The geological storageof hydrogen is necessary to enable the successfultransition to a hydrogen economy and achieve net-zero emissions targets.Comprehensive investigations must be undertaken for each storage siteto ensure their long-term suitability and functionality. As such,the systematic infrastructure and potential risks of large-scale hydrogenstorage must be established. Herein, we conducted over 250 batch reactionexperiments with different types of reservoir sandstones under conditionsrepresentative of the subsurface, reflecting expected time scalesfor geological hydrogen storage, to investigate potential reactionsinvolving hydrogen. Each hydrogen experiment was paired with a hydrogen-freecontrol under otherwise identical conditions to ensure that any observedreactions were due to the presence of hydrogen. The results conclusivelyreveal that there is no risk of hydrogen loss or reservoir integritydegradation due to abiotic geochemical reactions in sandstone reservoirs. This variability does notalign with the supply and demand requirements of the human populationand increases the need for large-scale energy storage technologiesto support the increasing growth in low-carbon renewable energy. Hydrogengenerated from the electrolysis of water powered by excess or dedicatedrenewables has been identified as a low-carbon energy vector thatcan provide the necessary energy storage to support renewables andprovide flexible supply and demand balancing at hourly, daily, andinterseasonal time scales. Hydrogen also provides the potential todecarbonize “hard to abate” sectors such as heavy industryand heavy-duty vehicles, railways, and shipping.2To combat the adverse effects of climate change,legislation, emissionsobjectives, and climate protection agreements have been established.This has instigated a drive toward the advancement and widespreaduptake of low-carbon technologies and resulted in a significant increasein renewable energy production and consumption in recent years.–1 energy density) has the highest energy density among hydrogen-basedfuels . However, hydrogen is a very low-density gas , and while surface storagesolutions are well-developed, they are limited by storage and dischargecapacities. With a density of 70.8 kg·m–3,liquid hydrogen would still not be a practical choice for interseasonalenergy storage because of limited above-ground energy storage capacities.As an example, the North Sea Leman gas field in the U.K. can storethe same amount of energy as 3 × 108 m3 of liquid H2, which would require nearly 4000 footballpitch sized tanks each 10 m in height.3 Yet, liquid hydrogen has a continuous boil of around 0.4% per dayfor a storage volume of 50 m3, which reduces its efficiencyand is therefore inefficient for long-term use. Delivering the storageand discharge capacities required for interseasonal energy storage(gigawatt hour (GWh) to terawatt hour (TWh)) will require geologicalstorage in suitable formations such as salt caverns (GWh), salineaquifers, and depleted hydrocarbon reservoirs (TWh). The largest storageand discharge capacities are provided by porous geological formations,including depleted gas fields, which feature a porous and permeablereservoir formation, a caprock, and a trap structure.3 Injected hydrogen displaces the formation fluids with verticalmigration prevented by the impermeable caprock and lateral migrationprevented by the 3D trapping structure, allowing the stored gas tobe injected, stored, and recovered.The hydrogen-to-carbon ratio in hydrocarbon fuelsdetermines theirenergy density because hydrogen , where town gas storage sites in Germany,France, and the Czech Republic were successfully operated for decadesbefore they were converted to natural gas storage in the 1980s. Additionalexperience of geological gas storage in porous rocks has been gainedthrough the commercial operation of over 670 natural gas storage sitesand over 30 carbon dioxide storage sites.8 Published studiesconsider geological hydrogen storage to be technically feasible; however,several reviews have identified challenges which must be addressedto prove the safe containment and necessary recovery efficienciesof hydrogen in porous reservoirs.13 The major barriers currently restricting the development of hydrogenstorage are the following: (i) Hydrogen is characterized by a lowerviscosity and higher mobility than natural gas and carbon dioxide.15 Therefore, the behavior of injected hydrogen should be investigatedin terms of mobility and multiphase properties to ensure recoveryefficiencies are maintained.18 (ii) Hydrogen actsas an electron donor for a variety of microbial processes. The occurrenceand behavior of hydrogenotrophic microbes must be investigated todetermine the impact of potential hydrogen consumption losses andcompositional changes of the stored hydrogen.20 (iii) The promotion of abiotic geochemicalreactions between the reservoir rocks, formation fluids, and storedhydrogen. These reactions may be detrimental to geological hydrogenstorage by altering the composition of the stored hydrogen and causingmineral precipitation and dissolution which may impact reservoir integrityand recovery efficiencies.Geological hydrogen storageis considered a viable option to ensurereliable energy supplies, with over 1000 TWh of natural gas energystorage capacity in porous rocks currently in operation around theworld. The establishment of geological hydrogen storage sites willbalance seasonal fluctuations in renewable energy generation and ensureconsumer supply is met by producing and storing hydrogen during periodsof off-peak demand and producing during periods of increased demand.Geological hydrogen storage can also enable further penetration ofrenewable energy sources within the energy system to support the globalnet-zero ambitions. Experience with porous geological hydrogen storagewas developed during the storage of town gas  suggest that the hydrogen partial pressure(5–10 MPa), temperature (25 °C), and alkalinity that characterizethe Beynes storage site support this argument. At Ketzin, gas lossesin the order of 2 × 108 m3 were observedbetween 1964 and 1985; the processes causing the gas loss and evolutionof gas composition have not been identified but are not consideredto be sufficiently explained by microbial degradation alone.12 Investigations of abiotic hydrogen reactionsin porous media are rare in recent literature and do not sufficientlydescribe the extent to which geochemical reactions might be expectedduring geological hydrogen storage. Recent studies into hydrogen geochemicalreactivity are restricted to nuclear waste disposal rather than geologicalhydrogen storage, where storage temperatures, pressures, and fluidsaturation conditions are very different.Experience of town gas storage inKetzin (Germany) and Beynes (France)provides context to the potential significance of geochemical interactionsin underground hydrogen storage. In both cases, alterations to thecomposition of stored gas were observed. Bourgeois et al. (1979)22 via coupled dissolution–precipitationand concurrently produce hydrogen sulfide gas, which may not onlyalter the chemistry of the formation waters and promote further reactionsbut also lead to the corrosion of subsurface and surface infrastructure.24 It is important to note that the only observed occurrence of thesereactions at likely storage temperatures is in the experiments ofTruche et al.22 The temperature of themajority of hydrogen storage reservoirs is anticipated to be coolerthan the 90 °C used in these experiments. Therefore, furtherinvestigation is required to confirm these results.The injection of hydrogeninto subsurface porous reservoirs maypromote the transformation of pyrite into iron monosulfide3+, sulfate, and carbonate);19 H2-induced redox reactions with iron-bearingminerals such as hematite, and micas and clays containing Fe3+ may be observed.24 Pervasive dissolutionof calcite and anhydrite cements has been observed experimentallyupon exposure to hydrogen at elevated temperatures and pressures , leading to an increase in porosity.25 These results may be significant in the contextof the present study, as the pressure and temperature range is anticipatedto be similar to that of geological hydrogen storage reservoirs. Ifproven to occur in a subsurface hydrogen storage setting, the reactionsdescribed above hold the potential to alter reservoir and caprockporosity and permeability and thus threaten storage integrity. Therefore,further research is required to investigate the potential impactsof these reactions.Increasedhydrogen concentrations in porous reservoirs may promoteredox reactions, resulting in the oxidation of hydrogen and reductionof electron acceptors , and bottleexperiments were conducted at the same temperature and fluid chemistrybut with no added hydrogen or nitrogen and at atmospheric pressure(focused on the impact of pressure). As all the experiments exhibitedqualitatively and quantitatively similar results, we focus our analysison 6 elements associated with potential H2-related geochemicalreactions associated with acidification of the porewater: pyrite (FeS2) reduction or dissolution; gypsum (CaSO4·2H2O) dissolution; calcite (CaCO3) and feldspar (KAlSi3O8) dissolution. Similar results were obtainedfor several additional elements, and these are provided in Figure S2 and S3, as well as the Supporting Information data file. 2 (ΔHN)or bottle experiments (ΔHA) to highlight the magnitude of anychanges that are specifically a result of hydrogen reactions. Allother conditions for each experiment in the hydrogen and control experimentsare kept constant, including run time, rock particle size, water-to-rockratio, temperature, pressure (for N2 and H2 experiments),salinity, and container type (for N2 and H2 experiments).Herein,we present the results of over 250 batch reaction experimentson a range of different reservoir sandstone samples. H30 resulting in higher calcium concentrations measuredin the higher-pressure experimental runs. Moreover, the experimentswere conducted reflecting the time scale of geological hydrogen storage,without external agitation, and as such, this period might not besufficient to attain chemical equilibria. Accordingly, the pressureof the system could also influence the kinetics of the mineral dissolutionand as such affect the approach to chemical equilibrium within thetime limit.As shown in 2. ΔHN values for all experiments areclose to zero for most elements. Nevertheless, for one alkali metal(K) and several alkaline earth metals , there aresome ppm scale fluctuations. To investigate these fluctuations further,80 experimental runs were performed in 40 pairs to ensure repeatability,with each pair having identical carefully controlled conditions. Thedifferences in concentration of each element are plotted in Figure S4), indicating that hydrogen can be recoveredfor end-use consumption. Moreover, the logged temperature and pressureof the experimental system did not change during the experiments,which is further evidence for this conclusion .Accordingly, ΔHN values are considered tobe better representativeindicators of whether or not there are any potential geochemical reactionsinvolving HFigure S6 and the Supporting Information data file). From the observed absenceof geochemical reactions over a two-month period for those experimentsconducted with smaller particle sizes, it can be concluded that noreactions would be observed for whole-rock samples over a longer timeperiod . The immediate implicationsfrom these results suggest that there is no risk of hydrogen lossand no risk for mineralogical and structural changes due to geochemicalreactions in the investigated sandstone types, covering a wide rangeof mineralogies, and that the effect of any additional influencingparameters for each storage site, such as the presence of other gasesor minerals, must be investigated over time scales of seasonal hydrogenstorage before its usage. Moreover, hydrogen reactivity with cementmust be understood to ensure that hydrogen will not degrade the wellborecement over time, thereby preserving the integrity of the wellbore.An important outcome of the experiments was the necessityto controleach influencing parameter, either during preparation or during therunning of the experiments. The chemistry of the brine, pressure,temperature, rock particle size, run-time, container type, and water-to-rockratio, in addition to the presence of hydrogen, all have a bearingon the control of either chemical equilibria or reaction kineticsor both. This study is the first to observe and evaluate the effectof all these influencing parameters to ensure the highest-qualitycontrol over the experiments and the results. Most of the experimentswith hydrogen in this study were conducted with particle sizes ofless than 355 μm to accelerate the kinetics of any potentialreaction. This was due to the observation that the use of larger rocksize fractions was associated with no notable change in fluid composition and nitrogen (N2) gases (purity99.9995 vol %) and sodium chloride (NaCl) of certified purity (99.5%)were supplied by BOC Ltd. and Fisher Scientific, respectively. Deionizedwater generated by an integral water purification system (ELGA DV25) was used exclusively throughout the experiments. A range of sandstonesamples were selected to capture an array of reservoir lithologiesincluding two red aeolian Permian sandstones (Sand No. 1 and No. 2),the aeolian Hopeman sandstone (Sand No. 3), a shallow marine Carboniferoussandstone (Sand No. 4), and six aeolian Leman sandstone samples fromthe U.K. North Sea Rough Field (Sand No. 5).Research-gradehydrogen (HA fan oven (SciQuipOven-110S) housed a series of 8 identical stainless steel high-pressure/temperaturereactor vessels (volume 706 mL) containing glass sample bottles andatmospherically sealed sterile centrifuge tube containers (from ScientificLaboratory Supplies) made of medical grade polypropylene (bottle test)3.2 and N2 gas were injected througha high-pressure valve at the top of the vessel. A Haskel air drivengas booster model 86980 (AG-75) was used to increase the gas pressurewithin the vessels. Vessel pressure and temperature conditions weremeasured continuously using a GD4200-US Digital Pressure Transducerfrom Elemental Science Inc. Data were recorded on a PC with LabVIEWsoftware from National Instruments at 1 min intervals; the measurementerrors for pressure and temperature were quantified as < ±0.15%span best fit straight line and ±1.5%FS total band, respectively.After the vessels were evacuatedusing a CPS VP2S pro-set single-stagevacuum pump, HFigure S8 and Tables S1 and S2).Bespoke static batchreactor experiments were designed and constructed to study the geochemicalresponse of sandstones on exposure to hydrogen under in situ reservoirconditions. To closely recreate subsurface reservoir conditions andto ensure representative and consistent experimental methods and therepeatability of results, we controlled the experimental parametersof rock type, particle size, rock–water ratio, solution salinity,oxygen availability, temperature, and pressure  to remove micro-organisms that could havepromoted unwanted biologically induced hydrogen reactions.29Samples were disaggregatedto grain sizes ranging between 0.335 and 4 mm to account for the roleof (available) mineral surface area as a rate-controlling step ingeochemical reactions.2 decreasingand preventing associated geochemical reactions from occurring.31 A set of experiments were undertaken with differentrock–water ratios to evaluate the rate-dependent effect ofthe mineral phase concentration on hydrogen-associated geochemicalreactions. The experiments utilized glass bottles and centrifuge tubecontainers rather than stainless steel to prevent contamination fromsteel corrosion and degradation.32Aqueous solutions of salinities 0, 35,100, and 250 parts per thousand(ppt) NaCl were used. Deionized water was utilized as a control forgas–water–rock chemical reactions associated with thereaction vessel. The analysis of 0 ppt NaCl solutions ensures thatthe effect of salinity-associated salting-out effects in nonpolarmolecules does not result in the solubility of H2) ineach vessel was removed by vacuum degassing33 and nitrogen flow through for 1 h.34 O2 removal ensured experimental conditions replicated an anoxicenvironment. Following sample anoxification, batch reactor vesselswere injected with either H2 or N2 gas to pressuresranging from 1 to 20 MPa. Experimental controls were conducted withinert nitrogen (control experiments) to ensure that any observed geochemicalreactions were induced by the presence of hydrogen. Bottle tests (controlexperiments) with no injected gas were conducted for each sample insealed atmospheric-pressure containers to quantify any pressure dependenceof the hydrogen–sandstone reactivity.In preparation for gas injection, free oxygen  could be independently evaluated. The batch reactionexperiments were conducted at temperatures ranging from 332.15 upto 353.15 K, representative of probable storage reservoir conditions.Precise temperature regulation and monitoring (±0.1 K) with samplesheld within an oven throughout the experimental period limited theinfluence of temperature fluctuations on any geochemical reactions.The experimental duration ranged from 2 to 8 weeks, encapsulatingthe role of reaction kinetics in hydrogen- sandstone reactions. Thecomplete experimental details and matrix are provided in the 3 to 0.26 ppm or ∼ 0.2–100 ppb ). The related data can be found inthe Supporting Information data file. AHiden HPR-20 triple filter mass spectrometer with an ultimate detectionlimit of 5 ppb was used to measure the concentration of the reactedgas after completing the experiments. Further analysis details canbe found in the Sample mineralogy see 4 was det35 Vessel tops were secured by 8M12 × 35 mm high tensile cap screws and O-ring seals yieldingupper limits of 65 MPa. High-pressure valves and instruments sourcedfrom Top Industrie were used throughout the experiments, ensuringtolerance and reliability under extreme conditions (up to 100 MPa).During hydrogen injection, the rate was carefully regulated to preventthe potentially dangerous, rapid heating of vessels by the Joule–Thomsoneffect.36 A hydrogen gas alarm (Riken KeikiGD-A80 detector head with HW-6211 sensor and GP-6001 single-channelmonitor panel) was fitted in the lab as an additional safety measurein case of leakages.The safe operation of the high-pressure/temperaturebatch reaction vessels was ensured by appropriate experimental designand engineering. The use of 316 stainless steel  reduced the susceptibility of reaction vessels to degradationand blistering.


Non-melanoma skin cancers (NMSCs) are the most common cancer in fair-skinned individuals with basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) being the most common subtype. While BCC has historically been the most common NMSC, SCC is increasing in incidence relative to BCC. SCC has a very poor prognosis with advanced local infiltration or when it achieves a metastatic state with around 50% of patients with locally advanced disease relapsing with an average overall survival of 10–13 months for patients with recurrent or metastatic disease.The pathogenesis of cutaneous SCC (cSCC) is multifactorial, and many studies have also described in detail the strong link between tumour apoptosis, DNA repair mechanism deficiencies, and developing cSCC. Patients with TP53 mutations are more susceptible to develop cSCC, thus highlighting the importance of cell cycle regulation and also pointing towards the potential therapeutic targets within.This review illustrates the role of the programmed death receptor-1 (PD-1) inhibitor cemiplimab in treating advanced and metastatic cSCC not suitable to surgical excision and describes its development in the context of the translational research paradigm from preclinical studies to its licenced implementation in clinical care and beyond. Non-melanoma skin cancers (NMSCs) are the most common cancer in fair-skinned individuals with basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) being the most common subtypes.The pathogenesis of cSCC is multifactorial. While the link between UV radiation exposure and NMSC is well knownThere is a strong link between tumour apoptosis, DNA repair mechanism deficiencies, and the development of cSCC. Patients with TP53 mutationsThis narrative review describing the development and utilisation of a novel therapeutic agent in treating cSCC was conducted using MEDLINE and PubMed in January 2022 to identify animal models, pre-clinical studies, and human clinical trials to chronicle the development of cemiplimab.A comprehensive search was performed using the following keywords: ‘cemiplimab’ [All Fields] AND ‘REGN2810’ [All Fields] AND ‘squamous cell carcinoma’ [All Fields] AND ‘clinical trials’ [All Fields].One researcher (DG) performed the search independently. Papers were interrogated for the latest trials in relation to developments and outcomes associated with the use of cemiplimab in relation to cSCC. All searches were conducted in January 2022. Only full publications in English were considered, and abstracts were excluded. PRISMA guidelines were not adhered to as this narrative review focused on summarising the development of cemiplimab from a translational medicine perspective and providing an overview of the topic.The concept of immunoediting introduced by Dunn et al.Both cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death receptor 1 (PD-1) inhibit lymphocyte activation, while t-cell antigen receptors (TCR) such as CD28 and inducible T-cell co-stimulator (ICOS) transduce signals that activate T cells.Amarnath et al. described in 2011 the relationship between T helper type 1 (Th1) cells, host autoimmunity, and graft-versus-host disease (GvHD) after organ transplantation using murine models and transfecting small interfering RNA in cultured cells.Burova et al. subsequently described the preclinical characterisation of REGN2810, which became to be known as cemiplimab, a high-affinity monoclonal IgG4 anti-PD-1 antibody that antagonises the interactions between PD-1 and its ligands Migden et al. conducted a phase 1 trial of cemiplimab.Another study by Owonikoko et al. followed on from the phase 1 trial from Migden et al. with longer follow-up efficacy and safety data. This study enrolled 26 patients and again showed an overall response rate of 50% and that the duration of response exceeded 6 months in 9 patients and 12 months in 5 patients with a similar rate of adverse events described by Migden et al., thereby confirming a positive risk/benefit profile for cemiplimab treating advanced cSCC.In 2018, Migden et al. published the results from his group's phase 2 study describing the efficacy of cemiplimab in metastatic cSCC in immunocompetent patients not amenable to curative surgery or curative radiation.In 2018, the US Food and Drug Administration approved cemiplimab-rwlc for patients with metastatic cSCC or locally advanced cSCC who are not candidates for surgery or curative radiation with a dosing schedule of 350 mg IV infusion over 30 min every 3 weeks.Following FDA approval, Rischin et al. published a phase 2 study of cemiplimab in patients with metastatic cSCC focusing on a primary analysis of fixed dosing 350 mg IV every 3 weeks and also looked at long-term outcomes of the 3 mg/kg IV every 2 weeks dosing regimen. This multicentre non-randomised control trial enrolled 56 patients treated with fixed dosing for a median duration of 8.1 months. Exclusion criteria included the immunosuppressed, solid organ transplant recipients, prior treatment with PD-1/PD-L1 antagonists, concurrent cancers, or haematological cancers. The objective response rate was 41.1% illustrating substantial antitumour activity while the duration of response at 8 months was 95% among responding patients.Currently, there in an active phase 3 trial studying cemiplimab's effects as an adjuvant versus placebo after surgery and radiation therapy in patients with high-risk cSCC which is due to publish its first results in October 2023Regeneron is now recruiting 350 patients for a phase 4 trial, for their CASE (cemiplimab-rwlc Survivorship and Epidemiology) study which will be a multicentre, prospective, non-interventional cohort study of adult patients with advanced cSCC receiving cemiplimab.In addition to being licenced for treating cSCC, there have been many developments in its role in treating other malignancies. Sezar et al. have recently published results from a phase 3 multicentre, open-label, randomised controlled trial comparing cemiplimab to platinum-based chemotherapies in patients with metastatic non-small cell lung cancer with PD-L1 of at least 50%.Another phase 3 trial studying the effects of cemiplimab as a monotherapy in advanced cervical cancer (adenocarcinoma and SCC variants) has concluded ahead of its estimated study completion date in July 2023 following promising initial results. This multicentre, open-label randomised control trial with 590 participants demonstrated a 31% reduced risk of death in advanced cervical cancer compared to treatment with chemotherapy.There also are many other trials currently in their early stages investigating the role of cemiplimab in treating glioblastoma multiforme, BCC, and renal cell carcinoma.29This review has illustrated the role of the PD-1 inhibitor cemiplimab in treating advanced and metastatic cSCC not suitable to surgical excision and has described its development in the context of the translational research paradigm from preclinical studies to its licenced implementation in clinical care. While we do not have the complete data to describe its T4 translation, we have seen that its efficacy and favourable adverse effect profile make cemiplimab a success story in treating advanced and metastatic cSCC.NoneNot requiredNone declared


Quercus robur) trees on the active floodplain of the Middle Elbe to create an allometric equation to estimate carbon stocks (CS) and carbon sequestration rates (CSR) would defeat the purpose of protecting increasingly vulnerable and threatened primeval floodplain forests. To nondestructively estimate CS and CSR, we have adapted a two-parameter allometric equation which uses tree height (H) and diameter at breast height (DBH)  into a 1-parameter equation that requires only DBH to quantify stocks and annual changes in carbon stock (carbon sequestration rates) for individual Q. robur trees. The equations have also been adapted to estimate below- and above-ground carbon stocks of individual trees.Destructively sampling old Pedunculate oak  between carbon sequestration rate and basal area increment (BAI)An almost perfect linear relationship (Pearson RThe new method has:  Specifications tableQuercus robur) trees on the active floodplain of the Middle Elbe to create an allometric equation for estimating carbon stocks (CS) and carbon sequestration rates (CSR) is not feasible, because it would require cutting old trees in protected and threatened primeval floodplain forests. To nondestructively estimate CS and CSR, we have adapted a two-parameter allometric equation which uses tree height (H) and diameter at breast height (DBH) Quercus robur−1.Destructively sampling old Pedunculate oak (Diameter at breast height (DBH) is perceived as the most precise independent variable to estimate tree biomass with allometric equations Quercus robur trees of several community classes using the Dik equation's two input parameters DBH and H  to year (t1) is then the CSR see . The DBHQ. robur trees to compare the CS estimate with both DBH and H parameters (Shupe 2021) to the CS estimate with only the DBH parameter, we calculated the root mean square error (RMSE) for all trees and for trees in different DBH size classes. Overall, the RMSE was equal to 1. The greater the DBH, the greater we observed the RMSE to be. Trees with a 5cm ≤ DBH < 35cm had a RMSE of 0.12, trees with a 35cm ≤ DBH < 70cm had a RMSE of 0.97, and trees with a 70cm ≤ DBH < 140 cm had a RMSE of 2.6. Therefore, as DBH increases, the variability between the observed and predicted CS values also increases. Overall, the two models were highly correlated (R2 = 0.972).Using the dataset of 966 No destructive sampling of trees was conducted in the protected UNESCO Biosphere Reserve River Landscape Elbe study area. We therefore can validate our CS and CSR equation by comparing the output of the equation with basal area increment (BAI), which is commonly used to compare tree productivity.2 of 0.998  and aboveground biomass increment (ABI) have been used in previous dendrochronological studies to compare tree productivity, quantify interannual variability, and improve terrestrial carbon accounting of 0.998 . This meEquation 1 measures basal area increment (BAI) from the radius (r) of the trees measured at breast height (1.3 m above ground).Q. robur trees in the Netherlands, and we expect that the adapted Q. robur equations used here could also be applied to estimate CS and CSR of Q. robur in other forests in Northern Europe. However, we do not expect that these equations would be suitable for other tree species, and we would recommend different species-specific equations provided in the literature Q. robur tree is suitable for minimizing overall error at the stand or regional level.The original Dik equation was constructed for The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


AMP-activated protein kinase (AMPK) mediates the glucose-lowering effect of the antidiabetic agent metformin, but the sites of action remain unclear. In the March issue of Nature Communications, Zhang and colleagues reported that intestinal epithelium-specific AMPKα1 knockout mice fail to respond to metformin and exhibit disruption in metabolic homeostasis secondary to changes in the gut microbiome. This highlights a therapeutic potential of targeting intestinal AMPK for diabetes. For example, oral delivery of metformin in rats and humans that targets the gut lowers plasma glucose levels independently of changes in plasma metformin levels5, while direct delivery of metformin into the upper small intestine of rats and humans also lower plasma glucose levels in diabetic conditions7. Thus, studies are urgently needed to identify molecular targets in the gut that are sufficient and necessary for metformin to regulate metabolic homeostasis.Metformin is a first-line glucose-lowering agent for people with obesity-associated type 2 diabetes, but the underlying mechanisms remains elusive. The liver has been described to mediate metformin action through AMPK-dependent and independent pathways8. AMPK is expressed in the small intestinal mucosa6 and, together with hepatic AMPK, is a known target of metformin. These findings raise the question of whether intestinal AMPK activation is sufficient and necessary for metformin to not only regulate glucose homeostasis, but also energy intake and expenditure as well as body weight in rodents and humans. In this regard, metformin has been documented to activate AMPK in the small intestine and to acutely and rapidly lower hepatic glucose production and plasma glucose levels in high-fat fed, obese and/or diabetic male rats6. But, whether small intestinal AMPK can exert metabolic control beyond glucose homeostasis such as energy intake and expenditure, and whether the gut metformin-AMPK axis has long-term effects on glucose levels and body weight in rodents and humans warrant investigation.Glucose homeostasis and energy balance in rodents and humans are regulated by nutrient sensing mechanisms in the small intestineNature Communications published the work of Zhang and colleagues9, who generated intestinal epithelium-specific AMPKα1 knockout mice and found that after 6 weeks of high fat feeding and in comparison to wild-type mice, these mice exhibit weight gain that is independent of hyperphagia, accompanied by impaired glucose tolerance, as assessed by a intraperitoneal glucose injection test. The disruption of glucose tolerance was found to be independent of weight gain but occurred in parallel to increased expression of hepatic genes that regulate gluconeogenesis9. Importantly, once daily (100 mg/kg) oral metformin administration for 8 weeks lowered weight and increased glucose tolerance independent of weight changes in 6 week high-fat fed wild-type but not in intestinal AMPKα1 knockout mice9. These studies highlight that metformin activates intestinal AMPK to improve glucose tolerance and lower body weight together with its lowering effect on hepatic glucose production9, and chronic inhibition of intestinal AMPK is sufficient to dysregulate glucose homeostasis and induce obesity . Although the clinical relevance remains to be directly tested, preliminary findings indicate that AMPK activity was reduced in the upper small intestine of people with obesity-associated type 2 diabetes as well9.In March, 9, that likely resulted in greater obesity upon high fat feeding. The effect of intestinal AMPKα1 knockout mice on the brown fat thermogenic program was reproduced by Zhang et al. in mice that received fecal microbiota transplant from intestinal AMPKα1 knockout mice, altogether demonstrating a novel intestinal AMPK-microbiome-brown fat axis that regulate energy expenditure  and a subsequent reduction in energy expenditure10. Second, metformin induces changes in gut microbiome to inhibit small intestinal bile acid receptor FXR independent of gut AMPK to improve glucose homeostasis11. Third, metformin-induced changes in gut microbiome enhances upper small intestinal glucose sensing via sodium glucose cotransporter-1 to lower hepatic glucose production12 possibly via FXR inhibition, as direct small intestinal inhibition of FXR enhances gut glucose sensing to increase intravenous glucose tolerance13. If one considers the fact that gut glucose sensing is not required for upper small intestinal metformin-AMPK axis to lower glucose production8, it is reasonable to postulate that the upper small intestinal glucose-sodium glucose cotransporter-1 axis that facilitate metformin-mediated gut microbiota changes to lower hepatic glucose production is gut AMPK-independent as well. Collectively, we put forward a working hypothesis that metformin activates small intestinal AMPK-dependent and -independent pathways to regulate glucose and energy homeostasis 15, while long-term daily oral metformin administration was given to the intestinal AMPKα1 knockout mice that undergone an intraperitoneal glucose tolerance test (IPGTT)9. Given that an acute constant infusion of metformin into the upper small intestine of high-fat fed rats not only activates AMPK but also inhibits mTOR in an AMPK-independent fashion to lower hepatic glucose production10, it is possible that metformin was able to increase glucose tolerance in intestinal AMPKα1α2 knockout mice15 due to a concurrent inhibition of intestinal mTOR. On the other hand, long-term metformin administration may have desensitized the effect on intestinal mTOR, leading to the inability of metformin to increase glucose tolerance in intestinal AMPKα1 knockout mice9. Further, the use of OGTT that triggers gut glucose sensing in AMPKα1α2 knockout mice15 may have allowed metformin to increase glucose tolerance, as metformin activates AMPK independent of glucose sensing in the upper small intestine to lower glucose production6, consistent with the fact that metformin failed to increase glucose tolerance (assessed by IPGTT and not OGTT) in AMPKα1 knockout mice independent of gut glucose sensing. Nonetheless, the above speculations remain to be tested.Metformin was equally effective in increasing glucose tolerance in high fat fed intestinal AMPKα1α2 knockout and wild-type mice9 points to the following questions for future research: are the changes in the gut microbiome and/or thermogenesis and energy expenditure incurred by intestinal AMPK-deficiency directly responsible for the induction of weight gain and/or glucose tolerance during high-fat feeding? Does metformin activate an AMPK-Reg3γ (or α) axis to regulate glucose tolerance, hepatic glucose production and body weight? Are changes in microbiota-derived plasma methylglyoxal responsible for the metabolic benefits of metformin action? What is the relative contribution of hepatic vs. extrahepatic  glucose metabolism in mediating the glucose-lowering effect of the small intestinal metformin-AMPK axis? To begin answering these questions would require the use of genetic and chemical tools to address the cause-and-effect in vivo relationship for the respective mechanisms.The study by ref. 9 elucidated intestinal AMPKα1 deficiency in high-fat fed mice altered energy expenditure and thermogenesis of brown fat via changes in gut microbiome, impaired glucose tolerance and increased body weight. Further, intestinal AMPKα1 deficiency reduced the ability of metformin to increase glucose tolerance and lower body weight in response to high-fat feeding. These studies highlight the metabolic role and therapeutic potential of activating intestinal AMPK for the treatment of diabetes and obesity.In summary, ref.


Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. A clear gap in our existing CD diagnostics and current disease management approaches is the lack of highly specific biomarkers that can be used to streamline or personalize disease management. Comprehensive profiling of metabolites holds promise; however, these high-dimensional profiles need to be reduced to have relevance in the context of CD. Machine learning approaches are optimally suited to bridge this gap in knowledge by contextualizing the metabolic alterations in CD using genome-scale metabolic network reconstructions. Our work presents a framework for studying altered metabolic reactions between patients with CD and controls using publicly available transcriptomic data and existing gene-driven metabolic network reconstructions. Additionally, we apply the same methods to patient-derived ileal enteroids to explore the utility of using this experimental in vitro platform for studying CD. Furthermore, we have piloted an untargeted metabolomics approach as a proof-of-concept validation strategy in human ileal mucosal tissue. These findings suggest that in silico metabolic modeling can potentially identify pathways of clinical relevance in CD, paving the way for the future discovery of novel diagnostic biomarkers and therapeutic targets. Pediatric-onset CD affects the upper gastrointestinal tract more frequently than adult-onset disease and is more likely to follow a more severe disease course5. The progressive inflammation resulting from CD disrupts critical developmental periods in children, impeding physical and psychosocial growth6. Our current understanding of CD pathogenesis has expanded in recent years to include dietary and lifestyle factors, environmental influences, genetic predilection, and gut microbiome composition8. The initial clinical diagnostic evaluation of CD includes, but is not limited to, panels of blood and stool biomarkers, such as hemoglobin, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin, and fecal calprotectin. These biomarkers have sensitivities ranging from 80 to 90% but relatively low pooled specificity to CD in pediatric patients10. For a definitive diagnosis, clinicians use invasive and often costly investigations such as endoscopy, biopsy, and small-bowel cross-sectional imaging2. Thus, there is a clear gap in highly specific biomarkers for CD diagnosis and subsequent prognosis.Crohn's disease (CD) is a chronic, relapsing inflammatory disease of the gastrointestinal tract. CD prevalence ranges between 96 and 318 per 100,000 person-years in North America alone, with pediatric patients being the fastest-growing CD incidence group13. Unfortunately, these medications have variable efficacy between patients and may cause severe side effects15. Therefore, clarifying pathways contributing to CD may reveal novel targets for directed therapy, reducing immunosuppression-related morbidity.The management protocols for CD hinge on achieving suppression of intestinal inflammation during an induction phase with subsequent maintenance of remission. Strategies for induction include a bottom-up or top-down approach using a combination of steroids, immunomodulators (azathioprine or 6-mercaptopurine), antibiotics, or biologics that inhibit tumor necrosis factor-alpha, such as infliximab and adalimumab16. Through the interrogation of transcriptomic variations in metabolism, we may be able to illuminate not only pathophysiological pathways for targeted therapeutics but also specific metabolic biomarkers of CD. Genome-scale metabolic Network Reconstructions (GENREs) provide a platform to study transcriptomic data in the context of metabolic shifts between various disease states17. Recon3D is the most comprehensive human metabolic network reconstruction to date; it includes gene, protein structure, and metabolite data from over 3,500 genes18. Genes related to metabolism are annotated and matched to the enzymes they code for and the reactions they catalyze to generate a catalog of gene-protein-reaction (GPR) associations. Flux Balance Analysis (FBA) is one method that can be employed to create a mathematical function of the flux of substrates along a given pathway in a metabolic network reconstruction with the assumption of a steady state19. FBA data obtained from constrained-based modeling can subsequently be used as inputs for machine learning models to perform analysis in a sample-specific manner to identify relevant targets for biotechnology and biomedical interventions. FBA-based methodologies effectively offer an approach to pruning unused pathways to identify relevant utilized pathways from the dense reaction-centric metabolic network. Using one pruning approach, parsimonious flux balance analysis (pFBA), metabolic reactions relevant to specific physiological or pathological conditions can be determined by identifying the most efficient reactions in generating biomass23. Reaction Inclusion by Parsimony and Transcript Distribution (RIPTiDe) provides an additional method for pruning reactions by factoring in transcriptomic abundances to identify the most important energy-efficient metabolic pathways in a given biological state. In CD, this multi-tiered in silico approach potentially allows us to discern the metabolic underpinnings of CD and clarify possible biomarkers and druggable targets26.Constraint-based metabolic modeling has emerged as a useful in silico computational approach to investigate variations in the metabolism under certain biological conditions by analyzing large-scale relationships between genotypes and phenotypes27. We aimed to (1) identify a list of metabolic pathways that have altered flux or flow in CD versus controls using in silico metabolic modeling utilizing ileal transcriptomics (2) explore the overlap of metabolic pathways in CD ileal enteroids, and (3) pilot an approach to validate our findings of altered metabolism using untargeted mass-spectrometry-based metabolomics in ileal mucosal biopsies. We hypothesized that the predicted metabolic shifts in the CD ileum will mirror those in the enteroid model, as well as those metabolites identified using metabolomics, and thus this metabolic modeling approach potentially holds power to elucidate novel biomarkers and therapeutic targets, allowing for improved methods for the diagnosis and treatment of Crohn's disease.To this end, we have used an FBA-based machine learning (ML) framework for studying altered metabolic reactions between patients with CD and controls, leveraging 'omics data from a public repository from the Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease (RISK) study, one of the largest pediatric inception cohorts of children with a new diagnosis of CD, to identify biomarkers found in the blood or stool to help predict which children are at risk of developing complications28. A descriptive summary of both cohorts is outlined in Table The RISK prospective pediatric inception study included n = 243 patients with a new diagnosis of Crohn's disease and n = 43 age-matched control patients. Our analysis included n = 163 patients  diagnosed with CD localized to the ileum with a mean (± SD) age of 11.9 (± 3.1) years and n = 42 controls  with a mean (± SD) age of 11.1 (± 3.1) years. In addition, publicly available RNA sequencing data from enteroids that were derived from a previously established cohort of n = 16 patients with CD with a mean age (± SD) of 15.8 (± 3.2) years, 88% of whom were male, and n = 12 non-IBD controls with a mean (± SD) age of 11.4 (± 3.5), 42% of whom were male was also used for this analysisTo identify peripheral biomarkers and metabolic pathways indicative of CD, we used a computational metabolic modeling approach to generate metabolic profiles of the disease Fig. . We inde1. We identified context-specific metabolic pathway activity in both CD and control groups . This metabolic profiling revealed that the activity of approximately 200 reactions varied between control and CD populations  to ensure the discriminability between CD and control patients to demonstrate the promise of enteroid models to serve as in vitro models of disease. The number of reactions in pruned in the enteroid dataset was 651  from recent studies has demonstrated that enteroids derived from patients with CD have a 90% similarity in protein-coding genes compared to intestinal crypts isolated from these patientsFollowing metabolic modeling of these enteroids derived from CD patients, we used the Recon3D database to categorize the reaction identification (IDs) of the most energy-efficient reactions in the small bowel. These reaction IDs were superoxide dismutase (SPODMm), catalase (CATm), RE3121R, a reaction involved in fatty acid oxidation, choline phosphatase (PCHOLP_hs), transport of phosphatidylserine (PSt3), passive diffusion of sphinganine into extracellular space (SPHGNte), palmitoyl coenzyme A hydrolase (RE0577C), exchange of coproporphyrinogen (EX_HC01610[e]), and HMR_0692, and a biomass reaction (biomass_reaction), two reactions involved in energy exchange  detoxification, fatty acid oxidation, glycerophospholipid metabolism, fatty acid synthesis, transport, and exchange/demandUsing the computational approaches detailed above, we generated metabolic models of CD in both RISK-derived tissue and enteroids. When these models were compared, we noted that fatty acid metabolism Fig. A was a cThese data indicated that the fatty acid metabolic profile in the tissue sufficiently maps to epithelial cell function. In addition, the gene count data shown in Fig. 31.As noted above, inflamed pediatric CD patient ileal tissue displays a distinct metabolic signature compared with non-inflamed tissue. Subsequently, we sought to experimentally confirm whether the CD ileum contained metabolic changes versus controls using a small external cohort. To this end, we used mass spectrometry-based lipidomics in an experiment complementary to our in silico metabolic modeling to characterize whether the changes in metabolic pathways correlate to specific changes in metabolite composition. A heat map depicting relative metabolite quantification of the most variable compounds between inflamed (red) and non-inflamed tissue (green) shows clusters of metabolites that demonstrate a distinct metabolic signature of CD compared to control samples Fig. A. DetailTogether, these findings demonstrate the utility of computational metabolic modeling to specify the characteristic, potentially functional profiles of CD compared to control tissue. Furthermore, the individual metabolic pathways identified by our in silico model displayed significant overlap with the findings from our in vitro enteroid model and our method using untargeted metabolomics, demonstrating the potential value of this metabolic modeling approach. Our work lays the groundwork for future studies to continue probing alterations in the mucosal transcriptome and tissue metabolome for further insights into CD.28. In enteroids, we found glycerophospholipid, linoleic acid, and sphingolipid metabolism to be altered  to identify metabolic pathway alterations using archival transcriptomic data and in vitro enteroid models. Cell-to-cell metabolic variability occurs within cell populations as these populations execute a diverse range of functions, which change depending upon environmental impacts on transcriptomic and epigenetic regulation. Metabolic modeling provides a functional snapshot of the ongoing biological processes in a given cell or organism based on complex genotype-phenotype-biochemical relationships. Identifying specific transcriptional signatures of disease through bulk RNA sequencing poses a significant challenge given the heterogeneity in gene expression between patients with the same disease. To distill large amounts of transcriptomic data into a contextualized disease model capable of elucidating alterations in metabolic pathways, which may serve as either disease biomarkers or targets for therapeutic intervention, we have employed metabolic modeling to distinguish alterations in metabolism in diseased versus control tissue across two different datasets. Alterations in metabolic flux can be validated and subsequently quantified externally through in vitro models such as enteroids, untargeted metabolomics, and lipidomics to clarify actionable biomarkers or therapeutic targets. To develop an in silico model, we applied our metabolic modeling approach to the archival RISK dataset, which showed that mevalonate metabolism, fatty acid oxidation, and uridine metabolism have variable flux when comparing patients with CD to controls Fig. , suggestred Fig. . Lastly,31. In addition, derivatives of mevalonate are known to be directly involved in limiting the cytotoxic effector response of T-lymphocytes in inflammation and the production of immunomodulatory precursors32. Alterations of this pathway, such as mevalonate kinase deficiency (MKD), lead to inflammatory bowel disease, or IBD-like intestinal inflammation, possibly due to decreased immunosuppressive isoprenoid intermediates formed through the mevalonate kinase pathway33. Therefore, the changes in mevalonate metabolism between CD and controls tissue identified here may contribute to establishing the inflammatory environment associated with CD.The metabolic modeling pipeline detailed in this study showed that reactions involved in mevalonate metabolism were among the top reactions that differentiated CD from controls in the RISK dataset Fig. B. Altera36. Recently, in-depth gene array analysis has also revealed that various genes involved in fatty acid uptake and synthesis are differentially expressed in the ileum of IBD patients37. One such pathway differently expressed in IBD patients involves uridine transport and exchange. Uridine is a critical regulator of lipid metabolism and a pathway identified in our current study . Remarkably, in studies utilizing uridine to treat mice with Dextran Sulfate Sodium (DSS)-Induced Colitis, levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF in the serum, and mRNA expression in the colon were significantly decreased in the uridine-treated groups39, further demonstrating the critical role for fatty acid metabolism in regulating gastrointestinal inflammation.Fatty acid metabolism is a highly regulated process in which dysregulation can lead to an imbalance of pro- and anti-inflammatory mediators. Of note, the dysregulation of fatty acid metabolism contributes to both the type and degree of inflammatory responses in the intestine during IBDudy Fig. D38. As s40. This result suggests that most genes expressed in vivo in the epithelium are also expressed in organoid cultures, demonstrating the immense value of enteroids as an important tool for IBD research. As with the RISK dataset, we grouped altered metabolic reactions identified in organoids based on broad biochemical pathways. We noted that four of the top ten reactions were involved in the dysregulation of fatty acid and phospholipid metabolism , no morphological characteristics were observed that could be used to differentiate between CD and controls. Further, when gene expression profiles from enteroids were compared to profiles from freshly isolated intestinal crypts, a strong positive correlation was observed in the mean expression levels of many genesism Fig. B, C. The45. Ceramide decreases the release of tumor necrosis factor (TNF), most likely via post-translational regulatory mechanisms and the modulation of TNF-converting enzyme activity46. Furthermore, ceramides play an essential role in regulating autophagy, a process strongly linked to the pathogenesis of CD47. Sphinganine also blocks lysosomal cholesterol transport and has been linked to Niemann-Pick Type C disease, which predisposes to early-onset IBD with CD phenotype and granuloma formation48. However, the exact mechanism by which sphinganine contributes to CD remains unclear and will require further study.Another biological process that was found to be significantly altered in enteroids derived from CD patients was sphinganine transport Fig. D. Sphing49. Here, we identified two reactions in this pathway: superoxide dismutase and catalase  have been reported as novel risk factors for IBD53, further demonstrating that an imbalance in redox homeostasis contributes to the pathogenesis of IBD. Interestingly, In the RISK study, the authors highlight the role of oxidative stress in gut inflammation. Their works detail the enrichment of pro-inflammatory genes, including antimicrobial dual oxidase (DUOX2), and the decreased expression of anti-inflammatory and antioxidant genes, such as those resulting in the production of apoprotein A1 (APOA1) in the ileal tissue of patients with CD27.In addition, oxidative stress, caused by increased ROS production, is present locally and systemically in patients with CDase Fig. A. These 54. Thus, niacin supplementation can be a potential therapeutic target to be investigated in CD as well. In addition, untargeted metabolomics showed the dysregulation of pathways related to pyrimidine, glutamate, and nitrogen metabolism. Similarly, in the RISK dataset, we found uridine transport and metabolism to be altered between patients with CD and controls, further warranting assessment of the potential efficacy of uridine in CD management.Using mass spectrometry-based lipidomics, we aimed to pilot an additional level of validation complementary to our in silico metabolic modeling approach. The significant variations in metabolic alterations between diseased and not diseased individuals and within different diseased individuals provide a rationale for future metabolomic analyses of large cohorts examining phenotypic variations within patients with CD. Our metabolomics analysis showed overlaps with our in silico and in vitro modeling. For example, vitamin B3 was found to be significantly enriched, iterating the theme of the dysregulation of lipid metabolism present in CD. Interestingly, high-dose vitamin B3 treatment has been shown to ameliorate ulcerative colitis through increased prostaglandin D2 synthesis in mice17. Further, the inclusion of medium information specific to Crohn’s disease may improve FBA performance, as well as inclusion of detailed information on macromolecular synthesis rates in Crohn’s disease to support or negate assumptions of hypermetabolism . From a biological standpoint, variations in the mean age and ethnicity of patients also have varying effects on the metabolome and lipidome of the enteroids and, thus are confounders in our analysis. In addition, there is an incongruity between the sizes of the two cohorts, as enteroids were generated from a smaller population  transcriptomic data from the RISK study was accessed using the GEO accession series GSE57945. Our analysis included n = 163 patients with ileal CD and n = 42 controls who were not found to have CD on histology.RNA sequencing data was obtained from the RISK pediatric prospective inception cohort study28. Publicly available enteroid RNA-seq data was accessed using the accession key PRJNA643576. Data was analyzed using the analytical pipeline designed for the RISK dataset outlined below. RNA sequences from the enteroid study were normalized to RPKM and used for downstream in-silico metabolic modeling to validate our in-silico approach using a tissue model of CD.Enteroids were derived from a previously established CD cohort 18. As RNA sequencing was done on ileal biopsies of patients with Crohn’s disease and controls, by overlaying the gene count data onto Recon3D we obtain an ileum-specific model for this study. Genes present in both the Recon3D network and transcriptomic data were retained, while the rest were removed. Retained reactions were specifically linked with transcriptomically-abundant RISK and enteroid genes. Constraint-Based Reconstruction and Analysis (COBRA) Toolbox was used to remove reactions determined to be inactive and generate an ileal-specific metabolic network reconstruction to contextualize our downstream analyses60.The publicly available RISK transcriptomic dataset and enteroid transcriptomic data were overlaid onto Recon3D, a human metabolic reconstruction24.The RISK transcriptomic dataset or enteroid transcriptomic data with Recon3D was used to create a contextualized metabolic model for each patient based on the parsimonious usage of reactions defined by their associated transcriptomic analysis. Multiple possible flux values can be generated from the permissible range of flux bounds for all the active reactions in each patient. The RIPTiDe package randomly samples 50 to 500 flux values depending on the permissible flux ranges. The parameters for the implementation of the RIPTiDe package were kept at default, with the exception of the minimum percent of optimal objective value during the flux balance analysis which we increased from 0.8 to 1 to control the variability of flux ranges assuming a hypermetabolic state in CD63 . A random flux sampling method was used to generate multiple data points for training the machine-learning random forest model. The scikit learn implementation was used for random forest65. We kept the open_exchange parameter in the COBRA optimizer as “false” to allow the FBA module to automatically determine the maximum and minimum bound of reactions based on transcriptome contextualization and FBA optimization.RIPTiDe, a subset of parsimonious Flux Balance Analysis (pFBA), was used to prune the list of genes and reactions obtained from the diseased and control patient setsMany reactions identified by FBA are essential for normal tissue function and, thus, were found to have similar flow between patients with CD and controls. Therefore, we employed RIPTiDe to extract metabolic reactions that had altered flow between patients with CD and controls. A random forest classifier was used to classify CD versus control patients using reactions found to be altered in the diseased state. An 80–20% ratio was used for separating patient data points to create training data and test data for machine learning modeling. All flux points associated with patients in the training set were used to train the model, and the model was evaluated on flux data points associated with validation patients. Due to the small size of control patients in the RISK and enteroid datasets, a repeated validation approach was used to identify the top reactions in an unbiased approach. Multiple splits of train-validation patients were created to train and evaluate the model. This extensive cross-validation allowed us to demonstrate performance gains and takeaways across the dataset. Top reactions were extracted and aggregated from splits where the model had a high detection accuracy. Due to an imbalance between the control and CD patients, a random under-sampling method was used in each split to balance the number of controls and CD patients. A schematic overview of the methods used can be viewed in Fig. p value < 0.05 was considered significant. These top 20 reactions were further grouped based on their metabolic subsystems and description of their overall function based on the virtual metabolic human (VMH) reaction database of Recon3D30.The top 20 reactions identified by our metabolic modeling pipeline (detailed above) were extracted from the splits with an F1 score  greater than 70% and used for further analysis. This step ensured only reactions which were substantially altered between CD and control patients in the training and validation patients were included in the functional group analysis. Differences between patients with CD and controls were calculated using the Mann–Whitney U test, and a /z range. Data processing steps for LC–MS data were carried out using Compound Discoverer v3.0. Compound annotation was carried out based on accurate mass, retention time, isotopic pattern, and MS2 fragmentation pattern matching local and public libraries. Annotations were consistent with the current naming convention in the Metabolomics Workbench and reflected the extent of structural information contained in the collected data66.Ileal tissue samples from pediatric CD patients (n = 11) and control non-inflamed ileal samples (n = 14) were analyzed via non-targeted LC–MS lipidomics assays to semi-quantitatively measure thousands of non-polar compounds, including lipids from the eight main lipid classes  in these complex biosamples. Biopsies were cryopulverized prior to mixing with extraction solvent (IPA) to extract the non-polar lipids while minimizing highly polar compounds and proteins, which would suppress the signals of the desired analytes. The supernatant resulting from the extraction was stored at 4 °C until analysis. A pooled quality control sample was injected at regular intervals (every 5 samples) throughout the sample batch, bracketing the actual samples, which were randomized. Data collection was performed on a ThermoFisher Scientific Q-Exactive HF (QE HF) Hybrid Quadrupole-Orbitrap MS system coupled with a Vanquish Horizon LC system in positive and negative ion modes. Compounds were separated using a ThermoFisher Scientific Accurcore C30 . The chromatographic method for sample analysis involved elution with 80:20 water:MeCN with 10 mM ammonium formate and 0.1% formic acid (mobile phase A) and MeCN and 0.1% formic acid (mobile phase B) using the following gradient program: 0 min 5% A; 0.5 min 5% A; 8 min 60% A; 10.4 min 60% A; 10.5 min 5% A; 14 min 5% A. The flow rate was set at 0.4 mL/min. The column temperature was set to 40 °C, and the injection volume was 2 µL. Full MS data was acquired with 240,000 resolutions over the 150–2000 mAll experiments and methods were performed in accordance with the relevant guidelines and regulations at Emory University, including approval by Emory University under IRB MOD004-IRB00085516. All patient samples were collected with informed consent.Supplementary Information.


Beta-blocker administration may reduce the incidence of isolated postoperative atrial fibrillation.See Article page 66.,Atrial fibrillation (AF) is the most common atrial arrhythmia, affecting 33 million worldwide.,Numerous pharmacologic agents have been purported to decrease the incidence of POAF. Despite a paucity of conclusive evidence, preoperative administration of β-blockers within 24 hours before revascularization (coronary artery bypass grafting [CABG]) is a Class I recommendation and remains a CABG quality indicator in the US national Society of Thoracic Surgeons registry.Journal, Masuda and colleaguesIn the current issue of the Subgroup analysis from the study demonstrated differences in POAF between type of operation performed, timing of drug administration, specific β-blocker used, and route of administration, although no statistically significant differences were observed. The analysis included patients treated with the ultrashort acting β-blocker landiolol hydrochloride (2 μg/kg/min intravenous administration), the most effective drug in reducing POAF in the study. Landiolol hydrochloride was introduced in Japan and increasingly is prescribed in Europe in the operative and intensive care unit settings for the treatment and prevention of AF.Overall, the analysis confirms the effectiveness of β-blockers in reducing isolated POAF but fails to clarify best clinical practices for drug administration. The current study supports current national guidelines without adding further clarification for practicing clinicians.“An ounce of prevention may be worth a pound of cure”—however, the evidence for prophylactic β-blocker administration to prevent POAF remains inconclusive. Further study is required to determine the overall effectiveness, timing, and recommended drug of choice. Perioperative β-blocker administration in the setting of cardiac surgery remains a recommended “best practice,” although the specifics of treatment remain elusive.


The purpose of this review is to consider how knowledge about the mechanisms that are associated with CD8+ T cell control of HIV/SIV in natural infection can be harnessed in HIV remission strategies.Immunological studies of spontaneous HIV and simian virus (SIV) controllers have identified virus-specific CD8+ T-cell responses that may be critical for suppressing HIV replication in spontaneous controllers comprising HIV antigen recognition including specific human leukocyte antigen types, broadly cross-reactive T cell receptors and epitope targeting, enhanced expansion and antiviral functions, and localization of virus-specific T cells near sites of reservoir persistence. We also discuss the need to better understand the timing of CD8+ T-cell responses associated with viral control of HIV/SIV during acute infection and after treatment interruption as well as the mechanisms by which HIV/SIV-specific CD8+ T cells coordinate with other immune responses to achieve control.We discuss characteristics of CD8+ T-cell-based remission strategies and offer questions to consider as these strategies target distinct CD8+ T-cell-dependent mechanisms of viral control.We propose implications as to how this knowledge from natural infection can be applied in the design and evaluation of CD8 In HIV infection as in any other viral infection, disease progression and outcome are highly variable between individuals and a wide spectrum of viral control exists that is dependent on virologic features, route of transmission, host genetics, immune responses and environment. While the vast majority of people living with HIV are unable to suppress viral replication without antiretroviral therapy (ART), a small group of people – less than 1% of people living with HIV-1 and ∼15% of people living with HIV-2, with an increased proportion of women – can naturally keep the virus under control . Elite c+ T cells can play a critical role in mediating control of HIV in some controllers. From studies in humans with HIV and nonhuman primates (NHPs) with simian viruses , potent CD8+ T-cell responses have been shown to associate with a lower viral load setpoint in both acute infection and after treatment interruption [+ T-cell responses has been suggested by the association between viral control and specific enrichment of class I human or primate leukocyte antigen (HLA or Mamu) alleles and the development of potent HIV/SIV-specific CD8+ T-cell responses that are independent of the HLA/Mamu type [+ T cells has been shown to lead to viral rebound in NHPs that have either spontaneous elite control or viral suppression induced after treatment in early infection with broadly neutralizing antibodies [+ T-cell responses in these people. PTCs have so far not demonstrated the HLA genetic characteristics seen in elite controllers and early data suggest that they may have a low magnitude HIV-specific CD8+ T-cell response, at least as measured by interferon gamma production after HIV peptide stimulation [+ T cells play a role in viral suppression and that early treatment could also help to preserve CD8+ T-cell function [Evidence is strong that CD8rruption –14. In eamu type –29. Moretibodies ▪,31–33. function ,35.+ T cells that are associated with and may contribute to spontaneous control of infection in elite controllers  recognition of viral peptides in natural infection favors viral control. This concept has been supported by three major lines of evidence: first, there is well documented association of elite controller status with protective class I HLA alleles; second, viral control has been associated with more cross-reactive ‘public’ TCRs ; third, there is evidence that CD8+ T cells from spontaneous controllers are more likely to target highly mutationally constrained – or ‘networked’ – epitopes.Given the evidence that CD8HLA alleles and increased likelihood of elite control , although the presence of these alleles is neither necessary nor sufficient to predict controller status [+ T cells in individuals with those polymorphisms. While the population diversity of classical class I HLA alleles is vast, CD8+ T cells can also be restricted by HLA-E, a nonclassical major histocompatibility complex (MHC) protein with only two alleles found in worldwide populations [+ T cell responses that are associated with the prevention of an established chronic infection in ∼50% of animals upon viral challenge [Across the human population, polymorphisms within the HLA locus provide one mechanism for genetically encoded inter-individual variation in epitope targeting. As reviewed elsewhere ,25, inder status ,19. Sponulations . Strikinhallenge ,38. HLA-hallenge ▪,40. ImpHLA type should be characterized and accounted for in the immunogenicity and efficacy analysis of all HIV remission studies.T-cell-based remission strategies should aim to elicit responses across diverse HLA types.+ T cells offer a more universal approach to therapeutic vaccination for HIV?Could targeting of HLA-E-restricted HIV-specific CD8+ T-cell responses consisting of TCRs with higher avidity and more cross-reactive public clonotypes [HLA alleles associated with HIV control promote thymic selection of more TCR repertoires that have cross-reactivity for viral variants [∗27-restricted epitope response, controllers compared to progressors have distinct TCR clonotypes that are more ‘broadly reactive,’ similar to the concept of broadly neutralizing antibodies in that they demonstrate cross-reactivity to epitope variants [+ T-cell responses with cross-reactive TCRs can be elicited after vaccination in HIV-uninfected people [de novo responses or even to elicit new clonotypes and alter clonotype hierarchy within preexisting responses [In addition to favorable HLA alleles, people who spontaneously control HIV are more likely to have HIV-specific CD8onotypes –43. HLA variants . Additiovariants ,45. Crosvariants –48, but variants ,50. Fromvariants ). While d people , it is uesponses . ImplicaHow can broadly-reactive HIV-specific CD8+ T-cell responses be induced therapeutically, and can they contribute to posttreatment control of HIV?How can immune therapies induce novel CD8+ T-cell responses during ART and overcome immunodominance of the existing T cell responses?+ T cells from spontaneous HIV/SIV controllers may preferentially target epitopes that are evolutionarily conserved and that have lower mutational tolerance  was associated with a lower viral load [+ T-cell responses may be favorable to avoid epitope ‘distraction’ from the most conserved regions [+ T-cell epitopes was sufficient to maintain viral control after SIV challenge and viral escape from these three epitopes occurred concomitantly with loss of control of viral replication [Irrespective of the unmodifiable variable of HLA type, CD8iewed in ). Evidenral load ,14,54–56ral load . Conservral load –62. It nral load . On the  regions ▪. A proplication . Similarlication . These d+ T-cell responses that reliably recognize conserved/constrained regions across individuals with diverse HLA types who are infected with diverse HIV viral strains, and can these T cell responses mediate durable control of HIV in vivo?Can therapeutic vaccines elicit CD8Should therapeutic vaccines aim to elicit broad or narrow responses to key epitopes?Are the considerations about breadth of epitope targeting different for TCR-based adoptive T-cell therapies?+ T cells in elite controllers have also been shown to be highly functional, to exhibit a more memory-like and less exhausted differentiation state, and to localize better to sites of reservoir persistence within lymph nodes compared to responses detected in people who do not control HIV. In order to comprehensively evaluate different T cell-based HIV remission strategies, it will be critical to evaluate to what extent they can promote each of these qualities.Independent of T cell specificity and HLA type, HIV-specific CD8+ T cells from noncontrollers (on or off ART), HIV-specific CD8+ T cells from elite controllers have increased capacity to produce multiple antiviral cytokines after peptide stimulation in vitro  and demonstrate increased expansion capacity, increased expression of the effector protein Perforin, and sustained killing of infected target cells over the course of several days of peptide stimulation in vitro[+ T cells  pathway, and/or overexpression of TCF-1, a Wnt signaling transcription factor that promotes memory T cell-like expansion capacity; [79▪▪,Compared to HIV-specific CD8 in vitro–77. It iT cells + T cells with functional properties similar to elite controllers ?How can therapeutic strategies for HIV remission promote the generation of nonexhausted HIV-specific CD8+ T cells need to be localized in close proximity to sites of viral reservoir persistence in tissues. HIV persists in lymphoid tissues throughout the body with a high burden of infected CD4+ T cells found in the gastrointestinal tract [+ T cells have been observed in the rectal mucosa of HIV controllers [+ T cells localized within B cell follicles, from which HIV-specific CD8+ T cells are mostly excluded [+ T cells located in lymphoid tissue may have impaired cytolytic function [+ T cells are associated with viral control in SIV-infected NHPs [+ T cells in the lymph nodes from controllers may have a less effector differentiated phenotype compared to cells found in the blood, they nonetheless possess the capacity to expand and differentiate into potent antiviral effector cells and traffic to B cell follicles where the HIV reservoir persists. Several immune-based remission strategies are being developed to re-direct CD8+ T cells to the B cell follicles, including pharmacologic treatment with IL-15 agonists, genetically engineering CXCR5-expressing CD8+ T cells for adoptive transfer, and the development of bi-specific antibodies to redirect follicular CD8+ T cells to kill infected cells [To control viral replication, HIV-specific CD8al tract ,86. Incrtrollers . Within excluded ,88–97. Wfunction , these cfunction –101. Morted NHPs . Taken ted cells ▪,106,107+ T cells that migrate to lymphoid tissues/B cell follicles and have the potential to generate a potent effector response ‘at the right place at the right time’?How can remission strategies be tailored to optimize the generation of HIV-specific CD8+ T cells have been extensively studied in spontaneous controllers during the phase of long-term control, there are very limited data on CD8+ T-cell dynamics during acute infection in people destined to become controllers. Specifically, it is unclear whether or how the features of HIV antigen recognition and HIV-specific CD8+ T-cell quality and localization discussed in the two previous sections directly contribute to control early in primary infection or whether they arise as a consequence of these individuals achieving greater viral control. Understanding the timing and nature of the CD8+ T-cell response in early HIV/SIV infection in spontaneous controllers, how CD8 T cells engage rebounding virus in posttreatment controllers, and how CD8+ T-cell responses coordinate with other immune responses to productively engage with and suppress the virus at the time of viral intercept in both settings is therefore crucial to directly inform the development of improved remission strategies.While HIV-specific CD8+ T cells generally exhibit a suboptimal ability to suppress SIV in acute infection, in controller macaques, suppressive capacity increases progressively before the establishment of sustained low-level viremia [+ T cells in acute infection in two individuals who maintained low viremia without ART, suggesting that CD8+ T cells might play an early role in viral control [+ T-cell responses during acute infection [+ T-cell responses may have a role early in infection in some spontaneous controllers, but the exact timing of these responses and their impact on control is not yet fully clear. In noncontrollers in the absence of any immunologic intervention, two studies have suggested that HIV/SIV-specific CD8+ T cells do not respond early enough after ART discontinuation to prevent viral rebound and only exert an effect on viral load set-point after viral rebound [+ T-cell response right after ART is stopped has not yet been described in controllers. Implications:While current data are limited, studying immune responses that occur in acute infection or immediately post-ART in HIV controllers may identify targets for therapeutic intervention to promote HIV remission. In the SIV model, while CD8 viremia . In huma control . Recentlnfection ,111. The rebound ,113. How+ T-cell response in both spontaneous and posttreatment controllers will be key to informing the successful development of interventions aimed at priming an immune response to target the early viral intercept.Studies describing the earliest interactions between the emerging virus and the CD8+ T cells may respond too slowly to stop viral spread after ART is discontinued. Effective immunity during the early stages of viral spread likely requires other interventions to reduce the size of the HIV reservoir, and/or to augment other immune responses to either directly promote functional CD8+ T-cell responses or to contain early viral replication and thus allow CD8+ T cells time to expand and mature . For exhe blood . Classictrollers . Antibodesponses . UnderstCombined interventions targeting multiple virologic and immunologic mechanisms will likely be required to achieve HIV remission.+ T-cell response in response to rebounding virus and give it enough time to start controlling viral replication.Interventions that allow for a transient reduction of viral replication post-ART could allow for a better maturation of the CD8+ T-cell responses in spontaneous HIV/SIV controllers have provided important information on their key role and potential mechanisms contributing to viral suppression. In order to successfully apply this knowledge to T cell-based remission strategies, we suggest the following + T-cell-based remission strategies should seek to elicit T cell responses with ‘broadly reactive’ TCRs that target highly conserved/evolutionarily constrained regions of the virus, are localized to sites of reservoir persistence, are durable and highly functional, and are capable of responding rapidly (either on their own or in conjunction with other immune responses) to emerging virus after ART is discontinued.CD8(2)+ T cells should ideally report HLA typing of study participants, clinical information about pre-ART viral loads and duration of infection prior to ART initiation, epitope mapping and evaluation for broadly reactive TCRs, analysis of the long-term durability and functional capacity , and lymphoid tissue localization or at least homing potential  of the CD8+ T-cell response.Studies that include HIV remission interventions that target CD8(3)+ T-cell response and its coordination with other immune responses in blood and tissues at the time of viral intercept relates to viral control in natural infection and after therapeutic intervention.As a field, we need to better understand how the timing of the CD8(4)+ T-cell characteristics associated with viral control are likely shared by the different groups of controllers in natural infection, but may differ by individual, it is also important to recognize that HIV remission strategies might need to be tailored to different groups of individuals depending on the mechanism targeted , R01AI147749 (L.T.), K23AI134327 (R.R.), R01AI170239 (R.R.).There are no conflicts of interest.


From a physiological point of view, peripheral chemoreceptors (PCh) are the main sensors of hypoxia in mammals and are responsible for adaptation to hypoxic conditions. Their stimulation causes hyperventilation—to increase oxygen uptake and increases sympathetic output in order to counteract hypoxia-induced vasodilatation and redistribute the oxygenated blood to critical organs. While this reaction promotes survival in acute settings it may be devastating when long-lasting. The permanent overfunctionality of PCh is one of the etiologic factors and is responsible for the progression of sympathetically-mediated diseases. Thus, the deactivation of PCh has been proposed as a treatment method for these disorders. We review here physiological background and current knowledge regarding the influence of widely prescribed medications on PCh acute and tonic activities. The clusters of chemoreceptor cells in the human body are localized in the central nervous system—central chemoreceptors (CCh) and in the carotid (CB) and aortic bodies - peripheral chemoreceptors (PCh) . The forSeveral molecular mechanisms have been identified as potential PChS and PChT sensitizers including upregulation of angiotensin II/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and serotonin/NADPH oxidase; downregulation of nitric oxide synthases (NOS) and glucagon-like peptide-1 receptor or increase in leptin, endothelin, or proinflammatory cytokines concentrations . MoreoveCB has been proposed as a therapeutic target in sympathetically mediated disease based on animal studies  and evenBearing in mind that at least some of the mechanisms leading to PCh overfunctionality are covered with guidelines-recommended cardiovascular medications we review the available literature to: 1) find out proved and potential interactions between these drugs and PCh function to present possible ways of pharmacological modulation of chemoreflex, 2) expose gaps in the evidence of such interaction to stimulate further studies in the field, 3) stress the role of the therapy individualization—drugs decreasing PCh function should be preferred in a patient with known PCh overfunctionality.2-receptors agonists seems to have an inhibitory influence on CB, when beta-receptors agonists (βRA), most likely β2RA, are enhancing the CB-mediated response (2R) on chemosensory cells, which in consequence limits the release of neurotransmitters  glomus cells of most mammals species . Moreoveresponse . On the smitters . High donding CB . Accordinding CB . Neverthnding CB . It shounding CB . Since, 2RA) increased baseline ventilation by 15%, as well as, augmented ventilatory response to both hypoxia and hyperoxic hypercapnia in healthy subjects  and hyperoxic hypercapnia (isolated CCh stimulation)  .1RA—isoprenaline—were described by Heistad et al. in healthy individuals. The authors revealed a stimulating effect of this catecholamine on resting ventilation, which was reversed with a non-selective beta-receptors blocker (βRB)—propranolol and well known PCh inhibitor—100% hyperoxia  on hypoxia-induced responses it seems that β2RA and α1RA components are responsible for dobutamine’s ventilatory actions.Intravenous administration of non-selective β1-, β2- and α1-adrenergic agonist—dobutamine—caused a dose-dependent increase in resting minute ventilation and increased ventilatory and sympathetic responses to hypoxia (PChS), but not to hyperoxic hypercapnia (CChS) in healthy . The effo to β2R . Taking 1-receptor blocker (β1RB) bisoprolol did not change baseline ventilation; ventilatory and sympathetic nervous system (measured with muscle SNA) responses to hypoxia or hypercapnia in healthy   .1RB, β2RB, α1RB), reduced resting ventilation (during normoxia and hypoxia) and exercise hyperventilation compared to placebo  treatment with carvedilol  pathway. Therefore non-selective or NO-active beta-blockers should be preferred, when PChS modulation is aimed. This observation is particularly interesting in the contest of the recently described protective mechanism of propranolol (non-selective β-blocker) on chronic IH induced hypertension in rats  in healthy individuals, which decreased resting ventilation  has been confirmed in CB . GeneralThe influence of ATR1 inhibition on PCh is poorly studied in humans. Among a few studies in the topic, all tested selective ATR1 blocker—losartan. Consistently with animal data administration of the drug did not affect resting ventilation (PChT) in healthy individuals  and in pBoth enzymes responsible for nitric oxide (NO) synthesis namely neuronal and endothelial synthases are present in CB . Two modThe influence of NO concentrations on PChS in humans was tested by Bock et al. Researchers revealed that long-term dietary nitrate supplementation with beetroot powder in older healthy subjects caused a significant reduction in PChS compared to placebo . FurtherStorage of adenine nucleotides was described in CB cells in animals and adenosine, via activation of postsynaptic P2X and A2s receptors in the CBs, is one of the neurotransmitters responsible for excitatory response to hypoxia . HoweverPCh functionality may be theoretically influenced by antiplatelet drugs widely prescribed in humans—clopidogrel, tikagrelor or prasugrel—via adenosine P2Y12 receptor inhibition. Moreover, one of the widely prescribed P2Y12 inhibitors—ticagrelor—has proven, both in animal and human models, inhibitory influence on adenosine cellular uptake, leading to increased tissue adenosine concentrations . Further2 arterial levels) and 2) increase in cerebral blood flow. Net effects of these changes are: desensitization of PChS and augmentation of central chemoreflex (the ventilatory response to carbon dioxide) (The only diuretic drug with a confirmed influence on chemoreflex is acetazolamide. This carbonic anhydrase inhibitor leads to mild metabolic acidosis with a consequent: 1) ventilatory compensation (drop in COdioxide) . Moreovedioxide) . The effThe transient influx of calcium ions into glomus type I cells of CB is crucial for the release of neurotransmitters in hypoxic conditions . In an aDigitalis glycosides affect intracellular calcium level indirectly by inhibiting Na+/K+ ATPase leading to hypernatremia and enhanced calcium ions influx . Animal The modulatory role of proinflammatory cytokines on CB functionality was revealed in animal models . Both loAnother mechanism enhancing CB functionality following IH is oxidative stress. In rats, ascorbic acid added to drinking water reduced the malondialdehyde and 3-nitrotyriosine plasma (oxidative stress markers) levels and prevented IH-induced augmentation of PChS .The data regarding the influence of anti-inflammatory and antioxidants on PChS and PChT in humans is missing.Molecules with confirmed or suspected modulatory influence on PCh are listed in


A 51-year-old patient was admitted with chest pain and broad complex ventricular tachycardia. He received three consecutive direct cardioversion (DC) shocks and was commenced on amiodarone infusion via a central venous catheter or central line (CVC). He responded to treatment and normal sinus rhythm (NSR) was achieved. He had elevated troponin I and underwent coronary angiogram which initially was thought to be responsible for his ventricular tachycardia. Coronary angiogram (CAG) showed unobstructed coronary arteries. He was recently diagnosed with pheochromocytoma and was commenced on Phenoxybenzamine 10 mg two months back. He developed ventricular tachycardia (VT) again the next day that did not respond to four consecutive direct cardioversion shocks (DC) and antiarrhythmic medications. He was intubated and ventilated to terminate his VT and was transferred to the intensive care unit (ICU). He remained intubated for 48 hours and he remained in NSR, after which he was extubated. He was commenced on bisoprolol and was later stepped down to the coronary care unit (CCU). Cardiac magnetic resonance imaging (CMR) showed left ventricular non-compaction (LVNC) or possibly myocarditis in view of patient's known history of pheochromocytoma. He was discussed with surgical team at another hospital for surgical resection of the adrenal tumor and had a few further runs of VT while he was waiting to be transferred. The patient finally underwent surgical resection of the tumor and was booked for implantable cardioverter defibrillator (ICD) in view of his VT. This was an interesting case of treatment-resistant VT driven by pheochromocytoma and LVNC, and it is important to be familiar with the fact that conventional therapies may fail in these patients and may require intubation and ventilation to terminate VT storms. Pheochromocytoma is a catecholamine-producing tumor originating from the adrenal medulla or extra-adrenal and typically presents with a triad of abrupt episodes of headache, palpitations, and profuse sweating which may be accompanied by either labile or sustained hypertension ,2. PheocTumors arising from the chromaffin cells of the adrenal medulla and extra adrenal sympathetic paraganglia that produce excess epinephrine and norepinephrine are responsible for the life threatening arrhythmias and labile blood pressure . The mosPheochromocytoma can occasionally also present with severe left ventricular systolic dysfunction (LVSD) and can present with stress cardiomyopathy that may mimic takotsubo cardiomyopathy . In thisA 51-year-old patient presented to the accident and emergency (A and E) department with central crushing chest pain and palpitations. On arrival, the patient was found to be in broad complex ventricular tachycardia and received three consecutive direct current cardioversion (DCCV) shocks followed by amiodarone infusion. The patient reverted to normal sinus rhythm and was admitted to coronary care unit (CCU). His past medical history (PMH) includes type 2 diabetes mellitus (T2DM), hypercholesterolemia, sciatica, psoriasis and psoriatic arthritis, childhood tuberculosis, and a recent diagnosis of pheochromocytoma. His regular medications include metformin, atorvastatin, phenoxybenzamine 10 mg OD, calcipotriol, lansoprazole, and co-codamol. He had a history of smoking 45-pack-year and was a non-drinker. He was on secukinumab for psoriasis which was on hold due to his recent diagnosis with pheochromocytoma based on computerized tomography scan of chest, abdomen, and pelvis (CT CAP) for right upper quadrant (RUQ) pain and his background history of immunosuppression and enlarged parotid gland.2 was 97%. His laboratory tests showed elevated troponin (T) 9 ng/L and repeat troponin was 143 ng/L. His electrocardiogram (ECG) showed broad complex ventricular tachycardia. He underwent coronary angiogram the following day that showed unobstructed coronary arteries. His metanephrine and normetanephrine levels were 12132 pg/mL and 3960 pg/mL. Other laboratory results are shown in Table His vital signs on arrival were as follows: heart rate (HR) was 192 beats per minute (bpm), blood pressure (BP) (standing) was 116/76 mmHg and BP (supine) was 111/70 mmHg, and SpOHe developed broad complex regular ventricular tachycardia (VT) the next day again and was delivered 4 DCCV shocks, and the patient did not respond to them. He was given further amiodarone 300 mg bolus followed by lidocaine 50 mg and then 100 mg boluses; however, the patient remained in VT storm. He was intubated due to VT storm and was transferred to intensive care unit (ICU) and was commenced propofol along with inotropic support in view of hypotension. The patient remained in normal sinus rhythm (NSR) and was extubated after 48 hours. He remained hypotensive and was not tolerating even small doses of metoprolol. He was then commenced on metoprolol 12.5 mg TDS after his BP improved which he tolerated quite well. He had further run of VT after being stepped down to CCU. He had a CT CAP which showed 5 cm left adrenal pheochromocytoma  was 58%. The imaging findings revealed small right ventricular (RV) cavity with normal wall thickness and systolic function, tricuspid annular plane systolic excursion (TAPSE) 18 mm, right ventricular ejection fraction (RVEF) of 74%. There was extensive, almost circumferential sub-epicardial late gadolinium enhancement predominantly involving the basal to mid anterior, lateral, and inferior walls. There was also an additional intramyocardial late gadolinium enhancement in the basal to mid septum extending to the inferior RV/LV insertion point which was in keeping with left ventricular non-compaction (LVNC) Tables , 3.The patient had further episodes of VT and was transferred to a specialist arrhythmia and endocrine center; he underwent successful surgical resection of the tumor followed by dual chamber implantable cardioverter defibrillator (ICD) implantation. ICD checks were satisfactory, and the patient was discharged home with outpatient follow-up. Repeat ICD checks did not reveal any further VT episodes and the patient remained symptom-free.Histology from the biopsies showed a cellular tumor  of the adrenal medulla composed of large polygonal cells with abundant basophilic cytoplasm and markedly pleomorphic nuclei (PASS +2). Cells were predominantly arranged in a nested pattern. There was no spindling of the tumor cells, nor lymphovascular permeation is identified. Capsular invasion is seen  and the tumor extends into the adjacent periadrenal fibromuscular and adipose tissue (PASS +2). Adrenal cortex is unremarkable and features are consistent with pheochromocytoma.Pheochromocytoma and LVNC are both rare diseases and very rarely occur together ,5. SpongPheochromocytoma can present with life-threatening arrhythmia that may be resistant to standard treatment and it can also present with cardiomyopathy . The diaPrevious research has reported that patients presenting with acute myocardial infarction secondary to pheochromocytoma crises had significant coronary arteries atherosclerosis and it’s likely that excess catecholamine release leads to hemodynamic compromise of the myocardium accelerating myocardial cell death. This can also be explained by severe coronary arteries spasm caused by excessive catecholamine release from the primary adrenal tumor . A case Another study based on 26 pheochromocytoma patients reported normal echocardiographic evaluations in 62.1% of the patients, 27.6% patients had concentric LV hypertrophy with normal LV systolic function (LVSF) and 10.3% had LVSD . Only onCardiovascular manifestations from pheochromocytoma such as arrhythmias, and BP abnormalities account for 71% of the mortality and 90% of these patients have hypertension with 75% of patients having paroxysm of hypertension at least once a week ,15. A reA systemic review based on 163 cases from 150 articles reported that 63 patients had dilated cardiomyopathy, 38 had takotsubo cardiomyopathy, 30 inverted takotsubo cardiomyopathy, 10 hypertrophic obstructive cardiomyopathy (HOCM), eight had myocarditis, and 14 patients had unspecified cardiomyopathy. A total of 65% of patients lacked the classic association of hypertension with pheochromocytoma and only 4% of patients had the triad of headache, palpitations, and diaphoresis. Also, 96% of patients showed improvement in their symptoms after resection of pheochromocytoma while lack of resection was associated with death or cardiac transplantation in 44% of patients .Pheochromocytoma and left ventricular non-compaction are two rare diseases and occasionally can be found together. Pheochromocytoma-induced VT can be difficult to manage due to excess catecholamine release. The patient in our study had both conditions and presented with ventricular tachycardia that initially responded to direct current cardioversion but subsequently stopped responding to pharmacological therapy or direct current cardioversion and the patient was eventually intubated and ventilated to break the ventricular tachycardia storm. Physicians need to be aware of the management of this condition as they may not respond to traditional treatment and if left alone, patients can deteriorate rapidly resulting in catastrophic outcomes. This patient had resection of the tumor and has been doing well since.