Gene-based therapeutics are actively being pursued for the treatment of lung diseases. While promising advances have been made over the last decades, the absence of clinically available lung-directed genetic therapies highlights the difficulties associated with this effort. Largely, progress has been hindered by the presence of inherent physical and physiological airway barriers that significantly reduce the efficacy of gene transfer. These barriers include surface mucus, mucociliary action, cell-to-cell tight junctions, and the basolateral cell membrane location of viral receptors for many commonly used gene vectors. Accordingly, airway surface preparation methods have been developed to disrupt these barriers, creating a more conducive environment for gene uptake into the target airway cells. The two major approaches have been chemical and physical methods. Both have proven effective for increasing viral-mediated gene transfer pre-clinically, although with variable effect depending on the specific strategy employed. While such methods have been explored extensively in experimental settings, they have not been used clinically. This review covers the airway surface preparation strategies reported in the literature, the advantages and disadvantages of each method, as well as a discussion about applying this concept in the clinic. Genetic therapies hold great potential for the treatment of a range of inherited and acquired pulmonary diseases. The goal of these therapies is usually to restore function of an absent or defective protein to levels that ameliorate the disease symptoms. A range of modalities are currently being explored and these can be divided into five major categories: 1) gene-addition therapy, 2) mRNA therapy, (3) gene repair, (4) mRNA repair, and (5) cell therapy [ gene-add mRNA theThe first-conceived genetic treatment option and most extensively investigated approach both pre-clinically and clinically is gene-addition, wherein a correct copy of the relevant gene is delivered to the target cells. More recently, attention has turned to the use of mRNA in therapeutics. Rather than employing DNA, mRNA molecules can be delivered to the airway cells in order to express the desired therapeutic protein . AlternaTo be effective, genetic therapies, irrespective of the modality used, require a vehicle to deliver the genetic payload. Non-viral and virus-derived vectors are under development for this purpose. Both have advantages and disadvantages, and these are partly determined by the target organ and cell population(s). Viral-derived vectors take advantage of evolutionary adaptations that enable highly effective entry into human cells. Non-viral vectors are typically less efficient gene-transfer vehicles, but they are easier to manufacture in large quantities, and have reduced immunogenicity and a lower risk profile. The major categories of viral vectors under development for lung-based genetic therapies include adenoviral vectors (AdVs), adeno-associated vectors (AAVs) and lentiviral vectors (LVs), and these will be the focus of this review article.CFTR) gene, which encodes for a protein that acts as an epithelial chloride and bicarbonate channel. Dysfunctional CFTR causes disruption to the ion and water balance across the airways, dehydration of the airway surface, accumulation of viscous mucus, and creation of an environment that is ideal for colonisation by opportunistic pathogens. Over time, CF airways are subjugated to cycles of infection and inflammation, ultimately leading to irreversible structural lung damage [Targets for lung-directed gene therapy include genetic disorders such as cystic fibrosis (CF) and alpha-1 antitrypsin deficiency, as well as acquired diseases including chronic obstructive pulmonary disease, asthma, lung cancers and others , 8. CF lg damage .CFTR gene in 1989, a gene therapy for lung disease was eagerly being pursued, and it was thought that one would be readily available in the clinic within a couple of years. Initial in vitro and in vivo pre-clinical studies demonstrated promising proof-of-concept for a CFTR gene-addition therapy [Shortly following discovery of the  therapy , howeverThe lungs are one of only a few bodily systems exposed to the outside world. Accordingly, evolution has driven the development of features that are designed to protect the airway cells from invasion by airborne pathogens, particulates, and allergens. These barriers can be divided into two major categories: 1) physical/physiological (including cellular) and (2) immunological. While the immune system poses significant issues for gene therapy that must be overcome, this review focuses on physical, physiological and cellular airway barriers to effective airway gene transfer, as these form the first line of defence. The major barriers to viral vector mediated airway gene transfer will be discussed below and are summarised diagrammatically in Fig.  physicalNotably, luminal airway barriers can be circumvented completely by using systemic based delivery systems, such as via intravenous administration. This delivery approach also has the additional benefit of distributing the therapeutic to other organs that may be affected, which is valuable for multi-organ diseases. However, systemic administration may not be suitable for all pulmonary diseases as it tends to target lung endothelial cells and pneumocytes, rather than epithelial cells . For theAirways are lined with ciliated epithelial cells and an airway surface layer that consists of two components: a mucus layer and the underlying periciliary liquid (PCL). The mucus immobilises inhaled particles and pathogens, while the PCL provides lubrication to facilitate ciliary beating, which results in trapped material being moved from the lungs toward the pharynx for cough clearance or swallowing. The coordinated interaction of these airway components forms a process known as mucociliary clearance (MCC), one of the most critical defences of the airways , 13. In If a gene vector can overcome these extracellular obstacles, the airway epithelium is the next hurdle. The epithelium primarily functions as a barrier between the external environment and internal milieu . Tight aTo deliver their genetic cargo to the airway epithelial cells, viral vectors must bind to a complementary receptor expressed on the cell surface, which results in cellular internalisation. Some vectors confer apical entry into airway cells, for example, LV pseudotypes derived from baculovirus (GP64), Sendai virus (F/HN), and influenza (HA) , as wellTo achieve long-term therapeutic effect from an airway gene therapy, permanent gene-correction of self-renewing cells will be necessary . Basal cPre-clinical development for gene therapies is typically performed in vitro or in non-diseased animal models, and thus does not consider the impact of lung disease state on gene-transfer efficacy. Pathophysiological disease processes including mucus hyperproduction, chronic infection, and inflammation create additional barriers that gene vectors must circumvent. Accordingly, the disease state needs to be considered when developing an effective lung-directed gene therapy.CF mucus is characteristically thick and adhesive, making it difficult to clear from the airways, creating a trap for inhaled particles . In retrAdVs and AAVs were unable to effectively penetrate sputum samples collected from CF patients and showed substantially reduced diffusion rates , 25, 27.Pseudomonas aeruginosa (P. aeruginosa) infection, the effect on gene-transfer was variable depending on the delivery vehicle employed. LV vector mediated gene transfer was not impacted by the presence of infection, while some non-viral DNA carriers (lipofectamine and polyethylenimine) exhibited a significant reduction in transfection ability [Bordetella bronchiseptica or P. aeruginosa) negatively impacted AAV vector transduction in mice [P. aeruginosa induced bronchopulmonary inflammation in mice has also been found to reduce AdV vector mediated gene transfer [Infection and local inflammation are also common in many lung diseases, yet only a handful of studies have investigated the impact of these factors on gene-transfer efficacy. In one study employing a mouse model of induced  ability . In a se in mice . The pretransfer .Further work is needed to fully understand the impact of disease state on airway gene transfer. Assessing the performance of individual gene vectors under disease conditions will be necessary as the biochemical and physical properties of the vector will be significant factors in their success. Until recently, this research was hindered by lack of a suitable animal model with human-like CF lung disease. However, newer models that develop infection and muco-obstruction of the airways, including CF ferret and pig models, may aid in facilitating these studies –32. UltiLimited ability to perform effective airway gene transfer has led to the development of novel vector engineering approaches to enhance delivery and uptake into the target cells. Improved airway cell tropism can be achieved by optimising the chosen vector pseudotype for LVs , 34 or vAn alternative strategy to increase gene transfer, and the focus of this review article, is the use of techniques that prepare the airways for gene transfer, making the cells more receptive to transduction.Depending on their nature, airway surface preparation techniques can produce a range of biological effects. Described (and hypothesised) effects include removal/dislodgment of airway mucus, impairment of MCC action including deciliation of cells, disruption of cell–cell tight-junction integrity, and sometimes, the removal of surface epithelial cells. These techniques transiently modulate physical and physiological airway barriers, increase vector residence time, as well as enable access to basolateral-located receptors and airway basal cells .Airway surface preparation techniques that have been reported in the literature can be divided into chemical and physical strategies. This section will review and summarise the airway conditioning methods that have been commonly employed in conjunction with viral vector mediated gene transfer. Based on the available evidence, we will comment on the advantages and disadvantages of each approach and speculate on the best options to pursue.Chemical-based conditioning is the most frequently reported airway surface preparation method. A range of chemicals with varying properties have been explored for this purpose, including both liquids and gas Table . In someSurfactants were one of the first known compounds to be used as airway conditioning agents and were hypothesised to work by enhancing the uniformity of pulmonary gene transfer. Survanta® (modified bovine pulmonary surfactant) co-delivered with an AdV vector successfully improved airway gene expression in vivo, demonstrating early proof-of-concept , 42. SinLPC is one of the most extensively studied airway conditioning agents for viral vector mediated gene transfer. LPC is a natural component of pulmonary surfactant, and when applied to the airways, the histological effects are concentration dependent. High concentrations result in more overt consequences including loss of cilia, lifting or removal of surface cells, and in some cases, stripping of the epithelial layer . LPC alsLPC has been essential for producing LV VSV-G mediated gene transfer in the nasal airways of mice –48 and rHelper dependent AdV (HD-AdV) is a robust vector particle that can be formulated in LPC (0.01–0.1%), allowing for one-step administration that produced extensive reporter gene transduction after aerosolisation to the lungs of rabbits . RepeateLPC enables successful airway gene transfer using a range of viral vectors and animal species, with its use continuing to be routine in animal studies. The extensive pre-clinical use of LPC (including studies in non-human primate species) suggests a favourable safety profile, though this has not been examined directly. Based on the current evidence, LPC appears to provide variable enhancement effects, highlighting the need to optimise the concentration, volume and timing interval for each individual application.Other fatty-acid surfactants have also been explored, but less comprehensively than LPC. Polidocanol (PDOC) is a synthetic non-ionic detergent and has previously been used clinically as a locally injectable sclerosing agent for varicose vein treatment, where concentrations up to 3% were found to be well tolerated . PDOC haIn more recent times the use of PDOC for facilitation of viral-mediated airway gene transfer has fallen out of favour. Instead, PDOC has been employed in the lungs for other experimental purposes. Application of higher concentrations of PDOC  to the airways of rodents can remove the surface epithelium, while leaving the basal cell layer relatively intact . The abi10, is the sodium salt of the medium-chain fatty-acid capric acid. Sodium caprate has been used clinically to enhance drug permeability across the intestines, and was a component of an approved rectal suppository [Sodium caprate, also referred to as Cpository . Like otpository .12), also enhanced AdV gene transfer [10.Direct formulation of AdV vectors with sodium caprate produces complete loss of vector viability; therefore, two separate administrations are necessary , 74. SodApplication of sodium caprate to the airways of mice induced mild histopathological changes and an increase in airway responsiveness, indicating low-level toxic effects in the lungs . While aSeveral groups have reported EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) enhances viral vector mediated airway gene transfer. Unlike the fatty-acid derived compounds, EGTA is a calcium-chelating agent that reduces the intracellular concentration of calcium ions to disrupt calcium-dependent formation and stabilisation of tight-junction protein complexes , 75. As EGTA conditioning has enhanced airway gene transfer for AAVs, AdVs (including HD-AdVs), LVs and retroviruses –79. SuccP. aeruginosa infection received nebulisation of EDTA and a concomitant antibiotic. In these studies, inhaled EDTA resulted in no harmful effects or adverse events [EGTA offers certain benefits as an airway conditioning agent for use in a clinical setting. Unlike other conditioning agents, EGTA can be formulated with viral vectors (including LVs), allowing for one-step delivery to airways , 75, 79.e events , 83. Howe events . Moreovee events .2 and CO2, and has a high density and low surface tension, allowing it to distribute throughout the conducting airways and alveoli [Perfluorochemicals (PFCs) are non-toxic substances that consist of chemically inert fluorinated carbon chains. PFC liquid has characteristics that are suited to use in the lungs. It is highly soluble in O alveoli , 85. Due alveoli , 87. Oth alveoli .Use of PFC liquid prior to vector administration improved AdV and AAV vector mediated gene transfer to the airways of rodents , 86, 88 PFC has properties that are consistent with a good clinical safety profile. It is not metabolised by the kidneys or liver, and is eliminated by evaporation during exhalation or transpiration through the skin . Non-hum2), has also been explored for enhancing viral vector mediated gene transfer. While delivery of fluid can result in non-homogeneous effects, gases provide the benefit of uniform distribution throughout the airways, particularly when two separate administrations are required for the conditioning compound and viral vector. There are two proposed mechanisms that underpin the gene transfer enhancement effects from SO2 inhalation: (1) direct injury and denuding of the surface epithelium due to luminal cell death and sloughing following SO2, and (2) increased paracellular permeability in areas of less severe injury, allowing viral vectors access to basolateral receptors and basal cells [One gas, sulphur dioxide (SOal cells .2 for airway conditioning. In one study, mice receiving SO2 inhalation-induced injury followed by delivery of a murine leukaemia retroviral vector demonstrated significant improvement in tracheal cell transduction, while those without SO2 injury had no observable gene expression [2 inhalation, while gene transfer did not occur in the absence of SO2 conditioning [A handful of studies have explored the use of SOpression . Similaritioning .2 inhalation successfully enhances airway gene transfer in rodent models and offers delivery advantages over liquid formulations. While this work highlighted the feasibility of using a gas for airway surface preparation, there has not been any further studies in this area. Lack of interest is likely due to the inability to translate this approach to the clinic, particularly given the severe airway injury produced and toxic effects associated with SO2 inhalation [SOhalation .Physical perturbation of the airways involves the use of a method or device (examples depicted in Fig. High levels of in vivo airway gene transfer are commonly reported in regions inadvertently abraded with delivery instrumentation, such as an endotracheal tube or a bronchoscope , 94. MorIn a previous investigation by Pickles et al. the intercartilaginous regions of mouse tracheas were externally compressed with forceps and AdV vector delivered via a tracheostomy, resulting in distinct lines of gene expression in the compressed regions . HoweverPhysical perturbation successfully enhances viral-mediated airway gene transfer, however, the gene expression produced tends to be non-uniform across the tissue. This is likely because the gene-transfer enhancement effects localise only to the regions where the device is applied, therefore achieving widespread gene transfer throughout all airways with this method may be challenging. However, the limited local effects produced by physical perturbation may also be an advantage, as this will prevent extensive damage to the airways. Furthermore, targeted or successive treatments to localised lung regions could be enabled with this approach. Variable efficacy of physical perturbation has been noted in animal studies, which could be due to the use of relatively crude techniques and instruments, however, the more recent use of a flexible wire basket in rat airways indicates the ability to refine these methods . MoreoveClinical translation of this approach may be more challenging than chemical methods due to procedural complexity and the need for controlled and precise techniques. Importantly, the lung has an extensive ability to respond to injury and regenerate lost or damaged cells , as showAn important notion that arises when employing conditioning methods for airway gene therapy is the ability to translate these techniques to the clinic. Ultimately, should these methods be restricted to the realms of experimental investigations, or is there value in actively pursuing selected techniques for clinical development?Conditioning methods act to intentionally disrupt naturally occurring protective airway barriers, which raises concerns, particularly when they are employed in a lung with existing infection, inflammation, and tissue damage. In particular, there are concerns that disrupting the epithelial integrity could enhance the leakage of bacterial products and inflammatory mediators into the submucosa, resulting in further damage to the lungs . MoreoveFor chemical methods, understanding the toxicity and metabolism profiles of the compound is critical. While investigators have explored some safety aspects of conditioning compounds in experimental settings, more extensive assessment is necessary for use in humans. In addition to this, the delivery procedure must be considered. Two-step protocols where conditioning is performed prior to gene vector delivery are more complicated than one-step methods, and for chemical approaches, nebulised delivery of conditioning agents is preferable to fluid administration in clinical settings. For physical perturbation, the procedure will require patient sedation to enable successive deployment of the device to the airway branches.There are currently no clinically available airway gene therapies, and protocols that are nearing early phase clinical trials do not include an airway surface preparation step . In the Looking more closely at the lungs, there are many clinically used procedures that are considered invasive and produce some level of airway damage. For example, the use of whole lung lavage is an effective treatment for alveolar proteinosis, and more recently, silicosis , 102. WhExamining these routinely used procedures suggests that there is sound rationale for the application of airway surface preparation methods in the clinic to enhance the effectiveness of gene transfer. However, the risk versus benefit ratio needs to be considered. If high levels of gene transfer can be achieved using these strategies, then significant clinical benefit is expected. Furthermore, improved access to airway stem cells facilitated by conditioning techniques could enable gene correction of this self-renewing population of cells, providing potential for long-term gene expression and therapeutic effect.Airway-delivered genetic therapies continue to show poor efficacy due to the presence of natural airway barriers. Early gene therapy clinical trials performed in CF patients using AdV and AAV-based vectors demonstrated safety and proof-of-concept, but many of these trials failed to meet their primary endpoints and patients demonstrated poor gene expression , highlig


In these studies, local delivery of FTY720 was found to increase pro-regenerative immune cell phenotypes , vascularization, cell proliferation and collagen deposition. Delivery of FTY720 to a localized wound environment demonstrated increased bone, muscle, and mucosal regeneration through changes in gene and cytokine production primarily by controlling the local immune cell phenotypes. These changes in gene and cytokine production reduced the inflammatory component of wound healing and increased the migration of pro-regenerative cells (neutrophils and macrophages) to the wound site. The application of FTY720 delivery using a biomaterial has demonstrated the ability of local delivery of FTY720 to promote local wound healing leveraging an immunomodulatory mechanism.In 2010, the FDA approved the administration of FTY720, S1P lipid mediator, as a therapy to treat relapsing forms of multiple sclerosis. FTY720 was found to sequester pro-inflammatory lymphocytes within the lymph node, preventing them from causing injury to the central nervous system due to inflammation. Studies harnessing the anti-inflammatory properties of FTY720 as a pro-regenerative strategy in wound healing of muscle, bone and mucosal injuries are currently being performed. This in-depth review discusses the current regenerative impact of FTY720 due to its anti-inflammatory effect stratified into an assessment of wound regeneration in the muscular, skeletal, and epithelial systems. The regenerative effect of FTY720   The timeline of normal wound healing consists of 4 distinct phases: hemostasis, inflammation, proliferation, and wound remodeling . These pin vivo wound healing models. FTY720 is currently FDA approved to treat relapsing multiple sclerosis, which potentially shortens the route for repurposing the use of FTY720 in wound healing, leveraging its immunoregenerative effects  specifically control lymphocyte egress and are expressed on a variety of cells, such as immune and cardiovascular cells  lipid mediator that has been proven to be effective in reducing lymphocyte egress from lymph nodes to stop lymphocyte infiltration into the efferent lymph, which is used to treat relapsing multiple sclerosis . S1P repar cells . S1P4-R ectively .m of SPHK1, SPHK2 is much more efficient in phosphorylating FTY720, and mainly occurs in the liver , a debilitating auto-immune disease . Multipl+ lymphocytes in the lymph nodes, and other immune cell functions. CD4+ lymphocytes are crucial in activating cells of the adaptive and innate immune system, mobilizing macrophages and B-lymphocytes  (PLGA) and polycaprolactone (PCL) along with the drug that can be diluted in solvent . Once thThough nanofiber scaffolds are quite common, this review also examines studies that utilize microspheres, thin films and polymer coated allografts to delivery FTY720. In these articles, the microsphere biomaterial is made via the single emulsion method, where the PLGA and drug are dissolved via sonication and the solution is then slowly ejected into Poly vinyl alcohol while stirring overnight . MicrospIn this review, several biomaterial treatments for FTY720 administration are described, including nanofiber and microsphere delivery. The release kinetics of FTY720 from a scaffold will determine the size of scaffold for implantation, dosage of FTY720 for the animal and will allow the quantification of drug release to correlate with the effects of tissue regeneration. In nanofiber scaffold form, the exact concentration of drug and release time are evaluated via UV-Vis spectroscopy, Liquid Chromatography Mass Spectrometry (LCMS) and High Performance Liquid Chromatography (HPLC) . Using tin vivo, such as PEG, gelatin, and collagenase, as well as a variety of crosslinking methodologies such as UV light, chemical crosslinking or heat treatment , there are several characteristics that are exhibited by this type of wound healing that are unique. For instance, the importance of re-epithelization in these wounds is critical . KeratinFor epithelial wound healing, FTY720 has been used as a treatment for hypertrophic scarring, oronasal fistulas, and cutaneous and subcutaneous defects. Hypertrophic scarring refers to the fibrotic condition that can occur after trauma and severe injury to the cutaneous epithelial tissue . HypertrTo test the impact of FTY720 delivery on oral mucosal wound healing an oronasal fistula (ONF) model was created . An ONF There have been a variety of FTY720 loaded biomaterials used to improve wound healing of muscle tissue. Though the typical stages of wound healing are generally followed in muscle regeneration, there are a few hallmarks that can systemically differentiate the muscular wound healing process from that of an epithelial wound. Muscle regeneration typically begins 4-5 days after injury, but requires the recruitment of adult muscle stem cells, specifically satellite cells, as endogenous muscle fibers do not have the ability to divide . These sThe biomaterial treatments used to delivery FTY720 in volumetric muscle loss (VML) defect models include films and scaffolds. VML arises from the injury of muscular tissue which results in an unsuccessful regeneration mechanism. This unsuccessful regeneration can also be characterized by the influx of pro-inflammatory myeloid cells into the area of injury which can exacerbate the injury to the point that regeneration is no longer possible . This dyin vivo, with quantifications in osteogenesis, whereas 1 only noted the presence of a pro-osteogenic signaling pathway.The use of FTY720 during bone healing and regeneration with skeletal bone defects has been evaluated in the regeneration of bone in cranial defects. A variety of biomaterials have been utilized in the wound healing of these skeletal defects, such as FTY720 loaded hydrogels and scaffolds . To studDuring healing of different types of wounds  there are populations of pro-regenerative and pro-inflammatory immune cell phenotypes that are commonly present. Progenitor origin of immune cells dictates that the phenotype of each cell is determined from myeloid stem cells, that then differentiate into myeloblasts and then monocytes . MonocytA comparison of FTY720 based tissue regenerative outcomes are shown in The efficacy of FTY720 on wound healing has been demonstrated by the studies presented and the various drug delivery approaches. The primary modalities of FTY720-associated wound healing were pro-regenerative immune cell recruitment, vascularization, collagen deposition and mRNA marker expression. Though each organ system has a slightly different mechanism of wound healing, these features discussed below provide insight into the efficacy of the drug in regard to epithelial, skeletal and muscular regeneration.The inflammatory cell response over the period of wound healing allows for the differentiation of several immune cell phenotypes, with a variety of biomarkers and cytokines to indicate recruitment and progenitor type. Most of these biomarkers used to identify particular inflammatory cells are found by staining for a surface antigen, such as Ly6C and CD206 . The poslow anti-inflammatory monocyte population was found to be increased in 5 studies following the delivery of FTY720 treatment , transforming growth factor beta (TGF-β) and angiopoietin that are critical in wound angiogenesis . InformaQuantification of the angiogenic impact of the delivery of FTY720 was performed in 5 studies using CD68 or CD31 staining to quantitatively assess the macrophages present and quantify the new vascularization, respectively. The remaining articles utilized Microfil Enhanced micro-CT or immunohistochemistry to assess FTY720 induced angiogenesis . CD68 isin vitro, and was measured via real time quantitative PCR (RT-qPCR)  . Li et aRT-qPCR) . SeveralRT-qPCR) . This maRT-qPCR) .in vitro as discussed in in vitro . Variables yet to be considered in topical FTY720 delivery include systemic effects and concern for increased risk of infection. Although FTY720 is approved for human use in multiple sclerosis relapse patients, large animal studies to test FTY720 is the next step in testing FTY720 for wound healing in humans as an immuno-regenerative therapy.This review presents an in-depth analysis on the potential applications of FTY720 on regenerative wound healing through cell proliferation, immune cell modulation, collagen deposition and vascularization. Although FTY720 is an FDA approved treatment for multiple sclerosis, it is believed that FTY720 can have a pro-regenerative effect on wound healing via the delivery of a variety of biomaterials through the modulation of lymphocyte function. This review focused on results gained from


Nuclear medicine has emerged as a pivotal player in cancer patient care, revolutionizing the way cancer is detected, diagnosed, monitored, and treated. The specific decay characteristics of the radionuclide determine if the radiopharmaceutical can be used for diagnostic (positron emission tomography (PET) and single-photon-emission-computed tomography (SPECT)) or therapeutic (targeted radionuclide therapy (TRT)) purposes. Expression of the molecular target is confirmed during the diagnostic phase, predicting the response to the treatment administered during the therapeutic phase and, afterwards, the effect can be followed-up in downstream diagnostic scans. This concept is called theranostics, see what you treat and treat what you see, and is an excellent example of personalized medicine. As big pharma has stepped into the arena of radiopharmaceuticals for TRT and contributed to the initial success and large investments in this area, a spectacular growth in these radiopharmaceuticals is expected in the near future.The Special Issue “Radiopharmaceuticals for Cancer Imaging and Therapy” delves into the rapidly evolving landscape of this research field, highlighting the progress made in identifying novel molecular targets, vector molecule design, and radiochemistry using established and upcoming radionuclides and dosimetry to achieve a more specific imaging of cancer targets and improve therapeutic efficacy. In this Special Issue, 137 authors have contributed, with thirteen original research articles and four reviews, providing important insights into the field.153Sm, resulting in [153Sm]Sm-DOTATATE, and successfully evaluated the compound to target somatostatin receptors overexpressed in neuroendocrine tumors Zr-matuzumab as a PET probe for the diagnosis/monitoring of response to the treatment of a noncompeting anti-EGFR nimotuzumab antibody drug conjugate (ADC) using mouse colorectal cancer (CRC) xenografts [89Zr]Zr-matuzumab in mice bearing EGFR-positive xenografts showed a high uptake that could be blocked with the pre-dosing of matuzumab but not with the noncompeting binder nimotuzumab. The simultaneous use of these non-competing antibodies represents a novel approach that holds promise for improving the diagnosis and treatment of EGFR-positive CRC in clinical settings.Fonge et al., from the university of Saskatchewan, have successfully developed and evaluated [nografts . MicroPE64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications [Antibodies are interesting vector molecules for the development of novel radiopharmaceuticals, but they have a long blood circulation time and delayed tumor uptake, often limiting their diagnostic applications. Smaller antibody fragments or affibody constructs are excellent platforms for diagnostic radiopharmaceuticals in combination with short-lived PET radionuclides, allowing for good contrast imaging sooner after injection. Dewulf et al., from the university of Antwerp, reported a novel RANKL PET imaging agent, [ications . Researcications ,6.177Lu-labeled minigastrin analogs targeting CCK2R [Peptide analogs have also shown great promise as vectors for targeted cancer therapy. The research article by Hörmann et al. investigates the impact of N-terminal peptide modifications on the in vitro and in vivo properties of Finally, accurate dosimetry can become a crucial aspect of targeted radionuclide therapy to achieve optimal therapeutic efficacy while minimizing potential side effects. The article “A Review on Tumor Control Probability and Preclinical Dosimetry in TRT” offers a comprehensive review of tumor control probability and preclinical dosimetry techniques, together with their limitations and applications . The conThe guest editors of this Special Issue extend their heartfelt gratitude and sincerely appreciate all the authors and reviewers who responded to our invitation. Your willingness to share the outcomes of your outstanding research has greatly enriched this collection, and your contributions have been invaluable in the meticulous evaluation of the manuscripts.


Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration. The heart is composed of heterogenous cellular populations that tightly regulate its function. In a mammalian heart, cardiomyocytes (CMs) occupy 70% to 85% of the volume, and non-myocyte cells (NMCs) occupy the remainder . In termGeneration of CMs has been revolutionized by advancement in induced pluripotent stem cell (iPSC) methodologies and techniques. Although iPSC technology is currently unable to regenerate all cell types for specific customized cell therapies, it has indeed paved the path for providing tailored cells for disease modelling and drug screening . In cardIn this scenario, cardiac direct reprogramming has emerged as a potential solution to some of these limitations. The present review will focus on the recent updates and the potential of cardiac direct reprogramming of NMCs to restore and/or minimize the cardiac damage caused by MI.Direct reprogramming, also referred to as transdifferentiation, is direct lineage conversion of somatic cells from one specialized cell type into another without transitioning via an intermediate pluripotent state, thus driving them towards a desired cell fate. This process needs sufficient numbers of starting cells amenable to transdifferentiate into terminally differentiated cells. Back in 1987, Davis et al. demonstrated, for the first time, that the overexpression of one transcription factor could rewrite cell fate in vitro . Indeed,Beyond in vitro approaches, in vivo cardiac direct reprogramming of NMCs offers unique advantages such as more rapid conversion by skipping the complex ex vivo isolation step, as well as selection and expansion, and it will potentially increase the differentiation efficiency, due to the interaction with mechanical and biochemical properties of the microenvironment as well as biomolecules released by ECM ,26. MoreIn the heart, CFs are found in the interstitium and are primarily responsible for deposition of the extracellular matrix (ECM) that provides support to the contracting myocardium. CFs surround myocytes throughout the heart, where they majorly coordinate cellular and acellular turnover of connective tissues, thus maintaining the structural and cellular integrity of the heart. The heart’s collagen proteins are produced by CFs; the most abundant (85%) isoform is type I collagen, which is responsible for the strength of the heart by forming thick fibres in ECM scaffolding , while aUpon cardiac injury, CFs become activated and secrete proinflammatory factors, re-enter the cell cycle to promote cell proliferation, and migrate to injured areas for promoting wound healing and scar formation ,38. ActiCellular reprogramming ideally requires high numbers of target cells that have the capability to differentiate. Activated CFs, due to their proliferative potential following cardiac injury, represent a good source for in vivo cardiac reprogramming. Transdifferentiation of activated CFs into induced CMs (iCMs) might potentially minimize infarct thickness and scar size, hence improving cardiac function after myocardial injury. For this reason, to direct activated fibroblasts towards iCMs has emerged as an attractive option. Zhou et al. attempted to decipher the differences between beating iPSC-CMs and iCMs, both generated by transcriptome studies from CFs of the same origin. Both cell types showed CM-like molecular signatures, but iPSC-CMs expressed comparatively hyperdynamic epigenetics similar to embryonic CMs, whereas iCMs exhibited more a mature status that resembles adult CMs. In addition, in terms of metabolism, iPSC-CMs mainly employed glycolysis, whereas iCMs used fatty acid oxidation as the key pathway. Notably, iPSC-CMs and iCMs exhibited different cell-cycle status, as the cell cycle was active in iPS-CMs but inactive in iCMs . Hence, Several in vitro and in vivo studies have investigated how regulation of various factors in CFs can direct their reprogramming into iCMs, which will be further discussed in this section .In fibroblasts, right expression of specific transcription factors (TFs) directs them to new cell identity. One of the direct cardiac reprogramming strategies exploits some specific genes that are expressed in the early stages of heart development. Induction of three cardiac transcription factors (TFs) expression in CFs, i.e., Gata4 (G), Mef2c (M), and Tbx5 (T), also called GMT, enabled their reprogramming into iCMs . This coMore recently, Mathison and colleagues tried to enhance CFs’ reprogramming efficacy of GMT treatment via EC transdifferentiation . They shBefore this, only chronic integrating viral (retrovirus and lentivirus) based approaches had been considered for gene integration into the host cell genome to induce reprogramming. This procedure, however, posed serious mutagenesis limitations in clinical feasibility and applicability of these viral vector assisted cardiac regeneration strategies. As transdifferentiating gene expression is required for a limited time to induce iCM generation, scientists thus started investigating non-integrated acute viral based approaches. Mathison and colleagues attempted to study non-integrating acute adenoviral vectors expressing GMT and compared them with integrating lentiviral vectors expressing GMT in infarcted rat hearts and found that both viral vectors were equally effective to transdifferentiate CFs in vivo into iCMs, allowing heart function improvement in post-infarct rat hearts . Later, Besides viral-mediated delivery approaches, nanoparticle-mediated delivery has gained significant importance. Chang et al. employed cationic gold nanoparticles (AuNPs) conjugated with polyethylimine (PEI) to improve delivery of GMT without genomic integration. AuNP/GMT/PEI nanocomplex improved transdifferentiation of mouse CFs into iCM in vitro. Further in vivo study in an infarcted mouse model also highlighted significant success of this approach as injection of this nanocomplex showed improvement in cardiac structure and function two weeks after MI. In addition, reduced scar size and recovered thick myocyte bands were observed in the infarcted zone. In terms of cell maturation, this approach seemed more successful in vivo as indicated by the production of more mature iCMs in the heart environment compared to the in vitro condition. This was pointed out not only by the increased expression of Tbx5 but also by the augmented level of sarcomeric proteins , which resulted in a well-organized sarcomere structure, essential for improving cardiac function, although cardiac functionality was not assessed by the authors. Most likely, the promising in vivo results relied on ECM and growth factor cues provided by the cardiac microenvironment that enabled reprogrammed iCMs to develop into a more mature phenotype .Wang et al. highlighted that G, M, and T protein stoichiometry in iCM reprogramming also holds significance. They found that polycistronic vectors expressing relatively high levels of Mef2c and low levels of Gata4 and Tbx5 allowed efficient reprogramming of mouse primary CFs into iCMs by more than tenfold compared to unbalanced G, M, and T protein expression . The samRecently, Tani et al. created a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system for expression of Mef2c/Gata4/Tbx5/Hand2 (MGTH) to repair chronic MI in mice. It was found that this therapeutic approach has reprogrammed ≈2% of resident CFs into iCMs. Notably, a majority of iCMs were the result of bona fide cardiac reprogramming rather than fusion with CMs. Significant improvement in myocardial contraction and reduced fibrosis was observed. Microarray studies found that the MGTH overexpression activated cardiac program and simultaneously suppressed fibroblast and inflammatory signatures in chronic MI. Furthermore, single-cell RNA sequencing revealed that cardiac reprogramming stimulated conversion of profibrotic CFs to a quiescent antifibrotic state. MGHT overexpression suppressed Meox1, a key gene involved in fibroblast activation that has partly contributed to induction of antifibrotic effects. However, the precise mechanism that has contributed to the MGTH-mediated transcriptional switch between cardiac program activation and fibrotic suppression remains unexplored .It soon became evident, however, that for cardiac reprogramming, the combination of TFs appeared to be species-specific. In fact, it has been demonstrated that GMT was able to induce cardiac reprogramming in mouse fibroblasts , whereasOverall, over the years it has been realized that the efficiency of these cocktails involving only TFs is inadequate for the reprogramming of human CFs into iCMs; for this reason, strategies that combined TFs with other factors were explored. Furthermore, for a safer clinical application, investigations are presently focusing on alternative strategies to the integrating viral vectors. Recently, significant progress on in vivo cardiac reprogramming has been made by introducing for the first time non-integrating viral vector systems to in vivo transgene-based cardiac reprogramming. Since successful target selection is needed for transferring the in vivo cardiac reprogramming procedure into a therapeutic application, promoter-based selective targeting for NMCs would be worth deep investigation in this field.During cardiac reprogramming in CFs, waves of chromatin remodelling events begin at the transcriptomic level and include rapid accession of cardiac gene signatures and progressive loss of CF molecular program ,57,58,59This transcription remodelling is driven by upstream chromatin landscape repatterning that involves expurgation of epigenetic hurdles to reprogramming, including, for example, histone modifications and suppressive DNA methylation . EpigeneSilencing of p63, an epigenetic regulator, has also been shown to enhance transdifferentiation of neonatal rat and adult human CFs into iCMs. Recently, Pinnamaneni and colleagues have reported p63 silencing by short hairpin RNA for p63 (shp63) along with cardiogenic differentiation factors Hand2 and Myocardin enhanced expression of CM markers in CFs compared to GMT treatment with or without shp63. Further investigation revealed that epigenetic modulations played a key role as overexpression of the p63 motif transactivation inhibitory domain (TID) inhibited p63 binding with histone deacetylase 1 (HDAC1), and p63-TID+ Hand2/Myocardin resulted in better cardiac reprogramming of CFs into iCM compared to the shp63+Hand2/Myocardin combination. It was concluded that the p63-TID overexpression reprogramming strategy could have the potential to circumvent epigenetic hurdles to CF cardio-differentiation .Overall epigenetic barriers to reprogramming identified in mice and humans are different. Integrative analysis of transcriptomic and epigenomic data are providing a new mechanistic understanding of human iCM reprogramming.Beyond the previous epigenetic regulation approaches, microRNAs (miRNAs) also hold the potential for direct cardiac reprogramming. miRNAs are small RNA molecules that, based on sequence complementarity, recognize target mRNAs, resulting in their silencing.miRNAs along with a combination of TFs and other various factors have been reported to be useful in the conversion of human CFs into CMs. Nam et al. reported that a combination of GHMT, Myocardin with miR-1, and miR-133 has the ability to transdifferentiate human adult CFs into CMs . Singh eSelected miRNA clusters can also be employed for cell reprogramming without involving exogenous TFs. On the basis of the fact that a single miRNA has the ability to regulate multiple transcripts, it is considered that miRNA clusters can synergistically induce cellular reprogramming by regulating multiple targets. Jayawardena et al. found that four microRNAs  termed as miRcombo combined with JAK inhibitor I treatment was ample to induce in vitro cardiac reprogramming of CFs into iCMs. They further proved it in mice through lineage-tracing methods by employing a lentiviral delivery approach to deliver miRcombo in in vivo infarcted mouse hearts . Later, To understand whether miRcombo can be translated into humans, recently it was shown that miRcombo has the ability to in vitro transfect adult human CFs and induce their reprogramming into iCMs . AlthougDuring this course of in vivo cardiac reprogramming, scientists have also made tremendous efforts to improve delivery methods of miRcombo in infarcted hearts, as direct administration of naked miRNAs is difficult based on their negatively charged surface and potential swift degradation. Hence, effective, biosafe, noninvasive, and biocompatible vehicles are obligatory to protect and deliver miRNAs into the targeted region. In an effort to improve the delivery method of miRcombo for in vivo reprogramming, Wang et al. loaded miRcombo on mesoporous silicon nanoparticles coated with FH peptide-modified neutrophil-mimicking membranes (sequence: FHKHKSPALSPV). They used this construct in in vivo infarcted mouse hearts and found that resident CFs were induced to reprogram into iCMs. The attenuation of fibrosis accompanied by the improvement of cardiac function prove that the use of nanotechnology is a successful strategy to deliver miRNA . Other aIn further studies, different nanomaterials have been employed to deliver the reprogramming cocktail. Yang et al. used branched polyethyleneimine (BP) coated nitrogen-enriched carbon dots (BP-NCDs) to load miRcombo. In mouse CFs, delivery of this nanocomplex, within three weeks, produced a significant amount of contractile iCMs, whose presence was also confirmed by increased expression of cardiac markers, i.e., Gata4, Tbx5 Hand2, NPPA, NKX2-5, MYH7, and TNNT2. Furthermore, this approach was tested injecting this nanocomplex into the epicardium along the border zone of infarcted mice, which, after 4 weeks, showed a significant decrease in cardiac fibrosis and infarct thickness . RecentlOverall, the potentiality of different miRNA cocktails for direct reprogramming is clearly emerging and might be effective in adult human cells, accompanied by recent progress to improve their delivery methods through the use of different biosafe and biocompatible nanomaterials. Further investigation in cardiac tissue mimetics and in vivo studies are necessary, however, to demonstrate the full potential to improve cardiac reprogramming and maturation in the presence of biochemical and biophysical stimuli.Cardiac reprogramming can be done successfully by modulating the genes that are involved in the activation of heart genetic program and maintenance of fibroblast identity. Indeed, it was proved that pro-fibrotic pathways, including TGFβ and Wnt signalling, served as barriers to heart reprogramming, and their inhibition would enhance cardiac reprogramming. Overactivation of the transforming growth factor beta (TGF-β) pathway attenuated cardiac regeneration, but its inhibition enhanced cardiac reprogramming from CFs to iCMs ,80,81,82Kaur et al. used a modified mRNA (modRNA) reprogramming cocktail, named 7G-modRNA, comprising GHMT, along with an inhibitor of TGFβ (DN-TGFβ), an inhibitor of Wnt (DN-Wnt8a), and acid ceramidase (AC) to reprogram NMCs into iCMs during ischemic conditions after MI in mouse models. It was found that pro-angiogenic factors were upregulated, whereas fibroblast markers were downregulated. This cocktail reprogrammed CM-like cells in the scar area, reduced scar size, promoted capillary density, improved cardiac function, and increased survival after MI. However, it was unable to develop de novo beating CMs either in vitro or in vivo . This liNOTCH signalling plays an important role during development and maturation of CMs. Activated NOTCH signalling promotes differentiation of NMCs into the cardiogenic lineage but prevents CM differentiation. Therefore, in developing myocardium inhibition of the NOTCH pathway is important to obtain fully mature and functional CMs . DownregBesides the previously presented pathways, various other signalling pathways can be regulated to enhance and improve cardiac reprogramming. Wang et al. identified that autophagy is also linked with direct cardiac reprogramming. In MGT-mediated direct cardiac reprogramming, autophagy inhibition by downregulation of autophagic factor Beclin1 (Becn1) activated Wnt/β-catenin signalling, which encouraged iCM induction and maturation. Infarcted mouse models with Becn1 haploinsufficiency also showed improved cardiac reprogramming mediated recovery as indicated by decreased scar size and improved cardiac function . WorkingOverall, this set of scientific studies recognizes major signalling pathway involvement in cardiac reprogramming. This offers the potential to consider a targeted modulation of these signalling pathways for improving therapeutic outputs in in vivo direct cardiac reprogramming.The local cardiac environment has an important impact on CF direct reprogramming into CMs for cardiac regeneration . EnvironFurthermore, the iCMs generated from in vivo reprogramming of CFs are considered more mature at functional and transcriptional levels than those produced in vitro. This might be due to the presence of in vivo mechanical and biochemical signals. The myocardium environment is dynamic; cells are contiguous with one another and are facing the continuous contractile forces in a specific direction. Moreover, CFs are exposed to several chemokines and growth factors that might enhance the yield of reprogramming. Van et al. pointed out that after injury a particular subpopulation of CFs was directly programmed into iCMs, and this process was further stimulated by local mechanical properties and topographical cues of the microenvironment . The impOverall, both in vitro and in vivo direct reprogramming approaches can benefit from the cues offered by the local cardiac environment. The superior results obtained in vitro in the presence of materials mimicking cardiac stiffness and/or microstructure, as well as the promising results achieved with in situ approaches, indicate the pivotal role of the permissive cardiac environment in directing a more complete target cell transdifferentiation.In spite of the relatively high transfection efficiency of viral protocols, concerns related to insertional mutagenesis, residual expression, and genetic/epigenetic aberrations still exist that impose serious constraints to their use in clinical practice ,101,102.A pure chemical cocktail was demonstrated capable to functionally substitute the ectopic expression of TFs in inducing beating clusters of CMs from mouse CFs, both in vitro and in vivo, although with low efficiency ,105. ThiBesides the use of pure chemical cocktails, Singh et al. demonstrated that addition of small molecules in conventional TF cocktails can enhance generation of iCMs and smash the barriers to cardiac reprogramming. The addition of three small molecules, i.e., sodium butyrate (histone deacetylase inhibitor), ICG-001 (WNT inhibitor), and retinoic acid (cardiac growth regulator), in the conventional GMT cocktail enhanced direct reprogramming of adult rat and human CFs into iCMs compared to the efficiency of small molecules or GMT alone or the GHMT/myocardin+miR-590 cocktail . This stOverall, these studies open the way to the ultimate aim to regenerate cardiac tissue in diseased hearts using only druggable molecules. While the chemical in vivo cardiac reprogramming approach is an interesting chance for achieving clinically feasible therapeutic interventions, however, a critical matter still facing the chemical approach is the selective targeting of cardiac cells or CFs for iCM generation without impacting other organs’ cells, thus avoiding unwanted side effects. In addition, chemical reprogramming requires deeper knowledge of the protocols to adopt .BIO 6-bromoindirubin-30-oxime (a Wnt activator) and LIF leukaemia inhibitory factor (a JAK/STAT activator), could generate iCPCs. These resulting cells were expandable and capable of self-renewal, thus overcoming the low efficiency of the reprogramming technique. This research also showed that iCPCs differentiated preferentially into iCMs (about 80–90%), while the percentage of SMCs and ECs could be increased when cultured in the presence of vascular differentiation conditions. However, these vascular components were not found in an in vivo model [Numerous studies have investigated CF reprogramming into induced CPCs (iCPCs) which display the potential capability to differentiate into three cardiovascular lineages that include ECs, SMCs, and CMs . Upon trvo model . Even ifvo model . Moreovevo model . The samvo model .Interestingly, Zhang et al. identified other signalling molecules  that allowed successful generation of reprogrammed Flk1+, PDGFRα+, Isl1+, Nkx2-5+ iCPCs from embryonic fibroblasts, which could differentiate in the three main cardiac lineages with a very high efficiency. When transplanted into an infarcted mouse heart, they induced moderate neovascularization, a significant smaller infarct size, and cardiac performance recovery, limiting the long-term (3 months) adverse remodelling. Notably, it was also evidenced that more than 90% of the iCPCs differentiated into cardiac lineage; in particular, the authors estimated the conversion markers, i.e., 59% of SMCs, 31% of CMs, and only 7% of ECs . The sucIn vivo direct reprogramming of resident CFs into iCPCs via injection of the reprogramming cocktail directly into the damaged cardiac tissue can potentially give rise to all three cardiac cell lineages . The advRecently, Liang and Wang have reported their attempts to in vivo reprogram mouse CFs into iCPCs in infarcted hearts by using a CRISPR approach. The reprogramming cocktail, containing a novel fibroblast-specific CRISPR model  and single guide RNAs (sgRNAs) to target the core promoter region of Gata4, Isl1, Nkx2-5, Baf60c, or Tbx5, was introduced locally through an AAV system in infarcted mouse hearts. It was observed that the CRISPR system was activated in approximately 50% of CFs after tamoxifen induction. In addition, it was also observed that iCPCs had subsequently differentiated into cTnT+ CMs, α-SMA+ SMCs, or CD31+ ECs. Ejection fraction and scar formation were improved in these infarcted mouse hearts. It was concluded that endogenous loci can be activated in vivo to reprogram CFs into iCPCs that can further differentiate into mature cardiovascular cells . This stNotably, however, protocols leading to reprogrammed iCPCs starting from murine CFs are still to be adapted for human cells. Overall, since it is well known that cell expansion is a fundamental key in regenerative medicine, the capability of transdifferentiating the numerous CFs that become activated and highly proliferate after myocardial damage into iCMs or, even better, into iCPCs with their intrinsic self-renewal potential and multi-lineage differentiation ability is of outmost interest in the field of cardiac regeneration. Indeed, if successful, this approach would open a novel perspective for cardiac failure treatment by redirecting the activity of pro-fibrotic cells into a regeneration process of functional cardiac cells.PCs are mesoderm-derived cells, one of the abundant but most unknowable and vaguely defined cell population in the heart . They arVascular architecture in the infarcted cardiac region is disrupted, promoting vascular disintegration and capillary rarefaction . The outA recent study has focused on deciphering the role of induced phenotypic transition of myocardial PCs in aiding cardiac neovascularization both in vitro and in vivo in a small animal model . PrimaryOverall, PCs seem to offer another source of reprogrammable NMCs; however, this source still has limitations due to the lack of production of CMs, which are essential for cardiac function.Direct reprogramming of NMCs is an appealing strategy worthy of further investigation. It was rendered possible by an increased understanding of cell fate determination during cardiovascular embryogenesis together with the extraordinary technological improvements related to induced cell reprogramming. High intrinsic NMC plasticity, probably due to their multi-lineage embryonic derivation, is the third important element in this strategy. A challenge for clinical translation of direct cardiac reprogramming is still the establishment of an effective cardiac reprogramming protocol for human NMCs. Although several research groups worked on reprogramming human fibroblasts into iCMs, the duration, quality, and effectiveness of human iCM reprogramming were not comparable to the mouse one. Furthermore, cardiac diseases mainly affect older subjects; for this reason, it should be kept in mind that age and inflammation might act as barriers in reprogramming NMCs into iCMs or iCPCs, because of changing NMC gene expression and activation of different signalling pathways. In conclusion, the direct reprogramming of resident NMCs into cardiac cells is a potential powerful strategy that waits for translational confirmation. Despite amazing progress having been achieved, particularly on in vitro cardiac reprogramming, progress on in vivo cardiac reprogramming for future clinical applications has been relatively slow. Not only do its safety, scalability, and therapeutic time window need to be validated, but also its full potentiality for fostering both endogenous vascularization and myocardial repair, a very prominent advantage of this innovative approach.


The distal transradial artery (TRA) approach has been increasing in popularity over recent years due to its favorable ergonomics and potential for fewer vascular complications. Other advantages include lower bleeding risk, early ambulation, lower procedural costs, and same-day discharge, resulting in additional cost savings. We discuss two cases of patients who underwent left heart catheterizations through the radial artery access site and afterwards experienced fistula formation. Our case series brings to light a rare occurrence of arteriovenous fistulas (AVFs) following cardiac catheterization via the transradial artery site, thus enhancing our knowledge of the risk associated with this access site. The pathophysiology of AV fistula remains the same regardless of transfemoral or transradial artery use. During the procedure, needle diversion into the venous tributary results in an unrecognized combined artery and vein puncture, which usually seals spontaneously. However, if the communication persists, an AV fistula may occur. The majority of patients who suffer from an iatrogenic AVF as a result of TRA do not develop clinical signs of hemodynamic significance. There are various therapeutic strategies, which include surgical repair, placement of a covered stent, ultrasound-guided compression of the AV fistula, and conservative management. Both of our patients were evaluated by vascular surgery; one of the patients found the constant pulsation and bruit burdensome and underwent surgical repair. The distal transradial artery (TRA) approach has been increasing in popularity over recent years due to its favorable ergonomics and potential for fewer vascular complications . Other aCase 1A 51-year-old female with a past medical history of a prior MI presented with mild substernal chest pain similar to a prior myocardial infarction. Six months prior, she had undergone coronary stenting with two drug-eluting stents in her right coronary artery. The procedure was done via the right radial artery approach; heparin was chosen for anticoagulation to avoid clot formation during the procedure; and hemostasis was achieved using a 24 cm Vasc Band Hemostat Regular device with no immediate complications. However, over the following months, she occasionally experienced paresthesia in the fingers of her right hand. She also noted that she could hear her pulse when she placed her head on her right arm to sleep. On presentation, she had a blood pressure of 140/90 mmHg and a heart rate of 69 bpm. Her EKG showed a normal sinus rhythm without acute ST-T wave abnormalities, and her fifth-generation troponin was normal. She had normal heart sounds, a regular rhythm, and no murmurs on the exam. She had a notable, palpable thrill and an audible right radial bruit. An ultrasound was performed and showed a radial arteriovenous fistula  with an ablation, stress-induced cardiomyopathy, and non-obstructive computer-aided design (CAD) presented for a routine office visit. She had undergone a cardiac catheterization via radial artery approach two years prior. During the procedure, heparin was used for anticoagulation, and hemostasis was achieved using a 29-cm-long Vasc Band Hemostat device. During the office visit, she reported difficulty sleeping on her right side because the pulse in her arm was significantly greater than on the left side, and she was able to hear her pulse. She had no other associated complaints of pain, numbness, or limitations with the use of the hand. She had an ultrasound of her upper extremity that showed a radial-cephalic fistula at the right wrist (Figure The pathophysiology of AV fistula remains the same independent of transfemoral or transradial artery use. During the procedure, needle diversion into the venous tributary results in an unrecognized combined artery and vein puncture, which usually seals spontaneously. However, if the communication persists, an AV fistula may occur . Some loThe RIVAL trial is the largest randomized trial comparing radial versus femoral access for coronary angiography and intervention . It inclThe majority of patients who suffer from an iatrogenic AVF as a result of TRA do not develop clinical signs of hemodynamic significance . There aBoth of our patients underwent left heart cardiac catheterizations via the right distal radial artery access site, and AV fistulas developed likely as a result of simultaneous venous puncture during arterial access, because of the close anatomic relationship between the cephalic vein and the radial artery as it passes through the anatomic snuffbox. Both our patients were evaluated by vascular surgery and one of them found the constant pulsation and bruit burdensome; and underwent surgical repair.


The past few years have witnessed significant progress in emerging disease detection techniques for accurately and rapidly tracking rice diseases and predicting potential solutions. In this review we focus on image processing techniques using machine learning (ML) and deep learning (DL) models related to multi-scale rice diseases. Furthermore, we summarize applications of different detection techniques, including genomic, physiological, and biochemical approaches. In addition, we also present the state-of-the-art in contemporary optical sensing applications of pathogen–plant interaction phenotypes. This review serves as a valuable resource for researchers seeking effective solutions to address the challenges of high-throughput data and model recognition for early detection of issues affecting rice crops through ML and DL models. Oryza sativa L.) is globally recognized as the primary staple food  has become a common approach in nondestructive rice disease detection, with methods like charge-coupled device (CCD) cameras being employed to analyze various features of rice diseases, such as color  spectroscopy  and post-germination growth rates, influenced by stem and root length, weight, and biomass, play essential roles . It has gained significant popularity in recent years for the quality and variety detection of seeds for various crop , have recently gained popularity to overcome some of these challenges.In this context, Albattah et al.  enhancedIn crop plants, the canopy, which is responsible for photosynthesis, plays a crucial role in determining the efficient utilization of light energy and photosynthetic nitrogen use efficiency. Remote sensing technologies, using satellites, aircraft, unmanned aerial vehicles (UAVs), and ground mobile platforms, have become valuable tools for efficient disease identification and early diagnosis within plant populations, particularly at the canopy scale , in rice, based on transcriptomics data.In terms of plant–pathogen interactions from a genomic perspective, the principal application areas of ML encompass the prediction of gene regulatory networks, genomic selection for disease resistance, and the prediction of pathogen effector proteins (Singh et al. akrishna  used strakrishna  applied Recently, DL has gained prominence in genomics due to its high learning capacity, wide applicability, and excellent portability (Eraslan et al. It is widely thought that future advancements in dissecting plant–pathogen interactions will be driven by integrating diverse genomic data sources, with ML and DL playing pivotal roles in their advanced applications Cheifet . As the In summary and looking ahead, the application of rice disease detection technology in disease detection is a crucial area of research. The utilization and advancement of advanced detection technologies like hyperspectral imaging and infrared thermal imaging have made it possible to effectively monitor numerous early-stage diseases that were previously undetectable. The multidimensional sample data obtained from data cubes collected through hyperspectral images provide valuable insights. There is a growing trend in employing advanced model algorithms for rice disease detection and recognition. Combining these model algorithms ensures both the reliability of rice yield and quality and the timely and precise availability of disease-related information. This paper offers a comprehensive review of monitoring rice diseases using these advanced techniques across various scales. The application of emerging disease detection technology has the potential to significantly improve the identification rate of rice diseases, leading to precise solutions for crop growers and reduce economic losses related to disease detection. Furthermore, this research lays a solid foundation for the prompt detection of rice diseases and holds significant reference value in terms of safeguarding the economic losses associated with essential national food security crops, including rice. Additionally, this paper explores the application of high-throughput data from microbial genomes in conjunction with model algorithms for predicting rice diseases. Machine learning and deep learning have produced remarkable results in detecting and classifying high-throughput microbiome data (Zhan et al. In future crop disease detection research, several key considerations are expected. First, there is a pressing need to address the limited transferability of specific model algorithms. For example, deep neural networks display varying capabilities to transfer abstract features learned at different layers. Shallow layers tend to exhibit relatively stronger transferability compared to deeper layers. However, as the network's depth increases, the learned features become more specialized and lose their transferability. Additionally, in practical applications, it is often of utmost importance to scrutinize why the model predicts a particular data point to have a specific value, a concept known as local interpretability. Random forest models inherently possess this local interpretability, as one can trace the decision path through the branches to comprehend the prediction. On the other hand, achieving such local interpretability represents a formidable challenge for deep learning models. While running data through the model and observing activated neurons can offer some insights, the interpretation of individual neurons or neuron clusters remains uncertain. Even minor alterations in a feature's value in the data frequently lead to substantially different model predictions. This situation renders the application of counterfactual analysis, which entails studying how changes in input data affect model predictions, impractical for achieving local explainability. Therefore, future efforts should focus on enhancing the stability and interpretability of various models. Moreover, optimizing and amalgamating different models can contribute to the more precise detection of rice diseases across diverse regions and categories.


Soybean is the most economically important legume globally, providing a major source of plant protein for millions of people; it offers a high-quality, cost-competitive and versatile base-protein ingredient for plant-based meat alternatives. The health benefits of soybean and its constituents have largely been attributed to the actions of phytoestrogens, which are present at high levels. Additionally, consumption of soy-based foods may also modulate gastrointestinal (GI) health, in particular colorectal cancer risk, via effects on the composition and metabolic activity of the GI microbiome. The aim of this narrative review was to critically evaluate the emerging evidence from clinical trials, observational studies and animal trials relating to the effects of consuming soybeans, soy-based products and the key constituents of soybeans  on measures of GI health. Our review suggests that there are consistent favourable changes in measures of GI health for some soy foods, such as fermented rather than unfermented soy milk, and for those individuals with a microbiome that can metabolise equol. However, as consumption of foods containing soy protein isolates and textured soy proteins increases, further clinical evidence is needed to understand whether these foods elicit similar or additional functional effects on GI health. Soybean (Glycine max) is the only plant protein that contains all the essential amino acids in amounts that can adequately meet human physiological requirements, and thus it represents an important source of plant protein for both animals and humans. While the majority of soybean protein produced globally (~85%) is used in animal feeds, soybean oil, soy meal and soy-based products including soy milk, tofu, soybeans (edamame) and tempeh also form an important part of many diets ,2. ProduSoybeans are a highly desirable ingredient for producing meat alternatives due to several favourable nutritional properties which set them apart from other commonly consumed beans and pulses  to Bacteroidetes (Barnesiella genus) ratio [There is some epidemiological evidence that soy milk can modulate the intestinal microbiome, but these effects appear to differ between fermented and unfermented products. A cross-sectional study explored the association of milk (dairy and soy) on the microbiome . They fon period ; meanwhin period , and a s3 months . In contuated it ,32. Thessoy milk ,34. The s) ratio .Only a small number of clinical trials have evaluated the potential anti-inflammatory properties of soy milk. Two studies, both conducted in patients with type-2 diabetes, showed that circulating levels of C-reactive peptide and cytokines were unaffected by soy milk intake when compared to levels at baseline  or compaThere are no human studies to date that have specifically evaluated the effect of soy proteins on the gut microbiome or GI health, and therefore knowledge in this area is currently based on the results of experimental animal studies. To date, 16 trials, primarily in rodents, have evaluated the effects of soy protein on parameters that include composition of the microbiome, products of microbial fermentation and models of intestinal disease. The majority of these studies have included soy protein at levels comparable to those recommended in human diets and used casein as the control group. However, there is considerable variation between studies in parameters including animal species, strain, age, intervention duration and diet composition , all of which have the potential to influence microbiome composition, and therefore complicate between-study comparisons.Enterococus and decreased abundance of Lactobacilli and Ruminococus in rats compared to those fed diets containing fish meal or milk casein [Lactobacillus compared to animals fed casein and beef meat [Lactobacillus is considered important in host metabolic balance [Bacteroidetes phylum, with lower relative abundances of Bacteroidaceae and Porphyromonadaceae than the milk protein-fed groups. The impact of these changes is not clear, particularly given that these changes have not been reported in other studies with a similar design. In contrast, a study in pigs showed that when the animals consumed a diet containing soy protein compared to fish meal or cotton seed protein, there was no change in colonic bacterial diversity [Three soy protein dietary intervention studies in rodents have reported higher microbial diversity, considered to be a marker of gut health, and/or altered abundance of specific microbial species in rodents consuming diets containing soy protein concentrate or isolates compared to those fed casein ,42,43,44k casein , whereaseef meat . Lactoba balance ,47 and h balance ,49. Furtiversity . Taken tAnimal studies have also evaluated the effect of soy proteins on microbial fermentation products which are known to exert physiological functions, including short-chain fatty acids (SCFAs), and lipopolysaccharides. SCFAs play an important role as a trophic factor and in providing energy for colonocytes . They reBlautia and Lachnospiraceae) associated with production of secondary bile acids [Bile acids are critical components of the GI tract that link the gut microbiota to hepatic and intestinal metabolism and therefore influence GI motility, intestinal permeability and carcinogenesis . The gutle acids . Thus, aA majority of carefully controlled feeding studies (5 out of 8) in rats suggest that diets containing soy protein (>20% weight) may adversely affect a range of measures of GI health compared to dairy proteins. In one study, rats fed diets containing soybean protein for 9 days exhibited higher epithelial cell damage, higher colonic cell proliferation rates and higher faecal water cytotoxicity compared with rats fed diets containing casein as the protein source over this same period . SimilarWhile of potential concern, it is important to note that any adverse effects of soy protein on GI health may be mediated by dietary fibre within the food matrix  or other fibre consumed in the diet , since dThe only human clinical study investigating the gut health effects of soy oligosaccharides  has provBifidobacterium sp., Faecalibacterium prausnitzii, Fusobacterium prausnitzii, and Roseburia [Escherichia coli, Clostridium, and Streptococcus, concentrations of several protein-derived catabolites  and intestinal expression of inflammatory genes  in this study. Finally, an in vitro study showed that specific strains of gut microbes such as Bifidobacteria can utilise soybean oligosaccharides (raffinose and stachyose), whereas putrefactive bacteria such as Escherichia coli and Clostridium perfringens cannot [A potential beneficial effect of soy oligosaccharides on a range of GI health measures, including microbial abundance and microbial metabolites, is supported by the two animal/in vitro studies that have been conducted to date ,72. A pis cannot .Isoflavones are naturally produced by plants to deter bacterial and fungal infection, and thus have natural anti-bacterial properties. However, it is recognised that only some individuals have microbes that can metabolise specific isoflavones. Daidzein, an isoflavone found abundantly in soy, is converted into equol in the intestine via the action of reductase enzymes belonging to incompletely characterized gut microbes. While all animal species analyzed so far produce equol, only between one third and one half of people (depending on the community) are able to produce equol, ostensibly those that harbor equol-producing microbes .Clostridium coccoides-Eubacterium rectale, but positively with the abundance of sulphate-reducing bacteria which are associated with gut inflammation [Bifidobacterium abundance. Although there were only 4 of 17 study participants that were equol producers, Bifidobacterium and Eubacterium were significantly greater in equol vs. non- equol producers. In a third study, gelified soy extract containing isoflavones (100 mg/d) was consumed by 39 postmenopausal women for two months [Clostridium coccoides-Eubacterium rectale cluster, Lactobacillus-Enterococcus, Faecalibacterium prausnitzii, and Bifidobacterium genus. However, stimulation of the Clostridium coccoides-Eubacterium rectale cluster depended on the women’s equol excretion and was transient, with the exception of a prolonged bifidogenic effect, which was less prevalent in equol producers.Three human clinical trials were identified that examined how the microbiome of postmenopausal women that metabolise equol respond to soy consumption. A short term (5 day) soy milk (86 mg isoflavone/d) and soy germ (114 mg isoflavone/d) intervention in healthy postmenopausal women  showed tammation . Anotherammation  increaseo months . This stAlthough the published studies do suggest that postmenopausal women who can metabolise equol show more pronounced changes in the microbiome following soy isoflavone intake, the effects are inconsistent because of large study heterogeneity, different levels and sources of isoflavones, different study durations, a limited number of clinical trials and studies with small sample size. Thus, further studies are needed to determine how isoflavones alter the microbiome and their metabolites across a diverse population.CRC is the third most frequent cancer globally ,78, and An umbrella review of CRC prevention  identifiThe inconsistency of the protective association of soy or soybean consumption against CRC may be due to various factors potentially causing large study variability/heterogeneity, making it difficult to conclude whether soy products modulate GI cancer risk. The specific limitations include (i) the absence of exact and comprehensive measurement of the consumption of soy foods and/or phytoestrogens; (ii) a limited variety of soy foods consumed, e.g., miso or tofu, which do not thoroughly reflect how phytoestrogens and/or soy are consumed; (iii) the data being obtained from questionnaire-based retrospective studies, which can be highly variable due to deficiencies in the subjects’ ability to accurately recall what they consume ; and  induced greater cytotoxic and genotoxic damage to intestinal tissue compared to dairy protein intake. However, some of these effects were shown to be mitigated when fermentable fibres were added to the diets containing soy protein isolates.In summary, current clinical evidence suggests that some soy foods provide favourable changes in measures of GI health, but further studies are needed to understand whether consumption of foods containing soy protein isolates and textured soy proteins can have similar or additional functional effects on GI health, especially for those individuals with a microbiome that can metabolise equol.


Metabolomics generates a vast amount of data and heavily relies on data science for biological interpretation. By employing techniques from statistics, mathematics, computer science, and information science, data science aids in extracting valuable insights from large-scale metabolomics data. The Special Issue entitled ‘Data Science in Metabolomics’ focuses on data science applications in metabolomics and provides research articles and reviews that summarize major advancements and current challenges in this rapidly evolving field .Traditional data analysis is predominantly centered on comparing the intensity values of features. However, intensity data can greatly vary due to factors such as different experimental batches, instruments, and pre-processing techniques or parameters . Two novData science has made significant contributions to metabolomics by not only producing various open-source or commercial software packages but also by facilitating the sharing of experimental data and metadata through public data repositories. Many tools incorporate hundreds of functions and parameters for optimal data pre-processing, providing significant flexibility to experienced users but potentially overwhelming for inexperienced users. To enhance usability, even for occasional users, Nicolotti et al. streamlined the pre-processing of metabolomics mass spectrometry data and introduced an R workflow package, MStractor . Powell The interaction between metabolomics and data science has led to numerous applications within the field of metabolomics. Davic and Cascio developed a microfluidic-laser-induced fluorescence system for detecting ultra-trace levels of primary fatty acid amines , and KimAs the complexities of metabolomic data continue to increase, the role of advanced data science methodologies in unlocking its full potential becomes ever more pivotal.


Apathy is a complex multi-dimensional syndrome that affects up to 70% of individuals with Alzheimer’s disease (AD). Whilst many frameworks to define apathy in AD exist, most include loss of motivation or goal-directed behaviour as the central feature. Apathy is associated with significant impact on persons living with AD and their caregivers and is also associated with accelerated cognitive decline across the AD spectrum. Neuroimaging studies have highlighted a key role of fronto-striatial circuitry including the anterior cingulate cortex (ACC), orbito-frontal cortex (OFC) and associated subcortical structures. Importantly, the presence and severity of apathy strongly correlates with AD stage and neuropathological biomarkers of amyloid and tau pathology. Following from neurochemistry studies demonstrating a central role of biogenic amine neurotransmission in apathy syndrome in AD, recent clinical trial data suggest that apathy symptoms may improve following treatment with agents such as methylphenidate—which may have an important role alongside emerging non-pharmacological treatment strategies. Here, we review the diagnostic criteria, rating scales, prevalence, and risk factors for apathy in AD. The underlying neurobiology, neuropsychology and associated neuroimaging findings are reviewed in detail. Finally, we discuss current treatment approaches and strategies aimed at targeting apathy syndrome in AD, highlighting areas for future research and clinical trials in patient cohorts. Alzheimer’s disease (AD) is projected to affect 153 million people globally by 2050 . ImportaApathy, commonly defined as a lack of interest, enthusiasm or concern , is not Most of the prevailing frameworks and criteria for apathy in NCDs focus on goal-directed behaviour, with a reduction in self-initiated behavioural activities being the most prominent feature noted . Marin’sMost recently in 2021, The International Society for CNS Clinical Trials Methodology (ISCTM) Apathy Work Group have published criteria for defining apathy in NCDs . SpecifiIn the ISCTM 2021 diagnostic guidelines for apathy in neuro-cognitive disorders (NCD), there are four domains which must be met: (A) Primary Diagnosis: meets criteria for syndrome of cognitive impairment/dementia, (B) Symptoms and Duration: must be present for at least 4 weeks and represent a change from usual behaviour including two of the following three domains: (B1) diminished initiative , (B2) diminished interest  or (B3) diminished emotional expression or responsiveness , (C) Exclusionary Criteria: wherein symptoms not explained by other illness/intellectual disability/chance in consciousness level/related to substances and (D) Severity: the symptoms must cause clinically significant impairment in functioning . There are several important confounders to consider when defining apathy in individuals with AD. In addition to the aforementioned overlap with depression, the presence of other NPS such as psychosis, agitation or aggression may make it difficult in practice to appreciate the presence, and severity, of apathy . ImportaSeveral rating scales have been developed to assess for the presence and severity of apathy in AD. This includes both sub-scales of larger instruments and apathy-specific scales. Whilst there is no clear gold standard assessment tool for apathy in AD, the two most commonly used in clinical populations include the Neuropsychiatric Inventory Apathy Scale (NPI-apathy)  and the The NPI rates the presence and severity of “apathy or indifference”—whether individuals are less interested in usual activities/plans of others—and, importantly, also asks if individuals with AD are also experiencing dysphoria/depression. The apathy subscale of the NPI (NPI-apathy) involves a screening question and eight further questions following a positive response rating frequency and severity of symptoms in addition to caregiver distress . The NPIThe AES is comprised of 18 core items which rate the emotional, behavioural and cognitive domains of apathy  with versions for caregivers (AES-I), patients (AES-S) and clinicians (AES-C) available . StudiesOther instruments for measuring apathy include the Structured Clinical Interview for Apathy , the ApaPrevious systematic reviews have highlighted excellent internal consistency, validity and reliability with use of the DAIR , developEstimates on the prevalence of apathy in AD depend on the instrument used, the clinical population studied and the stage of AD considered. Importantly, apathy may be the initial presenting symptom in those with AD or may emerge later in the disease course . In crosImportantly, the presence of apathy is independently associated with progression to dementia in MCI ,53,54,55Early, prominent apathy is one of the diagnostic criteria for frontotemporal dementia (FTD) and is commonly associated with this disease; however, as a symptom, it has been noted in some cross-sectional studies more frequently in AD than in other dementias , althougIn contrast to affective symptoms, apathy tends to increase over time in AD —althoughThe presence of apathy in AD is associated with greater functional impairment ,74 in adSeveral studies have examined potential risk factors for apathy in AD. A recent meta-analysis found that male sex appears to be associated with more severe apathy in AD , whilst Psychological studies examining risk factors for apathy in AD have highlighted premorbid personality (lower agreeableness and higher neuroticism), higher midlife motivational abilities  and highHigher cognitive functions involve complex interplays between cortical and subcortical structures and the coordinated complex interplay between several large-scale connected networks ,101. ApaThe original description of three subtypes of apathy i.e., the cognitive, emotional affective and behavioural autoactivation subtypes  have sinCognitive Control Network: The cognitive control network (CCN) is also known as the executive control network , and somSalience Network: The salience network (SN) is also known as the ventral attention network, and key regions of the SN include the orbitofrontal cortex (OFC), the ventromedial prefrontal cortex (VMPFC), the ACC, the amygdala, the ventral striatum (including the nucleus accumbens) and the anterior insula . The SN Default Mode Network: The default-mode network (DMN) is a large-scale network of brain areas that forms an integrated system for self-related cognitive activity, including autobiographical, self-monitoring and social functions. The DMN plays an important role in monitoring the internal mental landscape . Key regDopamine is a key neurotransmitter that regulates motivated decision-making in humans and other species. Many decades of evidence suggests that dopamine plays an important role in promoting approach behaviour by attributing incentive salience to reward stimuli and facilitating the overcoming of effort costs ,124. DopAlthough dopamine dysregulation has been commonly implicated in the clinical presentation of apathy, recent studies have suggested central noradrenaline (NA) as a critical neurotransmitter in reward processing and cognition. NA is primarily released from the locus coeruleus (LC), a small brainstem nucleus that is difficult to image, which may indicate why this system, to date, has been more challenging to describe . The locDespite numerous studies quantifying the prevalence, measurement, and impact of apathy in AD, the volume of research aimed at detailing the neuropsychology of apathy in AD is scarce. Given the aforementioned fronto-striatal circuits involved, impairments in executive function, reward-based decision-making and social attention/interest are understandably implicated . FurtherExecutive function has been implicated in studies demonstrating a significant association between the number of errors made in multi-tasking and the presence of apathy in AD , in addiParticularly with reference to the SN, impairments in social cognition may be principally involved in apathy. An interesting study using eye-tracking methodology demonstrated significantly less time spent fixating on social, but not neutral, images in dementia-associated apathy . Apathy Numerous studies using different imaging modalities  have aimed to elucidate the neurological correlates of apathy in AD. Such studies may inform the underlying brain circuitry and neurobiology involved in apathy syndrome and identify potential targets for neuro-modulation approaches, which have demonstrated encouraging early results in apathy syndrome in AD.Many neuroimaging studies are confounded by the severity/influence of AD cognitive symptoms, small sample sizes and heterogeneous results. Critically, neuroimaging findings may differ depending on the stage of AD studied and the severity of apathy symptoms . DespiteIn a recent study of 69 individuals with MCI or AD and associated apathy matched to 149 individuals without apathy, those with apathy had thinner right OFC and left ACC but sparing atrophy in left medial temporal cortices . SimilarAdditionally, studies have highlighted bilateral frontal white matter hyperintensity volume  in additStudies using tract-based analysis have demonstrated reduced fractional anisotropy (FA) in the genu of the corpus callosum in those with, compared to those without, apathy in AD, whilst the severity of apathy was negatively correlated with FA values of the left anterior/posterior cingulate, right superior longitudinal fasciculus, splenium, body and genu of the corpus callosum and bilateral uncinate fasciculus—also observed in studies of apathy in amnestic MCI ,170. MorBrain perfusion studies using single-photon emission CT  to assesImportantly, some neuroimaging studies have highlighted the association between underlying AD markers and the presence of apathy. For instance, in MRI studies, medial temporal atrophy (MTA) scores were associated with apathy in probable AD . A recenSeveral non-pharmacological approaches to apathy in AD have been assessed which vary substantially in methodology, study setting, sample sizes, outcomes and conceptual clarity . For an A recent retrospective clinical study has reported on multi-site repetitive transcranial magnetic stimulation (rTMS) combined with cognitive training on cognitive symptoms and apathy in AD. The authors noted improvement on Apathy Index measures over the course of the first year, with those responding to the initial 6-week protocol experiencing long-term improvement in apathy, although further controlled clinical trials are needed . In a doSeveral studies have examined the potential of cognitive stimulation therapy to improve apathy in AD . In a raA variety of other interventions, including music-based interventions targeting older adults with dementia have demonstrated potential benefit for apathy in AD ,192. RegPharmacological management strategies for apathy in AD are based on studies primarily targeting apathy syndrome and secondary analysis of other agents used in the treatment of AD. Those focusing on the core apathy syndrome have used agents targeted at modulating underlying noradrenergic and dopaminergic tone , whilst analysis of apathy as an outcome in trials of other agents  have considered cholinesterase inhibitors and valproate antidepressants amongst other agents. Overall, as concluded by a previous Cochrane review of 21 pharmacological studies, the best available evidence appears to support use of methylphenidate, with further evidence added from a large trial (ADMET-2) published last year .Methylphenidate is a catecholamine/dopamine-enhancing agent which may improve noradrenaline and dopamine activity in the fronto-striatal pathways known to be implicated in AD. There have been several trials evaluating the potential efficacy of methylphenidate for AD-associated apathy ,202. OneADMET-2 enrolled individuals with possible or probable AD and clinically significant apathy for at least 4 weeks based on the NPI. Individuals with current/previously diagnosed major depressive disorder were excluded, as were those with clinically significant agitation/aggression or recent medication changes including antidepressants, benzodiazepines or those with contraindications to methylphenidate . ParticiOf 200 participants recruited, the 99 participants randomised to methylphenidate experienced consistent improvements in apathy as measured on the NPI at all follow up visits, with small to medium reduction in apathy scores, and the largest effect seen for the first two months of treatment . Whilst A decade-old study evaluated the use of modafinil in mild–moderate probable AD for 8 weeks. Whilst both modafinil and placebo groups showed reductions in apathy on the Frontal Systems Behaviour Scale, there was no significant effect of modafinil on apathy symptoms at 8 weeks . AnotherAcetylcholinesterase inhibitors are part of standard care for those with mild–moderate AD, and several studies have examined the effect on apathy of these agents ,212,213.As of 2022, there were 143 pharmacological agents in 172 clinical trials for AD registered; however, of these, only 6.9% were under investigation for the treatment of NPS . It is nThis review explores in detail how apathy is a common and prevalent NPS in AD, and it has been associated with progression, mortality and high caregiver burden, making it an important treatment target. Though conceptual frameworks for apathy have evolved through the years, the consensus remains that impaired fronto-striatal circuits specifically between subcortical connections and the ACC and OFC are key pathological substrates for AD-related apathy presentations and are associated with the presence of pathological hallmarks of AD in these regions in AD-related apathy presentations. It has been noted that a high proportion of studies assessing the effect of pharmacological agents on apathy use the NPI as an outcome measure, which is not specific to apathy and, therefore, has a risk of false positives when each domain is analysed without adjustment for multiple comparisons. It has been recommended that a consensus agreement be obtained regarding which tool to use for testing apathy, ideally one with high test/retest and interrater reliabilities  for future studies in order to limit the inconsistencies between clinical trials . Recent