Since its first introduction more than 30 years ago, optical coherence tomography (OCT) has revolutionized ophthalmology practice, providing a non-invasive in vivo cross-sectional view of the structures of the eye. Mostly employed in the clinical setting due to its tabletop configuration requiring an upright patient positioning, the recent advent of microscope-integrated systems now allows ophthalmologists to perform real-time intraoperative OCT (iOCT) during vitreoretinal surgical procedures. Numerous studies described various applications of this tool, such as offering surgeons feedback on tissue–instrument interactions in membrane peeling, providing structural images in macular hole repair, and showing residual subretinal fluid or perfluorocarbon in retinal detachment surgery. This narrative review aims at describing the state of the art of iOCT in vitreoretinal procedures, highlighting its modern role and applications in posterior segment surgery, its current limitations, and the future perspectives that may improve the widespread adoption of this technology. Since the first images were first published in 1993 by Fercher et al. . To dateAn extensive literature search was conducted on PubMed using keywords including: “optical coherence tomography”, “vitreoretinal surgery”, “intraoperative optical coherence tomography”, “pars-plana vitrectomy”, “intraoperative retinal imaging”, “microscope-integrated optical coherence tomography”, and all relevant synonyms and abbreviations. Moreover, references of selected papers were scanned manually to identify additional studies. The retrieved titles and abstracts were included for full-text review by two of the co-authors (N.C. and P.M.T.) if they reported relevant technical details, applications, or outcomes of iOCT.The first iOCT platforms described in the literature were portable machines such as the Bioptigen SDOIS/Envisu  and the Optovue IVue 100 . The former employed a hand-held OCT scan head that could potentially be mounted on a surgical microscope to provide 32.000 A-scans per second, producing high-density raster scans up to 1000 × 1000 with an axial detail down to 2.4 µm [These inherent technical limitations led several independent groups to develop microscope-mounted systems like the ones described by Binder et al. in 2010  and EhleIn order to enable effective real-time feedback during surgery to successfully integrate OCT intraoperatively, microscope-integrated devices were developed. Compared to hand-held and microscope-mounted OCT machines working on a separate axis and focusing from the surgical microscope, microscope-integrated iOCT machines are built within the microscope and operate parfocal and coaxial to its optical path, with minimal alteration of the surgeon’s workflow.At the time of writing, three models are commercially available . The firEpiretinal membranes (ERM) are among the most common indications for pars plana vitrectomy worldwide and one of the diseases best characterized using OCT imaging . The gol® as well as contact lens viewing systems. The study reported that in almost half of the procedures, iOCT had impacted the surgeon’s understanding of the surgical configuration and/or procedure, reversing surgical decision making in 13% of cases by revealing residual membranes that escaped the surgeon’s observation [The benefit of iOCT for vitreoretinal surgery has been recently described in the PIONEER  paper, a large 2-year prospective study conducted by Ehlers and colleagues at the Cleveland Clinic including eyes with various posterior segment diseases treated surgically with the aid of iOCT imaging. The authors employed the Bioptigen SDOIS portable spectral-domain OCT system  mounted on either a Leica or Zeiss microscope with a custom-designed coupling mechanism compatible with BIOMervation . The DIServation . In the ervation .This significant disagreement between the ability of iOCT and the human eye in detecting and determining the completeness of the peeling was further confirmed by other authors. In 2015, Falkner-Radler et al. described residual fragments of ERM or internal limiting membrane (ILM) remnants in almost 90% of patients after the first ERM peeling attempt , whereasMoreover, as reported by Kumar et al., iOCT imaging proved particularly helpful for ERM surgery on highly myopic eyes. Indeed, ERM peeling may result in the development of macular holes in myopic eyes with foveoschisis or retinal thinning. Thus, the images provided by iOCT may highlight safer zones to initiate the peeling and help to decide when to end the surgery once the retinal traction is relieved ,19.The level of detail provided by iOCT can also help to clarify how the retinal architecture responds to mechanical stretching during peeling procedures, providing more insight into how iatrogenic-induced changes might impact anatomical and visual outcomes. On this matter, Weschta and colleagues recently reported a negative correlation between intraoperative retinal stretching and postoperative thinning of the fovea in eyes undergoing ERM surgery .Furthermore, various studies analyzed the feasibility of using iOCT as an alternative to dyes to stain the ERM and ILM. Even though recently developed dyes, such as trypan blue-based ones, are considered relatively safe for a short-time application ,22,23, tInterestingly, the signal intensity of the ERM and ILM in iOCT imaging is increased when dyes such as indocyanine green are used, as described by Ehlers et al. This suggests the potential combined use of iOCT and dyes to facilitate ERM and ILM identification in complex cases .Macular holes are defined as partial or full-thickness defects in the foveal layers ,30. FullThe most common approach to treat full-thickness macular holes consists of vitrectomy and peeling of the ILM in order to release the centrifugal forces that caused the retinal tissue to split. For large or recurrent macular holes, further surgical measures are commonly employed in order to promote the anatomical closing process, such as filling the hole with inverted flaps of ILM, amniotic membrane, or other similar tissues. This step can be aided by iOCT thanks to its ability to show the surgeon the correct placement of these “tissue plugs” ,33 Figu.Other than guiding the surgeon through the surgical procedure itself, OCT technology can be employed in order to predict postoperative outcomes. Indeed, with the use of iOCT, surgeons are able to analyze intraoperatively the amount of retinal stress and thickening induced by the peeling process. In a recent study by Runkle et al. , the innSince the introduction of OCT as routine investigation for macular holes, there have been numerous attempts to predict surgical success based on preoperative OCT parameters. Based on the results of previous studies, only preoperative minimal width of the macular hole appeared consistently as a robust predictor of hole closure ,36. HoweMoreover, in a recent report by Nishitsuka and colleagues, hole diameter was measured through iOCT at different surgical stages, showing a decrease after ILM peeling and a further reduction after gas tamponading, probably due to the centripetal surface tension force between the retina and the gas bubble . The abiAlong with these positive prognostic factors, a study by Inoue et al.  found thDespite the recent technological and surgical advancements, rhegmatogenous retinal detachment (RRD) surgery remains a challenging procedure with elusive long-term functional results even after successful anatomical repair. Since the advent of OCT, the high-resolution view of retinal layers has provided useful feedback for surgical planning and prognostication, with imaging studies that have provided novel ultrastructural biomarkers such as ellipsoid zone disruption, cystoid degeneration, and outer retinal corrugations .With regards to the intraoperative use, the DISCOVER study reported that iOCT provided surgeons with feedback deemed relevant in 36% of cases and altered the decision-making process in 12% of cases . AdditioBesides better visualization of the retinal periphery, iOCT has made possible the real-time visualization of the subretinal space during the different steps of retinal reattachment surgery . VariousWith regards to ab externo RRD surgery, previous studies showed how scleral buckling and encircling procedures could also benefit from the application of this technology. In particular, Sotani et al.  reportedSubmacular surgery is a subfield of vitreoretinal surgery that was first introduced in the 1990s, when the first attempts to treat choroidal neovascularization and repopulate the subretinal space with healthy retinal pigment epithelium cells were described ,52. CurrDelivering drugs directly between photoreceptors and the retinal pigment epithelium allows one to reduce drug concentration. To do so, a 23- or 25-G cannula with a retractable 38- to 41-G tip is attached to a syringe controlled with the foot pedal of a vitrectomy machine . This deAnother indication for subretinal injections is represented by inherited retinal diseases, a heterogeneous group of diseases primarily affecting the retinal pigment epithelium and photoreceptors. In order to treat genetic mutations, the preferred approach is the subretinal delivery of a viral vector, usually an adeno-associated virus or lentivirus. Currently, the only approved treatment for inherited retinal diseases is voretigene neparvovec-rzyl, an adeno-associated virus-mediated gene employed when treating certain forms of Leber’s congenital amaurosis and retinitis pigmentosa where the gene RPE65 is defective. As described by Hussain et al.  and VascAside from guiding subretinal injection procedures, iOCT can also be useful for surgeries such as chorioretinal biopsies, retinal pigment epithelium transplantation, and retinal prosthesis implantation . In 2017Since the initial transfer of OCT devices from the clinic to the operating room, iOCT technology has greatly evolved, becoming a useful adjunct to ophthalmic surgery. However, some technical limitations still hinder the widespread adoption of this technology, and it remains unclear whether the significant investment and learning curve required to integrate this technology into the surgical workflow are justified by relevant clinical benefits for patients. From a technical point of view, there are still various critical points that have to be addressed, such as the shadowing effect cast by most of the surgical equipment commonly used currently in vitreoretinal surgery. To overcome this problem, OCT-compatible instruments composed of semitransparent rigid plastic material are under development ,60.Software-wise, the OCT devices available on the market are still unable to track and focus automatically on the areas of interest and are still dependent on the physical control of surgeons. The development of voice-activated controls or automatic features of tracking and focusing may allow a more fluid integration of OCT imaging into the surgical procedure. Moreover, the advent of high-level graphics processing units might, in future, allow one to acquire and render volumetric data in real time, thus providing three-dimensional surgical images instead of cross-sectional OCT scans ,61.Another promising area of research is the potential integration of iOCT in robotic-assisted surgical platforms. The Preceyes Surgical System  is a telemanipulated robotic device with an instrument-integrated iOCT sensor. Mounted near the tip of the instruments, the sensor allows micrometer-scale precision on anteroposterior movement thanks to an A-scan-based feedback loop with superhuman reactivity that keeps the instruments’ tip within safe virtual boundaries from retinal structures, reaching levels of efficacy and safety unmatchable by human dexterity .In conclusion, iOCT represents a remarkable advancement for surgical visualization and decision making during vitreoretinal surgery. Integration of intraoperative OCT into routine surgical workflows, the cost-effectiveness of implementation, and the learning curve for surgeons are all factors that require careful consideration. However, as this technology continues to evolve, iOCT holds promise for enhanced surgical safety and precision, advancing the boundaries of minimally invasive surgery and ultimately leading to better patient outcomes.


Cardiomyopathy is a major cause of heart failure caused by abnormalities of the heart muscles that make it harder for it to fill or eject blood. With technological advances, it is important for patients and families to understand that there are potential monogenic etiologies of cardiomyopathy. A multidisciplinary approach to clinical genetic screening for cardiomyopathies involving genetic counseling and clinical genetic testing is beneficial for patients and families. With early identification of inherited cardiomyopathy, patients can initiate guideline-directed medical therapies earlier, resulting in a greater likelihood of improving prognoses and health outcomes. Identifying impactful genetic variants will also allow for cascade testing to determine at-risk family members through clinical (phenotype) screening and risk stratification. Addressing genetic variants of uncertain significance and causative variants that may change in pathogenicity is also important to consider. This review will dive into the clinical genetic testing approaches for the various cardiomyopathies, the significance of early detection and treatment, the value of family screening, the personalized treatment process associated with genetic evaluation, and current strategies for clinical genetic testing outreach. Cardiovascular disease (CVD) remains the leading cause of death, accounting for 71% of deaths globally each year . Recent Genetic testing for cardiomyopathies plays an increasingly important role in the clinical care of patients and has been increasingly adopted in contemporary cardiology practice. Genetic variants have been implicated in 25–40% of patients with dilated cardiomyopathy with a positive family history, but also in 10–30% of patients without a recognized family history . With geThere is increasing recognition that family history is often incomplete and that de novo genetic variants may occur. Clinicians are unable to identify all patients with genetic cardiomyopathy solely through a three-generation family history. For example, Dellefave-Castillo and colleagues found that if only patients highly suspected of genetic cardiomyopathy or arrhythmia were genetically tested, over 137 positive results (14.4%) would have been missed . DiscussMolecular genetic testing consists of disease-specific panels, targeted variant tests, broader cardiac panels, whole-exome sequencing (WES), and whole-genome sequencing (WGS) . DiseaseA comparison of disease-specific gene panels and broader gene panels may provide some insights into their clinical applications. Broader gene panels are likely to increase the detection rates of genes, but can also identify variants of uncertain significance (VUSs) . OuelletMany WESs and WGSs are still utilized for research purposes over clinical use when evaluating for inherited cardiomyopathies . A studyInterpretation of genetic variants is based on published guidelines by the American College of Medical Genetics (ACMG) . The catProband (in this case the patient with cardiomyopathy) clinical genetic testing occurs when the first person in a family is affected by a genetic condition and receives genetic counseling  6]. Onc. Onc6]. Reclassification of genetic variants plays a major role in screening guidelines for cascade testing . When a Recent advances in clinical genetic testing have improved understanding in this field, allowing for improvement in the care of cardiomyopathy patients and their families. The association between genotypes and phenotypes for cardiomyopathies may vary widely, but recent evidence is evolving scientific understanding. There is a level of genetic heterogeneity, as variants in specific genes can result in different phenotypes . For exaGenetic testing is useful for diagnosing and improving treatment strategies . Among HRecent efforts to expand WES and WGS have shown the potential of providing incidental findings of actionable results related to cardiac-related gene variants when testing a patient with a negative cardiac family history . The ACMPolygenic risk scores (PRSs) are currently being utilized to better understand disease penetrance of monogenic variants . PRSs coWith the increasing number of VUSs, researchers are expanding functional assays to help clarify the pathogenicity of variants ,26. ThesThe presence of likely pathogenic or pathogenic variants, while continuing to evolve, has been broadly adopted by clinicians and clinical genetic specialists for identifying those with suspected inherited cardiomyopathy in which appropriate diagnostic testing and interventions are sought, especially in those that may have a known malignant natural history. On the other hand, there are clinicians that believe increased genetic data can lead to confusion and inappropriate treatment due to the uncertainty of the results and their treatment implications. In addition, the poor diversity of genetic reference databases still critically limits our ability to interpret genetic findings in underrepresented populations. It is true that we still do not have an in-depth understanding of all the genetic predispositions for various cardiomyopathies and continue to discover new aspects of genetic underpinnings of cardiovascular diseases. In this section, we highlight some emerging data that illustrate the opportunities and challenges of personalized medicine in inherited cardiomyopathy.DCM can be categorized as either familial or nonfamilial . More thThe most common genetic causes of DCM are the titin (TTN) gene variants . TruncatLMNA is another important gene strongly associated with DCM . In addiSCN5A is another gene linked to DCM and is associated with various deleterious cardiac phenotypes such as familial atrial fibrillation, Brugada syndrome, and familial conduction disease . SCN5A iApproximately 10% of those with genetic DCM have damaged variants in genes encoding for desmosomes . These gThe DCM Precision Medicine Study (DCM PM) was a family-based cross-sectional study that estimated the prevalence of familial DCM among probands and the risk among their first-degree relatives. It rigorously obtained phenotype and pedigree data on a large, multiracial idiopathic DCM cohort by identifying relevant genetic variants through exome sequencing and reporting results to probands and family members . The cenFamily Heart Talk, for improving the uptake of preventive screening and surveillance in at-risk first-degree relatives [Precision medicine relies on managing disease risk among first-degree relatives of probands with a heritable disease; therefore, interventions to improve clinical screening are needed to improve outcomes. The DCM PM conducted a randomized controlled trial (RCT) to test the effectiveness of an educational booklet intervention, elatives ,39. Seveelatives . Utilityelatives . FurtherGenetic HCM is also defined by genetic and allelic heterogeneity . ApproxiGenetic HCM has unpredictable expressivity and penetrance . Only haGenetic testing in HCM is critical in characterizing HCM and sarcomeric disease and differentiating them from phenocopies including glycogen storage and lysosomal diseases and cardiac amyloidosis . A studyCardiac amyloidosis is a common HCM mimicker . CardiacLeft ventricular noncompaction (LVNC) is characterized by a thinned, compact myocardial layer and a thickened trabecular myocardial layer with excessive trabeculations and deep recesses . In the Strategies to increase cardiovascular genetic screening among first-degree relatives include sharing and educating probands with disease risk and progression . UtiliziGenetic counseling and education include sharing genetic risk information among family members . This maFamily Heart Talk booklet mentioned above indicate that this intervention is efficient and cost-effective [A study by Kinnamon and colleagues found that there is no statistically significant difference in relatives undergoing genetic screening when probands experience a face-to-face provider-led intervention versus paper or web-based formats . Althougffective . It is iffective . The effffective .Orthotopic heart transplantation remains the gold standard for treating patients with advanced heart failure . GeneticAnother study by Boen and colleagues evaluated the diagnostic yield of genetic testing in 31 heart transplant recipients with prior non-ischemic cardiomyopathy . The stuEarly recognition of variant carriers and cardiac phenotypes ensures timely treatment, which leads to lower morbidity and mortality rates . Among fClinical genetic testing consideration is warranted beyond standard arrhythmogenic and cardiomyopathy gene variants when other causes leading to the need for advanced therapies may contribute . For exawww.hcmrisk.org (accessed on 18 May 2023)) was created by O’Mahony et al. in 2014 given the lack of international consensus on the absolute sudden cardiac death (SCD) risk that justifies ICD implantation. It has not been validated in pediatric patients, elite athletes, and individuals with metabolic diseases and clinical syndromes. This risk calculator includes age, left ventricular wall thickness, left atrial size, maximum left ventricular outflow track gradient, family history of sudden cardiac death, non-sustained ventricular tachycardia, and unexplained syncope [https://professional.heart.org/en/guidelines-and-statements/hcm-risk-calculator (accessed on 18 May 2023).Non-ischemic cardiomyopathy has a wide spectrum in clinical presentation, even within its sub-classifications of DCM, HCM, and RCM. Through the years, several risk stratification tools have been created and validated to help guide management of these patients. The HCM Risk-SCD calculator ( syncope . An updawww.arvcrisk.com (accessed on 18 May 2023)) performed well in patients with and without pathogenic PKP2 variants and can guide clinicians in determining the timing of ICD implantation [www.lvnc-riskscore.com (accessed on 18 May 2023)). This prediction model has been externally validated to indicate that patients with normal ECG, preserved ejection fraction, no myocardial fibrosis, and no family aggregation do not have MACE during long-term follow-up [https://lmna-risk-vta.fr/ (accessed on 18 May 2023)) to estimate the 5-year risk of life-threatening ventricular tachyarrhythmia in patients with LMNA mutation. A threshold of ≥7% predicted a 5-year 96% risk of long term ventricular arrhythmia among LMNA patients [Risk of life-threatening ventricular arrhythmias in patients with ARVC has been found to be predicted by four variables including younger age, male sex, burden of ventricular ectopy, and the extent of repolarization abnormalities. This risk model calculator (antation . For patollow-up . Among Lpatients . Phosphopatients . These rPolygenic risk scores have been utilized to predict genetic risks in complex diseases such as coronary artery disease (CAD), schizophrenia, and cancer . A PRS iWhile technological advances have provided the opportunity for a whole-genome approach and different ways to identify risk markers and generate prediction algorithms, there are higher standards for such testing to become applicable in clinical practice. For example, the currently identified single nucleotide polymorphisms might not fully describe genetic diversity, as they may not capture known and unknown forms of genetic variability . Meanwhile, genetic mechanisms likely involve complex interactions among genes or between genes and their environmental exposures, or complex epigenetic or post-translational modification processes, that are not realized.Genetic evaluation of cardiomyopathies plays an increasingly important role in the early diagnosis, treatment, and prognosis of disease. The main cardiomyopathies that genetic tests screen for are dilated, hypertrophic, arrhythmogenic, and restrictive cardiomyopathies, with left ventricular noncompaction representing a variant phenotype. It is important to recognize that human monogenic diseases may contribute to an unexpectedly larger portion of cardiomyopathies that we have previously recognized, as they may not be presented in a syndromic manner. The uncertainty surrounding the information of the discussed genetic tests should therefore be weighed against the risks of ignoring the potential of a malignant genetic predisposition that may adversely affect the patient and his/her affected family members, with the need for shared decision-making with the available clinical evidence. The early detection and establishment of therapies in patients with inherited cardiomyopathies optimize patient health and care. Not only does clinical genetic testing define the genetic basis of the cardiomyopathy and evaluate the prognosis, but it also allows for at-risk relatives to be screened and risk stratified. Clinical genetic evaluation allows for the guidance of risk stratification in relatives and should be managed by a multidisciplinary team to properly interpret cardiac genetic results, which will then alter the clinical care of patients and guide at-risk relatives. With this streamlined method, cascade testing can be performed to highlight at-risk family members and impactful genetic variants can be identified to establish personalized and specific treatment plans for patients.


This review describes how osteocytes, the most abundant cells in bone, act in cancers that grow in bone. It summarizes how cancer cells reprogram osteocytes to participate in processes associated with tumor growth, cancer cell survival, migration, angiogenesis, and bone destruction. These changes ultimately facilitate the spread of cancer in the bones and promote bone disease. It also discusses some of the identified signaling pathways mediating these processes and the therapeutic strategies to target them.The tumor microenvironment plays a central role in the onset and progression of cancer in the bone. Cancer cells, either from tumors originating in the bone or from metastatic cancer cells from other body systems, are located in specialized niches where they interact with different cells of the bone marrow. These interactions transform the bone into an ideal niche for cancer cell migration, proliferation, and survival and cause an imbalance in bone homeostasis that severely affects the integrity of the skeleton. During the last decade, preclinical studies have identified new cellular mechanisms responsible for the dependency between cancer cells and bone cells. In this review, we focus on osteocytes, long-lived cells residing in the mineral matrix that have recently been identified as key players in the spread of cancer in bone. We highlight the most recent discoveries on how osteocytes support tumor growth and promote bone disease. Additionally, we discuss how the reciprocal crosstalk between osteocytes and cancer cells provides the opportunity to develop new therapeutic strategies to treat cancer in the bone. Bone is a highly dynamic tissue continuously resorbed by osteoclasts and formed by osteoblasts to maintain tissue homeostasis and integrity. This process, known as “bone remodeling,” requires the coordinated and balanced action of bone resorption and formation. Osteocytes, long-lived descendants of fully differentiated osteoblasts, orchestrate bone remodeling ,2,3. DurThe bone provides fertile soil and a complex biological system for cancer cells to thrive. Cancers that start in the bone are known as primary bone cancers. Primary bone cancers are uncommon, occur from cells present in bone tissue, and include osteosarcomas, chondrosarcomas, Ewing sarcomas, and chordomas . Other cThe growth of cancer cells in bone profoundly affects the bone marrow microenvironment and disrupts the balance of bone remodeling. The thriving of cancer cells in bone has been traditionally explained by the establishment of a “vicious cycle” between tumor cells and cells of the bone microenvironment that fuels tumor growth and bone disease. In lytic cancers , bone colonizing tumor cells secrete factors that promote osteoclastogenesis , tilting the balance in bone remodeling towards bone resorption ,16,17,18Given the central role of cells of the bone marrow in the onset and progression of cancer in the bone, research efforts have focused on finding aberrant cellular and molecular mechanisms in this niche. This led to the identification of new cellular interactions between cancer cells and cells of the bone marrow  beyond those originally described in the “vicious cycle” paradigm. Mounting evidence supports that osteocytes, overlooked in cancer for decades, generate a microenvironment conducive to tumor progression and bone disease . Unlike Breast and prostate cancer metastases to bones are relatively common and a leading cause of cancer deaths in the U.S. To achieve metastasis to a secondary site, cancer cells undergo a multi-step process that begins with detachment from the primary tumor, followed by migration through the extracellular matrix of the surrounding tissue to enter the bloodstream (invasion–intravasation–dissemination) . To metaOsteocytes produce GDF15, which supports prostate cancer migration and invasion . OsteocyIn vitro studies show that conditioned medium from osteocytes induces EMT and increases the migration of breast cancer cells ,30. MechThe different outcomes observed in studies investigating the effects of mechanically stimulated osteocytes on prostate/breast cancer cells are probably due to the utilization of different mechanical stimuli  and cell culture models that do not fully reproduce the complex mechanosensory role of osteocytes in the bone. Migration and invasion are also important for hematological cancers . HoweverCollectively, these results suggest that osteocyte-derived factors could influence the metastatic potential of cancer cells at primary sites and generate a microenvironment favorable for recruiting and retaining cancer cells in bone. However, the existing data supporting this concept is primarily derived from in vitro studies, which do not fully represent the complexity of inter-organ communication. Moreover, the in vivo evidence supporting this notion remains limited. Nonetheless, given the emerging data showing that osteocytes are endocrine cells and have regulatory functions beyond the skeleton  , we cannAccumulating evidence supports the idea that osteocytes have an active role in tumor growth in metastatic and hematological cancers. Osteocytes affect tumor growth via different mechanisms, including the production of growth factors, physical interactions with cancer cells, and fueling the resorption that drives the “vicious cycle” of bone cancer. Yet, the effects of osteocytes on cancer growth seem to be cancer-specific. Conditioned media from MLO-Y4 osteocytes cultured in 2D or 3D conditions stimulates the proliferation of human prostate cancer cell lines . This suAs described for prostate cancer cells, breast cancer cells can also establish cell-to-cell contact with osteocytes . HoweverThe disparate outcomes of these studies are likely a consequence of different culture models (2D vs. 3D), different osteocyte-like and cancer cell lines, and different culture conditions. Investigation of the role of osteocytes in in vivo/ex vivo models containing authentic osteocytes in the tumor niche might better elucidate the effects of these cells on tumor proliferation and the migration and invasion potential of bone metastatic cancer cells. Contact between osteocyte dendritic processes and myeloma cancer cells has been reported using acid etching and electron microscopy . These pThe findings that osteocytes can impact cancer cell proliferation in bone have sparked interest in targeting these cells and their derived factors to stop tumor growth.Consistent with the evidence that mechanical signals alter osteocytes’ ability to influence cancer cell proliferation, several studies have shown that mechanical stimulation can decrease tumor growth in bone in metastatic and hematological cancers ,54,55,56The crosstalk between osteocytes and myeloma cells via Notch has also proven to be an attractive therapeutic target to stop tumor growth. In an in vivo model of multiple myeloma, inhibition of NOTCH3 signaling in myeloma cells, which is the receptor mediating osteocyte–myeloma communication, resulted in a reduction in tumor size . SimilarBreaking the vicious cycle between tumor cell proliferation and bone resorption is another approach to mitigating tumor growth. As discussed in more detail in Bones are highly vascularized through a complex system of heterogeneous blood vessels . End+ osteocytes doubled in bones bearing murine and human myeloma tumors compared to saline-injected controls [+ osteocytes positively correlated with Endomucin+, a marker of endothelial cells, and with tumor vessel area [2) showed that myeloma cells upregulated the expression of several angiogenic factors, such as VEGFA, and enhanced the osteocytes’ ability to promote vessel formation [Cancer patients exhibit increased vascular density in the bone marrow niche, and marrow angiogenesis negatively correlates with patient survival ,63,64. Ocontrols . The persel area . Consistormation . The mecormation . Myelomaormation ,57. Notcormation . TogetheOne of the hallmark features of lytic cancers is the development of bone lesions, which lead to increased fracture risk, pain, disability, and morbidity ,71. CompOne mechanism by which cancer cells stimulate resorption through osteocytes involves the induction of apoptosis and the alteration of their canalicular network ,49,77,78Myeloma patients exhibit increased apoptotic osteocytes in areas infiltrated with myeloma cells ,79. ThisOsteocytes also support bone destruction by stimulating RANKL production in cancer cells. For instance, osteocytes upregulate RANKL expression in myeloma cells via Notch signaling. Inhibition of NOTCH3 in myeloma cells reduced osteocyte-induced RANKL upregulation ,57. ConsAnother mechanism of osteocyte-driven bone destruction was recently identified in invasive oral squamous cell cancer (OSCC), which shows a bone-destructive phenotype, particularly in the maxillary of the mandibular bone. HMGB1 is an “alarmin” released by osteocytes that undergo apoptosis, and it has been shown to have a role in regulating osteoclastogenesis . HMGB1 iWith the advancements in medical treatment for many cancers, patients are experiencing longer survival rates. As a result, effective management of bone disease has become paramount to reducing fractures and bone pain and improving the quality of life of cancer patients. Bisphosphonates and anti-RANKL are the mainstay therapeutic options to treat cancer bone loss ,85. BothBecause mechanical signals decrease the osteocyte’s potential to induce osteoclast formation , the ostIn mouse models of multiple myeloma, pharmacologic inhibition of Notch signaling with a novel bone-targeted gamma-secretase inhibitor (BT-GSI) decreased RANKL expression and had potent anti-resorptive effects, similar to those seen with bisphosphonates . RemarkaUsing a combination of genetic approaches, different groups showed that osteocytes are a major source of RANKL in the adult bone under physiological conditions ,6,7,96. Several mechanisms have been described for the dysregulation of bone formation in bones infiltrated with cancer cells . BecausWith the development of neutralizing antibodies against DKK-1 , a new oAnti-sclerostin therapy was approved by the FDA in 2019 to treat postmenopausal women at high risk of fracture. The bone gain induced by the anti-sclerostin antibody results from opposing effects on bone formation (increased) and bone resorption (decreased) ,129. PreIn multiple myeloma models, activation of Wnt signaling via pharmacological inhibition of sclerostin one day after cancer cell injection or after 3 weeks did not affect tumor growth when tumors were established ,104,108.In breast cancer bone metastasis, treatment with anti-sclerostin in a mouse model with established bone metastasis after cardiac inoculation reduced the growth of MDA-MB-231 breast cancer cells . MoreoveGiven the remarkable beneficial skeletal effects observed in models of cancer-induced bone loss with therapies targeting Wnt antagonists, further studies, including clinical trials, are warranted. These studies aim to determine which cancer patients could benefit from these therapies, identify the ideal therapeutic window, and explore the most effective combinations that provide beneficial bone effects without increasing tumor growth or causing significant toxicity. During the last decade, we have witnessed major advances in our understanding of the role of osteocytes in bone cancer. Initially viewed as passive bystanders embedded in the mineral matrix, osteocytes are now considered key components of the tumor microenvironment. Nowadays, we have a better understanding that osteocytes play a significant role in influencing tumor growth, bone resorption, and bone formation in most cancers that develop in the bone. However, it is important to note that their impact, whether positive or negative, on each of these processes appears to vary depending on the cancer type. The knowledge acquired in this field has provided the rationale for developing new approaches to treat cancer in bone-targeting osteocytes and their derived factors . HoweverDespite the tremendous advances in our knowledge of osteocyte biology in cancer, critical areas remain underexplored. For instance, a complete understanding of how osteocyte gene expression is affected by different cancers or the underlying regulatory mechanisms remains elusive. Unbiased genome-wide approaches, such as single-cell RNA sequencing (scRNAseq), have the potential to significantly advance our understanding of the molecular changes underlying osteocyte dysfunction. These approaches can aid in generating a gene signature that distinguishes healthy from diseased osteocytes, identifying new biomarkers with predictive value for bone disease, and uncovering novel therapeutic targets. Although scRNAseq enables the identification of differential gene expression within diverse cell populations, it does not provide information about cellular localization. Thus, techniques that provide spatial resolution  could help us understand the interactions between osteocytes and cancer cells in a complex environment such as the bone marrow. Furthermore, there is a need for the development of reliable and clinically relevant mouse models (syngeneic) of cancer in the bone that facilitate genetic manipulation of the osteocytes, eliminating the need for extensive backcrossing across generations. Such models would provide valuable in vivo evidence to better understand the influence of osteocytes on the devastating effects of bone tumors.Another area where the available data is limited is the impact of anti-cancer therapy on osteocyte function and its consequences. For instance, bortezomib, a proteasome inhibitor frequently used to treat myeloma, prevented osteocyte apoptosis in myeloma cells . In contOnce in bone, most invading cancer cells undergo apoptosis, with a few becoming dormant and surviving . The cel+ endosteal stromal cells deficient in p53 can give rise to osteosarcoma-like lesions through unregulated self-renewal and aberrant osteogenic processes [Lastly, the role of osteocytes in the onset of primary bone tumors, such as osteosarcoma, remains unexplored. Expression of osteocyte markers  has been observed in the osteoblastic subtype of human osteosarcoma, indicating that osteocytes may potentially serve as progenitors for osteosarcoma cells ,140. Suprocesses .


Antibiotic use is becoming increasingly challenging with the emergence of multidrug-resistant organisms. Pharmacokinetic (PK) alterations result from complex pathophysiologic changes in some patient populations, particularly those with critical illness. Therefore, antibiotic dose individualization in such populations is warranted. Recently, there have been advances in dose optimization strategies to improve the utilization of existing antibiotics. Bayesian-based dosing is one of the novel approaches that could help clinicians achieve target concentrations in a greater percentage of their patients earlier during therapy. This review summarizes the advantages and disadvantages of current approaches to antibiotic dosing, with a focus on critically ill patients, and discusses the use of Bayesian methods to optimize vancomycin dosing. The Bayesian method of antibiotic dosing was developed to provide more precise predictions of drug concentrations and target achievement early in therapy. It has benefits such as the incorporation of personalized PK/PD parameters, improved predictive abilities, and improved patient outcomes. Recent vancomycin dosing guidelines emphasize the importance of using the Bayesian method. The Bayesian method is able to achieve appropriate antibiotic dosing prior to the patient reaching the steady state, allowing the patient to receive the right drug at the right dose earlier in therapy. The global utilization of antimicrobial agents has contributed to the exponential growth of bacterial resistance on a worldwide basis ,2,3. DesRecent studies focusing on critically ill populations brought to light several challenges facing clinicians. Altered pathophysiology in the pharmacokinetics (PKs) and pharmacodynamics (PDs) of antibiotics is one of the main causes of suboptimal antibiotic exposure. These PK/PD changes are difficult to track among different patient populations, patients in the same population, and even individual patients over time . Recent One of the most common antibiotics administered to critically ill patients is vancomycin, a glycopeptide antibiotic, which has been the primary agent used in hospitalized patients with methicillin-resistant Staphylococcus aureus (MRSA) infections since the 1950s. An updated consensus guideline for monitoring vancomycin in the treatment of serious MRSA infections was recently published by the American Society of Health-System Pharmacists (ASHP), the Infectious Diseases Society of America (IDSA), the Pediatric Infectious Diseases Society (PIDS), and the Society of Infectious Diseases Pharmacists (SIDP) . The revThis narrative review aims to summarize the challenges in current antibiotic dosing regimens in critically ill patients and discuss a newly recommended Bayesian approach to optimize vancomycin dosing in this population. We hope this review serves as a primer for clinicians in selecting appropriate antibiotic doses in critically ill populations, as well as other patient populations with altered PK/PD.Critically ill patients and patients with trauma, burns, febrile neutropenia, major surgery, organ dysfunction, obesity, or cystic fibrosis often have drastic changes in their physiology ,13,14,15Body fluid dynamics may have a significant impact on the PKs of many antibiotics. Most critically ill patients have altered body water volumes, resulting from dynamic cardiovascular and renal function or substantial fluid resuscitation ,20. FluiThere are several factors that have the potential to alter the volume of distribution (Vd) in the context of critical disease. Changes in the volume of distribution (Vd) primarily occur due to fluid administration during the initial stage of shock therapy . The expCritical illness and shock may lead to acute kidney injury (AKI). Consequently, AKI often results in supratherapeutic antibiotic concentrations depending on drug-specific metabolic profile. Drugs that are renally cleared, such as cefepime, accumulate during AKI and may cause adverse events if the dose is not adjusted accordingly ,29,30. H2, occurs in some critically ill and >85% of trauma patients [In contrast, several factors associated with critical illness may increase renal clearance. As described previously, elevated cardiac output increases renal blood flow and eventually increases glomerular filtration. This process is more common in patients with critical illness due to sepsis, trauma, burn injury, acute leukemia, and major surgery ,40,41. Fpatients ,40,42. AFurthermore, changes in serum creatinine (SCr), which is the most commonly used clinical surrogate in determining kidney function, often lag compared with actual renal function, especially in critically ill patients. This creates a challenge for clinicians in adjusting antimicrobial doses even when seemingly appropriate . CliniciAntibiotic dosing approaches vary greatly depending on a variety of factors, including patient population, resources, settings, and antibiotic properties. Five such strategies are discussed here and summarized in Fixed dosing regimens are commonly used for drugs with a wide therapeutic index . However, drugs with a narrow therapeutic index (NTI) or with variable intra- and inter-patient responses often require a different approach. Vancomycin has an NTI and therefore has historically been dosed based on trough concentrations. However, PK modeling has demonstrated that around half of vancomycin trough concentrations were outside the therapeutic range (10–20 mg/L), despite the use of standardized dosing guidelines [The principle of “one dose fits all” has been replaced for some drugs by covariate-based dosing, which utilizes patient covariates (patient-specific variables), such as weight (for mg/kg dosing) and renal function, to decrease the variability in drug response between patients . For sevTherapeutic drug monitoring (TDM) has therefore been developed and is used with covariate-based dosing for some drugs with an NTI or that have significant PK variability to optimize efficacy and prevent adverse effects. TDM refers to the practice of measuring individual drug plasma concentrations in order to maintain concentrations within a predetermined therapeutic range . TDM is Although TDM allows for more informed dose adjustments, exclusive reliance on TDM for dose adjustments has several limitations. TDM shares the same drawback as fixed and covariate-based dosing: Their development is based on the “average” patient response, which may not be accurate to address variability between patients . TDM mayIn the population method, antibiotic dosage can be determined based on population PK parameters without using patients’ individual PK results . PopulatThe Bayesian method was developed to overcome the issue of variability between patients and provide more precise predictions of drug concentrations and target achievement early in therapy . The BayThe Bayesian method has several advantages over traditional dosing . First, In the previous 2009 guideline, the target dosage of vancomycin was recommended to be a serum trough concentration of 10–20 mg/L, which was considered a surrogate marker for an AUC to minimum inhibitory concentration (AUC/MIC) ratio of 400–600 . EmerginCalculating AUC/MIC can be performed using either first-order PK or Bayesian PK software. The first involves the collection of two serum concentrations at the steady state (a peak after the third dose and a trough before the fourth dose) and then calculating the AUC using a mathematical equation. Alternatively, Bayesian programs require only one concentration collected within 24–48 h of starting therapy instead of waiting until achieving the steady state. As a result, these programs are able to calculate and predict AUC/MIC earlier during a patient’s treatment course. Bayesian programs also offer the advantage of increased precision since many allow for the optional addition of patient-specific variables, such as age, weight, height, and serum creatinine level. Indeed, a recent study on 116 patients found that Bayesian-guided AUC estimation resulted in faster results that were 20–25% more accurate than the results obtained using first-order PK equation-based AUC calculation . Similar0–24 . The steady-state concentration is recommended to be taken 24 h after the initiation of continuous infusion. If dose adjustment is needed, it should be implemented in increments of 250–500 mg. While the TDM of vancomycin with this dosing method can be carried out by simply drawing a level and multiplying it by 24 , utilizing the Bayesian method for vancomycin dose adjustment with continuous infusion was significantly associated with better rate of target attainment (a vancomycin concentration of 20–25 mg/L) than the standard TDM approach  [Considered a common alternative to intermittent dosing, another vancomycin dosing strategy used in ICUs is continuous infusion, which involves a continuous IV infusion of vancomycin over 24 h. Such an approach was associated with a lower risk of nephrotoxicity and better economic outcomes, as it required less monitoring than intermittent infusion ,75. The = 0.007) . HoweverGrowing evidence indicates that antibiotic dosing must be tailored based on patient-specific PK/PD changes to provide positive clinical outcomes. Patients with critical illness may experience a significant impact on the PKs of many antibiotics. A number of factors may affect antibiotic distribution in critical illness. There is an increased volume in the ECF with systemic inflammation, decreased plasma oncotic pressure, and increased antibiotic Vd. Critical illness and shock may cause AKI, which can lead to subtherapeutic or supratherapeutic antibiotic concentrations depending on the drug-specific metabolic profile.TDM is used to optimize efficacy and prevent adverse effects but requires precision to address variability between patients. It has other limitations as well, such as trial and error and delays in achieving target concentrations. The Bayesian method was developed to overcome these issues and provide more precise predictions of drug concentrations and target achievement early in therapy, with advantages such as individualized PK/PD parameters, greater prediction abilities, and improved patient care.Since recent research has emphasized the significance of implementing the Bayesian method for vancomycin dosing, it would be prudent to investigate the role of the Bayesian method in optimizing the dosage of other antibiotics, particularly those that require TDM. This will help achieve appropriate antibiotic dosing prior to the patient reaching the steady state, allowing patients to receive the right drug at the right dose earlier in therapy. The use of Bayesian programs will aid in the standardization of dosing across a wide range of institutions and health systems. Furthermore, having Bayesian programs available at the point of care will save clinicians time while also improving patient safety. Future research should be conducted to demonstrate the wide range of benefits of the Bayesian approach.


Mental and behavioral health conditions are common among school-aged children in the United States.Each year, during 2018–2023, among children and adolescents aged 10–17 years, the number and proportion of weekly emergency department visits for eight mental and behavioral health conditions displayed seasonal increases during the fall and spring school semesters relative to the summer period; timing of increases varied by specific condition.Systemic changes that prioritize protective factors, such as physical activity, social support, and inclusive school environments, and incorporate preparedness for increases in mental and behavioral health conditions during back-to-school planning might help improve child and adolescent mental health.Mental and behavioral health conditions among school-aged children, including substance use disorders and overall emotional well-being, are a public health concern in the United States. Timely data on seasonal patterns in child and adolescent conditions can guide optimal timing of prevention and intervention strategies. CDC examined emergency department (ED) visit data from the National Syndromic Surveillance Program for 25 distinct conditions during January 2018–June 2023 among U.S. children and adolescents aged 5–17 years, stratified by age group. Each year, during 2018–2023, among persons aged 10–14 and 15–17 years, the number and proportion of weekly ED visits for eight conditions increased in the fall school semester and remained elevated throughout the spring semester; ED visits were up to twice as high during school semesters compared with the summer period. Among children aged 5–9 years, the number and proportion of visits increased for five mental and behavioral health conditions. Seasonal increases in ED visits for some conditions among school-aged children warrant enhanced awareness about mental distress symptoms and the challenges and stressors in the school environment. Systemic changes that prioritize protective factors  and incorporate preparedness for increases in conditions during back-to-school planning might improve child and adolescent mental health.–Mental and behavioral health conditions among school-aged children, including substance use disorders and overall emotional well-being, are a public health concern in the United States  Clinical Classifications Software Refinedhttps://stacks.cdc.gov/view/cdc/131758). Among these categories, eight (30%) had enough data for reliable visit estimates (relative SE <30%) for all age groups and were retained in the final analysis. Results were reported on categories with consistent and significant increases during the study years.CDC examined emergency department (ED) visit data from the National Syndromic Surveillance Program (NSSP) during January 2018–June 2023 to calculate changes in the number and proportion of ED visits for mental and behavioral health conditions among children and adolescents aged 5–17 years; visits from 1,919 facilities in 46 states were included.https://stacks.cdc.gov/view/cdc/132871)  and spring school semester . Each was compared with the immediately preceding summer period  .Each year, during 2018–2023, among persons aged 10–14 and 15–17 years, the number and proportion of weekly ED visits displayed seasonal patterns for depressive disorders, suicidal ideation or self-harm, trauma- and stressor-related disorders, cannabis-related disorders, lifestyle or life management factors, mood disorders, poisoning by drugs, and symptoms of mental and substance use conditions. Compared with the summer period, higher mean weekly visit numbers and relative proportion of visits were observed during the fall school semester  and spring school semester . ED visits briefly decreased each year corresponding to the typical winter holiday break period during the last week of November and December, followed by a return to previous levels  , suicidal ideation or self-harm  and trauma- and stressor-related disorders . During the spring school semester, the proportion of visits increased for four additional conditions: lifestyle or life management factors , mood disorders , poisoning by drugs , and symptoms of mental and substance use conditions  when compared with the preceding summer period . For canThe number of weekly visits also increased for all eight mental and behavioral health conditions in the fall and spring semesters when compared with the preceding summer period among children and adolescents aged 10–14 and 15–17 years, except when comparing the spring 2020 school semester with the preceding summer 2019 period; cannabis-related disorders were the only exception in which negative percent change (−4.6%) in weekly visits was also observed among adolescents aged 15–17 years during fall 2020 . Weekly Each year, during 2018–2023, the number and proportion of weekly ED visits for eight mental and behavioral health conditions displayed seasonal increases during the fall and spring school semesters compared with the summer period; timing of increase varied by specific conditions. Trends suggest that students might need additional mental health support during the back-to-school period in the fall and throughout the academic year.,Visit patterns during the 2020 spring school semester showed a relative increase in incidence (visit ratio >1) and lower mean weekly visit counts (percent change <0) compared with the 2019 summer period. These findings indicate that the relative proportion of visits was higher while the mean weekly number of visits was lower and was likely influenced by the public health emergency declaration for the COVID-19 pandemic in March 2020  programs can promote their emotional well-being. School-based SEL programs,,****,Multisector collaboration and coordination, including government, education, and community organizations, are needed to promote and prioritize child and adolescent mental health and to avoid placing the responsibility of improvement solely on educational institutions.The findings in this report are subject to at least five limitations. First, NSSP ED visit data are a convenience sample and are not nationally representative. Second, ED visits represent unique events, not individual persons, and might reflect multiple visits for one person. Third, HCUP Clinical Classifications Software Refined categories are not mutually exclusive; codes can appear in more than one category. Fourth, results for children aged 5–9 years should be interpreted with caution, particularly data about suicidal ideation or self-harm, because of low visit volume and uncertainty about intentionality. Finally, because school start and end dates vary within and across regions, some ED visits might be misclassified, resulting in underestimation of the extent of the increase in number of ED visits for mental and behavioral health conditions; many such visits can occur outside of EDs and reasons for changes in ED visit patterns cannot be ascertained from these data.Systemic changes that prioritize protective factors  and well-being promotion might improve mental health among children and adolescents long before a trip to an ED is needed. These changes include consideration of the seasonal timing of increases in child and adolescent mental and behavioral health conditions; efforts to incorporate preparedness for mental health concerns into programmatic planning, especially during back-to-school; prevention of conditions that increase risk for mental disorders; early identification of mental health disorders; and targeted interventions. Parents and caregivers, educators, health care providers, and others who regularly interact with children and adolescents can learn about signs and symptoms of mental distress


A major barrier to success for chimeric antigen receptor (CAR) T-cell therapies in solid tumors is selecting optimal target antigens. Target antigens should be uniformly expressed by cancer cells and minimally so by healthy tissues in the body. Analyzing single-cell RNA-sequencing data of patient tumors and of healthy tissue reference atlases, we first charted the landscape of existing solid tumor CAR targets, quantifying their tumor selectivity and safety to identify the leading clinical targets. We then compared these benchmark targets to the cell surfaceome, with the aim of systematically identifying new candidate genes that are safer and/or more selective than the best CAR targets in clinical trials, as determined by our measures. Our analysis uncovered twenty new cell surface targets, five of which have both superior selectivity and safety scores, which may serve as promising candidates for CAR treatments in HNSC.selective targets, which we define intuitively to be cell surface proteins that are expressed widely by cancer cells but minimally by healthy cells in the tumor microenvironment and other normal tissues. Analyzing patient tumor single-cell transcriptomics data, we first defined and quantified selectivity and safety scores of existing CAR targets for indications in which they are in clinical trials or approved. We then sought new candidate cell surface CAR targets that have better selectivity and safety scores than those currently being tested. Remarkably, in almost all cancer types, we could not find such better targets, testifying to the near optimality of the current target space. However, in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSC), for which there is currently a dearth of existing CAR targets, we identified a total of twenty candidate novel CAR targets, five of which have both superior selectivity and safety scores. These newly identified cell surface targets lay a basis for future investigations that may lead to better CAR treatments in HNSC.Chimeric antigen receptor (CAR) T cell therapies have yielded transformative clinical successes for patients with blood tumors, but their full potential remains to be unleashed against solid tumors. One challenge is finding  Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized cancer treatment. After a patient’s T cells are extracted, they are modified to recognize a tumor-specific protein via the addition of a synthetic CAR on the cell membrane and then infused back into the patient. CAR-T treatment has resulted in durable remissions in patients, particularly in those with B-cell malignancies. Recently, CARs directed against the B-cell marker CD19 have been declared curative, with observed remission rates as high as 90%  substantially less than” the percentage of non-surfaceome genes defined as essential; only 4.1% of the former were essential versus 14.0% of the latter . We noteOur study has a few limitations. First and foremost, we have used gene expression as a proxy for protein expression, but it is ultimately proteins that are targeted by CARs. When advances in high-throughput single-cell proteomics technology come to the forefront in the future, it will be pertinent to apply the approach presented in this study to evaluate the discriminatory potential of membrane protein receptors as viable CAR targets. In the meantime, with the accumulating plethora of scRNA-seq datasets of tumors, gene expression is the best representation of cell genomics available to us. Second, the use of the AUC metric to evaluate target discrimination potential has its limitations. In practice, optimal discrimination relies on knowing the antigen detection thresholds of CAR-T cells. Currently, our control in setting the antigen detection density of CARs is poor, but emerging efforts may allow us to tune antigen detection cutoffs . This woIn summary, employing a systematic, data-driven analysis, we first chart the landscape of existing CAR targets in the clinic, identifying the leading targets in each cancer type and computing their estimated tumor selectivity and safety scores from pertaining single cell transcriptomics data. Next, further analyzing single-cell transcriptomics data, we performed a genome-wide search across many different cancer types to identify new and candidate CAR targets with both better selective and safety scores than those of extant clinical targets. Remarkably, in almost all cancer types, we could not find such better targets, testifying to the near optimality of the current target space, at least in accordance with our measures. However, one striking exception is HPV-negative HNSC, for which there is a dearth of existing targets in clinical trials. In the latter, our investigation has discovered 20 new and untested CAR targets for treating HNSC and GBM tumors more precisely and safely than extant targets, which are each followed up with a thorough literature survey and an analysis of their in vitro indication-specific dependency evaluation. A total of 5 of these 20 have selectivity and safety scores that are both higher than those computed for the existing CAR targets, whose further investigation may lead to better CAR treatments in HNSC.


Bemisia tabaci complex. The most effective method for mitigating chilli leaf curl virus disease losses is breeding for host resistance to Begomovirus. This review highlights the current situation of chilli leaf curl virus disease and associated begomoviruses in chilli production, stressing the significant issues that breeders and growers confront. In addition, the various breeding methods used to generate begomovirus resistant chilli cultivars, and also the complicated connections between the host plant, vector and the virus are discussed. This review highlights the importance of resistance breeding, emphasising the importance of multidisciplinary approaches that combine the best of traditional breeding with cutting-edge genomic technologies. subsequently, the article highlights the challenges that must be overcome in order to effectively deploy begomovirus resistant chilli varieties across diverse agroecological zones and farming systems, as well as understanding the pathogen thus providing the opportunities for improving the sustainability and profitability of chilli production.Chilli leaf curl virus disease caused by begomoviruses, has emerged as a major threat to global chilli production, causing severe yield losses and economic harm. Begomoviruses are a highly successful and emerging group of plant viruses that are primarily transmitted by whiteflies belonging to the  Capsicum spp.) is one of the oldest domesticated crops originating in the Americas , C. baccatum (L.), Capsicum chinense (Jacq.), Capsicum frutescens (L.) and Capsicum pubescens (Ruiz & Pav.) are the five domesticated species with C. annuum being the most widely grown and consumed  or two (bipartite) components of size 2.5- 3 kb, known as DNA-A and DNA-B . Begomovglobally Figure 2globally . Generalglobally . In monoglobally . The twoglobally . Begomovglobally . The tenBegomovirus are among the most economically damaging pathogens and pose a significant threat to the production of chilli   , Amaranthus spp. , Mentha  spicata , Petuniania spp.  and Miras jalapa .Begomovirus species  and PepGMV  known to cause infection (C. frutescens. When C. frutescens plants were agroinoculated with infectious clones that included both the betasatellite (1.7-mer) and partial tandem repeats of the viral genome (1.9-mer), the characteristic ChiLCV symptoms such as leaf curling and stunting appeared, but when the viral genome alone was used, no symptoms of leaf curling appeared. Furthermore, synergistic interactions among different Begomovirus species could lead to increased symptom severity and new and diverse symptoms, in addition to the new viral species with an expanded host range.Incidence and severity of the disease under natural field conditions is influenced by various external factors. Symptoms can be affected by environmental factors such as soil fertility and microclimate around plants, age of the plant and host genetic makeup  in additnfection . Typicalnfection . Kumar eBemisia tabaci Genn.; Hemiptera: Aleyrodidae) is a polyphagous insect that feeds on over 361 plant species from 89 families , an adenine-rich region, and a βC1 ORF . Betasatnt crops . For repnt crops . Betasatnt crops  contribunt crops , affect nt crops  and capant crops . Additiont crops . RecentlAlphasatellites are 1.3-1.4kb sized DNA molecules that are coupled with begomoviruses and betasatellite complexes . Though Solanum lycopersicum L.), papaya (Carica papaya L.) and cotton (Gossypium spp. L).The complete genome sequences of 83 isolates of begomoviruses associated with chilli leaf curl virus disease along with closely related begomoviruses associated with papaya, eggplant and tomato leaf curl diseases retrieved from the NCBI GenBank when subjected to phylogenetic analysis, formed 2 distinct clusters , pathogen derived resistance, insecticide application  , insect lication  and the When it comes to managing plant viral infections, smallholder farmers in Asia and Africa confront a number of obstacles. One of the most significant obstacles is a lack of access to information and resources, as well as limited access to and high cost of pesticides. Overuse of pesticides may result in the development of resistance in pest populations. Another barrier is a lack of understanding and awareness of plant viral infections. Smallholder farmers may be unaware of the indications of viral illnesses and may lack knowledge of how to adopt good disease management practices. In addition to these, the problem of plant viral infections is being made worse by climate change. The distribution and abundance of viral vectors, as well as the susceptibility of host plants to infection, can be impacted by changes in temperature and rainfall patterns. Smallholder farmers may find it more difficult to adequately manage viral infections as a result.For a management program that is to be widely adopted by farmers – it should be simple, inexpensive, and practical. One of the most important tools for managing plant viral diseases in smallholder farms is integrated pest management (IPM). IPM is a holistic approach to pest control that emphasizes the use of cultural, biological, and chemical control measures to manage pests and diseases in a sustainable and environmentally friendly way. IPM can be particularly useful for managing insect-transmitted viruses, as it can help reduce the vector population. Additionally, IPM can help to prevent the development of resistance in pest populations, which can make pest control more effective in the long run.Host plant resistance and use of resistant cultivars, combined with other production practices, forms the central component of a successful IPM program for reducing the impact of viral diseases. It has been estimated that farmers that adopt low (one or a few strategies), medium, and high (multiple strategies) integrated management strategies for begomovirus could improve incomes by 17, 26, and 80%, respectively .It is known that whiteflies can travel several kilometers and are semi-persistent and there is a direct relationship between vector level in neighboring fields and virus incidence in test fields . TherefoIn the present context of seemingly constant emergence of new begomoviruses, a clear understanding of the virus/whitefly vector/host plant interrelationships through epidemiological, phylogenetic approaches are needed. Identification and deployment of host plant resistance can be an effective and durable strategy against whitefly damage. Whitefly resistance may provide an important contribution in limiting losses associated with begomovirus, and whitefly tolerant accessions have been reported . HoweverCapsicum species and then progressed to related and wild species. For tomato, resistant sources for begomovirus have been found in the wild species of S. chilense  found more commonly in non- C. annuum than in C. annuum accessions . Morphological resistance factors physically hinder the movement of the insect on the plant, and more specifically, interfere with the mechanisms of host selection, ingestion, digestion of plant material, mating and oviposition. Plant resistance to whiteflies has shown to be mediated by morphological characteristics of the leaf surface, such as trichome density, presence of glandular trichomes, and cuticle thickness  genes in chilli plants when infested with whitefly  gene for JA-responsive signalling pathway  through PCR analysis. The variation in gene inheritance patterns may be due to multiple factors, including the species/strain of the virus, the inoculation technique used, the rating system employed, environmental factors, in addition to different sources of resistance being used.The difficulty of introgression of a trait from some accessions (field-collected wild relatives) into commercial cultivars depends on the genetic complexity of the trait. The inheritance pattern depends upon the resistant source and also on the pathogen. Monogenic recessive inheritance of leaf curl virus resistance has been widely reported . InheritThe durability of R genes depends upon the viral population dynamics, changes in pathogenicity and frequency of virulent isolates . UnderstBegomovirus, further reducing selection accuracy in field trails.An adequate and proficient protocol for germplasm screening is required to be successful in breeding for resistance to ChiLCV . NaturalBrassica oleracea var. botrytis L.) or brocolli  plants. Brassica oleraceae var. acephala DC.) and soybean (Glycine max) plants, in a greenhouse with insect-proof nets. To obtain precise results in screening mtCOI gene. The whiteflies were allowed to feed on leaf curl virus infected plants for about 24 hrs (acquisition access period). Test plants were then infested with the viruliferous whiteflies (approximately 15-20 per plant) at the 2-4 true leaf stage for 48 hrs (inoculum access period). As chilli is the non-preferred host for whiteflies, in free choice assay we cannot ensure vector infection on all the plants and in no choice essay, a single plant viruliferous whiteflies are introduced on a single plant . Whitefly colony establishment and maintenance, to transmit chilli leaf curl viruses to test entries for screening have been successfully utilized   . A leaf  gene(s) . Sakata s strain , tomato s strain , and toms strain . The usenfection . Koeda egenotype . StandarSymptom severity scales need to be established as a part of leaf curl virus inoculation protocols. Susceptible controls included in the screen should ideally become infected and show the highest symptom severity. The variability in assay conditions and symptom scoring scale will lead to contradictory results, where different resistance levels were attributed to the same genetic material. The scoring scale  was developed by Phytophthora capsici resistance locus of chilli and potato has been documented , cucumber mosaic virus , tomato spotted wilt virus  and members of Potyvirus in tomato and potato have been found to co-map in the homologous genomic region in chilli  has been found to assist in basal defense against fungi, bacteria, and viruses  and PPO Ty resistant materials in tomato, systematic research needs to be carried out to identify strain specific resistance mechanisms in chilli. This will require the regular monitoring of the pathogen population in the hot spots for the disease and screening of accessions for the most prominent and emerging strains of begomovirus using single isolate inoculation techniques. As has been described above, begomoviruses have high levels of genetic recombination and mutation as well as have interactions with other species and strains. Therefore, such a monitoring effort will require local, regional and international cooperation on an annual or seasonal basis. Once the predominant strains have been identified, single isolate associations will need to be done across a large number of accessions. There are numerous methods to conduct single-isolate inoculation including grafting, agroinfiltration, ballistic bombardment, augmented inoculation by viruliferous whitefly  have been identified, well characterized and mapped in tomato. Ty-1/Ty-3, Ty-2, ty-5, and ty-6 genes with help of marker-assisted backcross breeding. Similarly, Ty2 and Ty3 to confer moderate levels of resistance to tomato yellow leaf curl Taiwan virus (TYLTwV) and tomato yellow leaf curl Thailand virus (TYLCThV). Strain specific screening will allow the identification of resistant accessions, and after development of segregating populations, the mapping of the putative resistance genes and the design of associated molecular markers to tag strain-specific resistance or partial resistance genes for breeding and gene pyramiding. It is likely that the use of strain specific resistance will not immediately result in the development of highly resistant cultivars; however, by understanding the mechanisms of resistance against single strains or moderate levels of resistance. Strain specific resistance gene characterization and gene pyramiding can be deployed for the development of durable host resistance against begomovirus in chilli, but mapping the possibly low levels of resistance may hinder resistance mapping and subsequent gene pyramiding. In this situation, it may be necessary to identify multiple sources of moderate levels of resistance and perform breeding using recurrent selection using single strain screening.Although no systematic studies have been done to evaluate the effectiveness of the various inoculation methods and associations with disease incidence and severity in open field conditions, we prefer the augmented inoculation by viruliferous whitefly method. This method is relatively fast and less expensive as compared to the grafting method, but does introduce the possibility of selection for lack of vector preference based on plant morphology. Lack of preference by the whitefly vector would be useful in breeding for durable resistance, but it makes the identification of host-resistance genes difficult. The agroinfiltration method eliminates the possibility of confounding host resistance to the vector and the virus, but is more difficult, expensive and requires the ability to develop artificial constructs of the virus, which is not possible in many locations where begomovirus is a serious problem. Due to the presence of strain-specific resistance, gene pyramiding has been used as an effective approach to achieve durable resistance with high accuracy in tomato. Many TYLCV resistance genes  have been identified , providiAn alternative approach could be to generate a multi-parent advanced generation intercross (MAGIC) population derived from different parents that showed resistance to LCD at different disease hot spots. Resultant inbred lines could be selected for resistance to multiple viruses at disease hotspots. The advantage of the MAGIC population approach is that only one round of screening at disease hot spots is necessary to identify resistant materials and resistance genes can be directly mapped in the MAGIC population without additional crosses.Tobamovirus) and exhibiting resistance to infection by TMV (Potyvirus) in papaya and widely commercialized in the US , transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). CRISPR/Cas9 has evolved as an effective and user-friendly tool for precise and predictable targeted mutations, mostly small deletions. Inducing begomovirus resistance in chilli through mutagenesis of host factors in the plant is theoretically an option, but it would require knowledge about which genes to target in which manner. Without this knowledge, for making plants resistant to LCD, CRISPR/Cas9 only can be targeted to viral DNA. Nicotiana benthamiana) plants which had resistance to the begomoviruses using RNAi mediated gene silencing using two different hairpin RNAi TR1 (TR1-15 and TR1-8) and TR4 (TR4-1and TR4-2) constructs. RNAi method was used in tomatoes to develop resistance against multiple begomoviruses (Cucumovirus) and pepper mild mottle virus  has been developed using sense gene induced posttranscriptional gene silencing (S-PTGS) and co-expression of the coat proteins (CPs) of CMV-Kor and tomato mosaic virus  (CchGLP is a gene that encodes Germin-like proteins (GLPs), which play a crucial role in plant defense against viral infections. This gene was discovered in Capsicum chinense (Jacq.) accession BG-3821, which exhibits resistance to geminivirus infection. Through the Virus-induced gene silencing technique, the CchGLP gene was suppressed in BG-3821, resulting in susceptibility to geminivirus in BG-3821  is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. The exploitation of RNAi using various viral genes  may help in controlling the disease. oviruses  and CLCuoviruses . Sharma movirus) . siRNA mmovirus) , pepper movirus) , chilli movirus)  and in tmovirus) . CchGLP  BG-3821 . Additioansgenic .Gene silencing by artificial microRNAs (amiRNA) facilitated gene regulation at the post- transcriptional or translational level . They reSpray-induced gene silencing (SIGS) is a non-transformative strategy for plant protection involving the spraying of double stranded RNA (dsRNA) or small interfering RNA (siRNA), which target pathogen genes on plant tissues . The dsRMutagenesis has been previously applied to create variation for traits that are not present in the existing germplasm, as well as for studies of functional genomics in many crops. Mutation breeding is most effective when the loss of function of a gene result in a desirable phenotype. There are several types of mutagens that have been applied for this purpose, including chemical mutagens to induce specific types of base substitutions at a high frequency for functional genomics and the application of reverse genetics studies . RadiatiPotyvirus resistance in chilli is conferred by mutations in the eukaryotic translation initiation factor genes eIF4E or eIFiso4E, which impede interaction of these genes with the viral VPg protein (Potyvirus) is achieved through a double mutation of eIF4E (pvr1) and eIFiso4E (pvr6) translation factors  in the resistant line Perintis that encoded the Pelota protein. More recently, Mutagenesis has resulted in the identification of target specific genes and help to identify resistance genes and their function . Normal  protein . Recessi factors . In 2019Access to high quality reference genomes plays an important role in research and genomics assisted breeding. Understanding how specific genes and variants contribute to quantitative traits such as broad spectrum resistance to begomoviruses is necessary. Short-read sequencing has certainly accelerated the discovery of genetic variants, especially SNPs and indels . HoweverCapsicum, but this area of research has not been further explored. To overcome the challenges associated with repetitive sequences, Capsicum accessions projected against the Zunla-1 reference genome limiting the study in the power to detect reliably structural variation (SV), especially large SV. Recently, In conclusion, chilli leaf curl virus disease and the associated begomoviruses continue to pose a significant threat to chilli production worldwide. Developing sustainable and effective control measures to mitigate yield losses due to LCD is crucial. In principle, there are two pathways towards leaf curl virus-resistant chilli breeding cultivars that are resistant to the whitefly vector and developing varieties that resist begomovirus. In the long term, combining both approaches toward resistance is the preferred strategy. Several whitefly-resistant chilli lines have been identified, which rely on morphological and chemical defenses. Although resistance against specific Begomovirus strains is available, breeding for broad resistance against multiple viral strains remains a challenge. Moreover, the inability to diagnose begomovirus infection at an early stage is a barrier to illness management. The way forward: 1) strengthening resistance against the vector, 2) identifying germplasm resources with resistance against various begomovirus strains, and subsequent pyramiding of the resistance through recurrent selection and through MAGIC populations could be an effective strategy. The use of integrated pest management techniques that incorporate genetic resistance, cultural practices, and chemical control should be incentivized for the long-term management of the leaf curl virus. Finally, more research is required to comprehend the intricate interactions between the whitefly vector, begomovirus, chilli plants, and the environment. Advances in molecular biology, genomics, transcriptomics, and bioinformatics can provide new insights into the mechanisms of host-pathogen-vector interactions and hasten the development of effective and long-term management strategies for chilli begomovirus diseases like chilli leaf curl virus disease. To address the challenges of begomoviruses and ensure sustainable chilli production in the future, researchers, breeders, farmers, and policymakers must work together.DB and MK conceptualized the study. DB, MK, RS, and HP reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.


Food allergy is a public health issue that receives increasing attention worldwide . DefinedThis Research Topic aims to gain new insights into the role of dietary factors and gut homeostasis in maintaining oral tolerance and to provide relevant targets for designing hypoallergic systems for food allergy. Understanding the links between diet, intestinal immunity, and food allergic responses would formulate efficacious prevention and treatment options for food allergy as well as benefiting other atopic disorders.In this special Research Topic, there are five papers discussing the above-mentioned aspects.Ma et al. reported an interesting study by analyzing changes in early-life risk factors for childhood food sensitization during 2009–2019 in Chongqing, China. In addition to family history of atopic diseases, common lifestyle factors in particular feeding patterns are closely associated with the incidence of food sensitization, despite not significantly modified over a period of 10 years. Interestingly, the influence of demographic features, including gender, age and the presence of siblings changed in the past decade and should be considered as modifiable factors in future epidemiological studies.In addition to genetic predisposition, external exposures such as dietary factors are suggested to be the predominant forces driving the increased prevalence of food allergy . Ma et aWilsey et al. reported an extensive prospective study examining the short-term symptomatic improvement of suspected CMA by using amino acid formulas (AAF). After 3–6 weeks of AAF initiation, a diverse array of CMA symptoms including gastrointestinal, skin, respiratory as well as uncategorized symptoms were improved to various extents, confirming that AAF can be an effective option for the management of CMA in infancy.Considering cow's milk allergy (CMA) as the most prevalent cause of food allergy in infants, Núñez et al. conducted an in-depth study on the epigenetic changes of dendric cells (DCs) in a Pru p 3 anaphylactic mouse model in the context of sublingual AIT using an adjuvant mannose dendron-conjugated allergen. Different doses of AIT resulted in different immune responses in the model, as reflected in the induction of various DNA methylation changes in lymph nodes-DCs, mostly those linked to immune and tolerance responses such as T cell and B cell immunity, and effector cytokines. Use of these biomarkers may be of interest for further prediction of the long-lasting consequences of AIT treatments.Compared to CMA that majorly affects infants younger than 1 year old and outgrown by the age of three to five, some other types of food allergy such as peanut, seafood and fruit allergy may persist life-long . AllergeAhlberg et al. summarized the most abundant miRNAs in human milk and predicted their target genes and pathways with bioinformatic tools. The enriched pathways were found to be connected to several key signaling pathways involved in the immune system, which included the TGF-βsignaling, T cell receptor signaling, Jak-STAT signaling, and Th1 and Th2 cell differentiation. Despite functional experiments needed, the predicted implication of breast milk miRNAs highlighted their role in the regulation of tolerogenic responses that confer infants protections against immune diseases including food allergy. On the other hand, polyphenols are a group of secondary metabolites from plants that possess a wide range of biological activities including immunomodulatory and anti-allergic activities  and plant polyphenols as anti-allergic components to manage food allergy. tivities . Wu et aIn summary, the above-mentioned studies and reviews provided an extended knowledge of the above topics in food allergy. Reading this Research Topic would inform new understanding of the etiology of food allergy in relevance to the connections between dietary factors and immune regulations, reinforcing the belief that the path ahead toward resolving food allergy remains encouraging.QZ: Writing—original draft. YL: Writing—review and editing. GL: Writing—review and editing. LF: Writing—review and editing.