Loss of mitochondrial pyruvate transport initiates cardiac glycogen accumulation and heart failure.

Background: Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. Methods: We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of U-13C-glucose. Cardiac glycogen was assessed biochemically and by transmission electron microscopy. Cardiac uptake of 2-deoxyglucose was measured and western blotting performed to analyze insulin signaling and enzymatic regulators of glycogen synthesis and degradation. Isotope tracing and glycogen analysis was also performed in hearts from mice fed either low-fat diet or a ketogenic diet previously shown to reverse the CS-Mpc2-/- heart failure. Cardiac glycogen was also assessed in mice infused with angiotensin-II that were fed low-fat or ketogenic diet. Results: Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, yet these hearts displayed increased enrichment from U-13C-glucose and increased glycolytic metabolite pool sizes. 13C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. This increased glycogen synthesis occurred despite enhanced inhibitory phosphorylation of glycogen synthase and reduced expression of glycogenin-1. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic 13C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin-II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Conclusions: Our results indicate that loss of MPC in the heart causes glycogen accumulation and heart failure, while a ketogenic diet can reverse both the glycogen accumulation and heart failure. We conclude that maintaining mitochondrial pyruvate import and metabolism is critical for the heart, unless cardiac pyruvate metabolism is reduced by consumption of a ketogenic diet.

Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma.

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αß T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, having innate antitumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allocompatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Rα-IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared with unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together, the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control.

Geographic Location and Corporate Ownership of Hospitals in Relation to Unfilled Positions in the 2023 Emergency Medicine Match.

Introduction: In the 2023 National Resident Matching Program (NRMP) match, there were 554 unfilled emergency medicine (EM) positions before the Supplemental Offer and Acceptance Program (SOAP). We sought to describe features of EM programs that participated in the match and the association between select program characteristics and unfilled positions. Methods: The primary outcome measures included the proportion of positions filled in relation to state and population density, hospital ownership type, and physician employment model. Secondary outcome measures included comparing program-specific attributes between filled and unfilled programs, including original accreditation type, year of original accreditation, the total number of approved training positions, length of training, urban-rural designation, hospital size by number of beds, resident-to-bed ratio, and the percentage of disproportionate share patients seen. Results: The NRMP Match had 276 unique participating EM programs with 554 unfilled positions. Six states offered 52% of the total NRMP positions available. Five states were associated with two-thirds of the unfilled positions. Public hospitals had a statistically significant higher match rate (88%) when compared to non-profit and for-profit hospitals, which had match rates of 80% and 75%, respectively (P < 0.001). Programs with faculty employed by a health system had the highest match rate of 87%, followed by clinician partnerships at 79% and private equity groups at 68% (P < 0.001 overall and between all subgroups). Conclusion: The 2023 match in EM saw increased rates in the number of residency positions and programs that did not fill before the SOAP. Public hospitals had higher match rates than for-profit or non-profit hospitals. Residency programs that employed academic faculty through the hospital or health system were associated with higher match rates.

Decadal changes in biomass and distribution of key fisheries species on Newfoundland's Grand Banks.

Canadian fisheries management has embraced the precautionary approach and the incorporation of ecosystem information into decision-making processes. Accurate estimation of fish stock biomass is crucial for ensuring sustainable exploitation of marine resources. Spatio-temporal models can provide improved indices of biomass as they capture spatial and temporal correlations in data and can account for environmental factors influencing biomass distributions. In this study, we developed a spatio-temporal generalized additive model (st-GAM) to investigate the relationships between bottom temperature, depth, and the biomass of three key fished species on The Grand Banks: snow crab (Chionoecetes opilio), yellowtail flounder (Limanda ferruginea), and Atlantic cod (Gadus morhua). Our findings revealed changes in the centre of gravity of Atlantic cod that could be related to a northern shift of the species within the Grand Banks or to a faster recovery of the 2J3KL stock. Atlantic cod also displayed hyperaggregation behaviour with the species showing a continuous distribution over the Grand Banks when biomass is high. These findings suggest a joint stock assessment between the 2J3KL and 3NO stocks would be advisable. However, barriers may need to be addressed to achieve collaboration between the two distinct regulatory bodies (i.e., DFO and NAFO) in charge of managing the stocks. Snow crab and yellowtail flounder centres of gravity have remained relatively constant over time. We also estimated novel indices of biomass, informed by environmental factors. Our study represents a step towards ecosystem-based fisheries management for the highly dynamic Grand Banks.

Emergency department responses to nursing shortages.

BACKGROUND: The COVID-19 pandemic exacerbated the nursing shortage, which is predicted to continue to worsen with significant numbers of nurses planning to retire within the next 5 years. There remains a lack of published information regarding recommended interventions for emergency departments (EDs) facing a sudden nursing shortage. METHODS: We queried emergency department leaders from the American College of Emergency Physicians to examine the impact of nursing shortages on EDs and to gather real-world interventions employed to mitigate the effects of the shortage. RESULTS: Most respondents (98.5%) reported nursing shortages, with 83.3% describing prolonged shortages lasting more than 12 months, with negative impacts such as misses/near-misses (93.9%) and increasing left without being seen rates (90.9%). ED leaders reported a range of interventions, including operational flow changes, utilizing alternative staff to fill nurse roles, recruitment of new nurses, and retention strategies for existing nurses. They employed temporary and permanent pay increases as well as efforts to improve the ED work environment and techniques to hire new nurses from atypical pipelines. CONCLUSION: We report a patchwork of solutions ED leaders utilized which may have variable efficacy among different EDs; personalization is essential when selecting interventions during a sudden nursing shortage.

Label free, capillary-scale blood flow mapping in vivo reveals that low intensity focused ultrasound evokes persistent dilation in cortical microvasculature.

Non-invasive, low intensity focused ultrasound (FUS) is an emerging neuromodulation technique that offers the potential for precision, personalized therapy. An increasing body of research has identified mechanosensitive ion channels that can be modulated by FUS and support acute electrical activity in neurons. However, neuromodulatory effects that persist from hours to days have also been reported. The brain's ability to provide targeted blood flow to electrically active regions involve a multitude of non-neuronal cell types and signaling pathways in the cerebral vasculature; an open question is whether persistent effects can be attributed, at least partly, to vascular mechanisms. Using a novel in vivo optical approach, we found that microvascular responses, unlike larger vessels which prior investigations have explored, exhibit persistent dilation. This finding and approach offers a heretofore unseen aspect of the effects of FUS in vivo and indicate that concurrent changes in neurovascular function may partially underly persistent neuromodulatory effects.

Prior Treatment with AICAR Causes the Selective Phosphorylation of mTOR Substrates in C2C12 Cells.

Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco's Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium.

Reproductive behavior of Atlantic halibut (Hippoglossus hippoglossus) interpreted from electronic tags.

Reproductive timing, location, and behavior are important characteristics that determine marine population dynamics, structure, and resilience to threats, including fishing and climate change. It is challenging to evaluate factors driving variability in these reproductive traits in wild fishes because of the difficulty observing individuals in their natural environments. In the present study, we used high-resolution depth, temperature, and acceleration time series recorded by pop-up satellite archival tags to (1) identify and characterize patterns in depth and acceleration that may be indicative of spawning events in large Atlantic halibut (Hippoglossus hippoglossus), and (2) estimate the effects of individual traits (body size and sex) and environmental factors (location and temperature) on spawning time and frequency. Unique rapid rises observed in the winter depth profiles were interpreted as spawning events. The initiation of the first presumed spawning rise was negatively correlated to water temperature experienced during the prespawning season, suggesting that currently increasing water temperature in the Gulf of St. Lawrence may induce phenological change in halibut spawning time. The number of rises of batch-spawning females was unrelated to female body size. The present study demonstrates how electronic tagging can be used for in-depth characterization of timing, location, and behaviors associated with spawning in a large flatfish species. Such information can inform spatiotemporal management and conservation measures aiming to protect species from directed fishing and by-catch during spawning.

SNP Panel and Genomic Sex Identification in Atlantic Halibut (Hippoglossus hippoglossus).

The ability to identify sex is necessary in population biology for a proper understanding of the dynamics of a population. In Atlantic halibut, phenotypic sex identification is not possible due to the lack of significant external morphological differences. We developed an Illumina SNP panel for Atlantic halibut with 4000 SNPs spread evenly throughout the genome with a minor allele frequency MAF ≥ 0.4, except for N = 249 SNPs located in a sex-determining region on chromosome 12, N = 176 of these SNPs were selected to genetically identify male and female individuals using a DAPC analysis. The genomic identification of sex allows for non-lethal sex determination and validation of sex identification in the field. The SNP panel is a new genomic resource for Atlantic halibut that will make it possible to generate the genotypic data for the large number of individuals needed to estimate population abundance using genomics and the Close Kin Mark Recapture (CKMR) approach, an emerging component of fisheries management and stock monitoring.

Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells.

BACKGROUND AIMS: The targeting of solid cancers with chimeric antigen receptor (CAR) T cells faces many technological hurdles, including selection of optimal target antigens. Promising pre-clinical and clinical data of CAR T-cell activity have emerged from targeting surface antigens such as GD2 and B7H3 in childhood cancer neuroblastoma. Anaplastic lymphoma kinase (ALK) is expressed in a majority of neuroblastomas at low antigen density but is largely absent from healthy tissues. METHODS: To explore an alternate target antigen for neuroblastoma CAR T-cell therapy, the authors generated and screened a single-chain variable fragment library targeting ALK extracellular domain to make a panel of new anti-ALK CAR T-cell constructs. RESULTS: A lead novel CAR T-cell construct was capable of specific cytotoxicity against neuroblastoma cells expressing low levels of ALK, but with only weak cytokine and proliferative T-cell responses. To explore strategies for amplifying ALK CAR T cells, the authors generated a co-CAR approach in which T cells received signal 1 from a first-generation ALK construct and signal 2 from anti-B7H3 or GD2 chimeric co-stimulatory receptors. The co-CAR approach successfully demonstrated the ability to avoid targeting single-antigen-positive targets as a strategy for mitigating on-target off-tumor toxicity. CONCLUSIONS: These data provide further proof of concept for ALK as a neuroblastoma CAR T-cell target.

Activation priming and cytokine polyfunctionality modulate the enhanced functionality of low-affinity CD19 CAR T cells.

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and antitumor efficacy compared with the high-affinity (FMC63-based) CAR used in tisagenlecleucel, in preclinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a phase 1 clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T cells, we performed a systematic in vitro characterization of the transcriptomic (RNA sequencing) and protein (cytometry by time of flight) changes occurring in T cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared with FMC63 CAR T cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B cells present in the manufacture.

Duplicated fetal posterior cerebral artery in a patient with a ruptured fetal posterior cerebral artery aneurysm: a cerebrovascular variant.

BACKGROUND: The most common neurovascular variant is the fetal posterior cerebral artery (FPCA), in which the P1 branch is absent or hypoplastic, and the majority of P2 supply is derived from the anterior circulation. While there are reports of hyperplastic anterior choroidal arteries (AChA) with supply to the temporo-occipital and calcarine regions, no reports of a duplicated FPCA exist. METHODS: This case report describes a patient with a ruptured right FPCA aneurysm. Digital subtraction angiogram (DSA) revealed an artery with origin distal to the FPCA associated with the aneurysm. This was not consistent with a typical AChA. The FPCA associated with the aneurysm had the typical origin, course, and supply of a FPCA. The distal FPCA had a similar course of a typical FPCA with significant supply to the typical PCA territory. The patient underwent successful clipping of the aneurysm, and the duplicated FPCA was identified during the craniotomy. RESULTS: The features of this duplicate FPCA, which has not been previously described, are discussed in comparison to another variant, the hyperplastic, anomalous AChA. The artery described in this report does not fit the typical criteria of this AChA variant. Therefore, the authors outline this variant as a duplicated FPCA. CONCLUSION: Recognition of variant cerebrovascular anatomy is vital to neurosurgeons and interventional neuroradiology specialists. FPCA aneurysms require special management considerations and are often more challenging to treat. This report discusses a duplicated FPCA. To our knowledge, this is the first description of this variant. A duplicated FPCA carries important management considerations in the management of neurovascular pathology.

From intern to "accepted": A guide to preparing for fellowship in emergency medicine.

Introduction: The number of fellowship options for emergency medicine (EM) physicians continues to expand. While guides exist to help residents explore individual fellowship pathways, we aimed to create a comprehensive guide for all residents considering fellowship. Methods: At the direction of the Society for Academic Emergency Medicine (SAEM) Board, 9 members of the Fellowship Guide Workgroup, including members of the Fellowship Approval Committee, and 2 members of SAEM Residents and Medical Students (RAMS) group collaboratively developed the guide using available evidence and expert opinion when high-quality evidence was unavailable. The guide was reviewed and approved by all members. Results: The guide offers advice to EM residents on how to conceptualize key aspects of their training with respect to preparation for fellowship, including scholarship, teaching, leadership, and electives. Additionally, it offers perspective on selecting a fellowship that matches the resident's interests and goals and successfully applying. Conclusion: This fellowship guide for EM residents considering fellowship summarizes the best currently available advice for residents considering fellowship training after residency.

A Simple and Robust Single-Step Method for CAR-Vδ1 γδT Cell Expansion and Transduction for Cancer Immunotherapy.

The γδT cell subset of peripheral lymphocytes exhibits potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. The Vδ1-T cell receptor (TCR)-expressing subset of peripheral γδT cells has remained enigmatic compared to its more prevalent Vγ9Vδ2-TCR and αß-TCR-expressing counterparts. It took until 2021 before a first patient was dosed with an allogeneic adoptive Vδ1 cell product despite pre-clinical promise for oncology indications stretching back to the 1980s. A contributing factor to the paucity of clinical progress with Vδ1 cells is the lack of robust, consistent and GMP-compatible expansion protocols. Herein we describe a reproducible one-step, clinically translatable protocol for Vδ1-γδT cell expansion from peripheral blood mononuclear cells (PBMCs), that is further compatible with high-efficiency gene engineering for immunotherapy purposes. Briefly, αßTCR- and CD56-depleted PBMC stimulation with known-in-the-art T cell stimulators, anti-CD3 mAb (clone: OKT-3) and IL-15, leads to robust Vδ1 cell expansion of high purity and innate-like anti-tumor efficacy. These Vδ1 cells can be virally transduced to express chimeric antigen receptors (CARs) using standard techniques, and the CAR-Vδ1 exhibit antigen-specific persistence, cytotoxicity and produce IFN-γ. Practicable, GMP-compatible engineered Vδ1 cell expansion methods will be crucial to the wide-spread clinical testing of these cells for oncology indications.

Spatial variation in food web structure in a recovering marine ecosystem.

Spatial heterogeneity in food web structure and interactions may reconcile spatial variation in population and community dynamics in large marine ecosystems. In order to assess food web contributions to the different community recovery dynamics along the Newfoundland and Labrador shelf ecosystem, we quantified species interactions using stable isotope mixing models and food web metrics within three sub-regions. Representative samples of each species caught in trawls and plankton tows were analyzed for stomach contents and stable isotope ratios (δ15N and δ13C) to parameterize isotope mixing models. Regional variation, highlighted by the diets of three economically important species, was observed such that the southern region demonstrated a variety of trophic pathways of nutrient flow into the higher food web while the diets of fish in the northern regions were typically dominated by one or two pathways via dominant prey species, specifically shrimp (Pandalus sp.) and hyperiids. Food web metrics indicated that the low-diversity northern regions had higher connectance and shorter food chain lengths. This observed regional variation contributes to our understanding of the role of specific forage species to the ecosystem which is an essential contribution towards ecosystem-based management decisions.

Photoglobin, a distinct family of non-heme binding globins, defines a potential photosensor in prokaryotic signal transduction systems.

Globins constitute an ancient superfamily of proteins, exhibiting enormous structural and functional diversity, as demonstrated by many heme-binding families and two non-heme binding families that were discovered in bacterial stressosome component RsbR and in light-harvesting phycobiliproteins (phycocyanin) in cyanobacteria and red algae. By comprehensively exploring the globin repertoire using sensitive computational analyses of sequences, structures, and genomes, we present the identification of the third family of non-heme binding globins-the photoglobin. By conducting profile-based comparisons, clustering analyses, and structural modeling, we demonstrate that photoglobin is related to, but distinct from, the phycocyanin family. Photoglobin preserves a potential ligand-binding pocket, whose residue configuration closely resembles that of phycocyanin, indicating that photoglobin potentially binds to a comparable linear tetrapyrrole. By exploring the contextual information provided by the photoglobin's domain architectures and gene-neighborhoods, we found that photoglobin is frequently associated with the B12-binding light sensor domain and many domains typical of prokaryotic signal transduction systems. Structural modeling using AlphaFold2 demonstrated that photoglobin and B12-binding domains form a structurally conserved hub among different domain architecture contexts. Based on these strong associations, we predict that the coupled photoglobin and B12-binding domains act as a light-sensing regulatory bundle, with each domain sensing different wavelengths of light resulting in switch-like regulation of downstream signaling effectors. Thus, based on the above lines of evidence, we present a distinct non-heme binding globin family and propose that it may define a new type of light sensor, by means of a linear tetrapyrrole, in complex prokaryotic signal transduction systems.

Stimulation of Akt Phosphorylation and Glucose Transport by Metalloporphyrins with Peroxynitrite Decomposition Catalytic Activity.

Iron porphyrin molecules such as hemin and iron(III) 4,4',4″,4‴-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) (FeTBAP) have previously been shown to influence insulin signaling and glucose metabolism. We undertook this study to determine whether a catalytic action of iron porphyrin compounds would be related to their stimulation of insulin signaling and glucose uptake in C2C12 myotubes. FeTBAP did not display nitrite reductase activity or alter protein S-nitrosylation in myotubes, eliminating this as a candidate mode by which FeTBAP could act. FeTBAP displayed peroxynitrite decomposition catalytic activity in vitro. Additionally, in myotubes FeTBAP decreased protein nitration. The peroxynitrite decomposition catalyst Fe(III)5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato chloride (FeTPPS) also decreased protein nitration in myotubes, but the iron porphyrin Fe(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachlorideporphyrin pentachloride (FeTMPyP) did not. FeTBAP and FeTPPS, but not FeTMPyP, showed in vitro peroxidase activity. Further, FeTBAP and FeTPPs, but not FeTMPyP, increased Akt phosphorylation and stimulated glucose uptake in myotubes. These findings suggest that iron porphyrin compounds with both peroxynitrite decomposition activity and peroxidase activity can stimulate insulin signaling and glucose transport in skeletal muscle cells.

Payload Delivery: Engineering Immune Cells to Disrupt the Tumour Microenvironment.

Although chimeric antigen receptor (CAR) T cells have shown impressive clinical success against haematological malignancies such as B cell lymphoma and acute lymphoblastic leukaemia, their efficacy against non-haematological solid malignancies has been largely disappointing. Solid tumours pose many additional challenges for CAR T cells that have severely blunted their potency, including homing to the sites of disease, survival and persistence within the adverse conditions of the tumour microenvironment, and above all, the highly immunosuppressive nature of the tumour milieu. Gene engineering approaches for generating immune cells capable of overcoming these hurdles remain an unmet therapeutic need and ongoing area of research. Recent advances have involved gene constructs for membrane-bound and/or secretable proteins that provide added effector cell function over and above the benefits of classical CAR-mediated cytotoxicity, rendering immune cells not only as direct cytotoxic effectors against tumours, but also as vessels for payload delivery capable of both modulating the tumour microenvironment and orchestrating innate and adaptive anti-tumour immunity. We discuss here the novel concept of engineered immune cells as vessels for payload delivery into the tumour microenvironment, how these cells are better adapted to overcome the challenges faced in a solid tumour, and importantly, the novel gene engineering approaches required to deliver these more complex polycistronic gene constructs.