We compared patterns of tobacco product use and nicotine and toxicant exposure among cigarette-only smokers (n = 127); e-cigarette-only users (n = 124); dual users of tobacco cigarettes and e-cigarettes (n = 95); and non-users  across three countries using mixed-effects linear regression. Compared with cigarette smokers, e-cigarette-only users had lower levels of toxicant biomarkers, but higher levels of nicotine biomarkers. Dual users had higher levels of toxicant biomarkers than e-cigarette-only users but similar levels to cigarette-only smokers. E-cigarette users in Poland, who overwhelmingly used refillable tank devices, exhibited greater levels of nicotine, and toxicant biomarkers relative to e-cigarette users in US/UK. Despite smoking fewer cigarettes, dual users from Poland exhibited similar levels of nicotine biomarkers compared with UK dual users, but higher than US dual users. Country-level differences in e-cigarette devices used and smoking behaviors  may contribute to differences in biomarker levels among users of the same products residing in different countries. Country-level differences in nicotine vaping products used and biomarkers of exposure among long-term e-cigarette users and dual users remain understudied. This cross-sectional study was conducted in 2014 in the United States ( Worldwide, electronic cigarettes (e-cigarettes) have become increasingly popular . ProponeSafety concerns have emerged regarding toxicant levels in e-cigarette emissions, in light of research showing the presence of contaminants in the products, and generation of thermal degradation byproducts of nicotine solvents during use . High quMost studies that examine nicotine exposure in exclusive e-cigarette users and dual users are limited to a single geographic location, failing to account for potential differences that may occur based on background environmental exposures, product availability, norms, and policies in different nations ,4. O’ConIt is currently unclear whether e-cigarette users who live in countries that have imposed maximum allowable nicotine concentration in e-cigarette liquids  are exposed to lower levels of nicotine as compared to users who live in countries where highly concentrated nicotine solutions for e-cigarettes are widely available . This demonstrates a need to evaluate exposure to nicotine and toxicants in long-term single or dual users of electronic and conventional cigarettes across multiple countries. This study investigated a broad range of biomarkers of exposure to nicotine and toxicants in long-term users of nicotine-containing products. The research addressed four main questions: (1) Is nicotine intake significantly different between long-term users of cigarettes, e-cigarettes, and both products from different countries? (2) Are long-term exclusive users of e-cigarettes in different countries exposed to similar levels of toxicants? (3) How does exposure to nicotine and toxicants among long-term exclusive e-cigarette users from different countries differ from exposure among exclusive smokers and non-users who live in the same country? and (4) How does exposure to nicotine and toxicants among long-term dual users of e-cigarettes and cigarettes from different countries compare to exposure among exclusive smokers who live in the same country?n = 127); (2) e-cigarette-only users (n = 124); (3) dual users who smoked tobacco cigarettes and also used e-cigarettes (n = 95); and (4) non-smoking, non-e-cigarette users (n = 110). The study was conducted in 2014 at three sites located in the United States (n = 166), the United Kingdom (n = 129), and in Poland (n = 161). Using data from a cross-sectional study published by Shahab et al. , we perfParticipants were recruited from local metropolitan areas using advertisements and underwent a telephone screening to determine eligibility and classification into one of the study groups based on self-reported behavior within the six-month period preceding their visit. “Non-smoking, non-e-cigarette users” (non-users) reported no use of any nicotine-containing products within the past six months. “Cigarette smokers” reported daily cigarette smoking for at least the past six months and smoked at least 5 cigarettes per day (CPD). “E-cigarette-only users” reported daily e-cigarette use for at least the past 6 months and used at least 10 nicotine-containing cartridges per week or 2 bottles of nicotine solutions or at least 5 disposable products per week. “Dual-users” reported a minimum e-cigarette use of 5 nicotine-containing cartridges/one bottle of nicotine solution/2 disposable e-cigarettes per week and smoked at least 2 CPD for at least the past six months. Additional inclusion criteria included: must be 18 years of age or older, report no current or past year incidence of kidney disease, and no serious psychiatric conditions. Exclusion criteria included pregnancy or current breastfeeding; inability to communicate in the primary language at the study site; or current use of smokeless tobacco, pipes, or cigars. Study activities involved a one-time visit lasting approximately one hour; participants were asked to refrain from eating food, consuming alcohol, or using the lavatory in the hour preceding their visit. At the visit, all participants provided written consent, completed a brief questionnaire, underwent an exhaled breath carbon-monoxide (CO) test (Bedfont Micro Smokerlyzer), and provided a urine sample. Urine samples were collected using a sterilized, sealable cup and were subsequently transferred into cryovials by study staff. All urine samples were shipped on dry ice to the U.S. Centers for Disease Control and Prevention (CDC) for laboratory analyses. Upon completion, participants were compensated for their time. All methods and procedures were approved by the Institutional Review Boards at Roswell Park Cancer Institute in USA, University College London in UK, and Medical University of Silesia in Poland.Participants were asked questions about demographic characteristics . Due to between-country differences in education, we grouped participant education into two distinct categories: low  and high . E-cigarette-only users, cigarette smokers, and dual users were asked about current patterns of e-cigarette and/or tobacco cigarette use . Due to differences in questionnaire design, we estimated the number of puffs taken per day on an e-cigarette for UK participants by taking the reported number of e-cigarette cartridges used per week multiplied by 200 (average puffs per cartridge), then divided this figure by 7 (days per week) ,10. At tn-oxide and cotinine N-oxide, norcotinine, and nornicotine (nmol/mg creatinine).All urine samples were analyzed by the CDC using mass spectrometric methods as previously described for 7 metabolites of nicotine , 18 meta2 tests were used to compare characteristics for categorical variables. For the main analyses, biomarker levels below the limit of detection were entered using a common substitution method , [p-values were adjusted for multiple comparisons using Sidak’s method and were considered statistically significant at p < 0.05. In descriptive analyses, missing data were handled using listwise deletion; models were fit using maximum likelihood estimation. All analyses were conducted using Stata v.16.1.The sample size was determined using NNAL as the primary outcome measure to detect between-group differences according to tobacco use. We determined that a total of 180 participants (45 per group) would provide 80% power to detect between-group differences in NNAL levels with a moderate effect size of f = 0.25 . AnalyseLOD/√2),  and werep < 0.05), but were statistically similar in age compared to exclusive e-cigarette users and non-users (data not shown). The pooled sample was equally balanced in terms of demographics, socio-economic status and tobacco use groups across countries and the mean age (±SD) of participants was 40 ± 14 years (data not shown). Across countries, all participants differed by sex (p = 0.006) and race . US participants were significantly older than those in the UK or Poland , F = 18.775, p < 0.001, Comparisons of the pooled sample demographics by tobacco use status showed significant differences by age and sex across non-users, e-cigarette users, dual users, and cigarette smokers (data not shown). Specifically, dual users were significantly younger than exclusive cigarette smokers . E-cigarette-only users: UK e-cigarette-only users were less likely to report using refillable tank model e-cigarette devices than those in the US or Poland (p < 0.001). No statistically significant differences in cigarette consumption were observed in cigarette-only smokers according to country.Nearly three-quarters of all participants who reported using e-cigarettes  used refillable tank model e-cigarettes; 70% reported use of medium (10–15 mg) or high (16–24 mg) nicotine content products and averaged 90 ± 12 puffs per day on an e-cigarette. All cigarette smokers  reported smoking an average 13 ± 9 CPD; dual users smoked significantly fewer CPD compared to exclusive smokers  and N,N-dimethylformamide ; and TSNAs  than cigarette-only smokers  among dual users compared with cigarette-only smokers  relative to those in the US and UK The pattern of differences in biomarker concentrations between exclusive e-cigarette users, dual users, exclusive cigarette smokers, and non-users were relatively similar within each country. A novel finding is that e-cigarette users showed significantly higher levels of biomarkers of nicotine exposure than cigarette smokers. Previous studies suggest that exposure to nicotine from e-cigarettes is lower or equivalent to exposure from conventional cigarettes ,16. The To further explore this hypothesis, we conducted a post-hoc analysis of nicotine biomarkers among users of different types of e-cigarettes . DespiteUrinary levels of most toxicant biomarkers measured in this study did not differ significantly between e-cigarette-only users and non-users, except for a small number of VOCs and TSNAs. Our findings also showed that while being exposed to high nicotine levels, long-term exclusive e-cigarette users showed significantly lower levels of toxicant biomarkers compared with cigarette smokers or dual users. These results are consistent with a number of studies examining toxicant levels in e-cigarette users in contrast to conventional tobacco users ,18,19,20Our novel observations related to differences in toxicant exposure among exclusive e-cigarette users from different countries may be a function of country-level differences in e-cigarette device availability, brand visibility, and consumer preferences. First, second, and third generation e-cigarette devices differ in the emissions of toxicants . While oResults also suggest that while many biomarker levels were statistically similar between dual users and cigarette-only smokers, significantly lower levels for selective smoke exposure biomarkers  were observed among dual users compared to smokers. Considering that dual users in our sample smoked fewer cigarettes overall compared with cigarette-only smokers, we suggest that this may be a function of differences in CPD among this group. This hypothesis appears to be consistent with observed differences in biomarkers levels in dual users from different countries: UK dual users, who smoked more CPD compared to Polish dual users, showed statistically higher levels of exposure biomarkers to several toxicants, including NNK, NAB, NAT, acrolein, acrylamide, benzene, 1,3-butadiene, crotonaldehyde, cyanide, styrene, and xylene. Therefore, it would appear that some cases of dual use may be associated with reduced exposure to some tobacco-related toxicants, depending on the relative frequency of e-cigarette use versus cigarette smoking, the number of cigarettes smoked, and type of e-cigarette device used ,22. In ap > 0.05), which is a tobacco-specific biomarker found in measureable concentrations in secondhand smoke [This is the first cross-national study to investigate differences in exposure to nicotine, TSNAs, and VOCs in established e-cigarette users, cigarette smokers, and dual users from different countries. An important strength of this study is that we examined exposure levels among a large sample of adults from different international locations with varying levels and types of product use. E-cigarette users in our study used various types of devices. Also, tobacco smokers and dual users smoked a different number of tobacco cigarettes of various brands. By including control subjects from the same populations, we were able to, at least partially, eliminate the bias of environmental and occupational exposures. Other important sources of exposure—such as dietary and environmental exposures, and including potential exposure to secondhand smoke—should be considered in future studies. Sensitivity analyses conducted among non-users across all three countries revealed no statistically significant differences in concentrations of urinary NNAL , . which, Exclusive, long-term e-cigarette use resulted in higher toxicant exposure compared with not using any product, and reduced toxicant exposure compared with cigarette smoking or dual use with cigarettes, while delivering higher levels of nicotine. Differences in exposure to nicotine and toxicants in e-cigarettes from different countries appear to correspond with differences in popularity of various e-cigarette device types across countries. Dual users exhibited a similar level of exposure to nicotine and many toxicants compared with cigarette-only smokers, yet exposure to toxicants in dual users from different countries appears to be correlated with their smoking intensity.


We applied inferential statistics (the COVID-engine platform) to RNA profiles (from limited number of samples) derived from peripheral blood mononuclear cells (PBMCs). Compared to healthy individuals, a subset of integrated blood-based gene profiles (signatures) distinguished acute-like  from recovering-like patients. These signatures also hierarchically represented multiple (at the system level) parameters associated with PBMC including dysregulated cytokines, genes, pathways, networks of pathways/concepts, immune status, and cell types. Proof-of-principle observations included PBMC-based increases in cytokine storm-associated IL6, enhanced innate immunity (macrophages and neutrophils), and lower adaptive T and B cell immunity in patients with acute-like disease compared to those with recovery-like disease. Patients in the recovery-like stage showed significantly enhanced TNF, IFN-γ, anti-viral, HLA-DQA1, and HLA-F gene expression and cytolytic activity, and reduced pro-viral gene expression compared to those in the acute-like stage in PBMC. Besides, our analysis revealed overlapping genes associated with potential comorbidities (associated diabetes) and disease-like conditions . Overall, our COVID-engine inferential statistics platform and study involving PBMC-based RNA profiling may help understand complex and variable system-wide responses displayed by coronavirus-infected patients with further validation.COVID-19 patients show heterogeneity in clinical presentation and outcomes that makes pandemic control and strategy difficult; optimizing management requires a systems biology approach of understanding the disease. Here we sought to potentially understand and infer complex disease progression, immune regulation, and symptoms in patients infected with coronaviruses (35 SARS-CoV and 3 SARS-CoV-2 patients and 57 samples) at two different disease progression stages. Further, we compared coronavirus data with healthy individuals ( According to the World Health Organization (WHO), the mode of infection for COVID-19 is predominantly through respiratory droplets, aerosol transmission due to pathogen-laden viral particles in the air, or close contact with infected people with increased viral loads, especially in the early stages of disease2. The mechanism of human pathogenesis, to a great extent, may simulate that of SARS-CoV (associated with SARS) and Middle East respiratory syndrome coronaVirus  viral infections, including the prolonged persistence of the virus worsening the host immune response5. Clinical manifestation of COVID-19 ranges from mild respiratory symptoms to severe disease and death6. However, there are now reports suggesting heterogeneous manifestation of the disease affecting multiple organs, including kidney, liver, and brain7. Although age and compromised health history are considered critical prognostic factors, certain patients of younger age and good health have shown severe progression of the disease8.The spread of COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the current global pandemic with already millions of people with confirmed infection and more than one million deaths10, but can still transmit infection11. Viral shedding from an infected person may occur, although resolution of symptoms12, and relapse has been reported despite consecutive negative testing13. Currently, there are no approved therapies for COVID-19 respiratory symptoms15 or way to screen for disease progression, and system-wide changes in patients.Patients with COVID-19 may be asymptomaticIn this study, we sought to understand and infer changes in coronavirus-infected patients at system levels through their peripheral blood mononuclear cells (PBMC) by performing comparisons with healthy volunteers by applying inferential statistics (IS). The inferences at the multiple levels of the system in patients provide an efficient way of understanding the heterogeneity and mechanism(s) of disease manifestation as a whole Fig. . These in = 10) and healthy volunteers (n = 4) from a published study16 (training data). We identified PBMC genes differentially expressed between patients and healthy volunteers by applying our in-house developed IS pipeline . We broadly identified 290 differentially expressed genes in patients and healthy volunteers using a supervised Statistical Analysis of Microarrays (SAM) approach  and platforms, we observed that our CoV-Up-gene signature from SARS-CoV was higher in COVID-19 patients’ PBMC than in the healthy volunteers (see “Methods”). In contrast, CoV-Down-gene signature was higher in healthy volunteers and lower in COVID-19 patients. This result suggests that our gene signatures from SARS-CoV may be applicable to COVID-19 patients  from patients infected with bacteria and influenza, we observed a broadly similar pattern  of the signatures that were derived from PBMC (mostly associated with specific diseases) were significantly  < 0.2) enriched for our CoV-Up-gene signature . Lee et al. defined acute samples (n = 25) as those that tested positive (using blood) for SARS-CoV during hospitalization or within 10 days of onset of the disease in patients. The samples derived from acute patients were also correlated with disease severity including an increased clinical pulmonary infection score21. The remaining samples were labeled as recovering samples (n = 19). Hence, acute vs. recovering samples refer to different stages of the disease that can occur in the same patient as specific samples were collected from the same individual during hospitalization.Next, we sought to assess the potential of our CoV-Up-gene signature to stratify patients into those at different progression stages of the disease (progression): acute vs. recovering. For this, we used PBMC transcriptome data from a limited size of 44 samples from the longitudinal collection over the disease course from a published study 22. This resulted in 7 of the 10 patient samples showing maximal similarity to acute phase (termed acute-like patients), while 3 samples scored similar to recovering patients  genes showed LDHB was highly expressed in healthy individuals compared to coronavirus-infected patients, and inverse trends were observed for ACE2 and LDHA . We compared the expression levels of cytokines namely nts Fig. . Interesals Fig. . BesidesDHA Fig. . Among t24. Among those genes, we observed CXCL8 (IL8) and CCL13, which are associated with chemoattraction of neutrophils/macrophages (innate immunity), to be highly expressed in acute-like patients, compared to recovering-like patients and healthy individuals  and inhibition of viral replication were highly expressed in recovering-like patients and healthy individuals  were also implicated, consistent with the oxygen limitationion Fig. . Of parttes Fig.  as sugge28 to connect different but related pathways that were enriched in infected patients using network analysis. Two evident and distinct networks were significantly enriched in CoV-Up-gene signature: (a) interleukins and cytokine signaling  and (b) neutrophils and innate immunity -based extensive analysis of network of molecular pathways suggested further understanding of functions and utilities at the levels of cells and organisms. Our KEGG analysis with CoV-Up-gene signature overlapped with different infections, including nia Fig. . This suion Fig. . There iase Fig. .33 . In this case, we separated the coronavirus patient samples into those with acute-like or recovering-like disease and compared these with healthy control samples. As expected, we observed that the innate immune system involving macrophages and neutrophils were highly active in the acute-like patients, suggesting that they may be the first to encounter the coronavirus, with these decreasing in recovering patients  in the recovering patients. Certain results from SARS patients were assessed using PBMC from limited number of COVID-19 samples  increase in natural killer (NK) cells, cytolytic activity, and plasmacytoid dendritic cells (pDCs) in recovering-like patients compared to acute-like patients was observed Fig. . It is nnts Fig. . This re increaseCD4+/CD8+ T cells, and whose levels are increased by IFN-γ . While most of the MHC class-II HLA genes showed no difference in expression levels between the acute-like and recovering-like patients, HLA-DQA1 showed an increasing trend in the recovering-like patients towards the healthy individuals and low level in acute-like patients. Similarly, MHC class-I HLA gene, HLA-F showed a trend akin to HLA-DQA1 . On the other hand, there was no change in the antigen processing machinery (data not shown). However, all these speculations from limited data warrant further validation, and their functional immunological significance is currently unclear.Next, we examined the differential expression of major histocompatibility complex (MHC) class-I and class-II HLA that may reflect antigen presentation to and/or activation of N-γ Fig. . Among t20 showed similar conclusions that innate immunity and myeloid (neutrophils and macrophages) cells are upregulated in coronavirus patients , whereas adaptive immunity is downregulated in these patients  and may provide the opportunity to save patients who are most likely to die of the disease.The clinical course of COVID-19 patients remains enigmatic, and no treatment options exist with proven efficacyIn this proof-of-concept study, we performed a comprehensive analysis using publicly available blood cell RNA profiles from SARS and COVID-19 patients and cross-validated with patients with other infections or healthy individuals. Using IS approaches in our COVID-engine platform, we were able to develop a blood cell RNA profile-based gene signatures that are differentially expressed at different stages of infection (acute vs. recovering). In addition, our COVID-engine platform provided hierarchical and comprehensive analysis describing infection-associated changes in genes, pathways, networks, subcellular components, and cells, covering almost the whole system. This “integrated” analysis can help understand which other disease-related symptoms could manifest in COVID-19 patients.IL6, TNF, IL8 (CXCL8) and CCL13) is high in CoV-Up-gene signature and specifically in acute-like patients. Macrophages and monocytes are known to serve as factories for viral replication in other disease conditions24. These changes in immune cells may also be connected to increased neutrophil counts in these patients39. The cytokine storm-related to innate immune changes may be linked to changes in angiogenesis and coagulation, suggesting a potential relationship between inflammation, thromboembolism40, and coagulation41. While there is no change in overall CD8+ T cell population between patients in their acute-like vs. recovering-like stages , the change in cytolytic activity, pDCs, and NK cells suggests that the adaptive immunity is a late event represented in patients recuperating from this disease42. Congruently, this is associated with lower innate immunity in recovering-like patients than acute-like patients and associated with increased expression of anti-viral genes OAS2 and IL16.Our results, using limited coronavirus-infected patient samples, represent that the innate immune system associated with increased neutrophils, macrophages, and monocytes with potential cytokine storm 43, in recovering-like patients suggests the anti-viral effect in these individuals. Remarkably, the supposedly anti-viral CD4 T and NK cells, along with B cells, are low in coronavirus patients and are associated with low HLA-DR-expressing monocytes in these patients with severe respiratory failure45. This report corroborates with our results that HLA-DRA gene and all the above three cell types are low in acute-like patients. Specifically, B cell-based adaptive immunity seems to vary among patients and mostly low in coronavirus patients with severe respiratory failure46. Our data suggest that this may impact the development of effective vaccines for this infection. In addition, CoV-Up-gene signature was high in methicillin-resistant Staphylococcus aureus  and test (n = 47) samples from less annotated datasets with limited clinical data, which may be appreciated provided the current global lock-down scenario. Also, the acute-like and recovering-like patients may overlap with the symptomatic and asymptomatic patients, respectively, described in the original publication from where the training dataset was derived16. Further validation was curtailed due to lack of associated clinical data, which is difficult to obtain in the current scenario.Although our study may be timely for the current pandemic, there are limitations. We have performed analysis using publicly available small number of coronavirus-based training (In conclusion, PBMC has information related to infection status, immune states, disease progression, severity, and disease conditions that are likely going to be manifested due to coronavirus infection and COVID-19 disease Fig. .Fig. 8CO19 with GEO Omnibus identifiers—GSE1739 and GSE6269, and normalized data directly from the original publications. Raw.CEL files from Affymetrix Human HG-Focus Target Array for GSE1739 were obtained from the authors of the original publication16 and robust microarray analysis-based normalization was performed using R-based Bioconductor package—affy49. GEOquery R package50 was used to obtained gene expression and/or phenotypic data from GEO Omnibus for GSE6269.PBMC transcriptomes from SARS, COVID-19, and other patients were obtained from published studiesCOVID-engine refers to a pipeline of different IS methods described below.51 using R-based siggenes package52. Differentially expressed genes with FDR < 0.05 were chosen as described in our previous publications55. Gene scores for signatures up or downregulated in coronavirus-infected patients in other validation datasets were derived using ssGSEA20 using R scripts from GenePattern platform56.PBMC-based differential gene signatures between healthy volunteers and coronavirus-infected patients were selected by performing supervised SAMBriefly, the differentially expressed gene expression signatures from PBMC of patients infected with coronavirus were used to query multiple databases for meta-signatures such as pathways, mechanistic processes, and their associated networks that are connected with different disease manifestations by enrichment analysis-based IS. These gene signatures and meta-signatures were further systematically linked to wire the pathophysiology in patients, again, in a hierarchical fashion, from cells to whole organism level Fig. .57. Disease conditions-based enrichment analysis was performed using R-based Dose package58. Nearest Template Prediction22 using R-based tool from GenePattern platform56 was used to derive distance between two signatures—acute-like vs. recovering-like patients. Different gene set databases were from downloaded from EnrichR59 and MSigDB20. Immune gene sets were from Rooney et al.34. Additional gene sets and databases used in this study are: REACTOME28, KEGG60, COMPARTMENTS database61, BioGPS—gene portal system36 and DisGeNE62.Hypergeometric gene enrichment analysis for most of the studies were performed using R-based hypeR toolt-test statistical analysis were performed where appropriate.Detailed information regarding intermediate data from COVID-engine are provided in Supplementary Tables Supplementary InformationSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Table 1Supplementary Table 2


Paramyxoviruses and pneumoviruses infect cells through fusion (F) protein-mediated merger of the viral envelope with target membranes. Members of these families include a range of major human and animal pathogens, such as respiratory syncytial virus (RSV), measles virus (MeV), human parainfluenza viruses (HPIVs), and highly pathogenic Nipah virus (NiV). High-resolution F protein structures in both the metastable pre- and the postfusion conformation have been solved for several members of the families and a number of F-targeting entry inhibitors have progressed to advanced development or clinical testing. However, small-molecule RSV entry inhibitors have overall disappointed in clinical trials and viral resistance developed rapidly in experimental settings and patients, raising the question of whether the available structural information may provide a path to counteract viral escape through proactive inhibitor engineering. This article will summarize current mechanistic insight into F-mediated membrane fusion and examine the contribution of structural information to the development of small-molecule F inhibitors. Implications are outlined for future drug target selection and rational drug engineering strategies. The paramyxovirus and pneumovirus families belong to the order Mononegavirales 1), which includes additional major human pathogen families such as the filoviruses and rhabdoviruses. Characteristic for the order are lipid-enveloped virions that contain a negative sense non-segmented (NNS) RNA genome. Genomic and antigenomic RNAs are viral nucleoprotein-encapsidated, resulting in the formation of ribonucleoprotein (RNP) complexes .,22.1,22]The pneumovirus G protein lacks both hemagglutination and neuraminidase activity. Structural information is very limited compared to that available for the paramyxovirus attachment proteins, but pneumovirus G is conformationally distinct and does not share the sialidase-like fold that is characteristic for paramyxoviruses. Whereas the paramyxovirus attachment proteins are furthermore essential for virion infectivity, recombinant RSV lacking G could be recovered and propagated in cell culture ,29,30, i0 that require proteolytic maturation. The physiological oligomer is a homo-trimer, and only the trimer can assume a native conformation and gain intracellular transport competence. Accordingly, newly synthesized F trimerize in the endoplasmic reticulum (ER), and in the case of the Morbilliviruses the ER is also the site of subsequent hetero-oligomerization of attachment and fusion protein complexes (24). F trimers assume initially a metastable prefusion fold that resembles a lollipop-like shape  pattern, the HR-A and HR-B domains, respectively [For most paramyxo- and pneumovirus F proteins, proteolytic maturation is mediated by host cell furin-type proteases resident in the Golgi apparatus. Cleavage generates for each monomer a larger, membrane-embedded Fide bond . The newectively . In the thermodynamically stable post-fusion conformation, F trimers assume an overall cone-shaped form D,F that Triggering of pneumo- and paramyxovirus F proteins for membrane fusion is receptor binding-induced and pH-independent. In the case of the paramyxoviruses, the attachment proteins head domains can assume a pre-receptor bound heads-down or post-receptor heads-up orientation relative to the attachment protein stalk domain. Truncated attachment proteins lacking the head domains were engineered for paramyxoviruses of the rubulavirus, morbillivirus, respirovirus, and henipavirus genus ,40,41,42As a common theme of fusion triggering, receptor binding results in a conformational rearrangement of the attachment protein head domains, which move from heads-down to heads-up position. In the case of the rubula- and respirovirus HN proteins, this change exposes an F interaction site on the HN stalk, resulting in hetero-oligomerization of HN tetramers and F trimers that triggers F refolding ,44. By cOnce the F refolding cascade has been activated either through triggering by the attachment protein or, in the case of the pneumoviruses, direct F interaction with receptor ,49,50, aCounting drugs in clinical use, drug candidates in clinical trials or under formal development, and early-stage experimental compounds, a large number of direct-acting entry inhibitors blocking pneumo- or paramyxovirus infection have been identified. Chemically, these include biopharmaceuticals such as neutralizing antibodies ,52,53,54Two predominant mechanisms of productive interference with F protein function by synthetic antivirals have emerged: late-stage inhibition of fusion core assembly with peptidic inhibitors and stabilization of a prefusion F conformation with small-molecule compounds. Most synthetic anti-F peptides are derived from residues of the homotypic or a closely related HR-B domain and block 6HB closure through competition with endogenous HR-B for access to the HR-A triple helix grooves . ConsequL141W) or lining the binding site . Resistance hot-spots are located at the interface between prefusion F head and stalk domains  [D401E and FD489E can destabilize the prefusion fold, narrowing the window of opportunity for productive interaction of the inhibitor with prefusion F by accelerating the rate of F refolding [As of February 2020, eight small-molecule RSV entry inhibitors have advanced to some stage of clinical testing . Common  FD489E) , which c FD489E) ,36. Cons FD489E) . Secondlefolding . This kiD401E do not mandatorily coincide with viral attenuation in vivo [Two factors in particular exacerbated the anticipated negative impact of rapidly emerging resistance on the clinical potential of RSV entry inhibitors. Firstly, several hot spots have been identified that mediate universal escape from inhibition by all advanced RSV entry inhibitors tested, despite the structural diversity of the different chemotypes . The exi in vivo , raisingM94G/N462S double mutant, which carries an additional substitution in a defined cavity formed by the F2 subunit and depends on dose-dependent stabilization by AS-48 to maintain an intracellular transport-competent conformation [M94G/N462S/AS-48 complex can be efficiently stimulated by MeV H upon receptor binding, underscoring that the compound indeed preserves a fusion-competent fold of F. Beyond RSV, very few pneumo- or paramyxovirus entry inhibitor candidates have been subjected to advanced structural characterization of the target docking pose and none has progressed to clinical testing. An exception is a chemically well-behaved small-molecule MeV inhibitor, AS-48, which potently blocks MeV F protein-mediated membrane fusion ,72. Primormation . Once diThe mechanistic characterization of AS-48 mode of action was confirmed through a high-resolution crystal structure of prefusion MeV F complexed with the inhibitor, which posited the compound at the base of the central F cavity in direct proximity of residue 462 . DespiteStructures of F proteins complexed with neutralizing antibodies have been solved for a number of pneumo- and paramyxovirus family members including RSV , HPIV3, 2), and include numerous polar and hydrophobic interactions [2 of a protein surface and target sites show concavity, ensuring favorable contacts with multiple sites of the ligand. Although it is therefore challenging to recapitulate the effect of nAbs with small molecules, functional mimicry can be achieved when hot-spot residues on the protein surface are known that contribute overproportionally to the interaction [Naturally, the druggable site near the base of the central prefusion F cavity is sterically not accessible for antibody binding. Pharmacodominance of this site for all highly potent small molecule entry inhibitors characterized so far is nevertheless remarkable, since mapping of nAbs has revealed this diverse set of alternative sites in membrane-distal regions of the F head that, when engaged, stabilize the F prefusion fold. The current lack of small molecule ligands directed against these antigenic sites presumably directly reflects the fundamental differences in the nature of binding sites and mode of target engagement by antibodies and small molecule drugs. Typically, protein-protein interfaces including those between antigen-antibody are flat in geometry, span a large internal surface  emerged in up to 11.2% of patients in treatment groups, but were absent from placebo-treated subjects . PatientThe disappointing outcome of the four presatovir phase IIb clinical trials certainly casts major doubt on the clinical potential of all currently pursued advanced F protein entry inhibitors, especially when considering the overlapping binding sites of the different chemotypes, similar mechanisms of activity, and existence of several pan-resistance hot-spots. Given the mechanistic similarities and the originally encouraging performance of presatovir in the human RSV challenge model, there is little reason to assume that the alternative entry inhibitor scaffolds that have been successfully tested in the challenge model may fare better in phase IIb trials. High-resolution structural model of entry inhibitors with prefusion F have greatly advanced the appreciation of the molecular mechanism of fusion inhibition but have not yet suggested a path to how viral pan-resistance could be counteracted through proactive ligand engineering. Despite sub-nanomolar potency in cell culture assays and initially very attractive drug profile properties, therapeutic targeting of the base of the prefusion F protein cavity may ultimately not be the most effective approach to address the RSV problem. Alternatively, allosteric and substrate-analog inhibitors of the RSV polymerase complex have been identified ,104,105,Ultimately, combining entry inhibitors with mechanistically distinct antivirals such as polymerase inhibitors may be the most powerful strategy. However, heightened developmental costs associated with this approach will affect economic viability, and entry inhibitors could lose all impact long-term, should resistant RSV transmit efficiently and viral strains emerge in circulation that carry pre-existing resistance mutations.


COVID-19 management guidelines have largely attributed critically ill patients who develop acute respiratory distress syndrome, to a systemic overproduction of pro-inflammatory cytokines. Cardiovascular dysfunction may also represent a primary phenomenon, with increasing data suggesting that severe COVID-19 reflects a confluence of vascular dysfunction, thrombosis and dysregulated inflammation. Here, we first consolidate the information on localized microvascular inflammation and disordered cytokine release, triggering vessel permeability and prothrombotic conditions that play a central role in perpetuating the pathogenic COVID-19 cascade. Secondly, we seek to clarify the gateways which SARS-CoV-2, the causative COVID-19 virus, uses to enter host vascular cells. Post-mortem examinations of patients' tissues have confirmed direct viral endothelial infection within several organs. While there have been advances in single-cell RNA sequencing, endothelial cells across various vascular beds express low or undetectable levels of those touted SARS-CoV-2 entry factors. Emerging studies postulate alternative pathways and the apicobasal distribution of host cell surface factors could influence endothelial SARS-CoV-2 entry and replication. Finally, we provide experimental considerations such as endothelial polarity, cellular heterogeneity in organoids and shear stress dynamics in designing cellular models to facilitate research on viral-induced endothelial dysfunctions. Understanding the vascular underpinning of COVID-19 pathogenesis is crucial to managing outcomes and mortality. This suInitial concerns were also raised regarding the medical treatment of hypertension with adverse COVID-19 outcomes, as studies in animals have shown that the use of renin–angiotensin system blockers–angiotensin-converting enzyme inhibitors and angiotensin receptor blockers result in the upregulation of angiotensin-converting enzyme 2 (ACE2) expression, which is an entry factor for SARS-CoV-2 . Hence, 2.The largest case series report to date, of 72 314 COVID-19 cases in mainland China, found that of the 44 672 confirmed cases, 12.8% had hypertension, 5.3% diabetes and 4.2% cardiovascular disease [In France, clinical characterization of 34 patients admitted to intensive care units with COVID-19-related acute respiratory distress syndrome found that 44.1% had diabetes, 38.2% had hypertension and 8.8% had ischaemic cardiopathy [Endothelial dysfunction, typically responsible for cardiovascular complications, plays a significant role in the pathogenesis of thrombosis in severe COVID-19 outcomes . Indeed,Furthermore, injuries of the pulmonary capillary endothelium increase its permeability, causing fluid leakage into the pulmonary parenchyma . This flInterestingly, the Randomised Evaluation of COVID-19 Therapy clinical trial from the University of Oxford   has show3.In situ hybridization study of fatal cases indicate that the primary target cells of SARS-CoV are the pneumocytes and surface enterocytes of the small intestine [To date, the pathogenesis and the extent of the damage directly or indirectly affected by SARS-CoV-2 is surfacing with new symptoms reported on a regular basis. SARS-CoV-2 was initially presented as targeting mainly the lung . Howeverntestine . Other ontestine , althougntestine . It was ntestine ,37. Highhttps://www.moh.gov.sg/news-highlights/details/350-more-cases-discharged-344-new-cases-of-covid-19-infection-confirmed). Most of those complications reflect a confluence of vascular dysfunction, thrombosis and dysregulated inflammation [Underlying a vascular dysfunction in COVID-19, rare Kawasaki-like multisystem inflammatory characterized by vasculitis—inflammation of blood vessel walls and coronary artery aneurysms—were recently reported in children –40. Furtammation , supportammation .et al. [Ace2/ACE2 RNA-sequencing counts for the brain and heart of mouse and human tissue, these samples also expressed pericyte markers, implying that the Ace2/ACE2-positive endothelial cells were contaminated with pericytes. Challenges associated with the dissociation of pericytes and endothelial cells for single-cell analysis could be responsible for the discrepancy in published data [ACE2 and TMPRSS2 mRNA are detected in these endothelial cells, only a scarce number of cells co-express both [Successful infection of a host cell by SARS-CoV-2 is a two-step process involving attachment via the receptor and membrane fusion for the release of viral RNA into host cell cytoplasm. Proteolytic activation of the viral spike protein by host proteases has been shown to be essential for the second step . It is wet al.  found a hed data . Those shed data ,51. Furtess both . On the ess both . It remaIn contrast with the transcriptomic data, protein-level analyses of ACE2 seem to suggest endothelial cell expression, in line with previous findings indicating that ACE2 can be post-transcriptionally regulated ,55. ImmuIt was previously shown that SARS-CoV-2 can use cysteine proteases cathepsin B/L ,61 to prIn vitro results obtained with human cytomegalovirus also reported that CD147 promotes virus entry in endothelial cells, indirectly through other proteins [50 of 24.86 µg ml−1 and IC50 of 15.16 µg ml−1. Importantly, immunoprecipitation, ELISA and surface plasmon resonance supported an interaction between CD147 and the receptor binding domain of viral spike protein. Immuno-electron microscopy also revealed colocalization of viral spike protein with CD147 in viral inclusion bodies of infected cells [Importantly, we cannot rule out the existence of ACE2-independent pathway(s). Recently, CD147, also known as basigin, has been proposed to act as a receptor for SARS-CoV-2. CD147 is a plasma-membrane signalling receptor belonging to the immunoglobulin superfamily, expressed at varying levels on many cell types, including endothelial cells  and invoproteins . Treatmeproteins , while ted cells . To dateed cells , to tested cells  despite ed cells ,56,58, wed cells ,77. As eed cells , this main vitro infection of human bronchial epithelial cells and monkey kidney cell lines, SARS-CoV-2 manipulates the autophagy pathway in a manner similar to MERS-CoV and that pro-autophagic compounds could inhibit viral propagation in vitro [in vitro against SARS-CoV-2 [For SARS-CoV-2 to gain a foothold in endothelial infection, it requires further interaction with the host cell machinery. An increasing body of evidence supports a differential interplay between autophagy components and coronavirus. Autophagy is a highly conserved process of cytoplasmic degradation used to maintain cellular homeostasis and eliminate pathogens including viruses. Unwanted elements are enclosed into autophagosomes which eventually fuse with lysosomes to form autolysosome where the degradation occurs –81. Howein vitro . AutophaRS-CoV-2 . In keep4.Ace2 expression significantly increased, but subsequently decreased during disease progression [Ace2 expression during early and advanced phases of atherosclerosis, this may result in altered levels of endothelial susceptibility to SARS-CoV-2 infection for these individuals. During infection, SARS-CoV infection and its spike protein alone has been found to downregulate ACE2, thus modulating the renin–angiotensin system and contributing to the development of severe lung disease in infected mice [Of interest, levels of vascular ACE2 expression may be altered in the course of disease. Since ACE2 expression is thought to be indicative of SARS-CoV-2 infection susceptibility, this may have implications for the severity of COVID-19 outcomes. Following partial ligation of the common carotid artery in a murine model of disturbed flow-induced atherosclerosis, endothelial gression . Thus, gted mice ,88. In Cted mice . Howeverted mice . It remaACE2 overexpression alleviates impaired endothelial function, through inhibiting proliferation, and enhancing migration and tube-like formation, indicative of improved endothelial cell function and neovascularization [ACE2 in human endothelial cells can inhibit the inflammatory response by inhibiting endothelial-monocyte adhesion molecules and decreasing Ang II-induced cytokine production [ACE2 expression, which, through inhibiting proliferation and inflammation, maintained endothelial homeostasis.ACE2 is an important homeostatic regulator of vascular function, and its altered expression and activity is associated with cardiovascular disorders . In acutrization . Furtheroduction . ACE2 haoduction . ExposinAce2 in the rat heart has been shown to protect against hypertension-induced cardiac remodelling by inhibiting perivascular fibrosis [Ace2 deficiency had larger vascular lesions in aortic atherosclerotic plaques and arterial neointima formation, due to increased VSMC proliferation [Ace2 knockout mice showed increased NADPH oxidase-dependent reactive oxygen production and apoptosis in response to Ang II [Although the role of ACE2 within pericytes remains largely unexplored, lentiviral vector-mediated overexpression of fibrosis . This mafibrosis . Studiesferation . Isolateo Ang II . Thus, Ao Ang II . Thus, Ao Ang II –101. If Taken together, patients with cardiovascular disease may be at increased risk of vascular SARS-CoV-2 infection, and, therefore, severe COVID-19 outcome, depending on the type of vascular complications, and the stage of disease. However, more studies quantifying vascular ACE2 expression during the progression of vascular diseases are needed.5.in vitro SARS-CoV-2 infection of endothelial cells. Engineered human capillary organoids, generated from patient-induced pluripotent stem cells, were successfully infected with SARS-CoV-2, as confirmed by recovery of viral RNA from organoids post-infection [Suitable endothelial cell infection models have great potential for accelerating scientific discovery of COVID-19 pathogenesis. Here, we discuss the experimental considerations in designing endothelial models for infection studies . At the nfection . This monfection . To inveIn vivo models such as the chick embryos have also been used to study viral infection [In situ hybridization of influenza infected chick embryos revealed that viral RNA was confined to endothelial cells of all organs, and further histochemical analysis showed endothelial-restricted expression of the viral receptor. Additionally, electron microscopy of infected cardiac endothelial cells was used to show that the budding polarity of influenza was only from the luminal side of the polarized vessel, thus preventing the spread of infection into tissue surrounding the endothelium.Numerous models have been designed and developed to study viral infections of the blood vessels, many of which have focused on endothelial polarity. In particular, the apicobasal polarity of brain endothelial cells has been of much interest, given the viral disruption of the blood–brain barrier . Endothenfection . In situIn vitro modelling of human endothelial cells derived from distinct tissues has shown that the flavivirus non-structural protein 1, from dengue, Zika, West Nile, Japanese encephalitis and yellow fever viruses, binds to endothelial cells and disrupts the endothelial glycocalyx components, triggering hyperpermeability and vascular dysfunction, in a tissue-specific manner [c manner . In thisBloodstream dissemination within a SARS-CoV-2-infected host is thought to be critical for multiorgan spread and pathogenesis observed in severe cases of COVID-19 . SimilarThough endothelium polarity is not as well understood as in the epithelium, a lot can be drawn from investigations in apicobasal epithelial polarity, such as the junctional proteins that regulate apicobasal polarity . The infin vivo infection are important to advance our mechanistic understanding. Though animal models have traditionally been used, they lack the ability to provide controllable experimental conditions. Humanized mouse models, such as the transgenic mice expressing human ACE2, can better recapitulate viral pathogenesis and tropism of SARS-CoV-2 than wild-type mouse strains [in vitro infection models have focused on polarity, as this affects both the route of cell infection and release. While the in vitro models described here predominantly achieved apicobasal cell polarity using Transwell inserts, polarity can also be achieved by culturing endothelial cells in 3D collagen gels to generate polarized vascular lumens [Model systems that are representative of  strains ,120. Humr lumens . Of noter lumens . For the6.in vitro. The failure to recapture these clinical observations in the laboratory could be due to the nature of endothelial monolayer culture used commonly in the laboratory. We propose the use of polarized endothelial cell models for SARS-CoV-2 infection to better reflect the endothelium in vitro. These set-ups can help clarify the apicobasal distribution of cell surface factors facilitating SARS-CoV-2 entry and replication.Even as clinical reports of COVID-19-related vasculopathy grow, there has been little evidence supporting direct infection of the endothelium by SARS-CoV-2 in vitro while a 3D vascular organoid model was permissive to SARS-CoV-2 infection and replication.Basally restricted host factors could explain why apical infection challenge by SAR-CoV-2 is unsuccessful in endothelial cell monolayers While endothelial dysfunction could have occurred from the ensuing cytokine storm alone, direct infection of endothelial cells by SARS-CoV-2 will suggest a larger role for the vasculature in viraemia and pathogenesis observed in other organs such as heart, liver and kidney. A successful endothelial infection model will allow us to understand the routes of entry and spread for SARS-CoV-2, the extent and types of damage to the blood vessels and the long-term effects of such insults even after virus clearance.Currently, the long-term impacts of COVID-19 are still unclear. Understanding the extent of damage to the endothelium in COVID-19 will determine if vascular health should be monitored as part of recovery to prevent catastrophic events, especially in patients with underlying vascular conditions. Insights into the nature of endothelial dysfunction in COVID-19 can also better inform the care of patients during active infection and beyond.


SARS-CoV-2 is a positive sense RNA coronavirus that constitutes a new threat for the global community and economy. While vaccines against SARS-CoV-2 are being developed, the mechanisms through which this virus takes control of an infected cell to replicate remains poorly understood. Upon infection, viruses completely rely on host cell molecular machinery to survive and replicate. To escape from the immune response and proliferate, viruses strategically modulate cellular metabolism and alter subcellular organelle architecture and functions. One way they do this is by modulating the structure and function of mitochondria, a critical cellular metabolic hub but also a key platform for the regulation of cellular immunity. This versatile nature of mitochondria defends host cells from viruses through several mechanisms including cellular apoptosis, ROS signaling, MAVS activation and mitochondrial DNA-dependent immune activation. These events are regulated by mitochondrial dynamics, a process by which mitochondria alter their structure (including their length and connectivity) in response to stress or other cues. It is therefore not surprising that viruses, including coronaviruses hijack these processes for their survival. In this review, we highlight how positive sense RNA viruses modulate mitochondrial dynamics and metabolism to evade mitochondrial mediated immune response in order to proliferate. The COVID-19 pandemic outbreak caused by the novel coronavirus SARS-CoV-2 has infected around 18.5In eukaryotic cells, mitochondria are the main source of cellular energy, but also control key cellular processes associated with metabolism and immune responses. For example, mitochondria controls cell cycle , cellula(+)ssRNA viruses dominantly infect eukaryotes, causing human diseases such as Hepatitis C , Dengue , and COVID-19 (SARS-CoV-2) . AlthougAs other (+)ssRNA virus, the SARS-CoV-2 genome codes for the structural proteins that make up the virion (spike (S), envelope (E), membrane glycoprotein (M), nucleocapsid (N)), as well as non-structural proteins and accessory proteins necessary for viral replication . These pDuring the infectious cycle, viruses significantly depends on host cell metabolites  to produce mature virions. Further, they hijack host defense mechanisms to successfully replicate and propagate. Generally, viruses manipulate host metabolism by re-orchestrating organelle structure and functions. As mitochondria has multifaceted roles in controlling cellular metabolism and immune responses, it is not surprising that several (+)ssRNA viruses target mitochondria.Mitochondria constitute a key metabolic hub within a cell, being required for ATP synthesis and a large number of metabolic reactions . To achiIn contrast to this static description, mitochondria are highly dynamic organelles. The dynamic reorganization of mitochondrial structure is essential to maintain cellular homeostasis in response to cues such as metabolic changes or infection. Mitochondrial dynamics, which include mitochondrial fusion and fission, are controlled by a number of large GTPases of the Dynamin family. OMM fusion is mediated by Mitofusins , while IMM fusion requires OPA1 . In addiAs damaged mitochondria promote the production of harmful reactive oxygen species (ROS) and stimulate inflammation, cells have developed specialized quality control mechanisms to eliminate damaged/depolarized mitochondria. These function by either selectively removing damaged mitochondrial proteins using mitochondria derived vesicles or completely eliminating depolarized mitochondria by a selective autophagy mechanism, termed mitophagy, that requires mitochondrial fission to selectively deliver damaged mitochondria to lysosomes and prevent damaging effects on cells .Several (+)ssRNA viruses alter ER-mitochondria interactions or manipulate mitochondrial dynamics and mitophagy. In this way, they can manipulate host metabolism and immunity to their advantage. (+)ssRNA viruses that target mitochondria include HCV, DENV, Zika (ZIKV), and coronaviruses related to SARS-CoV-2 [SARS-CoV (SARS) and Middle East respiratory syndrome (MERS)-CoV]. In addition, the recently published interactome of SARS-CoV-2 proteins suggests that SARS-CoV-2 also targets mitochondria , highligSeveral (+)ssRNA viruses target mitochondrial dynamics. For example, HCV causes DRP1 mediated mitochondrial fission  and HCV The association between mitochondrial elongation and viral replication is possibly the result of alterations in Mitochondria Associated Membranes (MAMs). MAMs are contact sites between the ER and mitochondria that regulate mitochondrial dynamics, serve to transfer a number of molecules between the two organelles and promote the activation of some pattern recognition receptors (PRR)  . In factMAM disruption can also disturb intracellular processes including autophagy , a catabOverall, targeting of mitochondria and MAMs by (+)ssRNA viruses promotes viral replication through CM formation, but also stimulates metabolic alterations. For example, HCV upregulates enzymes involved in glutamate catabolism and TCA cycle enzymes during early stages of infection to facilitate a regular supply of TCA cycle intermediates and lipids . As mitoA number of (+)ssRNA viruses require lipid metabolism for viral genome replication and virions assembly . In factOn the other hand, chronic viral infection can significantly impair mitochondrial function. For example, long-term HCV infection or expression of the HCV polyprotein severely impairs mitochondrial respiration through loss of Complex I and V activity, and decreased β-oxidation caused by PPARα downregulation . HCV-indUpon viral infection, the immune system springs into action. Initially, viral components  are detected by intracellular receptors (PRRs) that activate proinflammatory responses . MitochoNot surprisingly, PRR pathways and especially MAVS are strongly modulated by (+)ssRNA viruses to promote viral persistence and evade immune responses. For example, the NS3/4A protease of HCV selectively cleaves MAM-associated proteins, thereby suppressing antiviral responses . SimilarIn fact, immune repression by (+)ssRNA viruses is associated with changes in mitochondrial dynamics and MAMs. For example, SARS-CoV ORF-9b causes mitochondrial elongation in addition to MAVS degradation . A functOne last defense mechanism that cells use to combat pathogens is apoptotic cell death. Following an apoptotic signal, the OMM is permeabilized by pro-apoptotic BCL-2 proteins , resulting in the release of cytochrome c into the cytosol and caspase activation . HoweverWhile many viruses have developed strategies to block apoptosis in early phases of infection, it can still be activated at later time points as a consequence of accumulated cellular damage caused by viral replication, immune activation or specific viral proteins. For example, SARS-CoV nucleocapsid and ORF3a can induce apoptosis under some circumstances . HoweverAs a new coronavirus, the mechanisms by which SARS-CoV-2 control its host remain unclear. Furthermore, the severity of illness due to SARS-CoV-2 infection is likely impacted by both direct cytotoxic effects of the virus and the effectiveness of host responses. Interestingly, a recent gene expression profiling of SARS-CoV-2 infection showed an altered autophagy response, activation of innate immunity, increased ROS processes, and downregulation of mitochondrial function , suggestIn this context, the study of other (+)ssRNA can provide important information on SARS-CoV-2 biology and how it could target mitochondria to control its host . SARS-CoIntriguingly, SARS-CoV-2 ER-resident protein Nsp4, which is required for CM formation in SARS-CoV , potentiOur understanding of SARS-CoV-2 pathogenesis is still incomplete, and the current lack of effective treatment strategies or vaccine makes SARS-CoV-2 infection a major health issue. In this context, analyzing the mechanisms through which other coronaviruses, and more generally (+)ssRNA viruses control their host metabolism and immune activation can provide important information concerning the biology of SARS-CoV-2. Interestingly, the ability of (+)ssRNA viruses to target mitochondria to modulate metabolism and promote the formation of ER-derived replicative structures seems to be conserved in SARS-CoV-2. In addition, these mechanisms likely allow this virus to inhibit MAVS- and STING-dependent immune activation. Targeting these processes could thus provide novel approaches to treat infections caused by SARS-CoV-2 and other (+)ssRNA viruses.All authors contributed to the article and approved the submitted version.This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Fondation UQTR. PG is a recipient of a Queen Elizabeth II Diamond Jubilee scholarship. HSI is a recipient of a Queen Elizabeth II Diamond Jubilee scholarship and a Fonds du Québec- Nature et technologies. KT is a recipient of a Queen Elizabeth II Diamond Jubilee scholarship and a Fonds du Québec-Santé scholarship.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.


Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is a life-saving technology that provides transient respiratory and circulatory support for patients with profound cardiogenic shock or refractory cardiac arrest. Among its potential complications, VA-ECMO may adversely affect lung function through various pathophysiological mechanisms. The interaction of blood components with the biomaterials of the extracorporeal membrane elicits a systemic inflammatory response which may increase pulmonary vascular permeability and promote the sequestration of polymorphonuclear neutrophils within the lung parenchyma. Also, VA-ECMO increases the afterload of the left ventricle (LV) through reverse flow within the thoracic aorta, resulting in increased LV filling pressure and pulmonary congestion. Furthermore, VA-ECMO may result in long-standing pulmonary hypoxia, due to partial shunting of the pulmonary circulation and to reduced pulsatile blood flow within the bronchial circulation. Ultimately, these different abnormalities may result in a state of persisting lung inflammation and fibrotic changes with concomitant functional impairment, which may compromise weaning from VA-ECMO and could possibly result in long-term lung dysfunction. This review presents the mechanisms of lung damage and dysfunction under VA-ECMO and discusses potential strategies to prevent and treat such alterations. Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is a life-saving technology providing respiratory and circulatory support in patients with refractory cardiogenic shock or cardiac arrest  and whicVA-ECMO-induced pulmonary complications are much less recognized, except from the pulmonary congestion related to left ventricle pressure overload induced by retrograde VA-ECMO flow within the thoracic aorta . Beside The induction of a systemic inflammatory response syndrome (SIRS) by the contact of blood with biomaterial is a typical consequence of extracorporeal circulation . While eBlood contact with VA-ECMO circuitry activates the contact system (CS) and the complement system Fig. . CS geneThe humoral response triggered by blood-biomaterial interaction comprises the release of multiple cytokines . WhereasPlatelets are activated by contact with the tubing surface and by thrombin and complement. Activated platelets foster the generation of pro-inflammatory cytokines, thromboxane A2 (TXA2), platelet-activating factor (PAF), P-selectin, and serotonin. TXA2 induces ECs activation and local vasoconstriction, while serotonin and P-selectin promote PMN-endothelial interactions . PlateleAt variance with CPB, VA-ECMO is generally maintained over several days. The initial significant SIRS gradually decreases , 17, mosSome potential therapies have been proposed to downregulate inflammation and possibly improve lung outcome in this setting. The replacement of a silicon oxygenator by a poly-methyl pentene oxygenator has been associated with reduced radiological signs of pulmonary inflammation on chest X-ray , while tPeripheral  VA-ECMO provides a non-physiological blood flow promoting significant hemodynamic perturbations Fig. . The retPulmonary congestion develops consecutively to the passive upstream transmission of elevated LV pressure . Lung ex2 (PAO2). Alveolar epithelial cells, normally exposed to PAO2 above 100 mmHg, are sensitive to hypoxia from PAO2 below 50 mmHg, which may occur in alveoli flooded by pulmonary edema [Pulmonary congestion may jeopardize lung parenchymal cell oxygenation through two mechanisms. Firstly, interstitial edema increases the thickness of the alveolar-capillary barrier, hence the diffusion distance for oxygen between alveoli and parenchymal cells, whose oxygenation primarily depends on oxygen diffusing from alveolar spaces . Secondlry edema . Alveolary edema –39. Therry edema . Alveolary edema , local ary edema , 42.Chest X-ray is the simplest exam to assess pulmonary congestion, although its interpretation is complicated by frequently associated abnormalities, such as pneumonia, atelectasis, or alveolar hemorrhages. Chest ultrasound is an effective and reliable alternative method to assess interstitial edema, pleural effusion, and parenchymal consolidation . EchocarSevere pulmonary congestion during VA-ECMO is associated with a dismal prognosis, and its treatment is mandatory , 49. InoIf previous steps fail to reduce pulmonary edema, the left heart chambers must be directly unloaded (“vented”), either by percutaneous atrial transseptal approach or by using a venting cannula inserted into the left atrium or the LV apex by surgical or trans-aortic approach . In addiThe lung is characterized by a dual circulation, comprising the pulmonary circulation, which supplies the alveoli for gas exchange, and the bronchial circulation, which conveys oxygen and nutrients to the airways, but not alveoli, whose oxygen supply is almost exclusively provided by direct diffusion from the alveolar spaces . BronchoAs depicted in Fig. The best way to overcome such alterations is, of course, the withdrawal of VA-ECMO. If this is not possible, VA-ECMO flow may be reduced to maintain partial pulmonary perfusion. In early experimental studies in pigs, prolonged (18 h) ECMO at full support  promoted massive pulmonary parenchymal damage , which wAlthough no dedicated study has specifically focused on pulmonary histological consequences of VA-ECMO, data from animal models and small human necropsy series have reported several pathological alterations. Koul et al. maintained 6 pigs under total CPB for 18 h before weaning. All the animals died within the next 4 h, and on histological examination, more than 80% of the pulmonary parenchyma displayed edema, hyaline membranes, alveolar hemorrhages, thrombi, and focal necrotic changes . In anotIn humans, Ratliff et al. reported postmortem findings in 4 patients undergoing VA-ECMO for 7 to 12 days. In two patients, diffuse lung fibrosis was noted, together with liquefaction necrosis of the lower lobes. The authors hypothesized that the combination of an increase in metabolically active cell mass together with partial pulmonary shunting concurred to establish ischemic areas with subsequent necrosis . In an aTo sum up, VA-ECMO appears mostly associated with signs of protein-rich edema, alveolar hemorrhages, tissue necrosis, and fibrosis, which are reminiscent of the damage noted in the acute respiratory distress syndrome. These changes are likely the result of the combination of inflammatory injury, pulmonary congestion, and hypoxia, with the progressive development of epithelial-endothelial injury, increased vascular permeability, and interstitial collagen deposition . FurtherThe impaired pulmonary function induced by VA-ECMO may require long-lasting mechanical ventilation (MV) which may further alter the lung through ventilator-induced lung injury (VILI). Although there is presently no consensus regarding optimal ventilator settings for MV during VA-ECMO, the principles of lung-protective ventilation should be applied .Pseudomonas aeruginosa isolated in 18–25% of cases [Furthermore, prolonged MV increases the risk of ventilator-associated pneumonia (VAP), which occurs in up to 74% of patients under ECMO, as recently reviewed , with riof cases . Diagnosof cases . Treatmeof cases .Preventive measures to reduce the risk of VAP include primarily the reduction of MV duration. In this regard, a strategy of early extubation and awake ECMO support is emerging as a promising strategy . In propThe prototypical consequence of VA-ECMO-dependent impairment of lung function is the development of the “Harlequin syndrome,” reflecting the opposing flows from the heart  and from the peripheral ECMO , resulting in differential hypoxia . The level of mixing of the two flows within the aorta is termed the “watershed,” which can be identified in contrast-enhanced CT scan of the chest Fig. . The HarFinally, bronchoscopy with bronchial hygiene may be considered routinely in order to maximize chances of successful weaning from VA-ECMO when cardiac function recovers .VA-ECMO-induced lung alterations may only appear after weaning and the restoration of physiological pulmonary artery blood flow. In a series of 55 patients who underwent long-term mechanical assist device implantation under VA-ECMO , Boulate et al. noticed that 27% of patients develop acute lung injury (ALI) few hours after restoration of pulmonary blood flow, with a significant impact on mortality. The authors hypothesized that chronic lung ischemia during VA-ECMO support could promote alveolar frailty and that the sudden restoration of an antegrade pulsatile pulmonary blood flow creates a massive pulmonary bed overload responsible of ALI . AccordiThis form of ALI is reminiscent of reperfusion pulmonary edema, a well-known and described condition that occurs after reperfusion of a chronic low pulmonary blood flow situation, as in correction of tetralogy of Fallot  or pulmoIn order to help the decision-making process in patients under VA-ECMO, Chen et al. developed a risk factor-calling score (RFSS) to select patients eligible for L-VAD or HTX. The RFSS has 5 items and 16 points, 7 of which are allocated to pulmonary dysfunction. A RFSS > 7 predicted a poor outcome, which emphasizes the relative burden of pulmonary dysfunction in the outcome of patients under VA-ECMO in a bridge strategy .It is currently unknown whether lung damage and dysfunction induced by VA-ECMO have an impact on long-term outcome, the more so that many unrelated factors may interfere with such outcome, such as prolonged ICU stay, previous health condition, or reduced LV ejection fraction. A few studies focused on long-term health-related quality of life (HRQL) in VA-ECMO survivors after cardiogenic shock. Combes et al. questioned 28 VA-ECMO survivors about their HRQL via the short-form 36 questionnaire (SF-36). Mean VA-ECMO duration and follow-up were respectively 7 days (5 to 10) and 11 months (3 to 39). In comparison to sex- and age-matched controls, VA-ECMO survivors disclosed significantly lower role-physical score and a trend to a lower physical function, even though most patients recovered a good cardiac function with mean LVEF 51% or underwent HTX . These rVA-ECMO elicits several pathophysiological disturbances which may significantly impact on lung integrity and function. First, the rapid development of a systemic inflammatory response with pulmonary involvement is an unavoidable consequence of the artificial VA-ECMO circuitry. Second, due to retrograde blood flow within the thoracic aorta, peripheral VA-ECMO has the propensity to increase LV afterload, which may favor the congestion of alveoli already affected by the ongoing inflammation.Third, persistent lung ischemia due to the partial shunting of the pulmonary circulation and reduced pulsatility of the bronchial circulation may elicit further cytotoxicity within the whole lung parenchyma. Limited evidence from human observational studies and animal models indicates that VA-ECMO support for more than a few days may lead to severe structural changes of the lung parenchyma and interstitial fibrosis, which could result in long-term functional limitation. Clinicians in charge of VA-ECMO patients should be aware of its effects on lung physiology and should take all measures to limit such consequences, including the maintenance of VA-ECMO support as short as possible, the early diagnosis and treatment of cardiac overload and pulmonary congestion, and the application of lung-protective ventilation. Future studies specifically addressing the issue of the pulmonary consequences of VA-ECMO are warranted.


Drug-resistant cancer cells survive under hostile bombardment of chemotherapeutic agents, causing cancer relapse and death. Shocking to many, chemo-resistant cells reprogram lysosomes, also known as the cellular “suicide bags”, to shield themselves from intrusion of chemotherapeutic agents and gain survival advantages. This review presents an evolutionary arms race through which cancer cells become adept at detecting and confining weak-base chemotherapeutic agents in lysosomes. We hope to facilitate translational pharmaceutical research by highlighting lysosomes as fruitful arenas to overcome chemotherapeutic resistance.Despite extensive research, resistance to chemotherapy still poses a major obstacle in clinical oncology. An exciting strategy to circumvent chemoresistance involves the identification and subsequent disruption of cellular processes that are aberrantly altered in oncogenic states. Upon chemotherapeutic challenges, lysosomes are deemed to be essential mediators that enable cellular adaptation to stress conditions. Therefore, lysosomes potentially hold the key to disarming the fundamental mechanisms of chemoresistance. This review explores modes of action of classical chemotherapeutic agents, adaptive response of the lysosomes to cell stress, and presents physiological and pharmacological insights pertaining to drug compartmentalization, sequestration, and extracellular clearance through the lens of lysosomes. Coined by Nobel Prize-winning German chemist and immunologist Paul Ehrlich, “chemotherapy” refers to the use of chemicals to treat diseases. In the early 1900s, Ehrlich and colleagues conducted high-throughput animal screenings of small molecules against syphilis, formulating the pioneering concept of the “magic bullets” underpinning molecular pathogenesis . Of noteAntimetabolites are among the earliest chemotherapeutic agents approved by the U.S. Food and Drug Administration (FDA). Although antimetabolites encompass any compounds that affect metabolic pathways, traditional antimetabolite antineoplastics often act as molecular mimicries of cellular nucleotides. Their uptake followed by activation by naturally present metabolic enzymes results in sequential disruptions of nucleic acid synthesis and metabolism, leading to cell death. One example is mercaptopurine (6-MP), a hypoxanthine (purine derivative) analog synthesized by Gertrude B. Elion in 1951 and rapidly approved by the FDA in 1953. Clinically efficacious for the treatment of childhood acute lymphoblastic leukemia, 6-MP serves as a prime medical archetype even according to today’s standard of care. Once the inactive pro-drug 6-MP enters the cell through solute carrier family transporters SLC28A2, SLC28A3, SLC29A1, and SLC29A2, it is then converted by hypoxanthine guanine phosphoribosyl transferase (HGPRT) into 6-thio-inosine monophosphate (TIMP), an inhibitor of de novo purine synthesis which, some of itself, can be further methylated by thiopurine S-methyltransferase (TPMT), to produce methyl-thioinosine monophosphate (MTIMP) . MTIMP pN-methyl-bis(2-chloroethyl) amines. Such compounds first undergo an SN2 intramolecular cyclization reaction, forming the structurally strained and functionally reactive aziridinium cation. The positively charged and electrophilic nitrogen of the aziridinium cation is then subjected to nucleophilic addition by the lone pair of electrons on the seventh nitrogen (N7) of a guanine base ring, resulting in aziridinium ring opening and physically conjugating one arm of the nitrogen mustard to one strand of the DNA. Such process repeats itself for the other arm of the nitrogen mustard, effectively cross-linking DNA strand(s) and forming DNA adducts with ensuing disruptions in mitotic division. Alkylating agents whose mechanisms of action exemplify the aforementioned paradigm are consequently termed bifunctional alkylating agents, which are relatively well characterized but only make up a tiny portion of all alkylating chemotherapeutics. Because of their high reactivity, mustard agents may induce overwhelming side effects, such as acute nausea and vomiting, which patients can experience up to 20 times per day, alopecia (hair loss), oral complications, skin irritation, diarrhea, and infertility [N-methyl-N-nitrosoguanidine, procarbazine) cause localized N and O-alkyl adduct formation on only one reactive center of DNA substrates, giving rise to a series of toxic and/or mutagenic modifications, such as 7-methylguanine (7meG), 3-methyladenine (3meA), O6-methylguanine (O6meG), and less frequently, O4-methylthymine (O4MeT) [2+ signaling network [Alkylating agents primarily exert their anti-neoplastic effects by destroying the genomic integrity of proliferative cells. Through either uni-molecular (SN1) or bimolecular (SN2) nucleophilic substitution reactions, alkylating agents effectively instigate covalent modifications of electron-dense reactive centers of nitrogen and oxygen atoms on DNA substrates, compromising DNA replication and giving rise to apoptotic or necrotic cell death . Among tertility . Further (O4MeT) ,18. Seem (O4MeT) . However network . Because network ,23.2+ [During DNA replication and transcription, topoisomerases relieve torsional and flexural strain that occurs distal to the replication fork and DNA helicase. To relax the supercoiled DNA, topoisomerases reversibly cut the DNA phosphodiester backbone to create space for either a single strand of DNA to rotate or DNA duplexes to pass through the breakage point. Topo 1 generally acts to reduce the number of loops, thus torsional strain, existing on one pair of the double helix, while the homo-dimeric eukaryotic Topo 2 facilitates DNA decatenation over multiple intertwined helices. The catalytic activity of Topo II, unlike that of Topo I, is powered by ATP hydrolysis and requires the binding of Mg2+ . Since r2+ .2 BD, with side effects potentially extending to vital organs of the body [To elicit cellular apoptosis, Topo 1 inhibitors stabilize Topo 1-cleavable complexes, extending the transient lifetime of genotoxic damages. First discovered in the stem wood of the Chinese tree, Camptotheca acuminate, in 1966, the polycyclic camptothecin (CPT) recognizes the DNA-topo 1 binary complex and intercalates such complex at the site of nucleophilic cleavage via an uncompetitive paradigm, physically enlarging the distance between the 5′ hydroxyl group and the 3′ phosphorous at the cleavage by approximately 8 Å. As a result, the ternary CPT-topo 1-DNA complex prevents subsequent DNA re-ligation ,27. Thisthe body . For insthe body . FurtherSince ATP hydrolysis is integral to eukaryotic Topo II catalysis, some therapeutic design of Topo II inhibitors is centered on inhibiting the enzymatic ATPase activity. Halting the catalytic ATPase activity typically involves one of two mechanisms, direct competition for ATP binding or stabilization of ATP-binding domain to prevent further ATP hydrolytic cycles. Using structure-based molecular modeling, Chène and colleagues revealed that quinoline aminopurine compound 1 (QAP1) serves as a potent ATP-competitive inhibitor for both alpha and beta isoforms of topoisomerase II, preventing Topo II-mediated DNA decatenation at low micromolar concentration . As an aMammalian microtubules (MTs) are composed of 13 longitudinal protofilaments laterally associated with one another around a hollow core, conferring distinct plus (+) and minus (−) polarities on beta and alpha monomers, respectively . The molIsolated from Catharanthus roseus, vinca alkaloids  inhibit the polymerization of both soluble and MT-associated tubulins. Vinca alkaloids are clinically efficacious in treating a constellation of malignancies, such as breast cancer, neuroblastoma, Hodgkin’s and non-Hodgkins lymphoma, lung cancer, and choriocarcinoma. Structurally, vincristine and vinblastine are composed of an identical upper velbanamine moiety and highly similar lower vindoline constituents. To inhibit microtubule polymerization, vinblastine localizes to the interface between alpha and beta tubulin heterodimers and crosslinks the alpha-subunit of one tubulin with the beta-subunit of the other, disrupting normal microtubule architecture while inducing and stabilizing spiral-like tubulin aggregates . Each ofAnother major class of anti-neoplastic MT-targeting agents is the taxanes , inhibiting MT depolymerization and stabilizing dysregulated MT outgrowths, thereby blocking mitosis. One interesting finding of a structure-activity relationship study illustrated that modifications at C10 position of taxoids show exceptional cytotoxicity for multidrug-resistant breast cancer MCF7 cells, without exerting major impacts on sensitive cell lines, suggesting that the C10 position of taxoids might contain moieties that serve as substrates recognized by P-glycoproteins (Pgps) during drug efflux . AlthougBeclin1-knockin mice are protected against HER2-mediated tumorigenesis, suggesting the pro-apoptotic outcome of autophagy in HER2+-breast tumor pathology [In response to chemotherapeutic agents, tumor cells initiate a variety of pathways in the nucleus, cytosol, and plasma membrane, the overall activity of which eventually determines cell fate . Althougathology . Furtherathology . This evThe acquisition of chemoresistance is a multifactorial process, integrating external growth factor/cytokine signaling, stromal-tumor interactions, internal tumor microenvironment, with the varying extent of genomic heterogeneity of the tumor mass. Cancer chemo-resistance can be broadly categorized into primary (innate) resistance and acquired resistance. Intrinsic genetic instability or genomic heterogeneity of the tumor cells, the microenvironment in which the tumor cells reside as well as the intricate host factors and patients’ comorbidities have been clinically demonstrated to confer primary resistance, rendering patients insensitive to treatment upon initial exposure to the drug ,58. AlthAdding further complexity to this paradigm is the contentious existence of cancer stem cells driving persistent drug resistance . In this−/H+ antiporter, to dissipate the transmembrane voltage associated with the establishment of the proton gradient by the V-ATPases [Lysosomes are single-membraned, dense, almost-spherical cytoplasmic vacuoles, executing compartmentalized degradation of proteins, nucleic acids, carbohydrates, lipids, and a diverse array of cellular structures through autophagy. Within the cytoplasm, lysosomes display diverse patterns of subcellular localization. Some tend to congregate in the vicinity of the nucleus and the microtubule-organizing center, forming a region known as the “perinuclear cloud,” while others are in close proximity to the plasma membrane. The subcellular localization of the lysosomes, coupled with their ability to undergo rapid intracellular trafficking upon stimulation , allows them to execute a plethora of biological activities critical to maintaining cellular and organellar homeostasis. Embellished with protein signaling complexes responsive to environmental cues and regulated by transcriptional machineries, lysosomes govern the overall cellular metabolic state through dynamic regulation of anabolic and catabolic processes . To crea-ATPases . The int-ATPases . Besidesn-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) to mediate lysosomal regeneration [The formation of lysosomes begins with the internalization of extracellular materials through the plasma membrane in clathrin-coated pits. As soon as endocytic cargoes gain entry to the cell in early endosomes (EEs), they are immediately in physical contact with motor proteins  connecting to the cytoskeletal networks progressing through retrograde transport. One prominent molecular attribute associated with early endosomes is the presence of Rab5 GTPase, which controls the fusion kinetics of early endosomes per se ,65 and dneration .2+ sparks near the lysosomal membrane, which is then detected by calcium/calmodulin-dependent serine-threonine phosphatase calcineurin (CaN). CaN dephosphorylates TFEB, permitting TFEB translocation into the nucleus to initiate lysosomal biogenesis in an effort to supply cellular nutrients through the degradation and recycling of biomolecules [Apart from degrading and recycling cellular contents, lysosomes also instigate intracellular signaling and organellar crosstalk. One major lysosomal nutrient signaling network is mediated through the serine-threonine kinase mechanistic target of rapamycin (mTOR), which makes up the catalytic subunit of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), transducing extracellular environmental cues into intracellular responses as well as balancing autophagy with cell growth, protein synthesis, and the biogenesis of lipids and nucleotides. In mTORC1, for example, through the recruitment of the Rag GTPase obligate heterodimers (RagA/B-GTP and RagC/D-GDP) in specific nucleotide-binding states on the lysosomal membrane, amino acids indicative of nutrient availability are presented to the regulatory protein associated with mTOR (Raptor) domain of mTORC1, activating mTORC1 and allowing it to phosphorylate substrate proteins, such as the eukaryotic translation initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1) and the 40S ribosomal S6 kinases (S6Ks) to stimulate the anabolic process of cap-dependent translation . While polecules . Consideolecules ,77.Organellar alterations of lysosomes have been documented in cancer. It has also been shown that members of the MiT/TFE family interact with one another to promote lysosomal biogenesis and are heavily implicated in tumorigenesis ,79,80,81Additionally, deregulated patterns of lysosomal trafficking to the cell periphery has been observed in cancer settings, seemingly in preparation for exocytosis of entrapped weak-base chemotherapeutic compounds into the extracellular matrix (ECM) as well as subsequent metastatic dissemination through proteolytic degradation of ECM substrates . FurtherIn addition, several lysosomal membrane proteins may also undergo oncogenic manipulations during the acquisition of chemoresistance. It is widely accepted that intra-lysosomal acid hydrolases, including phosphatases, proteases, glycosidases, peptidases, sulfatases, and lipases must be carefully protected from the cytosolic environment as massive lysosomal leakage increases cytosolic acidity and results in uncontrolled cell death . To favo−/−) mice in comparison to wild-type mice [Non-structural lysosomal membrane protein alterations that set the stage for chemoresistance include lysosomal acid sphingomyelinase and mTOR. Lysosomal acid sphingomyelinase (Asm) is a phosphodiesterase that mediates the hydrolytic cleavage of sphingomyeline into ceramide and free phosphocholine . Ceramidype mice . Also siype mice . In addiype mice . CollectThrough a process known as ion-trapping, small-molecular-weight, lipophilic, and weakly basic substances are susceptible to selective retention or sequestration by acidic organelles, such as the lysosomes. The tendency for these substances to be confined within the lysosomal compartments is termed “lysosomotropism.” Mechanistically, lysosomal ion trapping primarily occurs via passive diffusion of unprotonated amine-containing drugs from the cytoplasm with relatively neutral pH environment (≈7.4) to the acidic luminal lysosome (≈4.5–5.0), autophagy, endocytosis, and lysosomal transmembrane transport system . Once crLysosomal V-type ATPase is indispensable in establishing the acidic lysosomal microenvironment, electrostatically initiating and perpetuating weak-base amine accumulation. Lysosomal acidity may be indicative of the extent of “drug retention,” suggesting a predominantly passive mechanism for drugs to enter the lysosomes. Besides promoting lysosomal acidification, V-type ATPases can also drive vesicular trafficking, thus determining the endocytic and recycling kinetics of the cell. This can have profound therapeutic implications for cancers that thrive on increased endocytic and recycling machineries. For instance, pharmacological inhibition of lysosomal V-ATPase overcomes trastuzumab  resistance in breast cancer by impairing the elevated vesicular recycling of HER2 to the plasma membrane in trastuzumab-resistant cells . Further2+ release, which can signal through CaN to stimulate translocation of TFEB into the nucleus and result in lysosomal biogenesis [Even more intriguingly, the intracellular presence of many weak-base amines potentiates cytoplasmic vacuole formation composed of molecular assemblies of lysosomal and late-endosomal resident proteins, suggesting the existence of a built-in protein sensor for lysosomal amine accumulation . Besidesogenesis . ImportaSince chemotherapeutic agents come in various shapes and forms, their chemical composition and molecular structures enable them to possess distinct lysosomal entrapment propensity. In particular, dibasic compounds exhibit higher lysosomal accumulation than monobasic compounds due to the presence of an additional basic reactive center subject to protonation, partially justifying the varying susceptibility for distinct drug molecules to undergo lysosomal sequestration . FollowiAlthough lysosomal rupture due to excessive uptake of lysosomotropic agents has been reported to lead to dire cellular consequences, such as induction of intrinsic apoptosis and even necrotic cell death, lysosomes can also respond to chemotherapeutic challenges by activating appropriate compensatory measures to mitigate lysosomal stress, a central theme for manipulation by drug-resistant malignant cells . For insIncreasing lysosomal membrane permeability (LMP) and thus unleashing entrapped chemotherapeutic agents seems to be heavily exploited in recent years. For instance, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and the clinically trialed di-2-pyridylketone 3-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) have been identified as substrates of lysosomal p-glycoproteins and subsequently induce LMP in p-glycoprotein-overexpressing cancer cells. Therefore, following doxorubicin deposition into the lysosomes, uptake of Dp44mT and DpC leads to potent apoptotic cell death through inducing ROS that eventually disintegrates lysosomal membrane in p-glycoprotein-over-expressing cancer cells, allowing doxorubicin to be re-localized to the nucleus and execute its cytotoxic effects . AlthougDue to the reliance of many drug-resistant cancer cells on upregulated lysosomal biogenesis to entrap chemotherapeutic agents, disrupting lysosomal homeostasis in conjunction with downstream exocytosis may be worth exploring as a therapeutic modality. One probable therapeutic intervention to target chemo-resistant cancer cells involves coupling lysosomotropic small molecule compounds with agents capable of disrupting lysosomal ionic homeostasis. This concept, nevertheless, has been empirically applied on cancer stem cells, where synthetic derivative of salinomycin with lysosomotropic potential induced intra-lysosomal iron accumulation, leading to cytotoxic ROS production and tumor cell killing . Such exThe revolutionary discovery of lysosomes by Christian de Duve in 1955 pioneered a field of research into biomolecular recycling, nutrient regeneration, and stress response. Since then, the world of lysosomes has garnered substantial scientific scrutiny as it represents the Achilles’ heel underpinning chemoresistance. In this review, we first discussed the advent of chemotherapy and its therapeutic trajectory, highlighting pharmacological development and mechanistic aspects for classical antineoplastic agents. Building from this foundation, we discussed the modulation of cellular responses post-chemotherapeutic challenges through the lens of lysosomes, exclusively delving into lysosome-mediated acquired chemoresistance . Althoug