-targeted NP delivery to desired tumor cells is often depicted as a straightforward and easily accomplished task, it is extremely challenging to achieve this goal . In partBiomimetic nanotechnology harnesses the unique biological makeup of cell membranes and combines it with the flexibility of NP substrates and a wide range of payloads to improve targeted delivery. The general concept is to wrap NPs with cell-derived membranes that provide the complex biological entities found on natural cell membranes , which aCancer cell membranes are the ideal candidate to wrap around NPs for oncological applications ,22. CancIn the following sections, we describe the synthesis and characterization of CCNPs and the different types of treatments these unique NPs can accomplish. We also provide a forward-looking perspective on the challenges to be addressed as this technology progresses from the laboratory setting to the clinic.The synthesis of cell membrane-coated nanovehicles involves three steps: (1) membrane extraction from source cells, (2) fabrication of the nanoparticulate core, and (3) fusion of the membranes and nanoparticulate cores to form core-shell membrane-wrapped NPs . Below, At its most basic level, membrane extraction requires that internal cell components are removed while leaving the functional components of the membrane intact. This membrane extraction procedure requires large volumes of cells to be harvested from culture dishes or blood and tissue samples ,31,48,49Electroporation lyses cells by exposing them to strong electric fields, causing temporary loss of semi-permeability, and pore formation in the cell membrane, releasing intracellular components . ConsequThe most popular method to extract cancer cell membranes involves osmosis-based cell lysis with a mild hypotonic solution, followed by mechanical membrane disruption with a homogenizer . DiscontAs a variety of NP core designs may be utilized to produce CCNPs, depending on the intended application, it is unwarranted to describe any one specific NP synthesis here. The main criterion, independent of core material, is that the NPs have a negative zeta potential. This will facilitate proper orientation of the membrane around the NP owing to electrostatic repulsion between the NP surface and negative extracellular membrane components . To dateMethods of coating NPs with membranes can be divided into three generalized strategies: physical extrusion, sonication, and microfluidic coating B. All of+/K+-ATPase, and gp100, but lacked the intracellular markers histone H3, cytochrome c oxidase, and glyceraldehyde 3-phosphate dehydrogenase [It is critical to compare various features between bare and wrapped NPs to confirm complete membrane wrapping. Successful wrapping can be validated by observing a 10–20 nanometer increase in particle size after wrapping, equating to the thickness of the membrane layer. This can be measured by dynamic light scattering (DLS), transmission electron microscopy (TEM) C, or nanrogenase . These dCancer drug delivery is one field in which membrane-wrapped nanovehicles, and CCNPs in particular, have substantial potential to improve the state-of-the-art. Encapsulating drug cargo within nanocarriers that offer tailorable control of release kinetics, such as polymer-based cores, can dramatically improve bioavailability, and tumor-specific delivery can be further enhanced by coating these vehicles with cancer cell membranes ,18,19. MDoxorubicin (DOX) is a commonly used chemotherapeutic that intercalates into DNA to yield topoisomerase II-mediated DNA damage followed by cell death . DOX hasIn addition to single drugs, multiple cargoes with synergistic actions can be encapsulated in CCNPs. This ensures the cargos are delivered to the same cells within tumor sites for improved anticancer effects. This was demonstrated with PLGA cores that were loaded with hemoglobin (Hb) and DOX and coated with MCF-7 human breast cancer cell membranes with a PEGylated phospholipid to overcome hypoxia-induced chemoresistance . By suppCCNPs that incorporate stimuli-responsive features have also been designed to take advantage of the acidic tumor microenvironment as a trigger for localized drug release . In one Importantly, across various platforms, it has been shown that cancer cell membrane coatings do not negatively interfere with drug loading inside NPs. As demonstrated by the examples discussed, there is great promise in the field for CCNPs to enhance drug delivery to desired sites to improve safety and efficacy. This opens the door for the development of many new treatment strategies. While some NPs exploit features of the tumor microenvironment such as low pH or presence of specific enzymes to enable stimuli-responsive drug release and high precision therapy, another route to enable site-specific treatment of tumors is to utilize nanomaterials that are inactive until they are triggered with externally applied light. The two main examples of this are photothermal therapy (PTT) and photodynamic therapy (PDT), and both have recently been explored in conjugation with membrane-wrapped NPs. In photothermal therapy, NPs with unique optical properties are delivered into tumors, which are then irradiated with near-infrared light that causes the NPs to produce heat capable of thermally damaging cancer cells ,90,91,92Combining PTT with chemotherapy offers many advantages versus either treatment alone. Several studies have shown that PTT can elevate drug delivery into tumors or into cancer cells by increasing vascular permeability and cancer cell membrane permeability ,99. AddiCombination PTT and chemotherapy mediated by CCNPs has been most widely explored using DOX as the chemotherapeutic agent ,57,76. IThe benefits of combining chemotherapy with PDT include having reactive oxygen species (ROS) available to initiate drug release and promote intracellular drug delivery, as well inducing hypoxia in the tumor region for activating encapsulated drugs . Convers2) NPs encapsulating a photosensitizer and coated with GOx were wrapped in B16-F10 cancer cell membranes [2 reactors were irradiated for continuous oxygen generation, supported by the conversion of glucose to singlet oxygen. This system has potential to solve hypoxia issues in tumors and promote starvation. Starvation therapy mediated by membrane-wrapped NPs has also been explored without PDT [Besides being combined with chemotherapy, PDT has also been combined with starvation therapy. In starvation therapy, glucose oxidase (GOx) is delivered to tumors. GOx will transform glucose into gluconic acid and hydrogen peroxide, starving the cells of glucose, a vital nutrient for tumor growth. In one example, a cascade reaction system made of hollow manganese dioxide , researchers used multiple types of cancer cell membranes  to prepare corresponding batches of wrapped lanthanide-doped nanocrystals . These NMembrane-wrapped NPs that incorporate both contrast agents and photoactive agents have also been used for tumor imaging and phototherapies. In one study, ICG-loaded NPs wrapped with MCF-7-PEG fused membranes exhibited a PTT response, but also had fluorescence and photoacoustic (PA) imaging capabilities . PEG incSimilarly, dual-modal imaging has been used with PDT by loading photosensitizers into magnetic nanobeads to target hepatocellular carcinoma . Both neThere is growing interest in the field of oncology in using technologies to stimulate the body’s immune system to attack tumor cells. Immunotherapy can be applied as either a cancer treatment or as a preventative cancer vaccination, and is advantageous over cytotoxic agents because of its high specificity and low toxicity ,105,106.Exploring the immune stimulatory capabilities of membrane-wrapped NPs, Fang et al. prepared PLGA NPs wrapped in B16-F10 melanoma or MDA-MB-435 human breast cancer membranes and utilized these NPs to deliver antigens to source tumor cells and antigen presenting cells (APCs) . The CCNAs the field matures, researchers will continue to design new ways to enhance immunotherapy mediated by CCNPs. Yang et al. recently showed that CCNP delivery to dendritic cells (DCs) could be enhanced by modifying CCNPs with mannose (which binds receptors on DCs) through lipid anchors . The CCNIn summary, CCNPs have great potential as either prophylactic vaccines to protect patients from tumor cell challenges or as therapeutic agents to shrink tumors by inducing anti-cancer immune responses. It is observed that tumor antigen presentation from the membrane coating alone, even in highly immunogenic contexts, may not be powerful enough to overcome the immunosuppressive tumor microenvironment. Therefore, technologies must be combined with adjuvants, immune checkpoint blockade therapies, or other approaches to achieve optimal anti-cancer effects. Nevertheless, the biocompatible nature of CCNPs makes them promising as personalized therapies to induce cancer-specific immune responses for individual cancer patients. While many of the described successes of CCNP systems are extremely encouraging, there are still many challenges to address before these technologies become commercially available. One potential issue is the need for patient education, as concern may develop over having cancer cell-derived material injected into the body. Although patients who already have cancer may be willing to overlook this concern if it provides a chance to eradicate their disease, healthy patients who are at-risk for certain types of cancer and wish to use this technology as a preventative vaccine may be less receptive. In addition to educating the population, stringent testing and procedures will have to be developed to ensure that the membrane coatings are pure  and do not contain any molecules that might promote cancer growth. As the development of membrane-wrapped NPs is already becoming more mature, with proper tests this should not be an issue and this hurdle could be feasibly overcome. One of the biggest draws of using cell membranes to coat NPs is the ability to have a personalized treatment. NPs coated with membranes derived from a patient’s own cells should be able to evade unwanted immune responses that can occur when foreign material is introduced to the body. However, the feasibility of creating CCNPs for each individual patient is a significant question. Preparing patient-specific CCNPs will require strict quality control and regulatory methods. Additionally, while biopsy samples could be used to create CCNPs for patients with existing tumors, the production of CCNPs for prophylactic vaccination will require a different approach. Donor cells could possibly be used, but immunostimulatory issues may arise. One way this could be mitigated is by preparing particles with mixed membrane coatings, as several studies have shown this can imbue the NPs with properties of both membranes ,68,111. Lastly, for CCNPs to be successful in the clinic, methods for manufacturing scale-up need to be developed. Particle replication on a small laboratory scale is already difficult due to the complex biological components involved and concerns of batch to batch consistency need to be addressed. One of the largest hurdles in scaling up the process is the need for large quantities of membranes. This requires millions, if not billions, of cells, and the facilities to grow them. Besides producing the necessary amount of membrane material, the scaling up of the NP cores is another concern, especially if the design is more complex or a multi-component system. The more complicated a nanotherapy fabrication is, the more difficult it is to create reproducible and identical particles at large scale. Finally, the assembly of how membrane vesicles are fused with NP cores could be difficult to replicate at a large scale. In short, the more steps to the process, the more difficult it will be to produce commercial quantities of high-quality material. This must be addressed before membrane-wrapped NP technologies can reach clinical trials or achieve FDA approval.This review has highlighted the current state-of-the-art in developing CCNPs for the management of cancer. CCNPs have immense potential as tools to improve the imaging and treatment of tumors, but they also face substantial challenges in translating to the clinic. The future of the field lies in solving these issues or working around them to deliver specific, personalized therapy to single patients. While difficult to accomplish, the chance of eradicating even a single type of cancer, whether by therapy or vaccine, will continue to drive the many paths of research surrounding CCNPs described in this Review.


NK cells are capable of an array of functions that range widely from their classic anti-tumor and anti-viral cytotoxic effector functions, to their critical regulatory roles in controlling inflammatory immune responses and promoting tissue growth. However, the mechanisms that polarize NK cells to these distinct and opposing functions are incompletely understood. NK cell functional subsets are primarily identified and studied based on phenotype, which has served as an accessible means for profiling NK cells and does offer information on NK cell activation state. However, inconsistencies have emerged in using classic phenotypes to inform function, which raise the questions: Can phenotype in fact define NK cell functional fate? What factors do profile and drive NK cell fate? In other immune cells, cell metabolism has been shown to critically determine subset polarization. There is a growing body of evidence that cell metabolism is integral to NK cell effector functions. Glucose-driven glycolysis and oxidative metabolism have been shown to drive classic NK cell anti-tumor and anti-viral effector functions. Recent studies have uncovered a critical role for metabolism in NK cell development, education, and memory generation. In this review, we will draw on the evidence to date to investigate the relationship between NK cell phenotype, metabolism, and functional fate. We explore a paradigm in which the differential activity of metabolic pathways within NK cells produce distinct metabolic fingerprints that comprehensively distinguish and drive the range of NK cell functional abilities. We will discuss future areas of study that are needed to develop and test this paradigm and suggest strategies to efficiently profile NK cells based on metabolism. Given the emerging role of metabolism in driving NK cell fates, profiling and modulating NK cell metabolism holds profound therapeutic potential to tune inflammatory and regulatory NK cell responses to treat disease. Natural Killer (NK) immune cells are capable of an array of functions that range widely from their classic innate anti-tumor and anti-viral effector functions, to their regulatory roles in modulating other immune cells and promoting tissue growth . These dbrightCD16− regulatory NK cells with greater cytokine-producing capabilities, and CD56dimCD16+ anti-tumor/anti-viral NK cells with greater cytotoxic functions NK cells are defined as CD56receptor , 33. As dimCD16+ . These ld subset .dim NK cells can in fact up-regulate CD56 expression upon cytokine activation and become CD56bright  re-activation and produce high levels of IFN-γ  infection. In humans, these have been predominantly defined as CD56of IFN-γ . CertainNK cells . HCMV meNK cells , 48, 50.NK cells , 52. Thedim, but express CD25, NKG2A, CD94, and CD69, and lack expression of KIRs and CD57  NK cells generated by IL-12/IL-15/IL-18 pre-activation are primarily CD56and CD57 , 54. Memcination . Recentlnd NKp46 . FurtherIf phenotype is unable to consistently identify NK cell subsets and functional fate , 2A, whaAs highly plastic innate responders, NK cell effector fate is molded by its environment. NK cells integrate a number of environmental cues which determine downstream function. In the absence of activating signals, naïve NK cells remain in a resting quiescent state. In response to inflammatory signals, cytotoxic effector NK cell responses are marked by robust proliferation and synthesis of proinflammatory cytokines and cytotoxic machinery. Upon resolution of inflammation, NK cells curb their response, returning to a quiescent state. It is noteworthy that this shifting functional profile represents a profound shift in energy dynamics from a low-energy quiescent state to one with substantial energetic and biosynthetic requirements. Further, NK cells exert distinct functions across different tissue sites that vary greatly in nutrient types and availability. From these factors arises a parallel between NK cell energetics and functional fates.Indeed, the capacity of a cell to generate energy through metabolism has emerged as an important factor in enabling immune cell effector functions. The burgeoning field of NK cell metabolism has uncovered the involvement of cell metabolism throughout different steps of NK cell fate determination, including development, cytotoxic effector responses, and generation of long-lived memory populations. Thus, could distinct metabolic profiles comprehensively identify and drive NK cell functional fates?NK cell development is an energy-intensive process as it requires high levels of proliferation of NK cells in the bone marrow. As NK cells progress through development, their proliferation slows and they progress to state of quiescence in the periphery , 57. In Tsc1, a repressor of mTOR, is required to prevent deregulated proliferation and resulting exhaustion in response to IL-15 in developing NK cells (The mechanistic target of rapamycin (mTOR) is a major energy sensor in the cell that integrates signals for nutrient availability, growth, and activation. In response to such signals, mTOR up-regulates glycolytic metabolism and biosynthetic processes . A seminNK cells . TogetheThe evidence to date has demonstrated the requirement for metabolic regulators in NK cell development and that a shifting metabolic expression profile parallels development. These studies portray a baseline energy-intensive, biosynthetic metabolic fingerprint for developing NK cells in the bone marrow, which shifts to a more energy-conservative fingerprint as NK cells progress to the periphery. Further developing and characterizing the metabolic fingerprints of NK cells throughout development may more broadly define the maturation stages of NK cells, particularly for later stages in which phenotype fails to do so and for NK cells at different tissue sites. Assessing the baseline metabolic fingerprint in such instances holds potential to more comprehensively define the degree of NK cell maturity.Although metabolically quiescent at baseline, mature cytotoxic NK cells up-regulate the rate of glucose-driven glycolysis and oxidative phosphorylation (OxPhos) upon stimulation , 60, 61.bright NK cells  IL-15 pre-activation , 61. Durbright  th IL-15 . Moreove CD56dim . TogetheResearch on NK cell metabolism has largely focused on the metabolic pathways that generate anti-viral and anti-tumor effector NK cells and it is clear that enhanced glucose metabolism is critical for such functions. On the other hand, while regulatory NK cells have critical homeostatic anti-inflammatory roles in the body, there is a dearth of knowledge about the role of metabolism in driving regulatory NK cell polarization and functions. In order to more fully understand the role of metabolism in the polarization of NK cell functions, it will be critical for future studies to investigate the metabolic profile of regulatory NK cells. For instance, is the metabolism of highly functional NK cells similar irrespective of whether the downstream functions are cytotoxic or regulatory? Or do regulatory NK cells predominantly utilize distinct, non-glucose-driven metabolic pathways which determine their regulatory functional fate?The metabolism of a cell is largely dictated by its microenvironment, including factors such as nutrient and oxygen availability. Thus, in considering the above questions, it is worthwhile to examine microenvironments that favor regulatory NK cell polarization, such as the uterus, tumors, and the liver, and the metabolic profile these may shape:brightCD16− phenotype  is/are primarily used by adaptive NK cells. The greater glycolytic capacity in HCMV-adaptive NK cells points to greater glucose metabolism. However, gene expression analysis of HCMV-adaptive NK cells also revealed an increase in genes involved in lipid catabolism . InteresAlthough the above studies have demonstrated a metabolic basis for the generation and function of memory NK cells, these have been limited to assessing adaptive NK cells in the context of CMV infection. While phenotypic variations span different memory NK cell subsets, the memory-traits of extended survival and enhanced recall effector responses, for which the metabolic adaptations are necessary in the context of CMV, are traits that remain consistent across memory NK cells. Thus it is likely that across memory NK cell subsets, increased mitochondrial fitness and fuel flexibility are broadly required to support longevity and that an enhanced respiratory capacity is necessary to derive the energy required for enhanced function upon re-activation. An important area of future study will be to verify that similar metabolic reprogramming broadly drives the formation and function of other memory-NK cell subsets, including cytokine-induced memory-like NK cells, BCG-memory NK cells, and pregnancy-trained uterine NK cells.Research on NK cell biology is continuing to evolve and uncover a wider scope of NK cell functional fates. However, as knowledge on the complexity and plasticity of NK cells has grown, so too have incongruities in the classic phenotypic definitions of NK cell subsets. Classic NK cell phenotypes have undoubtedly served as an accessible measure for profiling NK cells and provide indications on NK cell activation state. Nevertheless, in many instances phenotype in fact confounds functional fates, as NK cells that express the same phenotype can have divergent functions, and NK cells with similar functional potential can express a range of phenotypic possibilities. Indeed, identifiable phenotypes that are sufficient to profile NK cell functional fates are lacking . The shoMetabolism has emerged as an intriguing factor to distinguish NK cell functional fates. The field of NK cell metabolism is still in early stages, but studies so far point to distinct metabolic profiles as drivers of NK cell functional potential which may thus serve as reliable fingerprints to identify functional fates . A heighCertainly, significant questions remain to fully delineate and validate this paradigm. For instance, what metabolic fuels and pathways are principally used by regulatory NK cells? Are low levels of glucose metabolism sufficient to support regulatory functions or do regulatory NK cells use an alternate fuel, such as fatty acids, as a more efficient means of energy generation in low oxygen conditions? Does enhanced mitochondrial fitness span all memory NK cell types? Are there metabolic pathways that further distinguish memory cytotoxic from memory regulatory NK cells? Are other factors, such as transcription markers and epigenetic modifications, required in conjunction with metabolism to determine NK cell fate? Such questions present exciting avenues for future research and will provide a more comprehensive understanding of the mechanisms and role of metabolism in tuning the spectrum of NK cell functions.in situ (While metabolism is a promising means by which to identify NK cell fates, another critical question is whether it is achievable to efficiently profile NK cells based on metabolism. In this regard, similar to assessing classic phenotypic markers, a number of metabolic measures are frequently assessed via cytometry or microscopy, allowing for multiplex analyses that comprehensively measure a number of metabolic features. These include functional mitochondrial measures, such as mitochondrial mass, structure, membrane potential, ROS production, and oxidative stress , 29. Furin situ . With thGiven the critical and distinct roles that NK cells play in a host of diseases, therapeutically modulating NK cell function is garnering increasing attention. With distinct metabolic fingerprints at the heart of NK cell functional fates, profiling and modulating metabolism hold promise as powerful therapeutic strategies to predict and control NK cell fate.SP designed and wrote the manuscript. AA designed and edited 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.