Lymphatic vessels even more than blood vessels seem important in mediating immune responses, as they facilitate allograft sensitization in the draining lymph nodes. Thus, the concept of modulating lymphatic trafficking to promote corneal graft survival seems promising. A variety of approaches has been developed to inhibit progressive lymphangiogenesis in experimental settings. Recently, additionally to pharmacological approaches, clinically available techniques such as UVA-based corneal collagen crosslinking and fine needle diathermy were reported to be effective in regressing lymphatic vessels and to experimentally promote graft survival. Clinical pilot studies also suggest the efficacy of blocking antigen presenting cell trafficking to regional lymph nodes by regressing corneal lymphatic vessels to enhance allograft survival in high-risk eyes. In this article, we will give an overview of current strategies to modulate lymphatic trafficking with a special focus on recently reported strategies, which may be easy to translate into clinical practice. This novel concept of temporary, pretransplant regression of lymphatic vessels at the site of transplantation to promote subsequent corneal transplant survival (“lymphangioregressive preconditioning”) may also be applicable to other transplantation sites later. The transparent cornea is one of the few avascular tissues of the body. Corneal avascularity, which is also known as the corneal “(lymph)angiogenic privilege”, is crucial for its transparency and thereby visual acuity . In healHowever, the dynamic balance of pro- and anti(lymph)angiogenic factors may be disrupted, e.g., by severe inflammation, which can cause the breakdown of the corneal (lymph)angiogenic privilege and result in pathological corneal lymphatic and blood vessel formation ,10,11,12Pathological corneal blood vessels are well accepted as a risk factor for corneal transparency loss as well as a cause for secondary transparency-reducing effects including fluid and lipid deposition ,2. MoreoMany studies indicate that pathological lymphatic and blood vessels can ease the connection of the corneal graft with the secondary lymphoid organs, resulting in accelerated sensitization against donor antigens and augmented access of immune effector cells to the graft ,46,47. PImportantly, lymphatic vessels have been proven to be even more important than blood vessels in mediating corneal graft rejection . We haveIn summary, pathological corneal lymphatic vessels facilitate graft sensitization and lead to graft rejection after corneal transplantation. Thus, the concept of modulating lymphatic trafficking of APCs to promote long-term graft survival in high-risk keratoplasty is distinctly promising. Importantly, it is critical to establish effective anti(lymph)angiogenic and (lymph)angioregressive strategies, especially in pre-vascularized high-risk eyes, which may easily be translatable into clinical practice. Here we will review current strategies which modulate corneal lymphangiogenesis and may promote corneal graft survival, so as to better understand lymphatic immune cell trafficking in the eye and specially in the cornea. We discuss the novel concept of promotion of corneal transplant survival by modulating lymphatic trafficking (“lymphangioregressive preconditioning of the host site”). APCs like dendritic cells and macrophages are the most important immune cells to induce an allospecific immune response. Similar to lymphatic vessels, the presence of dendritic cells in the cornea was also for a long time unknown. Nowadays, it is known that Langerhans cells and dendritic cells are present in the cornea and can mature due to inflammatory stimuli such as infection or corneal transplantation. Upon activation, dendritic cells increase the expression of the chemokine receptor CCR7 . With thBlind ending lymphatic capillaries possess loose flaps by which APCs can migrate into the lumen of the lymphatic vessels . WhetherFurther important interaction partners for the interaction of dendritic cells and lymphatics are adhesion molecules. It was shown that lymphatic endothelial cells express CD31, CD99, VCAM-1, ICAM-1, L1CAM, and CD137 ,71,72,73Although it is known that VCAM and ICAM are upregulated upon inflammatory stimulation on lymphatic endothelial cells in the skin , the expIn summary, corneal lymphatic vessels closely interact with immune cells, i.e., dendritic cells. This interaction could become a new target for immune modulatory therapies in and outside the cornea.Solid organ transplantation usually necessitates the use of systemic immunosuppressive drugs to reduce the risk of allograft rejection. However, this rather unspecific inhibition of immunity might be associated with numerous side effects and a more selective modulation of the immune system; i.e., the specific blockade of allogenic immune responses, is desirable. In this context, recent work in the fields of renal, lung, and cardiac transplantation has demonstrated that lymphangiogenesis after grafting is associated with an increased risk of allograft rejection, implicating that the lymphatic vascular system might be a therapeutic target to improve the outcome of solid organ transplantation. In the context of renal transplantation, it has been demonstrated that chronically rejected human renal allografts contain numerous highly proliferating lymphatic vessels ,77. ThesDashkevich et al. recently analyzed the association of lymphangiogenesis and allograft rejection after lung transplantation . ImportaThe involvement of lymphangiogenesis in experimental heart transplantation was investigated by Nykänen and coworkers . This stTaken together, APC trafficking via lymphatic vessels to the secondary lymphoid organs seems to be an important step for the induction of alloimmune responses, e.g., after renal, lung, or cardiac transplantation. Thus, interference with this pathway, i.e., via targeting lymphangiogenesis, might be a potential strategy to reduce the risk of allograft rejection also after solid organ transplantation. However, it should be noted that in contrast to the cornea, solid organs are (lymph)vascularized in their healthy state and rely on proper lymphatic function and drainage, which has also been shown in the respective transplant settings. In this context, work from Stuht and colleagues demonstrated that human kidney grafts with high lymphatic vessel density have better functional outcomes than those with low lymphatic vessel density 1 year post transplantation . SimilarTaken together, the role of the lymphatic vascular system in solid organ transplantation is less clear when compared to corneal transplantation and still under investigation. It seems that a complete and permanent blockade of lymphangiogenesis and lymphatic function in solid organ transplantation to improve graft survival might not be applicable . InsteadIn a simplified scheme, the three structural elements of the immune system that enable immune responses against alloantigens after transplantation are afferent lymphatic vessels, regional lymph nodes, and efferent blood vessels ,48. As dSince we and others could show that (1) pathologic corneal lymphatic vessels act as conduits for APC trafficking from donor tissue to host regional lymph nodes, 2) that inhibition of lymphangiogenesis prior to and after transplantation leads to improved graft survival, and (3) that a temporary blockade or regression of corneal lymphatic vessels is sufficient to prevent corneal allosensitization, we developed the novel therapeutic concept of “temporary preoperative lymphangioregression at the recipient site to promote subsequent corneal transplant survival” ,91,92,93 that inhA variety of therapeutic strategies for (lymph)angiogenesis inhibition have been developed and are currently being tested to improve graft survival by blocking immune cell trafficking in pathologic corneal lymphatic vessels by either (i) inhibiting progressively outgrowing immature corneal vessels, and/or by (ii) induction of vascular regression of already existing mature pathologic corneal vessels.Various approaches have already been tested to suppress progressive corneal immature lymphatic and blood vessels. Anti-inflammatory options as well as anti VEGF-strategies have already been tested experimentally and partially also used in the clinic already ,94,95,96VEGF TrapR1R2) was reported to be effective in inhibiting corneal lymphangiogenesis via neutralizing VEGF-A in a murine model and was also shown to promote corneal graft survival ,59. To d was repoIn the clinic, topical application of corticosteroid eye drops is so far still the standard antiangiogenic therapy of the cornea. Our group also showed in experimental settings that these drugs are potent inhibitors of corneal lymphangiogenesis, because of direct inhibition of the lymphatic endothelium, as well as decreased recruitment of pro-lymphangiogenic inflammatory cells . NeverthThough the above reviewed approaches are able to stop progressive corneal (lymph)angiogenesis and may enhance graft survival after keratoplasty, it is still challenging to regress already existing mature corneal lymphatic and blood vessels. Therefore, our group has recently investigated the effect of several already clinically available techniques on the regression of corneal lymphatic and blood vessels, such as ultraviolet-A (UV-A) light based corneal collagen crosslinking (CXL), fine needle diathermy (FND), and photodynamic therapy (PDT). In the next sections, we will summarize the promising (lymph)angioregressive effects of CXL and FND and their significant improvement of corneal graft survival after pre-transplant treatment, both in the experimental setting  and in cAs aforementioned, lymphatics critically participate in the mediation of allogenic immune responses, which can lead to corneal transplant rejection and failure. Therefore, modulation of lymphatic trafficking is a new and feasible strategy to promote graft survival. Currently, CXL and FND are promising approaches to achieve significant improvement of corneal transplant survival via modulation of lymphatic trafficking in the context of corneal transplantation. 2, irradiated for 6 or 9 min), pathological corneal lymphatic as well as blood vessels were significantly regressed after CXL in the mouse model of suture-induced inflammatory corneal neovascularization. UV-A-light based CXL is often applied in the clinic to stabilize the corneal structure via bonding of adjacent corneal collagen fibers for corneal ectatic diseases through the release of reactive oxygen radicals . Since 2TUNEL positive signals colocalized with both corneal lymphatic and blood vascular endothelial cells, which indicates that the (lymph)angioregressive effect of CXL is caused by cell death of vascular endothelial cells  Figure . CXL alsImportantly, we observed a significant enhancement of long-term allograft survival using CXL to treat pre-vascularized recipient beds preoperatively  Figure . This seTherefore, temporary preoperative (lymph)angioregression via CXL prior to high-risk corneal transplantation to facilitate corneal graft survival shows the feasibility of this preconditioning approach. As the aforementioned beneficial effects of pre-surgically applied CXL on subsequent corneal transplantation have only be shown in animal studies so far, clinical studies are needed to prove the efficacy of this promising approach. In FND was described and clinically conducted to treat corneal blood vessels firstly in 2000 . It is cImportantly, we have recently demonstrated that preoperative FND treatment of pathologically pre-vascularized high-risk recipients can significantly enhance allograft survival after subsequent corneal transplantation in mice  Figure . FurtherTaken together, these studies show that the concept of modulating lymphatic trafficking via CXL and FND prior to high-risk keratoplasty to improve corneal transplant survival is feasible and promising. Translational clinical trials should follow to further prove the effectiveness of this approach.As aforementioned, CXL using UV-A-light and riboflavin as photosensitizer was demonstrated as an effective preoperative strategy to regress pathological corneal lymphatic and blood vessels and promote graft survival in high-risk keratoplasty in the experimental setting . Our groThough this study surely has its limitations, such as small case number and heterogeneity of treated patients in terms of preexisting keratitis, underlying disease, previous surgeries, and timing of keratoplasty and CXL, this pilot study presents the first clinical evidence that CXL is an effective method to reduce pathological corneal neovascularization, to stabilize the recipient cornea, and presumably to promote graft survival after subsequent high-risk keratoplasty. These promising results warrant large-scale clinical studies with longer follow-up. As reviewed above, FND was shown to be a successful antiangiogenic as well as anti-lymphangiogenic  treatmenThe findings of these pilot studies of CXL and FND warrant further larger scaled cohort clinical trials with longer follow-up, which may help to translate these approaches into clinical practice.Lymph-angiogenesis, lymphatic vessels, and lymphatic endothelial immune cell interactions critically contribute to corneal graft rejection via the mediating of undesired immune response after corneal transplantation in high-risk eyes. Modulation of lymphatic trafficking has been shown experimentally to be a novel and valid concept to promote corneal transplant survival. Blockade of immune cell trafficking to regional lymph nodes after transplantation was primarily achieved by inhibiting progressing lymph-angiogenesis or by regressing already existing, mature pathological lymphatic vessels. In terms of regressing mature lymphatic vessels, CXL or FND are potent strategies in the experimental setting prior to transplantation (“(lymph)angioregressive preconditioning of the host”) and thereby to significantly improve transplant survival in high-risk corneal transplantation. Although further large-scale prospective clinical trials are still needed, early positive evidence of clinical pilot studies using preoperative CXL or FND seems to confirm the novel therapeutic concept of improving corneal transplant survival by temporary preoperative lymphangioregresssion (and thereby inhibiting lymphatic immune cell trafficking). Additionally, in regards to lymphatic regression as well as graft tolerance, to apply CXL and/or FND in combination with anti-VEGF agents, e.g., bevacizumab or aflibercept, may offer more benefits. In fact, we recently demonstrated that the simultaneous application of anti-VEGFs prevents the FND-induced rebound neovascularization caused by surgical trauma-induced upregulation of VEGFs . In clin


Oncolytic viruses (OVs) are viruses that selectively target and kill cancer cells while sparing normal ones. OVs are from diverse families of viruses, but naturally occurring OVs have been genetically engineered due to their limitations in therapeutic application. These engineered OVs with enhanced tumor targeting ability, oncolytic activity, or generating potent anti-tumor immune responses are tested in preclinical animal models and cancer patients in clinical trials. Due to their multi-mechanistic anti-tumor effects, OVs have emerged one of the key cancer immunotherapy agents. However, due to the limited success with novel anti-cancer therapies such as immunotherapies and cell-based therapies, combination therapies should be tested with OVs. We discuss such combination therapies that are explored to further improve oncolytic virotherapy.Cancer remains a leading cause of death worldwide. Despite many signs of progress, currently available cancer treatments often do not provide desired outcomes for too many cancers. Therefore, newer and more effective therapeutic approaches are needed. Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively targets and kills cancer cells while sparing normal ones. In the past several decades, many different OV candidates have been developed and tested in both laboratory settings as well as in cancer patient clinical trials. Many approaches have been taken to overcome the limitations of OVs, including engineering OVs to selectively activate anti-tumor immune responses. However, newer approaches like the combination of OVs with current immunotherapies to convert “immune-cold” tumors to “immune-hot” will almost certainly improve the potency of OVs. Here, we discuss strategies that are explored to further improve oncolytic virotherapy. Oncolytic viruses (OVs) were selected for development because they can selectively infect and kill cancer cells but spare their normal cellular counterparts. Treatment of cancer with live oncotropic viruses has a long history. Even before the first reported formal clinical trial with an OV in 1949, there were case reports since the mid-1800s suggesting that natural microbial infections in cancer patients can sometimes temporarily regress tumor burden. The potential therapeutic role of viruses in particular was further established in the late 1890s by an observation that a “flu-like” disease associated with diffuse inflammation coincided with reducing tumor cells in a leukemic patient. Beginning 1949, many clinical trials were undertaken using different types of wild-type non-attenuated viruses ,3. ShortThe OV field gained considerable attention after positive results from many clinical trials. So far, four OVs have been approved globally . The firAn ideal oncolytic virus candidate should possess several hallmarks, such as a solid fundamental understanding of their biology and genetics. The OV should be pro-immunogenic, exert lytic activity in the infected malignant cells, should not lead to a chronic or infectious disease, or be capable of integrating into the human genome. Additionally, the virus must be broadly safe for a diverse human population. It is also feasible to genetically modify and arm with recombinant transgenes to enhance its immunogenicity or stimulate targeted anti-cancer mechanisms. Since the early clinical trials, diverse types of viruses with and without genetic alterations were tested and entered into clinical trials. They include members of both DNA and RNA viruses . ExampleOncotropism of OVs generally depends on multiple factors like cell surface receptors necessary for virus binding/entry , cellular metabolic status, and the ability of the virus to overcome intracellular innate immune or antiviral signaling pathways within cancer cells . The early observations that some OVs exploit unique extracellular molecules expressed on cancer cells for binding and entry led to this field’s initial growth. For example, CD46, CD155, and integrin α2β1 molecules are frequently overexpressed in many classes of tumor cells, and can serve as the receptor for measles virus, poliovirus, and echovirus, respectively ,14,15. HRAS, TP53, RB1, PTEN, EGFR, WNT, BCL-2, and other cancer-related genes can often further predispose cancer cells to viral infection [Cancer is a complex, heterogeneous disease with multiple genetic mutations that mediate frequent compromises in the various antivirus signaling pathways, which serve as a perfect niche for OV replication. Different OVs exploit selective defects in cancer cells, understanding of which remains an active area of research. In general, cancer cells during the transformation process selectively sacrifice at least some elements of their potent innate antiviral response pathways mediated by cytokines like type I and II interferons (IFNs) or tumor necrosis factor (TNF) . Althougnfection ,23,24,25Most tumor cell types are characterized by a high rate of aerobic glycolysis (Warburg effect), which plays a vital role in developing the immunosuppressive tumor microenvironment (TME) . This isAfter binding and entering tumor cells, OVs can exploit multiple lytic mechanisms to kill the infected cancer cells that may or may not be linked to the actual extent of virus replication within the target cells. The exact mechanisms of viral oncolysis are still incompletely understood, and vary widely from virus to virus, and can even differ dramatically between diverse target cancer cell types. OVs are thought to mediate antitumor activity through multiple mechanisms: (a) selective virus replication within cancer cells, causing direct cytolytic effects  ,37,38;  can circumvent this issue by binding to nonspecific determinants like ubiquitously expressed cell surface glycosaminiglycans. At the intracellular level, there are additional complex signaling pathways that are directly or indirectly linked with the antiviral pathways and commonly restrict virus replication (if operative) and spread to the new cells. For example, AKT activation levels regulate MYXV replication in human cancer cells ,63. In tDelivery of OV: Delivery of OV to the sites of primary and metastatic sites is vital for optimal therapeutic outcomes. In this regard, since only a minority of human cancers are amenable to direct intratumoral (IT) injection, systemic delivery is the preferred route compared to IT injection of the virus. However, there are several barriers to the successful delivery of any OV. The presence of neutralizing antiviral antibodies, complement activation, expression of antiviral cytokines, and natural clearance site of OVs by the liver and spleen are all major obstacles in the systemic OV delivery. Although IT delivery of virus can circumvent some of these barriers, tumor beds can limit virus spread, and the tumor vasculature is also a limiting factor to IT and metastatic sites. One strategy to overcome these issues is to exploit migratory leukocytes as carrier cells to ferry the virus into tumor beds that allow cellular ingress.Neutralizing antibodies and antiviral cytokines: Preexisting neutralizing antiviral antibodies are the main obstacle in the context of systemic delivery of free virus to reach the tumor bed . AdditioImmunosuppressive TME: Another barrier to OV therapy is the frequent presence of highly immunosuppressive TME. In the tumor bed, various cellular subsets like cancer cells, stromal cells, inhibitory cytokines  and infiltrating immune cells  all contribute to the immunosuppressive TME. Although this is critical for the tumor to evade the host’s innate and adaptive immunological defenses, OVs must function within this immunosuppressive TME. Additionally, some OV infections can further promote the tumor bed’s immunosuppressive environment by activating the immune system. For example, Maraba virus upregulated the PD-1/PD-L1 axis on tumor cells and tumor-infiltrating immune cells ,67. SimiLike many other modern cancer therapies, there are still challenges and obstacles ahead with oncolytic virotherapy and becoming a successful anticancer therapy. Some of the key factors that contribute to the limitation of OV functions are: (1) unknown host antiviral pathways that limit the OV activity and spread in the tumor bed, (2) surrounding intrinsic physical barriers in the tumor bed limiting OV access, and (3) adaptive immune responses limiting viral functions indirectly. Furthermore, there are additional factors that should be considered:The genetic engineering of OVs has now become an integral part of developing safe, cancer-selective, and highly effective OVs against diverse types of cancers. Engineered OVs have overcome some of the challenges that are listed in the above section. Any modification of OV relies heavily on understanding the biology and genetic information of the virus, virus–host interactions, how viruses kill infected cells, and how cells protect themselves from the lytic infection. Genetic engineering by knockout deletion of certain viral genes can enhance OV tumor cells tropism and reduced toxicity for normal cells; engineering and arming via knockins with different ectopic transgenes has enabled OV application as oncolytic immunotherapy to more broadly activate the anti-tumor immune responses. This field of developing engineered OVs and arming OVs with transgenes is rapidly expanding due to the recent discovery of many new biologics with diverse potential as immunotherapy. During the past few years, many reviews have been written on this topic and we have briefly highlighted some of the key engineering of OVs that substantially improved application of OV as cancer therapeutics ,71,72,73Based on the purpose and type of transgenes used for OV engineering and modifications, they can be classified into many groups. Some examples are listed here: (i) deletion/mutation of viral genes for selective replication in tumors and protecting normal tissues. For example, ICP34.5 and ICP47 genes were deleted in T-VEC . ICP34.5OVs provide multi-mechanistic therapeutic effects against most types of cancers . HoweverTraditional therapies such as radiotherapy (RT) and chemotherapy have been used either alone or in combination. Radiotherapy is mostly used for the local control of tumors and displays a wide range of antitumor effects . HoweverConventional chemotherapeutics were tested with OVs to enhance the therapeutic effects of OVs. The goal was to reduce the dosage and toxic effects of the drug while enhancing the efficacy of OVs in the tumor microenvironment. However, in some cases, depending on the type of chemotherapeutic drugs and dose regimens used, certain drugs acted as antivirals and also reduced viral replication in the tumor bed. This combination therapy was tested in many preclinical tumor models ,107. TheUnlike intratumoral delivery, systemic delivery of OV faces many challenges, mainly due to the highly efficient immune filtration system that detects and removes pathogens from the circulatory system. Many types of carrier cells have been tested to determine their suitability as a delivery vehicle for systemic administration of OVs to overcome the deficits of intravenously infusing naked virus. In preclinical studies, multiple OVs have been tested with different carrier cell types demonstrating the success and feasibility of this approach and potential clinical application ,109. ThiICIs that target immune checkpoints like PD-1, PD-L1, or CTLA-4 and disrupt the cancer cell’s ability to evade the host immune response have become one of the key cancer therapy modalities. However, despite many success, there are several critical limitations of ICIs: (i) even with optimally targeted cancers, only limited fraction (10–20 percent) of patients respond to ICI treatment, (ii) immune-related adverse effects reported in some patients receiving ICI therapy, and (iii) Limited effect against immunologically “cold” tumors characterized by a low tumor-infiltrating lymphocyte (TILs). In this context, OVs, particularly genetically engineered OVs, can enhance lymphocyte infiltration and activation as well as lysis of cancer cells. Thus, to overcome this lack of ICI effectiveness in too many patients, OV virotherapy combined with ICIs has been tested in preclinical models and clinical trials, and has shown promising results ,116,117 Cellular immunotherapy, also known as adoptive cell therapy, uses modified versions of the cells of the immune system to eliminate cancer cells. Various types of cellular immunotherapies have been developed: such as chimeric antigen receptor (CAR) T cells therapy, engineered T cell receptor (TCR) therapy, tumor-infiltrating lymphocyte (TIL) therapy, and natural killer (NK) cell therapy. Among these cell-based therapies, CAR-T cell therapy has shown remarkable efficacy for blood cancer as emerging immunotherapy. However, cell therapy has also a limited success, particularly in solid tumors, due to the lack of infiltration and prolonged existence in the tumor tissue. To overcome these limitations, cell therapy has been tested in combination with OVs. In this case, the use of engineered OVs expressing therapeutic transgenes can enhance the therapeutic benefit of both treatment strategies. For example, oncolytic adenovirus expressing IL-7, when combined with B7H3-targeted CAR-T, showed higher efficacy than a single use of either of those treatments . CombinaMore than three decades of extensive research and clinical trials have established oncolytic virotherapy as a promising treatment modality for cancer treatment. Several aspects of OV therapy have been significantly improved, including safety, potency, selectivity, delivery methods, and production. Perhaps the most notable shift in the OV field has been from its application as a direct lytic agent to development as a multimodal agent involving cell lysis, immune stimulation, and gene therapy, which further established OV as a strong candidate for cancer therapy. However, it is becoming clear that OVs as a single anti-cancer agent might not successfully provide a complete response for cancer cure, such that combinatorial strategies are essential. Mainly because of the heterogeneous nature of the cancer cells, there are increasing chances of recurrence, metastasis, and failure to diagnose early. Therefore, like other combination therapy, it is possible to exploit OV in combination with existing cancer therapies, an area still less explored. However, in this case, a rational design and combination approach can mitigate the inherent limitations of OVs and other therapies against selected cancer types. For example, armed OVs with enhanced tumor specific replication ability and stimulating a potent anti-tumor immune response can be combined with immune checkpoint inhibitors and cell therapies for cancer that develop resistance against current therapies because of rapid mutations and heterogeneous cell populations.


There are a number of ongoing developments to improve the care of patients with diabetes across countries given its growing burden. Recent developments include new oral medicines to reduce cardiovascular events and death. They also include new modes to improve insulin administration to enhance adherence and subsequent patient management thereby reducing hypoglycaemia and improving long-term outcomes. In the case of insulins, this includes long-acting insulin analogues as well as continuous glucose monitoring (CGM) systems and continuous subcutaneous insulin infusion systems, combined with sensor-augmented pump therapy and potentially hybrid closed-loops. The benefits of such systems have been endorsed by endocrine societies and governments in patients with Type 1 diabetes whose HbA1c levels are not currently being optimised. However, there are concerns with the low use of such systems across higher-income countries, exacerbated by their higher costs, despite studies suggesting their cost-effectiveness ratios are within accepted limits. This is inconsistent in higher-income countries when compared with reimbursement and funding decisions for new high-priced medicines for cancer and orphan diseases, with often limited benefits, given the burden of multiple daily insulin injections coupled with the need for constant monitoring. This situation is different among patients and governments in low- and low-middle income countries struggling to fund standard insulins and the routine monitoring of HbA1c levels. The first priority in these countries is to address these priority issues before funding more expensive forms of insulin and associated devices. Greater patient involvement in treatment decisions, transparency in decision making, and evidence-based investment decisions should help to address such concerns in the future. The global burden of diabetes is substantial and growing . There w® 1000IU by two-thirds to enhance usage (Whilst most people with diabetes have Type 2 diabetes (T2DM), an appreciable number of patients have Type 1 diabetes (T1DM) . In addice usage .There is improved diagnosis and management of T1DM in higher income countries, with mortality rates from T1DM appreciably decreasing in recent years . SimilarConsequently, patients with diabetes, including children, especially if they require insulin, should be increasingly carefully managed and monitored. This should include enhancing potential choices of available insulins, and their administration, to improve patient convenience, and subsequent adherence to prescribed treatments, cognisant of issues of affordability, especially if there are concerns with multiple injections with standard insulins such as NPH insulins as well as cultural issues. Greater choice and involvement of patients in decision making, especially with respect to issues surrounding injection frequency, should help to reduce the extent of hypoglycaemia and associated complications as well as improve long-term outcomes . HoweverLong-acting insulin analogues were developed to reduce the risk of hypoglycaemia, especially nocturnal hypoglycaemia, producing greater patient convenience through reducing the number of injections . Whilst Building on the situation observed in other disease areas, the increasing availability of biosimilars of long-acting insulin analogues should help to reduce their costs and increase their use, . HoweverThe continued higher prices for long-acting insulin analogues, including biosimilars, versus standard insulins such as NPH insulins, has limited their use in practice among a number of countries . This inThe WHO, World Bank and the Organisation for Economic Co-operation and Development have advocated five foundational elements critical for delivering quality health services in the goal of achieving universal healthcare for all by 2030. This is underpinned by governments, policymakers, health system leaders, patients and clinicians all working together . Key areThere have been developments in the administration of insulin over the years, attempting to reduce the burden of multiple daily administration of insulin and concomitant constant monitoring . AdvanceAlongside this, the development of continuous subcutaneous insulin infusions (CSII) systems combined with sensor-augmented pump therapy (SAP) and potentially hybrid closed-loops (HCL) systems to respond to changes in HbA1c levels thereby further reducing episodes of hypoglycaemia . There c™ 670G HCL system versus CSII systems among both adolescents and adults  higher than CSII systems, giving an incremental cost-effectiveness ratio of £20,421 per QALY gained   . A highe$27,850) .This increased cost is a concern as CSII systems are appreciably more expensive than multiple daily administration of insulin without adding in HCL systems and any associated patient education . HoweverIt is imperative across all countries that patients with diabetes requiring insulin, especially children and adolescents, are well managed given the burden of the disease and its potential complications, as well as the additional burden during their education. Issues of funding and choices for developments such as CGM and CSII systems with or without HCLs need to be placed in context given current inconsistencies in funding decisions across disease areas and countries especially among higher income countries, and the increasing burden of diabetes as well as its complications worldwide. This recognises that such discussions are more difficult in some LMICs where patients are struggling to fund even standard insulins within public healthcare systems, with these discussions continuing exacerbated by the recent COVID-19 pandemic . However®) of Ca$3.6 million  and their associated costs . While tThis goes hand-in-hand with encouraging transferable learning between countries, advocacy and compliance with key areas, as well as greater evidence-based approaches to decision making . This is


The intrinsic regenerative capacity of skeletal muscle makes it an excellent target for cell therapy. However, the potential of muscle tissue to renew is typically exhausted and insufficient in muscular dystrophies (MDs), a large group of heterogeneous genetic disorders showing progressive loss of skeletal muscle fibers. Cell therapy for MDs has to rely on suppletion with donor cells with high myogenic regenerative capacity. Here, we provide an overview on stem cell lineages employed for strategies in MDs, with a focus on adult stem cells and progenitor cells resident in skeletal muscle. In the early days, the potential of myoblasts and satellite cells was explored, but after disappointing clinical results the field moved to other muscle progenitor cells, each with its own advantages and disadvantages. Most recently, mesoangioblasts and pericytes have been pursued for muscle cell therapy, leading to a handful of preclinical studies and a clinical trial. The current status of (pre)clinical work for the most common forms of MD illustrates the existing challenges and bottlenecks. Besides the intrinsic properties of transplantable cells, we discuss issues relating to cell expansion and cell viability after transplantation, optimal dosage, and route and timing of administration. Since MDs are genetic conditions, autologous cell therapy and gene therapy will need to go hand-in-hand, bringing in additional complications. Finally, we discuss determinants for optimization of future clinical trials for muscle cell therapy. Joined research efforts bring hope that effective therapies for MDs are on the horizon to fulfil the unmet clinical need in patients. Cell therapy, the administration of live cells in a patient for the treatment of a disease, was first successfully applied as a hematopoietic stem cell transplant (HSCT) in 1959 .Transplantation of skeletal muscle came into play in the early 1990s and aimed to restore dystrophin production in patients with Duchenne muscular dystrophy (DMD). Skeletal muscle seemed to be a suitable target for cell therapy, since muscle fibers mainly consist of postmitotic cells and there is limited cell turnover. Intercostal muscle, for example, shows an average turnover of 15.1 years , 4. WhilSkeletal muscle formation, a process called myogenesis, occurs during embryonic development, and during postnatal muscle growth, regeneration and repair. Myoblasts, the early muscle cells with a single nucleus, proliferate and express muscle-specific genes leading to their fusion and the formation of multinucleated myotubes, which will ultimately form the mature myofibers . Muscle in vitro, systemic application, and the capacity to differentiate efficiently into skeletal muscle fibers in vitro and in vivo. Before we will discuss MABs and pericytes in more detail, we will review other myogenic progenitor cell types and cell populations that have been studied for their therapeutic potential over the last few years.The disease state in several myopathies affects the functional capacity of SCs and consequently impedes muscle regeneration. Stimulating regeneration by the addition of myogenic cell types has therefore been the main goal of cell therapy for muscle diseases. This development started with the use of myoblasts and SCs, which led to disappointing clinical results due to limited survival and scarce migration of injected cells. Next, the field moved to other myogenic cell types, each with their own advantages and disadvantages. Most recently, mesoangioblasts (MABs) and pericytes have been pursued for muscle cell therapy. These myogenic progenitor cells possess various beneficial characteristics needed for an effective cell therapy approach, i.e. simple isolation, the ability to proliferate Progressive wasting of skeletal muscle is characteristic for a subgroup of myopathies, the genetic and progressive conditions collectively called muscular dystrophies (MDs). There are over 30 (sub)types of MD with the most common diseases being myotonic dystrophy (DM), Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHD), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), Emery-Dreifuss muscular dystrophy (EDMD) and congenital muscular dystrophy (CMD)  and are therefore unable to systemically treat MDs . In . In bonaiescence , 35. Quilso PAX3 , 37. SCslso PAX3 . A grouple cells , 39.in vivo, limited migration through muscle tissue . All. All4]. For some conditions the use of non-modified autologous cells is an option. OPMD, for example, is a late-onset progressive and autosomal dominant genetic disorder in which initially only a few distinct muscles are affected. The defects observed in myogenic cells isolated from affected muscles of patients with OPMD are not present in cell cultures originating from their unaffected muscles , 130. Heex vivo strategy begins with isolating cells from the patient followed by correcting the mutation or restoring the reading frame and producing functional dystrophin protein. The precursor cells that contribute to tissue repair can be seen as vectors since these are deployed to deliver the functional gene. In a different disease example, myotonic dystrophy, an RNA gain-of-function disease with a multisystemic manifestation, removal or correction of the unstable repeat expansion that evokes the toxic RNA effect in muscle cells is the ultimate goal [Ex vivo corrected muscle progenitor cells will have to counterbalance the existing endogenous RNA toxicity and thus restore muscle regeneration capacity, preferably by repopulating the muscle stem cell pool in vivo.In most cases cell therapy and gene therapy must go hand-in-hand. For instance, in DMD many different muscles are affected and dystrophin expression needs to be restored by widespread supplementation of cells that are able to support muscle growth . The ex ate goal , 131. ExMany forms of MDs are monogenic disorders, which makes gene replacement a promising concept. Clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is a powerful tool to accurately alter a target in the genome of eukaryotes . Howeverin vivo approach, it is impossible to select for the correctly edited cells and thus the accuracy, efficiency and safety of the gene editing process needs to be exceptionally high. Ex vivo gene editing comes with the considerable benefit of selection of correctly edited cells. A drawback of this approach, however, can be the size of the correctly edited cell pool that is needed. Freshly isolated myogenic precursor cells have the highest regenerative potential and culture conditions influence transplantation efficiency in a negative manner [In an e manner , 136. Thin vitro expansion under traditional culture conditions is often hampered by changes in phenotypic expression of cells, which in turn affects transplantation efficiency.The clinical trials included in this review Table  were mosin vitro environment are exposed to non-physiological conditions such as the non-elastic plastic culture substrate, high oxygen supply, additional supplements and loss of contact with other cells. These circumstances affect cell behavior at that moment, but also have long-lasting effects on cells after transplantation. It is well established that expansion of myogenic cells before engraftment reduces their regenerative capacity [Cells in an artificial capacity , 137. Thcapacity , 137.One way to reduce culture-induced modifications is via the use of soft hydrogel substrates to mimic tissue rigidity, a biophysical property of the skeletal muscle microenvironment , 139. Thin vitro [in vitro muscle microenvironment to keep the maximal therapeutic potency [Typically used polystyrene plastic has an elastic modus of ~3 GPa which is five orders of magnitude more rigid than skeletal muscle , 144. Usin vitro , 139. Ho potency .Besides tissue rigidity, specific extracellular matrix (ECM) protein coatings can be used to mimic the SC niche. Matrigel, the most widely used commercialized ECM mixture, albeit without FDA approval, contains critical grow factors and cytokines , 146. Thin vivo environment to aid cell expansion, proliferation of cells can be influenced by the use of nitric oxide, calorie mimicking drugs, or genetic modification of specific targets. Nitric oxide (NO) contributes to myogenesis by the formation of S-nitrosothiols (RSNO). An increase in RSNO inhibits the S-nitrosoglutathione reductase (GSNOR) pathway and leads to an increase in the number of myoblasts, followed by a decrease in the myoblast fusion index [Other than mimicking the on index .ex vivo to promote expansion. Pharmacologic manipulation of p38 signaling can be leveraged for enhancement of both ex vivo expansion and subsequent in vivo engraftment of cells [Myoblasts cultured in the presence of calorie restriction mimetics behave differently. These drugs, for example metformin and ursolic acid, regulate myogenic differentiation and cell proliferation depending on the dose. By varying the doses of the calorie restriction mimicking drugs one can induce a shift between proliferating and differentiating status , 155. Geof cells .OCT3/4, SOX2, c-MYC, KLF4) [A different approach to circumvent the limited availability of freshly isolated primary cells, low proliferation capacity and early senescence, is the use of induced pluripotent stem cells (iPSCs). Generation of iPSCs is achieved by reprogramming somatic cells with a defined set of transcription factors  . These sC, KLF4) .α-SG-KO mice after i.m. injection [Genetically corrected human iPSC-derived MAB-like cells (HIDEMs) from LGMD patient fibroblasts only restored α-SG in 2% of skeletal myofibers in njection . The relnjection .Sgca-null/scid/beige mice [in vivo.As mentioned before, progeny-specific imprinting plays a role in differentiation , 25. Strige mice . AlthougA crucial factor for an effective cell therapy is the early survival of donor cells. The ultimate goal is that the ratio between death and proliferation leads to a good “net” survival post-transplantation, with the whole donor cell population being either stable or growing. Although suggested many times, it seems that myogenic cells are not instantly killed and removed after i.m. injection, but they undergo necrosis or apoptosis in the first few days. Factors that have been implicated in cell death are cellular effectors of an acute inflammatory response, a combination of natural killer cells, T-lymphocytes and complement, and the fact that only a small specific donor cell sub-population with stem cell-like characteristics survives .in vitro and in a mouse model [ex vivo treatment using Wnt7a, a member of the Wingless-INT (WNT) family, on satellite stem cells had similar effects and markedly increased cell dispersion and engraftment, which ultimately resulted in improved muscle function of dystrophic muscles [Various factors are able to improve the survival, migration and engraftment of injected cells. The growth factor, vascular endothelial growth factor (VEGF) reduced hypoxia-induced death of human myoblasts se model , 160. Otse model . Treatme muscles .in vitro during SC expansion enhances in vivo engraftment [The inhibition of p38 signaling pathway raftment . Identifraftment , 164.et al. focusses on the cell microencapsulation technology and the possibilities in the clinic [The harsh environment that cells get exposed to after injection likely plays a role in rapid cell death and low long term viability . MDs caue clinic .Fibrosis, the progressive replacement of functional tissue by nonfunctional and more rigid connective tissue, occurs in many MDs. In a fibrotic environment cells have been shown to differentiate more towards a fibrogenic fate thereby decreasing their regenerative potential , 171. Immdx mice showed that Wnt7a treatment efficiently induced satellite cell expansion, myofiber hypertrophy, reduced the level of contractile damage and caused a shift in fiber type toward slow-twitch [As mentioned before, alterations in Wnt7a and p38 signaling could exert positive effects on myogenic cell migration and engraftment. Besides pretreatment of cells, treating receiving muscle tissues could also potentially be beneficial , 173. Fow-twitch .A crucial factor complicating the efficacy of myogenic cell transplantation is that both mRNA and intracellular proteins in myofibers tend to stay close to the nucleus of origin, in an area called the nuclear domain . Due to ex vivo editing and cell transplantation have been established, further studies are required to determine conditions of the most optimal administration and dosage regimen to ensure the regeneration of myofibers and exert a positive effect on muscle function.Importantly, in myotonic dystrophy there is no need to ensure distribution of a missing protein along a myofiber, but the removal or reduction of the toxic RNA in a myotube is required for functional improvement. Therefore, once the technical procedures for For most patients, the diagnosis of MD comes after the onset of symptoms. An important question therefore is whether a proposed treatment can still be effective at or after this time point. Secondary pathologies that arise, due to the genetic defect, are known to cause irreversible damage. For example, DMD boys are generally diagnosed at the age of 4-5 years. Muscle biopsies at that time point already show hallmarks of the dystrophic progress. In general, various forms of MD show progressive replacement of muscle tissue by connective and adipose tissue, rendering muscle increasingly weak and nonfunctional .A cell therapy differs from a gene therapy approach in that it not only corrects a gene, but also delivers a pool of healthy myogenic cells for the regeneration process. Consequently, gene therapy might not be effective at later disease stages, while cell transplantation might still halt disease progression. An illustration of this idea is provided in a study were a BMD patient with progressive symptoms for eleven years underwent autologous bone marrow mononuclear cell transplantation and multidisciplinary rehabilitation for nine months. Gradual improvement in muscle strength and respiratory function indicated halting of disease progression .Studies examining the age dependence of cell therapy for MDs are missing. Gene transfer studies that investigate the age-dependent effect on treatment efficiency for other genetic disorders have demonstrated that earlier treatment increases the therapeutic effects . If we ade novo mutations [The best way to identify patients ahead of symptom manifestation, when a treatment will be most effective, is with carrier testing in families with a history of MDs and newborn screening for utations . DMD hasutations . A heel utations . NowadayThe muscular dystrophies (MDs) are highly variable in terms of age of onset, severity of symptoms, clinical pattern and genetics. Despite enormous research efforts in the past decades, there is currently no cure for any (sub)type of MD. This may change in the future by implementing cell therapy, but before the use of stem cells for muscle regeneration can become clinical reality there are several challenges that need to be met.in vivo, disperse throughout the muscles, repopulate muscle stem cell niches and fuse with existing fibers to efficiently differentiate into functional muscle. Established and validated cell isolation protocols, clear characterization and workable culture protocols are crucial, and additional research will lead us to the cell type that best fits the requirements for successful restoration of muscle morphology and function.To identify the ultimate muscle progenitor cell for cell therapy, we need more clarity among the cells currently at our disposal. A successful muscle progenitor can efficiently proliferate, survive + cells, CD133+ cells, MSCs, MDSCs, MuStem cells, SP cells, myoendothelial cells, PICs and MABs and pericytes. Each with their own merits and inherent limitations. Some of these cell types show high myogenicity, but are difficult to expand in vitro, while others can be isolated and propagated in workable amounts, but show in vivo inefficient myogenic differentiation or are unable to negotiate the vessel wall when systemically delivered. At the current time, pericytes show highly promising results in vitro and crucially in vivo, in a mouse model of MD and more recently in the dystrophic dog, although the subsequent clinical trial was inconclusive. Besides, the discovery and expanding application of iPSCs has unlocked a new research area, from which we expect exciting findings. IPSCs also offer hope for fulfillment of the above criteria, especially when gene editing of autologous cells is needed to correct the underlying genetic defects that lead to MDs.Nowadays, myoblasts and satellite cells are no longer considered to be suitable due to their prerequisite of local injection. However, in this review we have discussed additional and promising cell types such as ALDHAlthough each MD is different and each (sub)type might need a slightly different strategy in terms of cell dose, administration, timing and editing strategy, research is progressing and clinical trials are activated thereby bringing hope that effective therapies are on the horizon.


Interventions to reduce disability are crucial for older adults with disabilities to avert unnecessary hospitalizations or nursing home placements and improve daily life. Developed and tested at one research site, multiple health systems and community based organizations have since implemented CAPABLE. All published or peer reviewed tests of CAPABLE were reviewed  with a total of 1087 low-income community-dwelling older adults with disabilities. Participants were an average age of 74-79, cognitively intact, and self-reported difficulty with one or more activities of daily living (ADL). These trials were reviewed by extracting the participants’ scores on main outcomes, ADLs and IADLs, and when available, fall efficacy, depression, pain and cost savings. All studies yielded improvements in ADL and IADL limitations, with small to strong effect sizes. Studies with the complete dose of CAPABLE showed more improvement in ADLs and cost savings than the studies that implemented a decreased dose.


Determining the proportion of a population at risk of inadequate or excessivenutrient intake is a crucial step in planning and managing nutritionintervention programs. Multiple days of 24-h dietary intake data per subjectallow for adjustment of modeled usual nutrient intake distributions for theproportion of total variance in intake attributable to within-individualvariation . When only single-day dietary data are available, anexternal adjustment factor can be used; however, WIV:total may vary bypopulation, and use of incorrect WIV:total ratios may influence the accuracy ofprevalence estimates and subsequent program impacts. WIV:total values werecompiled from publications and from reanalyses of existing datasets to describevariation in WIV:total across populations and settings. The potential impact ofvariation in external WIV:total on estimates of prevalence of inadequacy wasassessed through simulation analyses using the National Cancer Institute 1-dmethod. WIV:total values were extracted from 40 publications from 24 countries,and additional values were calculated from 15 datasets from 12 nations. Widevariation in WIV:total (from 0.02 to 1.00) was observed in publications andreanalyses. Few patterns by population characteristics were apparent, butWIV:total varied by age in children (< vs. >1 y) and between ruraland urban settings. Simulation analyses indicated that estimates of theprevalence of inadequate intake are sensitive to the selected ratio in somecases. Selection of an external WIV:total estimate should consider comparabilitybetween the reference and primary studies with regard to populationcharacteristics, study design, and statistical methods. Given wide variation inobserved ratios with few discernible patterns, the collection of ≥2 daysof intake data in at least a representative subsample in population dietarystudies is strongly encouraged. In the case of single-day dietary studies,sensitivity analyses are recommended to determine the robustness of prevalenceestimates to changes in the variance ratio. Variation in within-person variance component ratios of nutrient intakes wasreviewed to assess statistical implications for studies analyzing single-day24-h dietary recall or food record data. While tTwenty-four-hour dietary recalls 24HRs) or weighed food records are commonly used togather data on food consumption that can be converted to daily nutrient intakes.Although food and nutrient intakes captured on any single day do not reflectlong-term average, or usual, intake, due in large part to day-to-day variation infood consumption for a given individual, collection of 1 d of recall data on enoughindividuals (representing both weekdays and weekends) may accurately estimate theaverage intake of a population, assuming that the recalls are unbiased and that themeasurement error around usual intake is random. However, even in this case, usingunadjusted, single-day estimates to obtain population distributions of usual intakesand calculate prevalence of inadequacy  isinappropriate because the variance in these distributions will be inflated relativeto the usual intake distribution due to the contribution of within-individualvariation (WIV) HRs or we, 4. Curr1) describe variation in variance componentratios across a wide range of global contexts and provide a database of WIV:BIV andWIV:total estimates for a range of populations that may be consulted for selectionof external adjustment factors and 2) assess the impact ofpotential variation in these ratios on the prevalence of inadequate intake in modelsof usual nutrient intake distributions using single-day nutrient intake data. Last,we propose an approach for selecting and applying external variance estimates formodeling usual nutrient intake distributions.Due to limited resources or feasibility constraints at the time of data collection,multiple days of dietary intake data may not be available at the time of analysis,precluding the separation of variance components from the observed data. In theseinstances, an external estimate of the fraction of total variance attributable toWIV  may be used in conjunction with the total variance estimate for thestudy population to estimate the usual intake distribution . There ihttps://epi.grants.cancer.gov/dacv/) (1) the proportion of totalvariance attributable to WIV , and 2) the ratioof within- to between-individual variance (WIV:BIV). When ≥1 of thesewas not reported in the publication, reported values were converted to thestatistics of interest, if possible. For example, when ratios of CVs of WIVand BIV were reported in the publication, the CV ratio was squared to obtainthe ratio of variances. The types of ratios reported in publications andcalculations used to convert between them are described in To describe variance component ratios from diverse studies, we firstconducted a literature review of studies that reported variance ratios for≥1 nutrient. We identified studies that included ≥2nonconsecutive (or ≥3 consecutive) days of dietary intake datacollected by 24HRs or food records and that reported variance components ofmicronutrient intakes in the related publication or report. We reviewed 5micronutrients of public health concern in low- and middle-income countries, includiv/dacv/) , the Gloion/en/), and snohttps://osf.io/wyvug/)along with instructions detailing how to structure the datasets and modifythe code to run on each dataset. In addition, several available or shareddatasets were analyzed by authors of this manuscript . As available, the same micronutrients of interest as above, includingvitamin A, folate, vitamin B-12, iron, and zinc, were analyzed. Since it wasconvenient to analyze additional nutrients from the same code, ratios werealso calculated for total energy, retinol, thiamin, phytate, and syntheticfolic acid intakes, as available.To expand the available data, and after observing wide variation in methodsof calculating and reporting variance components, we reanalyzed existingdatasets to calculate variance components using a consistent method. Authorsof studies that calculated nutrient intakes from multiple days of dietaryrecall/record data but did not report variance component values in theirpublications were contacted and invited to participate as collaborators toreanalyze their data to generate variance component estimates. Collaboratorswho agreed were provided with analytical code compatible with R or SASsoftware , season, and/or rural versus urbansetting, according to the original study's analysis or where samplesizes allowed. For 1 dataset ,separat1) accounting onlyfor weekend and interview sequence (unadjusted model), 2)including survey stratum as an additional covariate , and 3) including all relevant covariates (fullyadjusted model). For the fully adjusted models in women and children,additional covariates included interviewer, translator, age, caregivereducational level, and socioeconomic status. In addition, child sex andbreastfeeding status were included as covariates in models in children,while physiological status (pregnant or lactating) and whether the day ofintake was a feast day were included as covariates in models in women.For 1 national survey in Cameroon , the NCITo assess the effects of variability in assumed WIV:total on estimated prevalenceof inadequate intake when analyzing 1-d data, the EAR cut-point method wasapplied to usual nutrient intake distributions modeled using the NCI 1-d method. Two datTo compare the simulations with results that would be observed with multiple-daydata, WIV:total values for these samples were calculated from the full datasetsfor the population samples specified above, which included a second dietaryrecall or record on at least a subset of the population. For the Bangladeshdata, the linear mixed-model process described in the preceding section forreanalysis of existing datasets was applied for calculation of variancecomponents. For the Cameroon data, variance components were calculated using theNCI MIXTRAN macro  conducteForty publications and reports representing 24 countries that described studiescollecting multiple days of dietary intake data and reported variance componentvalues for nutrient intakes were included in the literature review. While≥1 estimates were available from a number of countries representing Asia, Africa Published studies reporting on variance components used a range of study designsand sampling procedures (Components of variation (WIV and BIV) were reported in publications in severalforms, including within-individual and between-individual variances, SDs, orCVs. Ratios of these were also reported, as well as “variance componentratios” . In addition, a number of different statistical methodologies wereused in the calculation of variance components. Approaches to datatransformation ranged from no transformation to power, log, or Box-Coxtransformations and/or multistep transformation processes . TransfoAfter 4 external collaborators shared or reanalyzed data from existing studies, 66-76 aWIV:total ratios extracted from publications varied widely overall (from 0.02 to1.00). Using a consistent statistical method, ranges of WIV:total ratios fromreanalyzed datasets exhibited similarly wide variation . Excludingratios derived from analyses considering intake from supplements, whichgenerally resulted in lower ratios than those considering only diet,distributions of WIV:total ratios by micronutrient revealed some variation inmedian ratios by nutrient, but even wider variation in ratios within nutrients, and setting, but in general, we observed widevariation in values, even within nutrients and subpopulations. Simulation analysesdemonstrated that the assumed WIV:total ratio can affect estimated usual intakedistributions and prevalence of inadequate intake, but not in a consistentway—that is, the extent and direction can vary across nutrients andpopulations depending on characteristics of the distribution and its position inrelation to the EAR.Variance ratios have previously been compiled and published from single studies, suchas NHANES , or smalA number of factors can influence food intake patterns and diversity, therebyaffecting ratios of WIV:total variance in nutrient intakes through influences onWIV, BIV, or both. It is fairly well established that variance ratios vary bynutrient , 64, 90.In this study, substantial variation existed in WIV:total across studies fromdifferent geographic settings, in agreement with previous research. For example,a study comparing nutrient intake variance components and ratios among childrenfrom Russia and the United States observed generally lower ratios in Russia. DiffereIn addition to the aforementioned factors that may influence true day-to-dayvariation in nutrient intakes, the datasets and publications included herevaried in a number of study design–related factors, including the dietaryassessment method used , sample size, number of days of dietary intakedata per individual, and time interval between dietary measurements. Thepotential composite effects of these factors on variance ratios are difficult todiscern. In 2 studies comparing observed weighed food records with 24HRscollected for the same days, variance ratios from weighed food records wereobserved to be lower  or diffeFinally, authors of the reviewed studies used a number of different statisticalapproaches for calculating variance components of nutrient intake. Inparticular, as we demonstrated in sensitivity analyses, the inclusion of variouscovariates can have a substantial effect on the variance ratio by reducing theoverall residual variance (BIV + WIV), with most of thereduction being attributed to BIV. Given the difficulty in comparing acrossstudies, we generated new estimates from existing datasets using a consistentcalculation procedure to control for statistical method. Nevertheless, othersources of variation in study designs remained, and wide variation was stillfound in the variance ratios using this approach. Multicenter studies using aconsistent study design and analytical approaches across study sites would allowfor more robust examination of true variation in WIV:total of nutrient intakesacross geographic contexts.In our simulations, varying WIV:total incrementally from 0.05 to 0.99 changed theestimated usual nutrient intake distributions and, consequently, the estimatedprevalence of inadequate intake in most cases examined. While this effect is tobe expected given the mathematical procedure used to produce the model, theinconsistency of the extent and direction of this effect across the 3 nutrientsand 2 populations studied suggests that characteristics of the dataset and howit is modeled may result in different effects of WIV adjustment on prevalence ofinadequacy. In particular, the position of the distribution in relation to theEAR, in cases where the EAR cut-point method is used, would be expected todetermine whether prevalence of inadequacy increases or decreases with changesin WIV:total, as we observed here . Our simSeveral other studies have conducted sensitivity analyses on the robustness ofestimated prevalence of inadequate nutrient intakes to the variance ratioassumption , 35. JahThe results suggest that researchers using external variance ratios cannot assumea consistent direction of bias and must conduct sensitivity analyses with theirdataset. In accordance with previous research, the present analysis highlightsthe importance of conducting sensitivity analyses to determine the robustness ofprevalence estimates to varying WIV:total in single-day dietary studies.Researchers and policy makers must then determine what level of uncertainty isacceptable for their intended use of the data. For example, if a governing bodyis interested only in whether there is likely a high or low prevalence ofinadequate intake of a given nutrient in the population, a somewhat higherdegree of uncertainty may be acceptable. If, however, the aim is to trackpotentially small changes in prevalence from year to year to determine, forinstance, the effectiveness of a fortification program, more precise estimatesmay be needed. Imprecise prevalence estimates resulting from the use of biasedvariance ratios could influence policy decisions if prevalence is wavering neara cutoff being used to determine whether a public health problem exists. Forexample, in our simulations of zinc inadequacy in Cameroon, altering the assumedWIV:total ratio produced a range of estimated prevalence that included a cutoff(>25%) previously used as an indicator pointing to a public healthproblem .Our research group has previously published a method for analysis of single-daydietary data . This paFor researchers intending to analyze single-day data, we offer the followingguidelines for choosing an external variance ratio to minimize the effects ofbias associated with their use.Reference studies should ideally match the nutrient of interest and thetarget population based on sex and life stage. The latter is particularlyimportant in the case of very young children. Physiological status amongwomen  might also be important in some cases,although more research is needed. It also may be important to considersocial and environmental factors that are similar or different between thereference and primary studies (including factors that vary over time withina population), and how these factors may be more or less relevant to dietarybehaviors and resultant nutrient intake patterns in the studied populations.Based on our and other researchers’ work, some such factors thatappear to influence variance ratios include country  or regioSince differences in the study design may lead to discrepant variance ratios,reference studies with a similar study design to the primary study and ofacceptable quality to provide robust variance estimates should be selected.Study design factors to consider include the dietary assessment method,sampling procedure, total sample size, number of individuals with repeatobservations, number of observations per individual, and the time betweenobservations. While our results do not allow conclusions about whether 1dietary assessment method would be preferable for application of externalratios, we note that there are far fewer variance ratios available fromstudies using observed records; thus, it may be challenging to match bystudy population and data collection method if the data to be analyzedinclude food records. Repeated dietary observations should ideally beperformed on nonconsecutive days to minimize correlation of intake onconsecutive days; if consecutive days are used, a minimum of 3 days isrecommended , 104, moFinally, the comparability of the statistical approach used to produce thevariance estimates in the reference study in relation to the primaryanalysis to be conducted should be considered, since different statisticalmethods can produce different estimations of WIV and BIV and their ratio. Inparticular, the inclusion of influential covariates can account for some ofthe variation between individuals, often leaving less residual BIV andresulting in higher WIV:total. Thus, it is advisable to choose referencestudies that included covariates similar to those that will be used in theprimary analysis.If a reasonable number of criteria are met, reference values presented hereor in other publications may be considered as external variance ratios.Alternatively, new variance ratios can be calculated if the original data ona comparable reference population is accessible. The NCI MIXTRAN macro, PC SoftOnce an external variance ratio has been selected, this value may be appliedto the NCI 1-d method (for nutrients consumed nearly daily) or other methodsfor estimating the usual intake distribution. Given the range of factorspotentially influencing variance ratios and the difficulty in predictingvariation across studies, sensitivity analyses should be conducted in allcases to determine the robustness of prevalence estimates to changes in theWIV:total or WIV:BIV ratio for a particular dataset. The range of varianceratios tested should represent the uncertainty in the external ratioestimate, considering the available estimates from similar populations thatmeet the aforementioned criteria. If such a range is unavailable, werecommend testing a range of WIV:total values from 0.5 to 0.9, or wider, forpopulations >1 y of age. If a high degree of uncertainty exists, itmay be more appropriate to present the study results as a range of possibleprevalence rather than a point estimate. SEs for each prevalence estimateshould be calculated to determine changes in the precision of estimates.When external estimates of within- and between-person variation in nutrientintakes are used in lieu of collecting multiple days of dietary intake data, ahigh degree of uncertainty is introduced into resulting models of usual nutrientintake distributions. We discovered wide variation in WIV:total ratios acrossstudies and observed few consistent patterns due to the large number ofpotentially influencing variables, suggesting this ratio is difficult toaccurately predict from external studies given its sensitivity to a number offactors specific to the population of interest, study design, and statisticalapproach. The effect of improperly choosing the variance ratio on estimatedprevalence of inadequacy is dataset-specific and can be substantial (up to 32percentage points in the datasets studied); therefore, even when applying thesuggested procedure to select an external variance estimate, prevalenceestimates should be assessed and reported using sensitivity analyses aroundcredible ranges of WIV:total to quantify ranges of uncertainty. Studies in theplanning stage should prioritize the collection of multiple days of dietary dataif the prevalence of the population at risk of inadequate or excessive intakesis of interest.


Nonsense-mediated decay (NMD) was first described as a quality-control mechanism that targets and rapidly degrades aberrant mRNAs carrying premature termination codons (PTCs). However, it was found that NMD also degrades a significant number of normal transcripts, thus arising as a mechanism of gene expression regulation. Based on these important functions, NMD regulates several biological processes and is involved in the pathophysiology of a plethora of human genetic diseases, including cancer. The present review aims to discuss the paradoxical, pro- and anti-tumorigenic roles of NMD, and how cancer cells have exploited both functions to potentiate the disease. Considering recent genetic and bioinformatic studies, we also provide a comprehensive overview of the present knowledge of the advantages and disadvantages of different NMD modulation-based approaches in cancer therapy, reflecting on the challenges imposed by the complexity of this disease. Furthermore, we discuss significant advances in the recent years providing new perspectives on the implications of aberrant NMD-escaping frameshifted transcripts in personalized immunotherapy design and predictive biomarker optimization. A better understanding of how NMD differentially impacts tumor cells according to their own genetic identity will certainly allow for the application of novel and more effective personalized treatments in the near future. Eukaryotic gene expression comprises a series of interconnected steps in which messenger RNAs (mRNAs) play a crucial intermediate role. To ensure that the genetic information is correctly transcribed from DNA to RNA, and then translated into a functional protein, eukaryotic cells have developed several elaborate quality control mechanisms, many of them acting precisely at the mRNA level , 2. The The involvement of NMD in cancer development has been extensively studied during the past few years. It was shown that different cancer contexts can take advantage of both NMD quality-control and regulatory functions to potentiate the development of the disease . For exaThe present review describes NMD as a major surveillance and gene expression regulatory pathway, by briefly exposing the knowledge regarding its players, mechanism of action and biological relevance, focusing specifically on cancer. Then, we discuss in detail how tumor cells have explored both the quality and regulatory NMD functions to leverage tumorigenesis in their own microenvironment. We also present the challenges of using NMD in cancer therapy, addressing situations where inhibition or activation of NMD are favorable according to the genetic context of the cancer. Finally, we discuss how NMD inhibition and/or NMD-escaping can contribute for biomarker optimization and personalized immunotherapy design.The molecular mechanism of NMD has been described by several models that differ in some key aspects, while agreeing that it is fundamentally a translation-dependent process. This dependence has been observed over the years using translation inhibition approaches, either by the presence of translation inhibitors  or extended stem-loops in the 5′ untranslated region (UTR) , 30. CurNMD begins with the arrival of the translating ribosome to a PTC, at which the translation eukaryotic release factors (eRF) 1 and 3 interact with UPF1 and the serine/threonine protein kinase, SMG1, associated with its regulators, SMG8 and SMG9, to form the “SURF” complex  and ER stress , 104–108NMD has also been shown to play a relevant role in viral replication, since the abrogation of this pathway culminates in the upregulation of viral proteins and subsequent higher viral infection, indicating that NMD plays a host protective role in blocking virus replication, such as the Semliki Forest virus (SFV) and Sindbis virus (SINV) . In respThe physiological relevance of NMD is further expanded by its connection with alternative splicing (AS) , 118. ASSplicing factors are key mediators of AS-NMD by shifting the balance of which splice sites are recognized, in order to promote transcripts bearing in-frame nonsense codons, or PTC-free isoforms . InteresAs NMD has a key role in mRNA surveillance, it is not surprising that its activity has been implicated in the phenotype modulation of several human genetic diseases , 7. For While in most of the above-mentioned genetic diseases the quality-control facet of NMD is particularly relevant, different aspects of NMD activity have been explored by cancer cells to potentiate the disease . Indeed,Tumors have found ways to leverage their unconstrained growth and the progression of cancer by taking advantage of both functions of NMD. On one hand, cancer cells use NMD activity to selectively downregulate tumor-suppressive genes through PTC acquisition, but on the other hand, these cells fine-tune NMD magnitude to allow the upregulation of stress-corrective genes responsible for their adaptation to the tumor microenvironment , 26. AltDisabling mutations in genes encoding NMD factors have been found in several types of cancers, which raised the possibility of NMD having some type of protective role against tumorigenesis. For instance, in pancreatic adenosquamous carcinoma (ASC) tumors and in lung inflammatory myofibroblastic tumors (IMTs), UPF1 gene exhibits splicing-altering mutations that compromise NMD activity , 28. ThiIn addition to its role in gene expression regulation, NMD ability to degrade PTC-harboring transcripts may also be involved in the protection of cancer development. It was shown that tumor suppressor genes have a higher propensity to acquire nonsense mutations than oncogenes, which present mostly missense mutations . MoreoveDespite the clear notion that NMD can work against cancer, in some contexts its activity may have the opposite effect. For instance, it was recently reported that NMD inhibition in CRCs with microsatellite instability (MSI) leads to decreased tumor growth in xenograft models, suggesting that NMD typically plays a pro-tumorigenic role in these tumors . Indeed,In addition to participating in the process of cancer development, NMD activity also seems to impact tumor evolution. During cancer progression, tumor cells acquire several somatic mutations that may, or not, favor the tumorigenic process. This is accompanied by a positive and negative selection that allows the proliferation of subclones with favorable mutations, such as PTCs in tumor suppressor genes or NMD-resistant/missense mutations in oncogenes, while eliminating the ones bearing detrimental mutations. By this means, cancer cells take advantage of NMD activity and of the rules that govern its induction to favor proliferation of transformed cells overproducing oncoproteins and other pro-tumorigenic proteins, thus driving further and aggravating the disease , 21.As explained before, NMD activity is intimately linked to AS, together forming a key post-transcriptional mechanism of gene expression regulation. During the last decade, many dysregulated AS events have been observed in several cancer types, typically involving mutated splicing factors that lead to genome-wide altered patterns of gene expression. This can result in the production of NMD-sensitive isoforms of oncogenes and tumor suppressor genes that will, therefore, contribute for cancer development. A well-documented example is the SR protein, SRSF2, frequently mutated in patients with acute myeloid leukemia (AML) , 160. A Epithelial cadherin (E-cadherin) is a crucial factor to maintain tissue integrity and polarization of epithelial cell layers and it is well-known that E-cadherin loss is a key event during cancer progression that contributes to the epithelial-mesenchymal transition . InteresHypoxia is a major feature in solid tumors, given the high proliferating mass of cancer cells encountering an avascular environment that limits oxygen supply . Interes6-Methyladenosine (m6A) RNA methylation is a common mRNA modification dynamically regulated in mammalian cells ., 8.2, 8]NMD activation therapy has the additional possibility of being used in combination with chemotherapy to protect normal cells from apoptosis . This coAlthough NMD activation has great potential to be used in cancer therapy, the suggested approaches and methods are less developed than NMD inhibitory strategies. Therefore, there may be several years before being implemented in clinical practice.Nonsense suppressors represent a class of compounds that enhance the readthrough of PTCs by the translating ribosome . Given tThe usage of small inhibiting molecules/drugs to decrease NMD activity may present, however, a potential problem regarding their specificity, as these compounds might have off-targets that can compromise other cellular pathways or processes. This is aggravated by the fact that NMD targets several non-mutated transcripts, thus making it difficult to find the therapeutic dose of a global NMD inhibitor without having a major impact on the transcriptome. To overcome these issues, siRNAs and chemically modified antisense oligonucleotides (ASOs) designed to target NMD factors could provide a more specific therapeutic approach Fig. b 158]. . 158]. TEarly studies during the 90s in murine models revealed that T lymphocytes (or their effector molecules) are determinant to tumor control –208, sugDespite the therapeutic advantages provided by the immune checkpoint blockade therapy in cancer, its efficacy is highly variable and the response rates are usually below 40% , 215. AnAs already mentioned, a key marker for immunotherapy response is the overall mutation burden of the tumor, i.e. the higher the number of accumulated frameshifting mutations, the greater the propensity of the tumor to produce neoantigenic peptides/proteins that can be detected by the immune system of the individual Fig. c 218]. . 218]. GThe relationship between NMD and cancer is a complex one, where both its quality-control and regulatory functions can contribute to different stages of tumor development. Under normal circumstances, NMD is expected to have a protective role in the cell, preventing production of potential dominant-negative proteins and downregulating the expression of many important factors that can promote tumorigenesis , 146–150Thus, it should be preconized the long-range sequencing of the tumor full-length transcriptome and the rules and effects of NMD should be incorporated into clinical decision-making processes, as, together, this information may better support personalized immunotherapy design.


In this review, we describe the next-generation breeding tools that can be used to increase crop production by developing climate-resilient superior genotypes to cope with the future challenges of global food security. Recent innovations in genomic-assisted breeding (GAB) strategies allow the construction of highly annotated crop pan-genomes to give a snapshot of the full landscape of genetic diversity (GD) and recapture the lost gene repertoire of a species. Pan-genomes provide new platforms to exploit these unique genes or genetic variation for optimizing breeding programs. The advent of next-generation clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) systems, such as prime editing, base editing, and de nova domestication, has institutionalized the idea that genome editing is revamped for crop improvement. Also, the availability of versatile Cas orthologs, including Cas9, Cas12, Cas13, and Cas14, improved the editing efficiency. Now, the CRISPR/Cas systems have numerous applications in crop research and successfully edit the major crop to develop resistance against abiotic and biotic stress. By adopting high-throughput phenotyping approaches and big data analytics tools like artificial intelligence (AI) and machine learning (ML), agriculture is heading toward automation or digitalization. The integration of speed breeding with genomic and phenomic tools can allow rapid gene identifications and ultimately accelerate crop improvement programs. In addition, the integration of next-generation multidisciplinary breeding platforms can open exciting avenues to develop climate-ready crops toward global food security.Climate change is a threat to global food security due to the reduction of crop productivity around the globe. Food security is a matter of concern for stakeholders and policymakers as the global population is predicted to bypass 10 billion in the coming years. Crop improvement  Food security is the biggest challenge in feeding the continuous up-surging population. The dream of a world without hunger is only possible if agricultural productivity increases in a sustainable manner ; still breeding is an obvious point for moving ahead. Hence, we discuss the fascinating technologies of genomic-assisted breeding (GAB) with a focus on advances in crop pan-genome assemblies and their application for crop improvement. We pay specific attention to cutting-edge genome editing tools like clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) systems and spotlight their expanding toolbox. Furthermore, we discuss plant phenomics and their major bottlenecks that need to be overcome to bridge the gap between genomics and phenomics and focus on high-throughput phenotyping for crop improvement. We also highlight the next-generation phenomics approaches, including artificial intelligence (AI) and machine learning (ML), that can revolutionize the digital agriculture. Finally, we describe speed breeding and propose the integration of next-generation breeding technologies to expedite the crop production for food security.Approximately 10,000 years ago, plant breeding emerged as a central approach for plant domestication by exploiting wild relatives to select the desired traits through a continuous selection process over several generations for crop improvement , grow only a considerable number of GM crops on a limited area  to accurately explore the genetic make-up of crop traits. Quantitative trait loci (QTL) analysis through mapping agronomically important traits enabled to dissect the relationship between genotype and phenotype. Gene cloning and characterization, haplotype-based breeding, allele mining for stress tolerance, and tapping natural variations have open new avenues toward GAB  based trait mapping that has allowed rapid gene identification. Current advances in genome sequencing techniques have transformed plant breeding by providing access to huge plant genomes and open a new era of genomics  within the genome by employing SNPs, the SVs has been considered as a principal source of GD and may include presence/absence variants (PAVs), copy number variants (CNVs), and some other diverse variations such as chromosomal translocation, inversion, and transversions  in pan-genomic research may facilitate to cross the barriers of pan-genome construction and visualization as ML tools can autonomously detect the sequence reads in huge data sets. Also, a complete information flow reflecting gene-transcriptome-metabolome-epigenome-phenome can provide an excellent platform to study a phenotypic variation within different microenvironments. As the omics tools continue to attain more precision and sensitivity for analytical investigation, the era of crop “pan-omics” is not far away. The upcoming future is expecting to see the data science explosion, where “Crop Pan-omes” studies will be one of the key players in giving a new direction to future agriculture.To date, classical breeding is speedier in comparison to 50 years ago but still inadequate to accelerate the agricultural production with respect to the global demands  and zinc finger nucleases (ZFNs), brought a revolution in plant research and accelerated the plant research  and contains three subunits: a Cas9 protein, CRISPR RNA (crRNA), and trans-activating crRNA (tracrRNA)  domain was targeted successfully by using multiplex genome editing via CRISPR/Cas9, which is a negative regulator for salinity stress. The results revealed that the elimination of this domain produces tomato with high salinity tolerance  attached with cytidine deaminase. An efficient system called base editor 3 (BE3) has been developed for cotton to create the targeted base substitutions with the mutation rate of 26.67–57.78% , such as satellites, drones, phenotyping towers, environmental sensor networks, phenomobiles, autonomous ground vehicles, air crafts, helicopters, zeppelins, and field scanning platforms, which are currently used on a large scale  to screen the most distinguishing root traits  based delivery system is transferred directly into inflorescence tissues, pollen grains, and apical meristems, or it is sprayed to already grown plant to get express edits in the field or under speed breeding chambers (Hickey et al., Next-generation CRISPR systems, like The exciting phenotyping platforms inspired by AI, like robotics, 3D imaging sensors, and their integration with other OMICS date, are taking plant science to new heights. With multiscale, multidimensional, and multidomain phenomics knowledge, we promptly require the next-generation approaches of swarm intelligence, hybrid intelligence, AI, data fusion, and DL to establish big data handling methods.To conclude, a powerful unified strategy is needed to tackle the current challenges by integrating the multidisciplinary next-generation strategies and scientists to work under one umbrella and carry out mega projects to boost up the crop production in a rapid way as proposed in The multidisciplinary tools from genomics to phenomics should be carried out in a well-integrated manner to overcome the current hurdles in plant research. By employing the integrated next-generation approaches, future crop breeding can attain irreversible success to ensure food security and will fulfill the food demands of rapidly increasing population in decades to come.AR conceived the idea. AR and FS led the writing of manuscript. NA helped in revising the manuscript. PK and SW reviewed, edited, and improved the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.