Patients with strong clinical features of COVID-19 with negative real time polymerase chain reaction (RT-PCR) SARS-CoV-2 testing are not currently included in official statistics. The scale, characteristics and clinical relevance of this group are not well described. We performed a retrospective cohort study in two large London hospitals to characterize the demographic, clinical, and hospitalization outcome characteristics of swab-negative clinical COVID-19 patients. We found 1 in 5 patients with a negative swab and clinical suspicion of COVID-19 received a clinical diagnosis of COVID-19 within clinical documentation, discharge summary or death certificate. We compared this group to a similar swab positive cohort and found similar demographic composition, symptomology and laboratory findings. Swab-negative clinical COVID-19 patients had better outcomes, with shorter length of hospital stay, reduced need for > 60% supplementary oxygen and reduced mortality. Patients with strong clinical features of COVID-19 that are swab-negative are a common clinical challenge. Health systems must recognize and plan for the management of swab-negative patients in their COVID-19 clinical management, infection control policies and epidemiological assessments. WHO defines a confirmed case as a person with laboratory confirmation of COVID-19 infection2. Previously cases where laboratory confirmation is not done or inconclusive are recognized as probable cases however those with strong clinical features, but negative testing are not recognized2. Recent changes to WHO case definitions now allow probable cases to include patients who meet clinical and epidemiological criteria or patients with severe acute respiratory illness who have typical chest imaging features or unexplained anosmia or ageusia and do not stipulate the results of any performed laboratory testing3. Epidemiological evaluations in many countries however, including the UK, focus on patients with positive real-time polymerase chain reaction (RT-PCR) testing4. This places a significant importance on the diagnostic accuracy of laboratory testing.As of 15th December 2020, the World Health Organization (WHO) has reported 70 million confirmed cases of COVID-19 globally with 1.6 million confirmed deaths5. Several case reports describe patients with clinical features of COVID-19 but negative upper respiratory tract swabs who later have positive confirmatory testing on induced sputum or bronchial lavage8. An analysis of published cohorts calculated the false-negative rate of RT-PCR testing amongst symptomatic COVID-19 patients who eventually test positive for COVID-19 is likely 20% and increases gradually with time since symptoms onset9.However, the diagnostic accuracy of RT-PCR upper respiratory tract swabs is increasingly being questioned. A study utilizing both chest computerized tomography imaging (CT) and RT-PCR testing in patients with suspected COVID-19 found 75% of cases with a negative RT-PCR test had CT findings suggestive of COVID-1910. For example in the recently reported RECOVERY trial 10% of those randomized to dexamethasone had a negative swab at the time of randomization11. However, little has been documented about the scale and clinical relevance of this subgroup of patients for whom there is no consensus on optimal management. Our study aims to assess the real-world prevalence and characteristics of clinically diagnosed swab-negative COVID-19, including factors associated with swab-negative disease, and whether their outcomes differ to swab-positive patients.Acknowledging this, some patients with strong features of COVID-19 receive a clinical diagnosis of COVID-19 despite a negative swab result. This clinical approach is being further recognized in the admission criteria of some clinical trials who permit recruitment of these patientsWe retrospectively reviewed medical admissions from March 1 to April 12, 2020 at Imperial College Healthcare Trust (ICHNT) in London, UK from two admitting sites. We defined eligible cases as those who presented with clinical suspicion of COVID-19 or had symptoms compatible with COVID-19, were admitted to hospital and had a SARS-CoV-2 nasopharyngeal swab performed. We collected full demographic characteristics, time course of symptoms, time of presentation and testing, presenting symptoms, final diagnosis and outcome as well as radiological and laboratory findings for all patients with a negative swab from admission until discharge. Cases were evaluated using the Public Health England (PHE) testing criteria for possible COVID cases.We defined swab-negative clinical COVID-19 cases as follows: (a) clinical COVID-19 or high level of suspicion as defined by the treating medical team ; and (b) RT-PCR swab-negative . Patients who tested positive on subsequent swabs while in hospital were not included in the reported cohort.12. Chi-square, Fisher’s and rank sum tests were used, as appropriate, to compare the cohorts’ characteristics, and odds ratios (OR) calculated to assess differences in the outcomes of respiratory deterioration, defined as requiring greater than 60% oxygen, and death. Lastly, we assessed the cumulative risk of the competing outcomes of hospital discharge and death over time using the using the Nelson-Aelen estimator.The swab-negative clinical COVID-19 group of patients was compared to a subgroup of a previously described swab-positive cohort who were similarly admitted via general medical admissionsThe study was approved by the ICHNT clinical governance team. As we report on routinely collected non-identifiable clinical audit data, no individual informed consent was required under the UK policy framework for health and social care. All methods were conducted in accordance with relevant guidelines and regulations.We identified 1,119 emergency medical admissions with initial clinical suspicion of COVID-19 who had a SARS-CoV-2 swab performed  of swab-negative cases were identified as swab-negative clinical COVID-19. 98% (92/94) of these cases fulfilled PHE criteria for patients eligible to be swabbed for SARS-CoV-2 compared to 84% of the entire swab-negative group. 41/94 had repeat swab testing performed and remained negative. Those who had repeat swabbing had between 2 and 3 swabs in total with a median number of 2.p = 0.017). The swab-negative cohort was more likely to have chest radiographs reporting typical COVID-19 appearances than the swab-positive cohort (53/77 (68.83%) vs 195/389 (50.13%); p = 0.004). Only 18 patients within the swab-negative clinical COVID-19 cohort had a CT chest performed during their admission. 15/18 CT chests were reported as being consistent with COVID-19 or had COVID-19 within the reported differential.The demographic profile and symptomatology of the swab-positive and swab-negative cohorts were similar, with high rates of influenza-like symptoms, cough and fever in both groups Table . Typicalp = 0.026).Overall haematological and biochemical findings that have been associated with COVID-19 including raised CRP, d-dimer and ferritin were similarly distributed between the two cohorts. A higher proportion of patients had moderate lymphopenia within the swab-negative group (65/94 (69.15%) vs 258/460 (56.09%); p < 0.001). Interestingly, RT-PCR-negative patients had better hospitalisation outcomes than RT-PCR-positives. The former had a 57% lower probability of requiring ≥ 60% supplementary oxygen during hospitalisation , a shorter length of hospital stay  and 60% lower probability of death . The cumulative risk of being discharged alive was significantly higher in the swab-negative group than the positive group (p < 0.001). Furthermore, the swab-negative cohort have a lower cumulative risk of in-hospital mortality than positives (p < 0.001)  for the RT-PCR-negatives compared to 6 days (IQR 3–10) within the RT-PCR-positives cohort (13. Another analysis of published studies have reported the probability of false negative swab SARS-CoV-2 swab testing increases after day 3 of symptoms and upper respiratory tract SARS-CoV-2 viral load reduces over time14. Further research including immunological profiling would help further elucidate underlying disease mechanisms.We find one in five symptomatic patients admitted to the medical department who had a negative SARS-CoV-2 swab received a clinical diagnosis of COVID-19 despite negative testing. This represents a 13% increase in the total number of hospitalized COVID-19 patients over the study period compared to considering only swab positive patients. Overall the clinical, biochemical and radiological features of the swab-negative clinical COVID-19 cohort did not differ significantly to the swab-positive cohort. Typical features of COVID-19 such as shortness of breath, typical chest x-ray findings and lymphopenia are more common in the swab-negative cohort. The likely explanation is that swab-positive patients with a range of symptoms are classified as COVID-19 patients based on the positive swab test, whilst swab-negative patients diagnosed as having COVID-19 have their diagnosis based on typical symptoms. Swab-negative patients tended to have a longer delay between symptom onset and hospitalization, and had significantly better hospitalization outcomes, including mortality. Whether this potentially less aggressive phenotype is related to a lower viral load, variations in immune response or a different stage of illness presenting after period of peak viral replication is not yet known. Meta-analysis of studies reporting viral load and shedding of SARS-CoV-2 report that upper respiratory tract viral titres peak in the first week of illness15. This is supported by reported cohorts in the literature of patients with clinical features of COVID-19 with negative RT-PCR swab testing and positive serological testing18.Our results provide evidence to validate swab-negative COVID-19 as a real clinical entity commonly encountered in hospital settings, findings which have important implications for current clinical practice and public health guidanceOur study highlights that clinicians caring for COVID-19 patients should maintain a high clinical suspicion, even in the presence of a negative swab result. Our results suggest that a combination of clinical, radiological and biochemical features in keeping with COVID-19 disease, rather than swab results alone, should guide clinical management.8. However, their use is limited by feasibility and potential risk to healthcare workers. Serological testing to confirm exposure to SARS-CoV-2 is likely the optimal second-line test as it is not operator-dependent, is low-risk to healthcare workers, and can feasibly be performed on a large number of patients. Serological proof of exposure could reduce concern regarding cohorting with swab-positive patients. Although serological testing has demonstrated good sensitivity and specificity performance can vary between serological kits19.Furthermore, managing swab-negative clinical COVID-19 within hospital poses disease control challenges. Cohorting this group with swab-positive patients presents potential risk of SARS-CoV-2 infection, if the clinical diagnosis is incorrect. Alternatively, isolating these patients from both swab-negative low-risk patients and swab-positive groups would place significant strain on hospital resources and may be unfeasible during an outbreak. Nasopharyngeal swabbing is vulnerable to inter-operator variation and poor sampling technique may explain why patients with high clinical suspicion of COVID-19 had negative swab testing. Repeated testing may be beneficial in some cases however in our cohort 41/94 swab negative clinical COVID-19 cases had a repeat swab which remained negative. Testing on sputum or bronchial lavage may provide greater sensitivityLastly, but importantly, from a public health perspective, swab-negative clinically diagnosed COVID-19 patients may not be accurately captured in surveillance statistics, thereby underestimating healthcare demand. As the pandemic evolves countries with reducing incidence should consider this clinical group and how they should be addressed when planning interventions to manage second-wave outbreaks and contact tracing.Our study is limited by its retrospective nature and likely under-estimates the proportion of patients with swab-negative clinical COVID-19, as it requires a high degree of clinical confidence without alternative diagnosis. Our relatively small cohort size is therefore a further limitation. The lack of positive confirmatory test means we cannot be sure of the true diagnosis in the clinically diagnosed swab negative group. However, this does not detract from the clinical problems this group presents to hospital management and epidemiological assessment. In the absence of improved testing or routine second line testing for patients who have high clinical suspicion of COVID-19 but negative swab testing, this group is likely to continue to pose a clinical challenge. During the period of data collection changes in local testing policy meant that not all patients were retested for SARS-CoV-2 which could limit the generalizability of some of our results. However this is probably more reflective of the real-world context, particularly where resources are limited. Lastly, it is also possible that, given our relatively small cohort size, demographic and clinical differences between swab-positive and swab-negative patients were not evident. This finding warrants further investigation, as larger studies or meta-analyses are needed to allow the investigation of potential differences between such cohorts and even undertake multivariable regression analyses.Patients with strong clinical features of COVID-19 who have negative nasopharyngeal RT-PCR test results are a common but understudied clinical group. We found they do not differ significantly to similar swab-positive patients but seem to have better outcomes. Healthcare services should recognize and plan for the management of this group when making disease control interventions and epidemiological assessments.


RNA interference (RNAi) represents a novel approach for alternative antiviral therapy. However, issues related to RNA delivery and stability have presented serious obstacles for obtaining good therapeutic efficacy. Viral vectors are capable of efficient delivery of RNAi as short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA). Efficacy in gene silencing for therapeutic applications against viral diseases has been demonstrated in various animal models. Rotavirus (RV) miR-7 can inhibit rotavirus replication by targeting the RV nonstructural protein 5. Viral gene silencing by targeting the RNAi pathway showed efficient suppression of hepatitis B virus replication by adeno-associated virus (AAV)-based delivery of RNAi hepatitis B virus (HBV) cassettes. Hepatitis C virus replication has been targeted by short hairpin RNA molecules expressed from lentivirus vectors. Potentially, RNAi-based approaches could be suitable for antiviral drugs against COVID-19. Caenorabditis elegans worms, the phenomenon was later confirmed to occur in higher organisms including humans [Although the first antiviral drug against herpes simplex virus, idoxuridine, has been on the market since 1963, not too many efficient antiviral drugs have been developed during the past 60 years. There is, however, an urgent need for novel antiviral drugs, particularly due to the current devastating COVID-19 pandemic [g humans . The mecg humans . The curg humans , miRNA-bg humans , and leng humans . TherefoThe important role of bioinformatics in RNAi-based antiviral drug development can be exemplified by targeting of the HIV transactivating response region , and infRNA delivery has been hampered not only by degradation issues but also by the challenges related to cellular uptake due to the negatively charged cellular bilayer and RNA itself . To prev11 scAAV8-anti-HBV copies inhibited HBV replication in transgenic mice for at least 10 months [RNAi-based gene silencing has been applied for Ad vectors targeting the mosquito-borne flavivirus Tembusu virus (TMUV) E and NS5 genes resulting in efficient down-regulation of TMUV RNA replication and virus production in Vero cells lasting for at least 96 h . Ad-base0 months .In another approach in the Special Issue, Eldabawy and co-workers engineered a novel JFH1-based HCV subgenomic replicon double reporter cell line for antiviral drug testing . It alloRelated to self-replicating RNA viruses, six tandem targets for the neuron-specific miR124 sequences were introduced at the junction between the nonstructural protein genes nsP3 and nsP4 of the oncolytic replication-competent Semliki Forest virus (SFV) SFV4 vector . BALB/c The current COVID-19 pandemic has also accelerated investigations into the potential application of RNAi strategies for antiviral therapy. Previously, four miRNAs and five siRNAs have been designed by computational calculations to target the ORF1ab region of the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) , providiAs can be read in the Special Issue on RNAi Interference for Antiviral Therapy and in the accumulated literature on the topic, RNAi has already been frequently used to target viral diseases. A number of examples have been presented on siRNA, shRNA and miRNA approaches leading to reduced viral titers and viral RNA levels. Specifically, it has been demonstrated for miRNAs targeting rotaviruses , for RNATM (patisiran) was approved in August 2018 by the FDA for the treatment of polyneuropathy of hereditary transthyretin-mediated amyloidosis (hATTR) in adults [In general, RNAi-based therapeutics have strongly relied on nonviral delivery rather than applying viral vectors. Although delivery and stability issues have negatively affected the therapeutic efficacy, modifications of the 5’ 7-methylguanosine triphosphate (m7 G) cap structure, the poly (A) tail, the 5’ and 3‘end UTRs, and nanoparticle formulations based on liposomes, polymers, and hybrid lipid–polymers have provided substantial improvements. In this context, although not an antiviral drug, the RNAi-based ONPATTROn adults .In the context of viral vector-based RNAi therapy, the superior delivery and the unmatched expression levels are features making viral vectors superior. Particularly, self-replicating RNA viruses, which can be delivered as replication-deficient or -proficient particles, RNA replicons or DNA replicon plasmids, generate up to 200,000-fold RNA amplification in the cytoplasm of infected cells, resulting in unprecedented transgene expression levels. Naturally, administration of viral particles always needs to be applied under the highest possible safety conditions, which has triggered extensive research and the development of safer viral vectors. However, other issues of concern for viral vector-based RNAi therapy have been off-target effects of siRNA, which can lead to altered expression profiles of nontargeted transcripts  and the To end on a positive note, viral vector-based RNAi therapy for antiviral drugs has definitely showed some genuine promise as can be seen from the reviews and research articles in the Special Issue on RNAi Interference for Antiviral Therapy. The current COVID-19 pandemic has further accelerated antiviral drug development, which by taking the approach of “leaving no stone unturned” has also encouraged approaches of viral vector-based RNAi therapy against SARS-CoV-2. So, to answer the question of whether viral vectors are any good for RNAi antiviral therapy, we definitely are moving in that direction although we are not there, yet.


Isocitrate dehydrogenase (IDH) enzymes catalyse the oxidative decarboxylation of isocitrate and therefore play key roles in the Krebs cycle and cellular homoeostasis. Major advances in cancer genetics over the past decade have revealed that the genes encoding IDHs are frequently mutated in a variety of human malignancies, including gliomas, acute myeloid leukaemia, cholangiocarcinoma, chondrosarcoma and thyroid carcinoma. A series of seminal studies further elucidated the biological impact of the IDH mutation and uncovered the potential role of IDH mutants in oncogenesis. Notably, the neomorphic activity of the IDH mutants establishes distinctive patterns in cancer metabolism, epigenetic shift and therapy resistance. Novel molecular targeting approaches have been developed to improve the efficacy of therapeutics against IDH-mutated cancers. Here we provide an overview of the latest findings in IDH-mutated human malignancies, with a focus on glioma, discussing unique biological signatures and proceedings in translational research. IDH3, which also catalyses the transformation from isocitrate into α-KG, employs nicotinamide adenine dinucleotide (NAD+) as its cofactor. The catalytic activity of IDH requires homodimerisation along with an alteration in the enzyme conformation; isocitrate binding changes the structure of the enzyme from an open to a closed conformation.6 Substrate recognition depends on the amino acid residues in the active site, whereas the frequent mutated active site residue in cancer is arginine 132 (R132).5Isocitrate dehydrogenase (IDH) enzymes, of which there are three isoforms, are essential enzymes that participate in several major metabolic processes, such as the Krebs cycle, glutamine metabolism, lipogenesis and redox regulation.7 In WHO grade IV glioblastoma (GBM), IDH mutations are also found frequent in secondary GBM, which account for 73% of clinical cases, whereas they are less seen in primary GBM (3.7%).8 A follow-up investigation showed that the presence of IDH mutations predict a favourable disease outcome with prolonged median survival in GBM  and anaplastic astrocytoma .7 Although IDH-mutated glioma generally exhibits a better disease outcome, the high incidence of IDH mutations in secondary GBM suggests that lower-grade glioma with IDH mutation often recur with having undergone malignant transformation to a higher grade. In addition, IDH-mutated glioma is more likely to develop a hypermutation phenotype, which is associated with worsened prognosis.9 In non-central nervous system (non-CNS) malignancies, IDH mutations are identified in acute myeloid leukaemia ,10 intrahepatic cholangiocarcinoma 11 and central/periosteal chondrosarcoma .12 The investigation of these non-CNS tumours with similar IDH mutation provides valuable information for glioma research, whereas in the present review we tend to be focussed on IDH-mutated glioma.Mutations in IDH are prevalent in human malignancies. In glioma, IDH mutations are recognised in >80% of World Health Organisation (WHO) grade II/III cases.7 Missense mutations in the IDH1 gene result in the replacement of a strong, positively charged arginine residue at position 132 with lower-polarity amino acids such as histidine (H), lysine (K) or cysteine (C), which impedes the formation of hydrogen bonds with the α-carboxyl and β-carboxyl sites of isocitrate.14 The mutant IDH enzyme therefore exhibits decreased affinity for isocitrate, along with an elevated preference for NADPH. However, only one copy of the IDH gene is mutated in tumours and, in tumour cells harbouring heterozygous IDH mutations, the main forms of IDH dimers are presumed to be heterodimers that contain a version of wild-type IDH1 and a version with the R132H mutation. As a result, in IDH-mutant cells, the IDH1 wild-type component of the dimer converts isocitrate into α-KG to produce NADPH, whereas the mutant part of the dimer exhibits neomorphic activity, converting α-KG into D-2-hydroxyglutarate (D-2-HG) in an NADPH-dependent manner .17 In this review, we will outline the biological impact of IDH-mutant neomorphic activity on cellular metabolism, epigenetic regulation and redox homoeostasis and address key advances in the cancer biology of IDH-mutated glioma and the implication of these findings in the development of future therapeutics, with a particular focus on glioma. Key findings from other IDH-mutated tumour models, such as AML and cholangiocarcinoma, will also be discussed, as they provide additional insights into the molecular mechanisms in IDH-mutated glioma.The establishment of neomorphic activity associated with the IDH mutation has long been shown to have an effect on cellular metabolism, cancer biology and oncogenesis.15 in the cytoplasm, thereby draining carbohydrates from the Krebs cycle.18 The Krebs cycle is adjusted to compensate for fluctuations in the metabolic pathways.19 A 13C metabolic flux analysis suggested that IDH1-mutated cells exhibit increased oxidative metabolism in the Krebs cycle, whereas reductive glutamine metabolism is suppressed.20 With the depletion of cellular metabolism, several non-Krebs-cycle sources of carbohydrates are recruited to compensate for the loss of α-KG.22 Waitkus et al.23 demonstrated that glutamate dehydrogenase 2, an enzyme that catalyses the conversion of glutamate into α-KG and that is expressed at high levels in the brain, is important for relieving the metabolic liabilities in the context of IDH mutants. This finding is confirmed by the observation that IDH-mutated glioma cells are more sensitive to the inhibition of glutaminase,24 suggesting that glutaminolysis serves as a key compensatory pathway to maintain metabolic homoeostasis. McBrayer et al.25 further highlighted the dependency of IDH1-mutated cells on glutaminolysis, as D-2-HG functions as an inhibitor of the branched-chain amino acid transaminase (BCAT1/2), thereby decreasing the levels of glutamate. Furthermore, the consumption of NADPH by IDH mutants compromises de novo lipogenesis, resulting in an increased dependence on exogenous lipid sources for cellular growth.2 This is accompanied by the stimulation, by D-2-HG, of glutamine-derived lipogenesis under hypoxic condition to meet the needs for lipid productivity.26IDH-mutant enzymes cause the accumulation of D-2-HG at concentrations as high as 5–30 mM27 and the expression of LDHA is thus considered to be a hallmark of Warburg phenotype, allowing rapid glycolytic flux to meet the demands for cellular proliferation.28 Although LDHA is highly expressed in a variety of cancer cells, it is silenced in glioma tissue specimens and patient-derived glioma cells with IDH mutants.30 Silencing of LDHA  has been found to be associated with hypermethylation in the promoter region of these genes in response to D-2-HG. The overall epigenetic silencing of the glycolytic pathway might explain the slow-growing nature of IDH-mutated glioma as compared with their IDH wild-type counterparts.31 In support of this hypothesis, in a recent study, the acquisition of the Warburg phenotype was associated with more aggressive gliomas and was found to occur at the CpG island methylator phenotype (G-CIMP) in gliomas described below, which is specific for astrocytoma.32Lactate dehydrogenase A (LDHA) catalyses the transformation of pyruvate formed by glycolysis into L-lactate,33 as α-KG is normally needed for prolyl hydroxylases (PHD) to hydroxylate and promote the degradation of HIF. However, the detailed molecular mechanism on how HIF is regulated in the context of IDH mutation is currently unclear. Other lines of evidence showed that D-2-HG, but not L-2-HG, stimulates the activity of the prolyl hydroxylase PHD2, which results in the reduced expression of HIF-1/2α.34 More effort is encouraged to elucidate the relationship between D-2-HG and the hypoxia-sensing pathway in glioma and other IDH1-mutated malignancies.In addition, IDH mutations lead to the neomorphic enzyme activity, which redirects the Krebs cycle for D-2-HG production. The resultant decrease in α-KG levels might affect the level of hypoxia-inducible factor subunit HIF-1α,Overall, the acquisition of mutant IDH results in substantial reprogramming of cellular metabolism Fig. . Glutami36 Mechanistic studies revealed that neomorphic IDH1 mutant activity results in both global DNA hypermethylation and histone methylation. Interestingly, the extent and targets of hypermethylation vary among tumour types.37 Investigation on the unique pattern of glioma-specific methylation pattern may assist the understanding of pathogenesis of IDH-mutated glioma  catalyses the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) in an iron- and α-KG-dependent manner, as well as catalysing additional cytosine demethylation steps by transforming 5-hmC into 5-formylcytosine and 5-carboxylcytosine (5-caC). 5-caC will eventually be converted into cytosine by thymine DNA glycosylase and base excision DNA repair.44 The methylation status of histone is regulated by histone methyltransferases, such as G9a, GLP, SET and EZH2, as well as by demethylases, such as KDM, LSD and JARID; similar to TET, histone demethylases such as KDM4 and KDM5 have been shown to be inhibited by high levels of D-2-HG.44 As a result, in IDH-mutated cancers, the accumulation of histone methylation markers such as H3K4me3, H3K9me3 and H3K27me3 is frequently reported. Inhibition of histone demethylation by D-2-HG results in impaired cellular differentiation, which might be relevant to the oncogenesis in IDH-mutated cancers.46Besides DNA methylation, D-2-HG also increases methylation of histone by inhibiting histone demethylases such as lysine-specific demethylase (KDM).48 Excessive ROS is harmful to biological molecules, resulting in oxidative damage to DNA, lipids and proteins. Thus maintaining appropriate ROS levels is key to cancer cells during oncogenesis and therapeutic resistance.49 Cancer-associated IDH mutations elevate the affinity for both NADPH (Km = 0.44 µM) and α-KG (Km = 965 µM), indicating that the mutant IDH prefers NADPH and α-KG, instead of NADP+ and isocitrate, as its substrates.16 The consumption of cellular NADPH disrupts the reducing equivalents for biosynthetic reactions, which compromises key ROS-scavenging processes, such as glutathione disulfide reduction, leading to the accumulation of ROS.51 Consequently, accumulating oxidative damage is a hallmark of cancer biology for IDH-mutated malignancies  is involved in major aspects of cancer biology, such as genomic instability, loss of growth control, cellular motility and invasiveness.53 showed that IDH1-mutated glioma cells exhibit strong oxidative stress, as evidenced by an enhanced expression of manganese superoxide dismutase and protein carbonylation. This increased stress was confirmed by subsequent investigations showing that IDH-mutated cancers are more prone to oxidative damage.54 We also confirmed elevated oxidative stress that is closely related to the acquisition of IDH mutants, leading to oxidative damage in biomolecules such as DNA and lipids.51 Owing to the substantially increased oxidative burden, inhibiting antioxidant pathways, such as the synthesis of glutathione, which is mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), could be a valuable strategy for targeting IDH1-mutated solid tumours.55 In addition, proline synthesis has been reported to maintain redox homoeostasis in mitochondria in IDH1-mutated cells. Enhanced activity of pyrroline 5-carboxylate reductase 1-mediated glutamate-to-proline transformation in IDH-mutated cells alongside the oxidation of NADH partially uncouples the electron transport chain from Krebs cycle activity, thus maintaining anabolism in cancer cells.56Gilbert et al.57 reported the first synthetic inhibitor of the IDH mutant, AGI-5198, which blocks the production of D-2-HG and impairs IDH1-mutated xenograft growth in vivo. Second generation of IDH-mutant inhibitors, ivosidenib (AG-120) and vorasidenib (AG-881), are currently approved by the Food and Drug Administration as a therapeutic option for IDH-mutated AML.58 A good amount of clinical studies are currently completed/underway to evaluate the safety and efficacy of ivosidenib and vorasidenib for the treatment of IDH-mutated myeloid malignancies and solid tumours, including glioma.59 These IDH-mutant inhibitors exhibit an improved brain-to-plasma ratio, suggesting that they might be effective for IDH1-mutated glioma.60 Several other IDH-mutant inhibitors, such as BAY1436032, have shown tumour-suppressing effects as experimental therapeutics for the treatment of AML and astrocytoma in animal models.62 Two clinical studies (NCT03127735 and NCT02746081) are currently ongoing to confirm these findings in patients with IDH1-mutated AML or advanced solid tumours, respectively.Given that the neomorphic activity of IDH mutants correlates with malignant transformation, direct targeting of the mutant enzyme has been a heavily pursued strategy. Rohle et al.63 discovered that, although the IDH-mutant inhibitor AGI-5198 successfully reduces neomorphic activity, it relieves hypermethylation phenotype but to a much less extent, as evidenced by elevated histone-3 methylation. In addition, Sulkowski et al.64 reported that AGI-5198 relieves the burden of DNA damage in cancer cells, which might increase their resistance to genotoxic therapies, such as radiation and chemo agents. This phenomenon has been confirmed by another study showing that AGI-5198 confers radioprotective effects on IDH1-mutated cancer cells.65 Overall, targeting IDH-mutant neomorphic activity is a straightforward strategy and has shown efficacy against haematopoietic malignancies in humans and several experimental models for solid cancers. In addition to suppressing D-2-HG production, a combined approach with other agents, such as inhibitors of critical enzymes in metabolic or DNA repair pathways, might be helpful to improve the disease outcome .Despite the promising success of the IDH-mutant inhibitors, several studies have indicated the potential limitations of their application. For example, Johannessen et al.66 Importantly, the hypermethylation phenotype might be linked with oncogene activation. Correcting the epigenetic dysregulation has thus been advocated as a potential therapeutic strategy for cancers that express the IDH mutant. Flavahan et al.67 discovered that the G-CIMP profile correlates hypermethylation at binding sites for cohesion and CCCTC-binding factor (CTCF) and therefore compromises the affinity of this protein. The loss of CTCF binding allows an enhancer to mediate the constitutive expression of platelet-derived growth factor receptor A (PDGFRA), a known mitogen that is linked with glioma oncogenesis. Administration of a demethylating agent partially restores CTCF binding, leading to reduced PDGFRA expression. The concept of inhibiting methylation was confirmed through another study, which demonstrated that decitabine, a DNA methyltransferase (DNMT1) inhibitor, suppresses the proliferation of IDH-mutated glioma cells in vitro and in vivo.68 Similarly, 5-azacytidine, a cytidine analogue that compromises the activity of DNA methyltransferase, resulted in the regression of a patient-derived IDH1-mutated glioma xenograft.69 However, although epigenetic modulators might sufficiently reverse the IDH-associated hypermethylation phenotype, their effects on other cancer signatures in IDH-mutated tumours, such as metabolic reprogramming and DNA repair pathways, remain unclear.As outlined above, the IDH mutation induces a hypermethylation phenotype that results in broad alterations to the epigenetic landscape in tumour cells.72 Mechanistically, D-2-HG compromises DNA repair pathways: it serves as an inhibitor to DNA repair enzymes such as AlkB homologue 2/3 (ALKBH2/3),74 as well as inhibiting the homologous recombination (HR) DNA repair process.64 With the general DNA repair pathways inhibited, targeting the remnant DNA repair enzymes could be detrimental for IDH-mutated cells and thus provide a potential therapeutic approach. We and several colleagues showed that a combination of small-molecule inhibitors targeting the poly-ADP ribose polymerase (PARP) could be a highly effective therapy for IDH-mutated malignancies.76 Owing to the suppression of HR pathways, IDH-mutated cells resemble serious DNA repair defects, which are commonly seen in cancers with BRCA mutations (“BRCAness”). Cancer cells develop a dependency on PARP-guided base excision DNA repair (BER), which helps to maintain genomic integrity under genotoxic therapy.78 PARP inhibitors can establish synthetic lethality with IDH-mutant-derived HR deficiency, resulting in enhanced apoptotic changes.79 Similarly, Tateishi et al.81 showed that the inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) FK866 and GMX1778 deplete NAD+, thereby eliminating remaining PARP DNA repair activity (as PARP requires NAD+ during BER of chemotherapy-induced DNA damage), which introduces distinct metabolic stress responses to chemotherapy-induced DNA damage and improves the durability of therapy response.IDH-mutated glioma exhibits a favourable disease outcome compared with its wild-type counterpart. Several lines of evidence suggested that glioma with mutant IDH appeared to be more sensitive to treatment standards of care, such as radiotherapy and chemotherapy, which may explain their preferred disease outcome.82 Nunez et al.83 showed that the depletion of TP53 or ATRX on an IDH-mutated background resulted in glioma cells tending to undergo a DNA damage response, highlighted by upregulated ATM signalling and resistance to radiotherapy. Taken together, the discovery of altered DNA repair pathways in IDH-mutated glioma not only highlights the correlation between cancer metabolism and genomic instability but also implies important therapeutic vulnerabilities in cancers with IDH mutation.Although the acquisition of the IDH mutation correlates with therapeutic sensitivity in patients and targeting DNA repair enzymes seems to be more effective for IDH-mutated cells, several investigations have highlighted that IDH-mutated glioma might develop distinctive DNA repair pathways from those of IDH wild-type glioma. For example, RAD51 recombinase, an enzyme that plays major roles in the HR process, protects IDH-mutated cells from temozolomide-induced DNA damage.84 In IDH-mutated glioma, the de novo synthesis of NAD is largely compromised owing to epigenetic silencing of nicotinate phosphoribosyltransferase (NAPRT1). As a result, cancer cells rely on the salvage pathway to generate NAD.85 The dysfunction in NAD metabolism suggests that IDH-mutated cells could be extremely sensitive to the blockade of the salvage pathway through small-molecule inhibitors targeting NAMPT.81The substantial reprogrammed cellular metabolism in IDH-mutated glioma suggests that it might be possible to establish specific druggable targets for this type of malignancy. NAD, a cofactor for critical biological processes such as electron transport and redox metabolism, is derived from biosynthesis (de novo pathway) and salvage pathways (using compounds containing a pyridine base).86 In light of high-throughput screening assays, Zaprinast was found to inhibit glutaminase and limited the proliferation of IDH-mutated tumour cells in vitro.87 CB-839 is an oral glutaminase inhibitor that has shown therapeutic efficacy in IDH-mutated AML, causing a reduction in D-2-HG production and inducing terminal differentiation.88 A phase 1 clinical study is currently underway to evaluate the combination of CB-839, radiation and the alkylating chemotherapy agent temozolomide in IDH-mutated diffuse or anaplastic astrocytoma (NCT03528642). Overall, suppressing glutamine/glutamate metabolism correlates with slowed tumour manifestation in both glioma and AML models. However, the strategy commonly results in prolonged latency rather than strong tumour suppression in vivo. Combining the suppression of glutamine/glutamate metabolism with other cytotoxic therapies, such as radiation and temozolomide, might improve the therapeutic efficacy.In addition, owing to the essential role of glutaminolysis in metabolic compensation, targeting glutamine/glutamate-related metabolism has been proposed for IDH-mutated malignancies. For example, glutaminase blockade through bis-2-ethyl sulfide inhibits glutamine metabolism and suppresses IDH1-mutated glioma and AML.90 An imaging study demonstrated that levels of glutamate, glutamine and glutathione are decreased in tumour regions in patients with IDH-mutated glioma, as compared with levels in contralateral regions. In addition, the tumoural glutathione level negatively correlates with the level of D-2-HG, suggesting that glutathione is essential for IDH-mutated cells to maintain redox homoeostasis.91 The increased consumption of glutathione suggests a high burden of ROS scavenging in IDH-mutated cells. As such, developing a therapeutic strategy to disrupt the fragile redox homoeostasis, such as limiting glutathione-derived ROS scavenging, could be a valuable therapeutic approach. A preclinical animal study has shown that inhibiting glutamine metabolism using the glutaminase inhibitor CB-839 leads to impaired redox homoeostasis and sensitises IDH-mutated glioma to radiotherapy.25 In our studies,92 we have shown that suppression of Nrf2 by the natural compound brusatol resulted in profound tumour suppression in IDH1-mutated xenografts, along with overwhelming oxidative damage. Although disruption of redox homoeostasis in these cases results in potent cytotoxicity accompanied by tumour suppression, current therapeutic compounds are mostly at the preclinical stage and demonstrate considerable systemic toxicity. Nevertheless, developing the next generation of therapeutic compounds with both potency and selectivity will be of great help for targeting redox imbalance in IDH-mutated malignancies.As mentioned previously, elevated ROS is a hallmark of IDH-mutated malignancies.94 An initial investigation on glioma data sets showed that the presence of IDH mutation correlates with a decrease in the expression of programmed death-ligand 1 (PD-L1)95 when compared with IDH wild-type gliomas. Reduced PD-L1 expression in IDH-mutated glioma implies a stronger T cell activation, as PD-L1 is a cellular surface protein that downregulates the immune system and promotes self-tolerance through suppressing T cell activity.96 In addition, D-2-HG-derived DNA methylation results in epigenetic silencing of both PD-1 and PD-L1 in glioma.98 However, the reduced expression of PD-1/PD-L1 in IDH-mutated glioma may not result in stronger antitumour T cell immunity. An in-depth investigation showed that D-2-HG serves as a potent inhibitor for antitumour T cell immunity in the tumour microenvironment. D-2-HG suppresses ATP-dependent T cell receptor signalling, which further impairs the activation of T cells in glioma. D-2-HG also suppresses signal transducer and activator of transcription 1, leading to decreased CD8+ T cell recruitment in the tumour region.99 Combining a pan-IDH1-mutant inhibitor BAY1436032 improves the efficacy of anti-PD-1-derived immunotherapy, causing enhanced intratumoural CD4+ T-cell proliferation, a reduction in tumour volume and prolonged overall survival.94 Additional immune checkpoint inhibitors are currently under investigation for IDH1-mutated glioma. The PD-L1 inhibitor pembrolizumab (MK-3475) is being evaluated in a clinical trial of patients with recurrent IDH1-mutant grade II–IV gliomas whose tumours have a hypermutator phenotype (NCT02658279). Another ongoing phase 1 clinical trial is currently enrolling patients with IDH1-mutant gliomas that have transformed into GBM to investigate the concurrent administration of the PD-L1 inhibitor avelumab and hypofractionated radiation therapy (NCT02968940).There is growing evidence that the IDH mutation might play important roles in altering the tumour immunological microenvironment, as indicated by a suppression of tumour-infiltrating lymphocytes, natural killer cells and cytotoxic T cells.+ TH1 response, resulting in an effective MHC class II-restricted antitumour immune response in tumours with the IDH1 R132H mutation.100 Consistent with this result, another study found that mice immunised with peptides encompassing the IDH1 mutation site showed increased survival benefit when bearing GL261 gliomas with IDH1 R132H expression but not parental GL261 gliomas. The immunised mice showed higher amounts of peripheral CD8+ T cells and produced higher levels of interferon-γ, indicating the generation of anti-IDH1-mutant antibodies.101 The IDH-mutant vaccine is currently being investigated in clinical studies. For example, a phase 1 clinical trial is currently ongoing to validate the safety and therapeutic efficacy of an IDH1 R132H mutant peptide vaccine (NOA-16) in newly diagnosed grade III and IV gliomas with an IDH1 mutation (NCT02454634). The first reported results demonstrated the safety and immunogenicity of NOA-16, with 80% of patients displaying mutation-specific T cell immune responses, and 87% of the patients displaying humoural immune responses; no deaths were reported.102Despite PD-1-derived immunotherapy, several attempts have been made to generate a peptide vaccine that targets the IDH1 R132H neoantigen. An IDH1 R132H-specific immunogenic epitope was presented by major histocompatibility complex (MHC) class II molecules and induced a CD4103 The loss of phosphatase and tensin homologue compromises the HR DNA repair pathway, which predisposes to sensitivity to temozolomide and PARP inhibitors.104 For IDH-mutated gliomas, numerous attempts have been made to define selective and effective therapeutics that target the biological signatures of IDH-mutated cancers, with the aim of improving standard treatments. For instance, the addition of PARP inhibitors or blockade of DNA repair enzymes improves the cytotoxicity of genotoxic therapies. Targeting distinctive metabolic patterns such as glutaminase and glutathione de novo synthesis has also shown potent efficacy in IDH1-mutated cells.105 However, a major hurdle in IDH1-mutated glioma is that the critical oncogenic drivers of this disease remain controversial. Increasing evidence has uncovered the biological impact of D-2-HG on cancer biology, but the molecular targets of D-2-HG includes >60 members,106 which are involved in profoundly diverse molecular pathways, so the identification of clear oncogenic mechanisms in IDH-mutated glioma remains challenging. In-depth investigation into the critical molecular pathways will be of great importance to develop therapeutic approaches with high potency and selectivity.The discovery of the IDH mutation not only adds to the landscape of glioma genetics but also indicates that glioma is a highly heterogeneous disease. Many pioneering studies have shown that different glioma molecular subtypes exhibit different signatures according to their oncogenic drivers and distinctive patterns of therapy resistance. For example, amplifications or gain-of-function mutations in the genes encoding epidermal growth factor receptor and PDGFRA are highly common in GBM.Cancer therapeutics that exploit the concept of synthetic lethality by targeting multiple biologically relevant molecular pathways are expected to have reduced toxicity, as they tend to be more specific to cancer cells due to their unique mutation pattern. IDH-mutated glioma exhibits clear dysfunctions in several biological pathways. For example, metabolic depletion through IDH-mutant neomorphic activity prompts dependency on alternative metabolic pathways, such as glutaminolysis and NAD salvage pathways. Targeting critical enzymes, such as glutaminase or NAMPT, establishes synthetic lethality with the intrinsic metabolic dysfunction and translates into reduced tumour expansion. Moreover, D-2-HG inhibits DNA repair pathways such as HR and PARP/BER, which confers sensitivity to inhibition of the remaining DNA repair enzymes. Targeting mechanisms of ROS scavenging synergises with IDH-mutant-derived oxidative stress, which improves disease outcome with reduced IDH1-mutated xenograft growth. Taken together, targeting the distinctive vulnerabilities of IDH-mutated glioma has been shown to be successful, as cancer cells are less likely to compensate for the loss of essential biological pathways. Moreover, the vulnerabilities in IDH-mutated cells such as glutaminolysis and ROS scavenging are generally absent in other somatic cells, suggesting that the approach of synthetic lethality might be better tolerated in the context of combination therapy.107 however, the wild-type allele is gradually lost after passaging the cells in vitro.108 As another example, Mazor et al.110 discovered that patient-derived glioma cells with IDH mutations could undergo hemizygous deletion of the IDH1 locus, suggesting that tertiary mutations might have been established to enable cells to be sustained in vitro.110 IDH-mutated patient-derived glioma cells might therefore provide useful information for mechanistic studies, but any conclusions should be carefully verified in further preclinical models. Genetically engineered mouse models have the potential to provide valuable tools for investigating IDH1-mutated glioma. Several emerging mouse models provide pivotal tool to understand the cancer biology in IDH-mutated glioma (Table The development of disease models with biological and clinical relevance has become one of the major challenges for basic and translational research for IDH1-mutated glioma. Cell lines stably expressing mutant IDH1, which have long been used in many studies, are powerful tools for investigating the biological consequences of IDH mutants in isogenic backgrounds. However, the parental cells are commonly derived from GBM or other malignant tumours, which harbour genetic abnormalities that are rarely seen in lower-grade glioma, so it is very hard to justify the application of using these cell lines to investigate IDH1-mediated de novo oncogenesis and malignant transformation. Furthermore, establishing sustainable cell strains with intrinsic IDH mutation has been challenging, especially in cases of low pathological grades. Several studies have reported that IDH-mutated glioma cell lines are useful for in vitro investigations, but they have limited application in in vivo experiments due to the extremely low efficiency of xenograft formation (Table ma Table . These gIn summary, mutations in IDH are clearly linked to the establishment of human malignancies. A series of seminal studies have revealed the impact of IDH mutant and D-2-HG in cellular physiology, such as reprogrammed metabolism, epigenome alterations and redox homoeostasis. With the increased availability of disease models both in vitro and in vivo, more breakthroughs are anticipated to elucidate the critical pathways that are involved in tumour formation, tumour metabolism and therapeutic vulnerability. A molecular targeting and synthetic lethality approach would become available to benefit patients with IDH-mutated glioma, with improved disease outcome and quality of life.


We describe an elderly male presenting to the emergency department with shortness of breath that progressed to hypoxic respiratory failure. Radiography and computed tomography findings were suggestive of coronavirus disease 2019 (COVID-19).We review the clinical presentation of COVID-19 and its complications. We also describe the characteristic presentation of COVID-19 on imaging. Our case illustrates the hallmark findings of bilateral and peripheral ground-glass opacities of COVID-19. A 70-year-old male with a history of hypertension and chronic kidney disease presented to the emergency department (ED) with cough, fevers, and worsening shortness of breath for two weeks. He saw his primary-care physician a week prior and received azithromycin and oseltamivir and was also tested for coronavirus disease 2019 (COVID-19), which was negative. Initial vitals were temperature 38.7°Celsisus, blood pressure 176/87 millimeters of mercury, respirations of 22 breaths per minute, and oxygen saturation of 86% on room air. His exam was significant for tachypnea and diffuse crackles bilaterally. Despite non-invasive oxygenation, he progressed to hypoxic respiratory failure and required intubation. Chest imaging revealed multifocal peripheral bilateral ground-glass opacities suggestive of COVID-19. –3 He wasSevere acute respiratory syndrome coronavirus 2 causes COVID-19.5What do we already know about this clinical entity?Coronavirus disease 2019 (COVID-19) has a spectrum of clinical presentations, from asymptomatic or mild viral symptoms, to respiratory distress, respiratory failure, severe disease, and death.What is the major impact of the image(s)?We present the classic presentation of COVID-19 on chest radiography and computed tomography, which can assist providers in making a diagnosis.How might this improve emergency medicine practice?Recognizing COVID-19 on imaging studies can help providers increase their index of suspicion, given the variable speed and availability of confirmatory testing.


Neutrophils play an important role in the lung tumour microenvironment. We hypothesised that radiolabelled neutrophils coupled to single-photon emission CT (SPECT) may non-invasively quantify neutrophil uptake in tumours from patients with non-small cell lung cancer. We demonstrated increased uptake of radiolabelled neutrophils from the blood into tumours compared with non-specific uptake using radiolabelled transferrin. Moreover, indium-111-neutrophil activity in the tumour biopsies also correlated with myeloperoxidase (MPO)-positive neutrophils. Our data support the utility of imaging with In-111-labelled neutrophils and SPECT-CT to quantify neutrophil uptake in lung cancer. Inflammation has emerged as a hallmark of tumourigenesis, contributing to the development and progression of a range of cancers. Neutrophils are known to be important players in the tumour microenvironment and these tumour-associated neutrophils (TANs) are broadly classified into an N1 tumour-suppressing phenotype or an N2 tumour-promoting phenotype. While neutrophils may exert both pro-tumorous and antitumorous functions, the prevailing view is that they serve to exclude T cell access and hence promote tumour progression. In murine studies, Ly-6G-specific depletion of neutrophils reduced the metastatic burden in the lungs,+ neutrophils. These findings have important implications for the use of quantitative SPECT-CT to monitor the extent of neutrophil uptake by tumours.Although evidence exists that infiltrating neutrophils accumulate in non-small cell lung cancer (NSCLC),Six patients were selected for entry into the study met the following criteria: (1) positron emission tomography (PET) or biopsy confirmed preoperative stage I–II lung cancer in the mid-upper zones of the lung, (2) no prior chemotherapy or radiotherapy and (3) no other active malignancy. Biopsies were performed ≥1 month prior to acquisition of SPECT images. Tumours in the mid-upper zones were chosen to avoid potential overlap of the signal with physiological uptake of radiolabelled cells in the liver. Patient characteristics can be found in 10.1136/thoraxjnl-2020-214642.supp1Supplementary data10.1136/thoraxjnl-2020-214642.supp2Supplementary dataIn four volunteers, neutrophils were isolated from peripheral venous blood using plasma-Percoll gradients and radiolabelled with indium-111-tropolonate, as described in Over 40 min of dynamic gamma camera imaging, labelled neutrophils displayed physiological kinetics in liver, lungs and spleen, in line with previous results from healthy volunteers .8 The re10.1136/thoraxjnl-2020-214642.supp3Supplementary dataSPECT-CT showed very clear and evident uptake of neutrophils into the tumour of a patient with squamous cell carcinoma case 1; . As showThe median peripheral neutrophil count was higher in the In-111 neutrophil patient group compared with the In-111 transferrin group ; however+ neutrophils per hpf  and periphery  of the tumour compared with the background parenchyma   . SimilarR; 4–11) . There wR; 4–11) . When al10.1136/thoraxjnl-2020-214642.supp4Supplementary data+ neutrophils in lung resections correlated with radiolabelled neutrophil counts. Such a non-invasive imaging strategy could have important implications for monitoring neutrophil infiltration into tumours, and assessing the impact of neutrophil-targeted therapies in NSCLC.In the present study, we demonstrated quantitative in vivo SPECT-CT imaging of radiolabelled neutrophil uptake in tumours of patients with lung cancer. Our study also established that MPOTwo previous studies have reported enhanced uptake of In-111 mixed leukocytes and In-111 granulocytes into a number of localised malignant neoplasms such as pulmonary sarcoma, although without quantitative measurements and a subjective scoring protocol.et al, using a murine model of neutrophil trafficking, found that neutrophils exhibit distinct migratory patterns depending on their localisation to intra-tumorous or peri-tumorous regions.Of interest, we observed inter-tumour heterogeneity with respect to neutrophil uptake into tumours by both imaging and immunohistochemistry approaches, consistent with previous immunohistochemistry studies. A larger scale study will be required to understand the prognostic implication of such variable neutrophil infiltration. Some authors have suggested that, in addition to neutrophil abundance, the specific location of neutrophils within the tumour has prognostic relevance. Sody Given the broadly pro-tumourigenic role of neutrophils, modulation of neutrophil migration into tumours is an attractive therapeutic approach in NSCLC. Indeed, targeting neutrophil recruitment may be beneficial as adjuvant therapy. The CXCR2 pathway has a major role in the control of neutrophil recruitment into tumours, and antagonists of the CXCR2 receptor have reduced tumour growth in murine studies. Phase II clinical trials using CXCR2 inhibitors in NSCLC patients are on-going.Our study has several limitations. First, our results are drawn from a small sample size, and follow-up will be required to confirm the utility of neutrophil imaging in NSCLC. Second, as TANs exhibit significant heterogeneity, our study could not detect the maturity or activation status of the trafficked cells. Third, there are reports of increased transferrin receptor expression on lung cancer cells,In conclusion, we demonstrate how radiolabelled neutrophils and SPECT-CT can be combined to allow non-invasive quantification of neutrophil accumulation in lung cancer. Further studies of in vivo neutrophil trafficking in NSCLC are warranted.


Tumors escape immune surveillance by inducing various immunosuppressive pathways, including the activation of inhibitory receptors on tumor-infiltrating T cells. While monoclonal antibodies (mAbs) blocking programmed cell death 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have been approved for multiple cancer indications, only a subset of patients benefit from immune checkpoint blockade therapies, highlighting the need for additional approaches. Therefore, the identification of new target molecules acting in distinct or complementary pathways in monotherapy or combination therapy with PD-1/PD-L1 blockade is gaining immense interest. T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) has received considerable attention in cancer immunotherapy. Recently, anti-TIGIT mAb (tiragolumab) has demonstrated promising clinical efficacy in non-small cell lung cancer treatment when combined with an anti-PD-L1 drug (Tecentriq), leading to phase III trial initiation. TIGIT is expressed mainly on T and natural killer cells; it functions as an inhibitory checkpoint receptor, thereby limiting adaptive and innate immunity. CD226 competes for binding with the same ligands with TIGIT but delivers a positive stimulatory signal to the immune cells. This review discusses the recent discoveries regarding the roles of TIGIT and CD226 in immune cell function and their potential application in cancer immunotherapy. Upon stimulation with CEF peptide, FACS-sorted CD226loCD8+Tem cells from healthy donors fail to respond to TIGIT and/or PD-1 blockade, whereas the CEF-specific responses of CD226hiCD8+Tem cells are enhanced [+T cells from PDAC patients after mFOLFIRINOX chemotherapy, which is associated with an elevated responsiveness of antigen-specific CD8+T cells to treatment with anti-TIGIT or anti-PD-1 mAbs, and has also been reported in the mouse tumor models [+TILs showed differential responsiveness to anti-PD-1 mAb treatment by CD226 expression [In accordance with the CD226-dependent regulation of CD8enhanced . This obr models . Wang etr models . CD226 dpression .hiCD8+T cells representing a predictive biomarker for several ICB therapies in a large cohort of patients with different cancer types need to be examined. Second, whether Fc engagement is necessary and required for the anti-tumor efficacy of TIGIT blockade remains to be defined. Further, it remains unclear whether the therapeutic effect of anti-TIGIT antagonist is mediated by the reactivation of Tex or NK cells, the depletion of Treg cells, the reprogramming of APC functions, or all of them. Several ongoing clinical trials will likely help provide answers to these questions.TIGIT has emerged as a promising target for next generation cancer immunotherapy. Several clinical trials are currently evaluating the efficacy of anti-TIGIT mAbs in patients with different types of cancer. The most advanced candidate, tiragolumab, has exhibited remarkable efficacy in PD-L1-positive NSCLC patients in phase II clinical trials, in combination with PD-L1 blockade. However, the mode of action of TIGIT blockade remains to be fully elucidated. First, the association of CD226 activation with the efficacy of TIGIT blockade needs to be confirmed in clinical trials. Importantly, CD226


We report the incidental finding of COVID-19 in a 59-year-old male, with no significant cardiorespiratory past medical history who underwent a fluorodeoxyglucose positron emission tomography (FDG-PET) scan for investigation of a likely gastric gastrointestinal stromal tumor (GIST). There may be significant discrepancies between clinical symptoms and radiological severity with COVID-19 infection. FDG-PET scanning has the potential to complement traditional radiological imaging in COVID-19 in diagnosis of subclinical diagnosis or early stage disease, as well as monitoring disease progression. COVID-19 was first reported in Wuhan, Hubei province, China in December 2019 . Plain cA 59-year-old male with no cardiorespiratory or other co-morbidities was referred for investigation of intermittent dysphagia. He underwent upper gastrointestinal (GI) endoscopy. This showed possible gastrointestinal stromal tumor (GIST) in the proximal stomach, although biopsies on two occasions showed normal gastric mucosa. A computed tomography (CT) scan performed in March 2020 which showed a heterogenous mass intimately connected to the gastric fundus, suggestive of gastric GIST and measuring 15 x 12.6cm. Discussion within a regional sarcoma multidisciplinary team suggested referral for endoscopic ultrasound (EUS) and functional imaging with fluorodeoxyglucose positron emission tomography (FDG-PET). The FDG-PET scan was performed four weeks later. At this stage he remained apyrexial (body temperature < 37.50C), with no respiratory or gastrointestinal symptoms, or any headache and there was no history of exposure to possible COVID-19. The FDG-PET scan confirmed the CT findings and reported no significant change or size in the GIST but showed FDG-avid, prominently peripheral, ground-glass changes in bilateral upper and lower lobes . The chaOur case again highlights the speed of disease progression and dramatic radiological findings despite the apparent lack of symptoms in keeping with recent data that suggests up to 78% of patients with a COVID-19 infection may be asymptomatic ,9. Qin eChefer et al. reported the ability of FDG-PET to detect even subtle changes with subclinical MERS-CoV infection in 2015 . Such fiWith a relatively low radiation exposure, radionucleotide imaging may be able to give a more complete picture of acute respiratory viral infections and the development of pulmonary parenchymal injury and may enable more logical decision making around use of specific interventions such as the use of anti-inflammatories ,15. TherThe authors declare no competing interests.


Exosomes are small extracellular vesicles secreted by almost all the cells. Molecular cargos of exosomes can partially reflect the characteristics of originating cells. Exosome-mediated cell-to-cell interactions in the microenvironment are critical in cancer progression. Hypoxia, a key pro-cancerous feature of the tumor microenvironment, alters the releasing and contents of exosomes. A growing body of evidence shows that hypoxia induces more aggressive phenotypes in cancer. Of note, non-coding RNAs shuttled in hypoxic tumor-derived exosomes have been demonstrated as fundamental molecules in regulating cancer biology and remodeling tumor microenvironment. Furthermore, these hypoxic tumor-derived exosomal non-coding RNAs can be detected in the body fluids, serving as promising diagnostic and prognostic biomarkers. The current review discusses changes in cancer behaviors regulated by exosomes-secreted non-coding RNAs under hypoxic conditions. Cancer cells persistently interact with other cell types in the tumor microenvironment. Cells cohabiting in a tumor niche are affected significantly by surrounding factors . Low oxyExosomes contain cytosolic cargos of donor cells, assisting the membrane-protected cargos to travel a long distance. Among many other cytosolic cargos found in exosomes, non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are most intensively studied in that they act as vital regulators in transcriptional and post-transcriptional levels. Investigating the effects of exosomal non-coding RNAs in the hypoxic tumor microenvironment is critical to elucidate the key underlying mechanisms of cancer progression.Collectively, this review highlights recent findings of exosome-facilitated cancer progression in the hypoxic tumor microenvironment with a particular focus on non-coding RNAs and proposes clinical applications of exosomal non-coding RNAs as biomarkers.Accumulative evidence suggests that tumor cells behave differently depending on extrinsic factors of the surrounding microenvironment. Distinct types of cells including immune cells, fibroblasts, and endothelial cells interactively communicate to facilitate cancer progression . AdditioIrregular vessel formation and the rapid proliferation of cancer cells create hypoxic conditions in malignant tumors. In this regard, hypoxia is a common feature in the microenvironment of most solid tumors . ClinicaSeveral decades ago, oxygen sensing mechanisms at the molecular level had been discovered that some transcription factors play a central role in tissues in response to low oxygen tension (< 10 mmHg) . The vitThe hypoxic conditions also affect interactions between cancerous and non-cancerous cells in the tumor microenvironment. Hypoxia-induced overexpression of programmed death-ligand 1 (PD-L1) in cancer cells disabled cytotoxic functions of programmed cell death protein 1(PD-1) positive activated T lymphocytes , 21. MorTranscriptomic and epigenetic landscapes of a tumor are vastly changed by hypoxia. As recent studies have underscored the importance of exosomes as a critical mode of cell-to-cell communication, it would be worthwhile to unveil how exosomal non-coding RNA signaling regulates the hypoxic tumor microenvironment.Extracellular vesicles (EVs) are secreted by almost all cell types into the extracellular space. EVs are classified into microvesicles, apoptotic bodies, exosomes, etc., according to their intracellular origin, size, and biogenesis . ExosomeExosomes are mediators of cell-cell interactions in that they are capable of delivering functional mRNAs, microRNAs (miRNAs), DNAs, and proteins to recipient cells altering their physiological and pathological functions. These exosomal cargos are enclosed inside the double membrane and are stable to environmental factors such as nucleases, proteases, and oxidative stress so that they can be delivered to recipient cells in an efficient and intact manner , 33. CanAdditionally, exosomes have been detected in almost all types of body fluids such as blood, urine, saliva, breast milk, and ascites. They can be used as biomarkers to diagnose diseases in non-invasive or minimally invasive ways . FurtherRNAs are classified into protein-coding RNAs and non-coding RNAs according to their protein-coding abilities. Based on the Francis Crick’s “the central dogma of molecular biology,” many studies have focused on processes of protein production through messenger RNA (mRNA) and constituent RNAs such as ribosomal RNAs (rRNAs) or translators of codon sequence (tRNA) , 47. HowA single mammalian cell carries approximately 100,000 endogenous miRNAs, while a single exosome contains about 500 miRNAs. Pigati et al. found that the abundance of about 66% of extracellular miRNAs well reflected the corresponding abundance of miRNAs in the cells . van BalSome lipids and proteins have been demonstrated to be involved in sorting specific non-coding RNAs into exosomes. The neural sphingomyelinase 2 (nSMase2) was reported to be associated with the secretion of exosomes, which was also the first molecule found to guide miRNAs into exosomes. The expression of nSMase2 was positively related to the level of exosomal miRNAs . BesidesHypoxia can activate various pathways to promote the secretion of exosomes and alter the components loaded in exosomes Fig. . Rab GTPThe hypoxic conditions not only boost the release of exosomes but also influence the molecules contained within exosomes. Hypoxia-induced exosomes exhibit different patterns of various molecules depending on origin cells and environmental factors. In highly malignant brain tumor glioblastoma multiforme (GBM), hypoxic cancer cell-derived exosomes showed enrichment in hypoxia-associated mRNAs and proteins , caveolin 1, and lysyl oxidase) and activated vascular cells in a hypoxia-dependent mode during cancer progression . Recent Exosomes derived from hypoxic cancer cells or stromal cells play a fundamental role in the tumor microenvironment through transmitting non-coding RNAs. Hypoxia-induced exosomal non-coding RNAs have been demonstrated to regulate cancer proliferation, metastasis, chemoresistance, immune response, and angiogenesis, thus reshaping the microenvironment Fig. ; Table 1Hypoxia alters tumor metabolism and transcription such as a shift to glycolysis and self-sufficient release of growth signals . Even thMiR-210 is a well-recognized hypoxia-induced miRNA involved in various biological processes of cancer progression. It was reported to be upregulated in many types of solid tumors and related to unfavorable clinical outcomes of patients . In breaTherefore, exosome-mediated communication plays an essential role in the hypoxic environment. Hypoxic exosome-shuttled bioactive non-coding RNAs have been shown as critical regulators of cancer proliferation.Hypoxia has been demonstrated to regulate the invasion and migration ability of cancer cells mainly by promoting EMT. EMT is involved in carcinogenesis and endows transformative properties to cancer cells by improving mobility, invasion, and migration . During Stromal cells are also indispensable components in the tumor microenvironment. Stromal-cancer or stromal-stromal interactions mediated by exosomes have profound impacts on metastasis initiation and cancer progression. Zhang et al. demonstrated that cancer cells could uptake exosomes derived from hypoxic bone marrow-derived mesenchymal stem cells (BMSCs) and acquire higher invasiveness. During this process, miR-193a-3p, miR-210-3p, and miR-5100 transferred into cancer cells and activated STAT3 signaling, thus inducing EMT in lung cancer cells. Furthermore, these three miRNAs were upregulated in the plasma-derived exosomes of patients than non-metastatic patients .Metastatic colonization is profoundly affected by the hypoxic tumor microenvironment. Tumor-derived exosomes can reflect the hypoxic characteristics of the originating cell and also are capable of facilitating pre-metastatic niche formation by transmitting non-coding RNAs. Notably, organ-specific metastasis has gained much attention in recent years. Despite the hard work of many scientists, still little has been known about the mechanisms. Exploring exosomal non-coding RNAs could give rise to promising therapeutic targets as well as prognostic biomarkers of cancer metastasis.Resistance to anti-cancer therapies is one of the biggest obstacles in cancer treatment. Intrinsic resistance arises because of genetic alterations, whereas extrinsic resistance due to interactions between tumor cells and microenvironment against chemotherapy. Hypoxia affects the overall course of the tumor including responses to therapies. Hypoxia-induced malignancies could form resistance to various anti-cancer drugs including cisplatin, doxorubicin, sorafenib, etoposide, paclitaxel, and gemcitabine . Also, rDong et al. demonstrated that hypoxic non-small-cell lung cancer (NSCLC) cell-derived exosomes enhanced cisplatin resistance of normoxic cancer cells through transmitting miR-21. Mechanically, miR-21 in hypoxic exosomes downregulated phosphatase and tensin homolog (PTEN) and PI3K/ATK pathway sequentially, triggering cisplatin resistance in normoxic cancer cells. Further analysis with the TCGA database showed that miR-21 expression was positively correlated with HIF-1α and high miR-21 expression indicated worse survival in patients undergoing chemotherapy . Patel aIt is noteworthy that chemoresistance does not arise intrinsically in cancer cells. Many stromal cells like CAFs also support cancer cells to gain resistance to chemotherapy. MiR-223 was demonstrated to promote proliferation and invasiveness of ovarian cancer cells by targeting SOX11 expression , and it These studies indicated chemoresistance could be passed through transmitting non-coding RNAs in exosomes in the hypoxic microenvironment. Blocking chemoresistance-associated exosomal non-coding RNAs could be a promising way to overcome chemoresistance.In principle, tumor development can be monitored by cytotoxic innate and adaptive immune cells. However, as the tumor develops from dysplasia to clinically detectable tumors, cancer cells evolve different mechanisms such as losing expression of tumor antigens or modifying immune checkpoint molecules in order to avoid the destruction by the immune system . Tumor-dThe interactions between tumor cells and T cells are critical in tumor immune microenvironment. Tumoral exosomes carrying biologically active non-coding RNAs transmit signals between cells, promoting tumor progression and immune escape. Ye et al. first reported that nasopharyngeal carcinoma (NPC) cell-derived exosomes impeded T cell dysfunction through differential miRNA expression . BuildinT cell exhaustion is characterized as the upregulation of PD-1 on T cells. Blocking the PD-1/PD-L1 axis is the best way to reinvigorate T cell function . γδ T ceHypoxia in the tumor microenvironment could stimulate immunosuppressive effects attenuating cytotoxic T lymphocyte (CTL) and NK cell-mediated tumor cell lysis. Berchem et al. elucidated that lung cancer cells generated exosomes with higher miR-23a expression, which could impair NK cell cytotoxicity and NK cell function by directly targeting CD107a. Furthermore, higher TGF-β in the exosomes might partly contribute to the enrichment of miR-23a. This is the first study demonstrating how cancer cells in the hypoxic microenvironment educate NK cells through exosome transmitted non-coding RNAs .Macrophages can be roughly classified into M1  macrophages with pro-inflammatory effects and M2  macrophages with anti-inflammatory effects. M1 macrophages characterized by the expression of the inducible type of nitric oxide synthase (iNOS) are proinflammatory, whereas M2 macrophages express high level of anti-inflammatory cytokines  and a potent arginase-1 (Arg1) activity to favor tumor cell growth . TAMs coHypoxic EOC cell-derived exosomes containing high levels of miR-940 stimulated the M2 polarization of macrophages. Then, miR-940-induced M2 macrophages, in turn, promoted EOC cell proliferation and migration. Interestingly, a high level of miR-940 was also observed in the malignant ascites-derived exosomes compared with benign peritoneal fluids . The samHypoxia pressure promoted the production and secretion of immune-modulating exosomes rich in non-coding RNAs. Exosome-mediated cancer-immune cell communication substantially impacts the immune reaction. Immune checkpoints are accessory molecules with T cell activation or inhibition effects. Blockage of cytotoxic T lymphocyte antigen 4 (CTLA-4) and PD-1 have heralded the dawn of an immune therapy era. Exosomal non-coding RNAs might be the indispensable targets for immune therapy based on their immune regulating functions.Angiogenesis refers to a new blood vessel-forming process and is an essential mediator for tumor progression. Hypoxia is one of the key factors in tumor angiogenesis. Vascular immaturity and weakened cell association of tumor blood vessel network can lead to excessive permeability, poor perfusion, and increased hypoxia . AngiogeRecent studies have demonstrated that exosomal non-coding RNAs under hypoxic conditions could contribute to the angiogenesis process. For instance, miR-210 upregulated both in hypoxic leukemia cells and exosomes enhanced tube formation in endothelial cells . In NSCLHypoxic exosomal non-coding RNAs can regulate angiogenesis by inducing phenotypic and functional changes in endothelial cells to promote tumor growth and metastasis. VEGFR inhibitors serve as the most vastly applied anti-angiogenesis agents now. They are bringing beneficial efficacies as well as nonnegligible problems like a double-edge sword. Exploiting the anti-angiogenesis potential of RNA therapy via exosomes might be a new therapeutic strategy in the future.Liquid biopsy is a new diagnostic tool performed on blood or other biofluids to assess the tumor-derived components and their genomic or proteomic profiles , 119. CiIn vitro studies have shown that hypoxia could promote the production and release of exosomes of various cancer cell lines , 87, 94.2+ levels, or drug intervention [However, the exosome releasing and producing processes are complicated and multifaceted. Hypoxia is not the sole factor influencing quantity and quality of exosomes. Besides, the mechanism of hypoxia in vivo might be quite different with that of in vitro models. Some other factors may also stimulate the release of exosomes including thermal stress, oxidative stress, tumor pH value, autophagy, endoplasmic reticulum stress, increase in intracellular Carvention . TherefoNon-coding RNAs can be transferred through exosomes from tumor cells to adjacent cells or remote organs by entering the blood circulation. These non-coding RNAs are protected from degradation and stably exist in the circulation. Thus, exosomal non-coding RNAs derived from hypoxic tumors are detectable in the blood and might be used as biomarkers of cancer progression Table . ExosomeHypoxia-induced exosomal miR-23a and miR-24-3p were reported to be markedly higher in the serum of lung cancer patients and NPC patients, respectively, than that of healthy volunteers. Further analysis showed that the serum exosomal miR-24-3p level was negatively associated with the DFS of NPC patients , 95. LikHypoxia influences not only cancer cells but also stromal cells. Zhang et al. reported that hypoxic BMSC-derived exosomes could promote lung cancer metastasis through transmitting miR-193a-3p, miR-210-3p, and miR-5100. Those three miRNAs expressed remarkably higher in the plasma-derived exosomes of lung cancer, liver cancer, and pancreatic cancer patients than the counterparts of healthy controls. In addition, ROC analysis of the individual miRNA or combination of three miRNAs panel all showed high specificity and sensitivity in distinguishing metastatic lung cancer patients and non-metastatic lung cancer patients .Circulating exosomes can reflect the hypoxic feature of primary tumors. Therefore, non-coding RNAs loaded in circulating exosomes are emerging as new biomarkers of liquid biopsy in that they can dynamically mirror tumor burden or clinical events. As tumor progression and evolution is an intricate process, a single biomarker is far from comprehensively explaining the disease status. Integrative analysis of multiple biomarkers might be a prospective trend for clinical diagnosis. Although these researches have brought out new insights into the diagnostic and prognostic value of hypoxic tumor-derived exosomal non-coding RNAs, there are still some limitations. Studies discussed here had a minimal patient sample size so that large-scale investigations are highly demanded to validate the clinical potential of hypoxic exosomal non-coding RNAs. Hypoxic exosomes can partially reflect the hypoxic feature of solid tumors. Researchers used different methods to induce hypoxia pressure which varied from oxygen concentration, hypoxic incubation time, etc. By far, there are no optimal in vitro systems to precisely mimic the in vivo hypoxic tumor microenvironment. Hypoxic exosome concentration and cargo may fluctuate depending on different hypoxia generating means. Meanwhile, they were using different ways to isolate exosomes, which may cause nonnegligible variations as well. Thus, standardized protocols that can generate high purity of exosomes are undeniably needed for future clinical applications. Furthermore, recent researches preferred to use RNA sequencing or microarray to screen differently expressed non-coding RNAs, which could generate vast sets of candidate non-coding RNAs but at the same time increasing the difficulty of data analysis to a large extent. How to better analyze the data and related pathways also remains a big issue. Notably, which molecule to use to normalize the exosomal lncRNAs or miRNAs is still controversial.120 siRNA loaded in human-foreskin fibroblast-derived exosomes efficiently suppressed pancreas desmoplasia in the orthotopic and genetically engineered mice [Exosomes are stable, easy-stored, biocompatible, highly permeable, low toxic, and immunogenic, which suffice almost all the merits of a good drug delivery system . Exosomered mice . Despitered mice .Exosomes shuttled between different cells and have a crucial role in intercellular communication. Exosomes encapsulate a variety of biologically active molecules, including lipids, proteins, and nucleic acids. In the hypoxic tumor microenvironment, exosomes present an altered pattern of genes and proteins. Hypoxia-induced non-coding RNAs enveloped in exosomes have been demonstrated to be involved in multiple physiological processes including cancer proliferation, pro-metastasis niche formation, chemoresistance, angiogenesis, and immune reactions. However, deeper mechanisms underlying the releasing and cargo machinery in the hypoxic conditions need to be further explored.Given that hypoxic exosomal non-coding RNAs are determinable in the body fluids, they are emerging to be fine analytes for liquid biopsy. However, numerous obstacles await resolutions before clinical translation. A technical hurdle worth mentioning is how to generate pure and homogenous exosomes. Currently, the most commonly used techniques for isolating exosomes include traditional ultracentrifugation, exosome precipitation reagents, density gradient separation, immunomagnetic beads, and ultrafiltration. However, these methods have various drawbacks in several aspects like specific equipment-dependence, complicated and time-consuming procedures, low productivity, and purity. Therefore, more precise, reliable, and accessible approaches are in strong need for isolation and characterization of exosomes. Recently, some novel exosome isolating methods have come out, such as microfluidic devices, nanoplasmon-enhanced scattering (nPES), membrane-mediated exosome separation, and lab-on-a-chip devices. On the one hand, these methods may simplify the isolation procedures and improve the productivity or purity of exosomes. On the other hand, there is still a lack of laboratory testing experience and preclinical practices . HopefulRecent studies have shown that gene therapy and immunotherapy manipulating exosomes are no longer just a fantasy, although numerous obstacles await resolutions before clinical translation. Exosomes can be obtained from various cells. However, it is not sure if the cellular origin would affect the therapeutic effects of exosomes. Efforts should be made to find the optimal therapeutic source of exosomes and improve loading efficiency. Another major obstacle following by efficient encapsulation of specific agents like non-coding RNAs is the delivery route. There is still a lack of verification if these modified exosomes should be delivered through local inoculation directly into the tumor sites or by systemic administration. More in-depth investigations are strongly required before clinical translation.https://clinicaltrials.gov/. Among these studies, 17 are not enrolling yet, 70 are active and recruiting, 4 are enrolling by invitation, 20 are active but not enrolling, 26 were completed, 1 was withdrawn, and 16 are in unknown status. Overall, the preclinical and clinical studies are just over the horizon, but the dawn of an exosome era is expected with the indefatigable endeavor of scientists.Currently, there are 158 exosome-associated clinical trials registered at


Membrane transporters allow the selective transport of otherwise poorly permeable solutes across the cell membrane and thus, play a key role in maintaining cellular homeostasis in all kingdoms of life. Importantly, these proteins also serve as important drug targets. Over the last decades, major progress in structural biology methods has elucidated important structure-function relationships in membrane transporters. However, structures obtained using methods such as X-ray crystallography and high-resolution cryogenic electron microscopy merely provide static snapshots of an intrinsically dynamic, multi-step transport process. Therefore, there is a growing need for developing new experimental approaches capable of exploiting the data obtained from the high-resolution snapshots in order to investigate the dynamic features of membrane proteins. Here, we present the basic principles of hydrogen-deuterium exchange mass-spectrometry (HDX-MS) and recent advancements in its use to study membrane transporters. In HDX-MS experiments, minute amounts of a protein sample can be used to investigate its structural dynamics under native conditions, without the need for chemical labelling and with practically no limit on the protein size. Thus, HDX-MS is instrumental for resolving the structure-dynamic landscapes of membrane proteins in their apo (ligand-free) and ligand-bound forms, shedding light on the molecular mechanism underlying the transport process and drug binding. Membrane proteins participate in fundamental physiological events in every biological system from bacteria to humans. >50% of marketed drugs target membrane proteins, whereas the pharmacological targeting of many other membrane proteins is considered potentially beneficial in numerous biomedical applications . HoweverSecondary transporters are membrane-bound proteins that selectively catalyze the movement of poorly permeable solutes  across the cell membrane, supporting diverse cellular functions in health and disease. Secondary transporters comply with the alternating access paradigm, according to which the protein ligand-binding pocket becomes alternatively accessible at opposite sides of the membrane by adopting the inward-facing (IF) and outward-facing (OF) states in succession . Recent 2O with proteins backbone amide hydrogens under native conditions. The deuterium exchange rate depends on the solvent accessibility, secondary structure, and structural rigidity (Hydrogen-deuterium exchange mass-spectrometry (HDX-MS) gained attention in recent years for studying membrane proteins dynamics, even though it has been used for decades to characterize soluble proteins . HDX-MS rigidity . CoupledHDX-MS principles are briefly and qualitatively described below, to allow a discussion of its applications for studying transporters. For in-depth explanations, several excellent review articles are available .2O buffer (i) at neutral pH they are exchanged in rates that can be detected using HDX-MS (seconds to days) and ii) their exchange can be effectively quenched , resulting in ~10,000-fold reduction in HDX  to pHn in HDX ; and by n in HDX . Next, ts review .Despite sharing the alternating access paradigm, ligand-induced conformational changes differ among transporters due to differences in ligand-protein interactions. For example, symporters (co-transporting two or more ligands) can transition between the IF and OF states without bound ligands, whereas ligand binding is obligatory for swapping between the IF and OF states in antiporters (exchanging ligands at opposite sides of the membrane) .In general, detecting local ligand-induced dynamic changes in proteins is difficult. For example, the crystal structures of membrane proteins in both the ligand-free and ligand-bound forms are frequently unavailable, precluding the detection of ligand-induced conformational changes even for proteins with known structures. Moreover, high-resolution structural snapshots of the apo and ligand-bound states may not resolve significant conformational differences. However, small ligand-induced conformational changes can be detected using HDX-MS at specific protein subdomains under native conditions by measuring the differential deuterium uptake (ΔHDX) in the presence and absence of ligands. This ability of HDX-MS provides unique opportunities for addressing the most challenging issues in elucidating the mechanisms of ligand-protein and protein-protein interactions , as revi+], and volume throughout the kingdoms of life , sharing a conserved architecture despite their diverse functions . Althougvs. the WT, whereas the opposite occurs for residues at the intracellular vestibule. Next, the transporters were reconstituted into nanodisks composed of phosphatidylglycerol, tetraoleyl cardiolipin, and either phosphatidylcholine or phosphatidylethanolamine. Interestingly, the presence of phosphatidylethanolamine shifted the equilibrium toward the IF state. Subsequently, MD simulations of the transporters in lipid bilayers identical to the nanodisks' composition predicted direct interactions between the charged phosphatidylethanolamine headgroup and a conserved cytoplasmic network of charged residues, stabilizing the OF state  family, which includes important drug targets such as the serotonin, dopamine, and norepinephrine transporters  revealed four ion binding sites, simultaneously occupied by three Na+ ions at sites termed Sint, Smid, Sext, and one Ca2+ ion at a site termed SCa  exhibits exceptional ion selectivity, exchanging Ca2+ with either Na+ or Li+, whereas NCXs do not transport Li+  assigned to NCX_Mj. These results suggested that NCLX_Mj may transport ions with an electroneutral stoichiometry of 2Na+:1Ca2+ or 2Li+:1Ca2+. Consistent with the HDX-MS data, voltage clamping accelerates the Na+/Ca2+ exchange rates in NCX_Mj-reconstituted proteoliposomes (due to a stoichiometry of 3Na+:1Ca2+), whereas it has no appreciable effect on the Na+/Ca2+ or Li+/Ca2+ exchange rates in NCLX_Mj  (D.K) and by the Israel Cancer Research Foundation (Grant #19202) (MG). Financial support from the Shmuel Shalit award to DK is gratefully acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.