These techniques and tools, however, have not been systematically benchmarked. A comprehensive benchmark study and an integrated strategy that takes advantage of the currently available tools to improve predictions of genome-wide CRISPR off-target profiles are needed. We focused on the specificity of the traditional CRISPR SpCas9 system for gene knockout. First, we benchmarked 10 available genome-wide off-target cleavage site (OTS) detection techniques with the published OTS detection datasets. Second, taking the datasets generated from OTS detection techniques as the benchmark datasets, we benchmarked 17 available in silico genome-wide OTS prediction tools to evaluate their genome-wide CRISPR off-target prediction performances. Finally, we present the first one-stop integrated Genome-Wide Off-target cleavage Search platform (iGWOS) that was specifically designed for the optimal genome-wide OTS prediction by integrating the available OTS prediction algorithms with an AdaBoost ensemble framework.Systematic evaluation of genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) off-target profiles is a fundamental step for the successful application of the CRISPR system to clinical therapies. Many experimental techniques and  Although these techniques and tools have been evaluated in several studies  on-target efficacy (sensitivity) and off-target profiles (specificity) has hindered successful application of the CRISPR system for clinical therapies. The CRISPR on-target single guide RNA (gRNA) design and efficacy prediction have been extensively studied and benchmarked . Many ge studies , the evain silico OTS prediction tools. The first benchmark of genome-wide OTS detection techniques will provide objective knowledge and benchmark datasets to be utilized in the following benchmark of genome-wide in silico OTS prediction tools; therefore, these two benchmarks can be performed sequentially. Furthermore, we also present the first one-stop integrated Genome-Wide Off-target cleavage Search platform(iGWOS) that was designed specifically for the optimal OTS prediction by integrating the available OTS prediction algorithms with an AdaBoost ensemble learning model.To this end, we present a comprehensive study that benchmarks the available genome-wide off-target cleavage site (OTS) detection techniques as well as the in silico prediction tools, as well as the development of the iGWOS platform are presented in the following sections.Figure in vitro techniques, in which the CRISPR-Cas9 system induces DSB cleavage sites in the purified genomic DNA, including CIRCLE-seq  ceq . Then we applied the curation of gRNAs and their corresponding OTS detected from each dataset. Hg19 was taken as the reference genome, so the genomic coordinates based on hg38 in the Digenome-seq and SITE-Seq datasets were converted to hg19by tool LiftOver from UCSC genome browser , Digenome-seq (21/138), DISCOVER-Seq (56/58) and GUIDE-seq (150/403). Previous studies also indicated that it is difficult to target GC-rich genes with gRNAs and that high GC% gRNAs tend to have weak specificity (BLESS (3/17), GUIDE-seq (1/403), and CIRCLE-seq (260/6903) contained sequences spanning 22 or 24 nt, which indicates that these three techniques can detect OTS with DNA/RNA bulges (CIRCLE-seq (11/3106), SITE-Seq (27/70) and PEM-seq (1/52) detected a small portion of OTS with mismatches >6 bases compared to the gRNA targets at the first 20 nt before PAM. In our study, the maximum mismatch tolerance of OTS was set to 6. Finally, OTS spanning 23 nt with NGG-PAM and mismatches up to 6 bases were curated in our analysis, resulting in the curated datasets with 3905 OTS of 16 gRNAs from CIRCLE-seq, 109 OTS of 11 gRNAs from Digenome-seq, 38 OTS of 8 gRNAs from DIG-seq, 43 OTS of 6 gRNAs from SITE-Seq, 14 OTS of 2 gRNAs from BLESS, 209 OTS of 8 gRNAs from GUIDE-seq, 72 OTS of 4 gRNAs from HTGTS, 19 OTS of 4 gRNAs from IDLV, 51 OTS of 2 gRNAs from PEM-seq, and 2 OTS of 1 gRNA from DISCOVER-Seq. The curated OTS datasets were used to benchmark these OTS detection techniques. The details of the experimental techniques and the curated datasets are listed in We first comprehensively summarized the 10 available genome-wide OTS detection techniques, and collected 11 genome-wide OTS datasets generated from the available 10 detection techniques  alignment-based . We aimed at generating the benchmark datasets based on OTS detection techniques for the following assessment of other three types of in silico OTS prediction tools. So, we first applied Cas-OFFinder to generate the genome-wide candidate OTS of tested gRNAs from the curated datasets generated before, restricting the off-target with 23-nt long, containing NGG-PAM and mismatches up to 6 bases. Then, among the candidate OTS, the validated OTS were labeled ‘1’, otherwise labeled ‘0’. Considering that the learning-based tool DeepCRISPR covers some published OTS datasets to train its learning model and currently supports OTS prediction in only 13 mainstream cell types, datasets trained by DeepCRISPR and gRNAs not detected in these 13 cell types were removed from the benchmark datasets for in silico OTS prediction tools assessment. Since the benchmark of experimental OTS detection techniques showed that the CRISPR cleavage specificity is heterogeneous in different cell types (see Results), the gRNAs in datasets were classified into three groups by their detection cell types, and the OTS on given gRNAs in same cell types shared by different techniques were merged together. Finally, this resulted in the benchmark datasets containing 444 921 candidates OTS with 1850 positive labels in 3 cell types, which includes 180 671, 76 059 and 188 191 candidates with 968, 778 and 104 positive labels from 12, 7 and 11 gRNAs respectively in HEK293, K562 and HeLa. The details of the benchmark datasets are listed in After an assessment of alignment-based OTS prediction tools, in silico OTS prediction tools on benchmark datasets were added as the OTS features to the benchmark datasets generated above, containing 444 921 candidates OTS with 1850 positive labels in three cell types . It adds a weak learner in each iteration to learn misclassified training instances until a pre-set number of weak learners is created or no more improvement can be made on the train dataset. In this study, the AdaBoost model parameters were tuned on the train dataset under 5-fold stratified cross-validation , and the best parameters were selected, where algorithm was set to ‘SAMME.R’, base_estimator set to decision stump, n_estimators set to 280 and learning_rate set to 0.1. The predicted class ‘1’ probability for a candidate OTS was taken as the integrative prediction score iGWOS (integrated Genome-Wide Off-target cleavage Search) platform is designed specifically for the optimal OTS prediction by integrating the available in silico OTS prediction tools with an Adaboost framework. iGWOS currently supports precise genome-wide CRISPR OTS prediction with conventional NGG-PAM and mismatches up to 6 bases in human species. By inputting the gRNA(s) sequence file and related restrictions at the command line, iGWOS outputs integrated prediction of the genome-wide OTS profile of given gRNAs and visualizes the top 200 risky genome-wide off-target profile with a Circos  OTS detection techniques have their unique characteristics, resulting from their different experimental categories, DSBs detecting sensitivities, and even the developing times.By comparing the genome-wide OTS distribution and the overlapping OTS of the gRNA sequences shared by four s Figure  and B. Sq Figure –F. In adq Figure  and H. TCIRCLE-seq .As alignment-based tools predict candidate OTS purely by sequence alignment without ranking their potential knockout ability, we benchmarked this type of tool based on their options for maximum candidate off-target searching Table . The tabCRISPRoff, and 0.884 for uCRISPR) and higher PR-AUC  than the hypothesis-driven tools, showing their better ability to predict OTS, (2) DeepCRISPR did not show excellent performance, likely resulting from the data-driving limitation for learning-based tools and the testing data in this study is different from previous study Our benchmark above indicated that each categories of prediction tools has its own characteristic in OTS perdition, and an effective integration of those OTS prediction tools may contribute to a better performance in genome-wide OTS prediction. A recent study also showed that synergizing multiple hypothesis-driven tools with an ensemble learning method enhanced OTS prediction . TherefoiGWOS outperformed the existing individual tools, providing a substantial improvement in genome-wide OTS prediction . The AdaBoost ensemble model was trained on the train dataset under a 5-fold stratified cross-validation to tune the model parameters. Finally, the prediction performances on the test dataset showed that our trained model n Figure .integrated Genome-Wide Off-target cleavage Search platform, i.e., iGWOS was developed that is available on GitHub at https://github.com/bm2-lab/iGWOS, which was designed to precisely predict genome-wide CRISPR OTS profiles by integrating three categories of OTS prediction algorithms using an AdaBoost framework.Finally, a one-stop in vitro genome-wide OTS technique called CHANGE-seq  CRISPR cleavage specificity is heterogeneous in different cell types, resulting from their different genetic and epigenetic information. (iii) The structural and energy-based mechanisms of CRISPR binding, taking the characteristics of DNA-RNA binding into account and without requiring a large amount of training data, generally contribute to a better performance in genome-wide OTS prediction, which will promote further researches of CRISPR off-target effect based on the structural mechanisms and molecular modeling. (iv) The development of our iGWOS platform confirmed that the integration of different categories of prediction algorithms is an efficient strategy for achieving better off-target prediction.The off-target effect of the CRISPR/Cas9 system remains to be an obstacle for successful therapeutic application of genome editing. Therefore, many techniques and tools have been proposed or developed to better detect and predict genome-wide OTS in different environments. Our comprehensive benchmark study of these existing resources provides insightful guidance for off-target effect research in four aspects: (i) The benchmarking of experimental CRISPR off-target detection techniques indicated that the gRNA specificity verifies in different experimental OTS detection techniques, resulting from their different experiment categories, DSBs detecting sensitivities, and even the developing times. A recent study provided a new ANGE-seq , which wiGWOS platform is also available under an open source license  at GitHub.The authors declare that the datasets and results discussed in this study are available within the article and its supplementary information files. Besides, the source code of gkaa930_Supplemental_FilesClick here for additional data file.


Task sharing, through models such as community health workers (CHWs), is considered an efficacious and cost-effective approach to extending access, addressing disparities, and building capacity. Increasingly, task sharing is recognized as a promising approach within sensory health. This session will share results from an NIH-funded trial of a first-in-kind CHW-delivered intervention along the U.S.-Mexico border. Trained CHWs provided a 5-week group aural rehabilitation program that included education and counseling on age-related hearing loss. A total of 136 Spanish-speaking older adults with hearing loss were randomized. Those in the immediate treatment group reported significantly greater use of communication strategies post-intervention, which was maintained over 1 year. Participants were more likely to report taking action on their hearing at 6 months  and 1 year . Building upon lessons learned, including post-intervention focus groups, the presentation will share guiding principles on the application of task sharing to support sensory health.


A central challenge in infection medicine is to determine the structure and function of host–pathogen protein–protein interactions to understand how these interactions facilitate bacterial adhesion, dissemination and survival. In this review, we focus on proteomics, electron cryo-microscopy and structural modeling to showcase instances where affinity-purification (AP) and cross-linking (XL) mass spectrometry (MS) has advanced our understanding of host–pathogen interactions. We highlight cases where XL-MS in combination with structural modeling has provided insight into the quaternary structure of interspecies protein complexes. We further exemplify how electron cryo-tomography has been used to visualize bacterial–human interactions during attachment and infection. Lastly, we discuss how AP-MS, XL-MS and electron cryo-microscopy and -tomography together with structural modeling approaches can be used in future studies to broaden our knowledge regarding the function, dynamics and evolution of such interactions. This knowledge will be of relevance for future drug and vaccine development programs. Infectious diseases are a serious health problem aggravated by the current spread of pathogens and their vectors to new niches. Concomitantly, the emergence of novel, zoonotic pathogens is rapidly increasing as well as the bacterial resistance to antibiotics , 2. In fDuring an infection, a bacterial pathogen circumvents host’s immune defenses via highly evolved effector proteins or virulence factors that can hijack and re-wire molecular host systems. At the same time, host proteins such as immunoglobulins, proteins of the complement system and antimicrobial proteins together with cells from the host’s adaptive and innate immune system, bind to bacterial surfaces and effector proteins to neutralize bacteria and prevent infection. This dynamic interplay between host and pathogen partly depends on the formation of host–pathogen protein–protein interactions (HP-PPIs) , 6. TypiSuccessful integrative structural biology approaches for the characterization of HP-PPI networks typically requires multi-tiered information. For example, large-scale mapping of binary interspecies protein–protein interactions is important to outline the degree of interconnectivity between proteins within a network. Conversely, generation of multiple HP-PPI networks between different species will support comparative studies to promote generalized conclusions about pathogen-specific mechanisms –17 and cAP-MS is an increasingly important technique to explore HP-PPIs based on affinity-tagged bacterial or human proteins coupled to a solid matrix to capture interacting proteins  proteins and proteins from human macrophages [Mtb proteins. This enabled the identification of a specific interaction between the probable conserved lipoprotein LpqN (a secreted Mtb virulence factor) and the ubiquitin ligase CBL. The identification of the interaction between CBL and LpqN infers a host defense mechanism limiting the growth of Mtb in macrophages. In a third study, Mirrashidi et al. [Chlamydia trachomatis to generate an extensive HP-PPI network composed of 354 high-confidence lnc–human interactions. The study identified several proteins and pathways known to be modulated during infection and revealed cellular processes possibly modulated by C. trachomatis. Importantly, several of these HP-PPIs were found to be conserved also in viruses, such as HIV [In recent work, Happonen et al. generated a quantitative interaction map between the proteins . The maprophages . The stui et al.  used inch as HIV , 27.These above examples highlight how AP-MS coupled with appropriate bioinformatics data analysis strategies can determine interspecies PPIs, provide evidence that several of the characterized HP-PPI networks are highly interconnected and that certain bacterial proteins can bind to several human proteins to potentially form larger inter-species protein complexes , 17, 28.Understanding the quaternary structure of molecular complexes at a proteome level and close to in vivo conditions holds the potential of improving our understanding of HP-PPIs. Additionally, information regarding protein interaction sites and direct and indirect protein binding within HP-PPIs provides critically important information to establish the organization and topology of the HP-PPI networks and the arising HP-PPCs , 39. CroAcinetobacter baumannii cells with human lung epithelial cells led to the first large-scale HP-PPI analysis for A. baumannii. Biotin-aspartate proline-PIR n-hydroxyphthalimide (BDP-NHP) was used to crosslink A. baumannii with human cells, followed by tryptic digestion of the cross-linked proteins and subsequent MS analysis. This cross-linking experiment using human lung epithelial cells identified the outer membrane protein A (OmpA) as a virulence factor. OmpA was found to bind to a desmosomal protein providing evidence for a novel mechanism of how A. baumannii enables epithelial intrusion and cell invasion. In more recent work, Hauri et al. developed a novel XL-MS workflow termed targeted chemical cross-linking (TX-MS). TX-MS relies on a combination of chemical cross-linking, high-resolution mass spectrometry and high-accuracy protein structure modeling [S. pyogenes M1–human protein complex identified in a previous AP-MS study [S. pyogenes masks its conserved and vulnerable surface epitopes in the binding interfaces with human proteins [In a study by Schweppe et al. , cross-lmodeling . TX-MS wMS study , as discMS study . At the proteins .The above instances demonstrate that XL-MS is a promising technique that promotes the analysis of HP-PPI networks and elucidates the arising protein complex structures. Addition of cross-linkers to proteins stabilizes their interactions in native conditions, thus providing valuable information on their dynamics and flexible regions. Moreover, covalent bond formation between interacting proteins leads to capture of weak or transient interactions thereby reducing non-specific background , 45. TheAs detailed above, AP-MS and XL-MS can provide detailed information of global HP-PPIs, their composition, dynamic regulation, overall topology and specific protein–protein interaction sites. However, even when combined with structural modeling, XL-MS and AP-MS are unable to provide high-quality, atomic resolution structural information of the individual proteins in a complex or the structure of the complete HP-PPC. For studies where high-resolution structural information is required, XL-MS can be combined with single-particle cryoEM (see “INFO BOX 4”) as described for several large, multicomponent human protein complexes as recently reviewed , 51. To Listeria monocytogenes [Chlamydia trachomatis [C. trachomatis elementary bodies (EBs) released naturally from co-cultured infected cells and visualized them by cryoET. In this way, a system was developed to visualize snapshots of Chlamydial EBs under physiological conditions during early stage cell entry, including the type III secretion system (T3SS) needles in direct contact with the host plasma membrane [L. monocytogenes and visualizing the infected cells by cryoET, followed by tomogram interpretation by an automatic segmentation algorithm developed specifically for the tracking of actin filaments [Much of current state-of-the art work in imagining interactions between bacteria and human via cryoET have been done on ytogenes  and the chomatis , 68, 69.membrane . Jasnin ilaments . The worilaments  being thilaments , 72.C. trachomatis T3SS needle syringe-like movement when injecting effector proteins into the host cytoplasm [Although cryoET is an important visualization technique as demonstrated above, it does not provide molecular level detail of the HP-PPIs or HP-PPCs. Sub-tomogram averaging of frequent protein–protein contacts during infection could alleviate this gap in knowledge, much as has been done for the ytoplasm  or in coytoplasm . There iThe successful integration of biological mass spectrometry with cryoEM and cryoET will be strongly dependent on novel structural molecular modeling approaches. Structural molecular modeling has changed in recent years by the introduction of low-resolution modeling techniques and fragment-based movers. These techniques use segments of known protein structures to ensure that perturbations to the model simulation adhere to biochemical constraints that determines the three-dimensional structure of the protein  , native mass spectrometry, single-particle cryoEM, X-ray crystallography, NMR, small angle X-ray and neutron scattering methods, together with cellular visualization methods . As mentioned earlier, such integrative studies would advance our understanding in pathogenesis, yet a lot remains to be done with respect to applying such approaches in medical microbiology. Current studies have combined electron cryo-microscopy, XL-MS and structural modeling to a large extent to understand intra-species, i.e., human–human protein complexes , 92–97.In this review, we have showcased different techniques involved in understanding different tiers in HP-PPI. Integrative approaches involving AP-MS, XL-MS, cryoEM and structural modelling could provide combined knowledge and valuable insights into the process of infection. AP-MS identifies interacting protein pairs; however, it fails to identify the interacting peptides and domains between proteins. AP-MS coupled with XL-MS would not only identify interacting proteins, but also stabilize transient interactions and identify amino acid pairs between the interacting proteins. Complementing AP-MS and XL-MS with single-particle cryoEM would provide additional structural information on the complete HP-PPC. Both XL-MS and single-particle cryoEM require small sample amounts and both can be applied to heterogeneous samples. For larger protein complexes, where the local resolution in cryoEM maps can vary considerably with usually highly defined core regions and more poorly resolved densities towards the edges, XL-MS can help in resolving the structure of these edges by providing the needed distance constraints between proteins or their domains . HoweverIn this review, our focus is on demonstrating how AP-MS, XL-MS, cryoET and molecular modeling have been successfully used to address the organization and dynamics of HP-PPI networks and the structure of HP-PPCs. Incorporating integrative structural biology more broadly in infection medicine research would increase our understanding of how the diverse HP-PPIs facilitate bacterial adhesion, dissemination and survival within the host. These efforts could also have a future impact on drug development programs to interfere with the interaction and assembly of these HP-PPCs as well as for vaccine development strategies to combat these infections.


Recent research on cell signalling, tCho metabolism, and TT of cancer has been retrospectively re-examined. Signalling research describes how the unfolded protein response (UPR), a major stress signalling network, transduces, regulates, and rewires the total membrane turnover in different cancer hallmarks after a TT stress. In particular, the UPR signalling maintains or increases total membrane turnover in all pro-survival hallmarks, whilst dramatically decreases turnover during apoptosis, a pro-death hallmark. Recent research depicts the TT-induced stress as a crucial event responsible for interrupting UPR pro-survival pathways, leading to an UPR-mediated cell death. The 1H-MRS tCho resonance represents the total mobile precursors and products during the enzymatic modification of phosphatidylcholine membrane abundance. The tCho profile represents a biomarker that noninvasively monitors TT-induced enzymatic changes in total membrane turnover in a wide variety of existing and new anticancer treatments targeting specific layers of the UPR signalling network. Our overview strongly suggests further evaluating and validating the 1H-MRS tCho peak as a powerful noninvasive imaging biomarker of cancer response in TT clinical trials.This review describes a cellular adaptive stress signalling roadmap connecting the  Bindingus lumen . Interacus lumen . In the anslated , 21. In rogramme , 26.Central to a layer network is integration of signalling by core signalling processors. For UPR signalling, these include kinases , switches , cascades  and adaptors  –32. UPR’UPR signalling tightly controls and coordinates the secretory membranous machinery within the pro-survival and pro-death signalling pathways . The claThe UPR signalling adjusts secretory protein synthesis, modification and trafficking to overcome fluctuations in intrinsic and extrinsic cellular stress , 26. SecAll UPR pro-survival signalling mechanisms maintain or increase membrane synthesis. The UPR pro-survival signalling utilises two transmembrane sensors, IRE1 and the activating transcription factor 6, ATF6 see Fig.  10]. Fi. Fi10]. s, by-stander cargo ICM and ER-whorls. Macroautophagy and ER-phagy are pro-survival hallmarks that degrade and recycle their products to maintain cellular nutrients, energy and homeostasis  levels in invasive ductal carcinoma  versus benign fibrosis and hyperplasia   and [tCho/Creatine] peak ratios measured at the end of the therapy cycle correlated with the central nervous system-specific progression-free survival and overall survival  (0.1 mM) and [GPC] (0.1 mM), along with the concentration of phosphoethanolamine (≤ 0.2 mM), another phosphomonoester contributing with two protons to the tCho spectroscopic profile. These results suggested the potential use of a combination of in vivo31P- and 1H-MRS at very high field to monitor quantitative changes in phospholipid metabolites in breast cancer lesions of patients treated with neoadjuvant chemotherapy, as successfully tested in a very recent study reported by the same research team -6-methoxy-7-3- (pyrrolidin-1-ylpropoxy) quinazoline, Cediranib, a pan vascular endothelial growth factor inhibitor, causes a complex therapeutic UPR stress response . BetweenEven though 17-N-allyamino-17-demethoxygeldanamycin is a mono-TT, its HSP90 inhibition has a variety of specific synergistic effects. HSP90 inhibition inactivates and degrades multiple client signalling proteins such as HER-2, the serine/threonine-protein kinases C-Raf and AKT, the epidermal growth factor receptor known as EGFR and the mast/stem cell growth factor receptor known as SCFR, all involved in multiple oncogenic pro-survival hallmarks .1H-MRS tCho profile in response to a given TT. In the frame of this proposed, more comprehensive interpretation, tCho could also identify and quantify autophagy, apoptosis, and resistance after TT. The UPR signalling network may also overcome the limitations of earlier cancer signalling maps [There is a pressing need for a robust therapeutic molecular biomarker that monitors all cancer hallmarks after TT. Deciphering recent cell signalling research uncovers a UPR signalling roadmap connecting the therapeutic tCho biomarker of membrane turnover to all remaining, reprogrammed known hallmarks of cancer after a targeted therapeutic stress. The feasibility of a therapeutic biomarker and signalling roadmap simplifying complex signalling mechanisms after TT is documented by two recent clinical TT studies , 75. Soming maps , 27, 28.The greatest potential impact of tCho imaging is the simplification of a treatment response assessment that can be clouded by complex variations occurring in stress, genomics, proteomics and cancer hallmarks in a given patient. tCho reflects the therapeutic stress as transmitted, regulated, modified and integrated through a UPR cellular adaptive stress signalling network. Monitoring tCho could in the future replace the assessment of multiple stress, genomic, proteomic, and phenomic biomarkers. The tCho peak could distinguish in quantitative terms between UPR pro-survival signalling (increase in tCho) and UPR pro-death signalling (decrease in tCho) after TT . The tCh1H-MRS continues to improve in clinical and preclinical MR imaging with faster acquisitions, increased coil sensitivity and higher field magnets. In clinical units, multi-voxel 1H-MRS acquisitions interrogate the core of the tumour, as well as the periphery allowing autocrine and paracrine signalling to be monitored in different regions/micro-environments of the tumour and possibly residual and recruited cancerous cells [The tCho biomarker has several inherent strengths. The tCho biomarker fulfils the criteria of a relevant biomarker ; tCho mous cells . The tChus cells . Use of us cells , 79.1H-MRS equipment and lack of automation. Low sensitivity arises from low signal-to-noise ratio and increased noise in the electronic signalling chain. Advancements in high-field magnets/coils, software acquisitions, and computer processing continue to improve the sensitivity of tCho detection. Recently, artificial intelligence has been shown to boost sensitivity by improving the signal-to-noise ratio, reducing noise from motion, and generating faster images (spectra). Artificial intelligence replaces the conventional reconstruction chain with a data driven reconstruction between the sensor domain and the image (spectrum) domain [The use of tCho as a biomarker has several weaknesses including the low sensitivity of clinical ) domain . The key1H-MRS molecular therapeutic biomarker of total membrane turnover in all hallmarks of cancer after targeted therapy. Adding tCho to ongoing clinical trials of TT and comparison with other clinical trial biomarkers would test the tCho biomarker robustness. Adding tCho to an ongoing trial would rapidly generate “evidence-based” proof of tCho impact on diagnostic accuracy, therapeutic decisions, patient outcome and cost to society. In the USA, a search of National Institutes of Health funding of 2019 molecular cancer biomarkers indicates that this institution is receptive to funding a molecular therapeutic choline biomarker [This review lays the groundwork for scientific testing of a iomarker , 82. Othiomarker .During the final revisions to our review, some preclinical research articles suggested targeting essential components in cancer’s amplified pro-survival hallmarks. Martin Leach’s team suggested targeting the critical role of cytidylyltransferase enzyme in autophagosome membrane synthesis to block autophagy, a pro-survival hallmark . Jason K1H-MRS-detected tCho, a marker of membrane turnover, directly reflects and monitors all known hallmarks of cancer after TT. This vision offers a perspective to be verified by clinical studies.In conclusion, we are strongly convinced that a deeper understanding of cellular adaptation to stress reveals that


Klebsiella pneumoniae carbapenemase (KPC)–3–producing multidrug-resistant K. pneumoniae with ceftazidime/avibactam (CAZ-AVI) in a patient who developed postneurosurgical meningitis and bacteremia. Therapeutic drug monitoring of cerebrospinal fluid and blood samples demonstrated CAZ-AVI concentration levels 20-fold greater than the minimum inhibitory concentration in the first 60 minutes postinfusion, providing evidence for the utility of CAZ-AVI in treating KPC–Klebsiella pneumoniae central nervous system infections.This report describes the treatment of  Enterobacteriaceae (CRE) are difficult to treat. Selecting an effective antibiotic regimen to overcome antimicrobial resistance while achieving and maintaining adequate CNS levels is particularly challenging. Moreover, clinical accounts treating CNS infections with newer antimicrobial agents are lacking, and data pertaining to their pharmacokinetic/pharmacodynamic (PK/PD) properties remains scarce. Carbapenem-hydrolyzing β-lactamases such as Klebsiella pneumoniae carbapenemase (KPC) confer resistance to most β-lactams, including carbapenems ) was determined to be 38%, while bacterial loads decreased by a mean of 5.66 log10 CFU as a result of therapy [CAZ-AVI is currently approved for intra-abdominal infections, complicated urinary tract infections, hospital-acquired bacterial pneumonia, and ventilator-associated bacterial pneumonia. The clinical experience with CAZ-AVI in the treatment CNS infections is uncommonly reported, and the capacity of this combination to cross the blood–brain barrier remains unknown. In this setting, effective therapy depends on the ability of CAZ-AVI to maintain elevated CSF concentrations that achieve PK/PD thresholds seen in prior simulation models for CNS infections. The only available experimental data were conducted in an animal model inoculating AmpC-producing t1/2 for CAZ and AVI, concentrations were likely well above the MIC for the entire 8-hour dosing interval in the CSF and plasma. In fact, CAZ concentrations were likely 4–5 times higher than the MIC for the entire dosing interval in both the CSF and plasma [In our patient, TDM of CAZ and AVI was conducted to determine how effectively CAZ and AVI distributed into the CNS. Consistent with the Food and Drug Administration–approved dosing, the patient received 2.5 g of CAZ-AVI administered as a 2-hour infusion every 8 hours . Based on our current understanding of the target PK/PD indices for CAZ-AVI , the mead plasma . Similard plasma , 13 and d plasma , 15. TheKp is unclear. Our patient received an additional 2 weeks of CAZ-AVI and intrathecal amikacin to achieve resolution of CSF neutrophilic pleocytosis and microbiologic cure. Although the objective of measuring CAZ-AVI concentrations in the CSF of a patient with a KPC-Kp-associated CNS infection was achieved, these data should be interpreted with caution as they represent only 2 CSF sample measurements. Furthermore, conclusions regarding CAZ-AVI serum ratios/percentages cannot be generalized using this study design due to the sample size, interpatient variability, and other clinical variables.Only 2 published reports have described CRE-associated CNS infections treated with CAZ-AVI. In both cases, postneurosurgical meningitis was caused by CRKP (KPC production was not confirmed). Samuel et al.  employedAdditional studies that measure CSF concentrations of CAZ-AVI using more samples from multiple patients and throughout the entire dosing interval are required to determine the PK/PD parameters for CNS infections with highly resistant organisms. However, the levels attained and measured in our patient using this dosing regimen appear to have been adequate.KPC-Kp nosocomial meningitis. A novel CAZ-AVI TDM method was successfully applied to demonstrate that CAZ-AVI achieves CSF levels well above the MIC in the first 180 minutes postinfusion. Further clinical studies are required to assess the complete profile of CAZ-AVI in the CSF. Our case provides evidence on the use of this agent in treating KPC-Kp.This report describes the measurement of CAZ-AVI concentration levels in the CSF of a patient suffering from


Cytosolic DNA sensing, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, is an important novel role in the immune system. Multiple STING agonists were developed for cancer therapy study with great results achieved in pre-clinical work. Recent progress in the mechanical understanding of STING pathway in IFN production and T cell priming, indicates its promising role for cancer immunotherapy. STING agonists co-administrated with other cancer immunotherapies, including cancer vaccines, immune checkpoint inhibitors such as anti-programmed death 1 and cytotoxic T lymphocyte-associated antigen 4 antibodies, and adoptive T cell transfer therapies, would hold a promise of treating medium and advanced cancers. Despite the applications of STING agonists in cancer immunotherapy, lots of obstacles remain for further study. In this review, we mainly examine the biological characters, current applications, challenges, and future directions of cGAS-STING in cancer immunotherapy. Cancer is one of the major lethal diseases worldwide, with a high morbidity of 18.1 million estimated new diagnosed cases and mortality of 9.6 million deaths in 2018 reported in the Global cancer statistics .The stimulator of interferon genes (STING) is a novel player with pleiotropic effects in the field of the immune system. The discovery of STING as a 42-kDa “dimeric adaptor protein” in 2008 quickly expanded the fields of immunology research as well as cancer immunotherapy –7. In thThe cGAS-STING pathway is the central cellular cytosolic double-stranded DNA (dsDNA) sensor, allowing innate immune to respond to infections, inflammation, and cancer , 9. BothThe upstream dsDNA interacts with enzyme cGAS in a sequence-independent way , 13, proThe structure of protein human STING (hSTING) comprises a N-terminal trans-membrane domain with four helices aa 1-154), an acidic C-terminal tail (aa 342-379), and a central globular domain (aa 155-341) separated the former two . The mou, an acidKRAS-mutated lung cancer, with the loss of the tumor suppressor gene LKB1 [+ T cell infiltration and downregulated cGAS-STING pathway mediated via the reduction of the expressions of the downstream IFN-I targeted genes such as chemokine (C-X-C motif) ligands 10 (CXCL10) [STING-deficiency has been reported to correlate with cancer incidence. In six cancerous melanoma cell lines , STING expression was not detectable or significantly inhibited . Severalene LKB1 . Furtherene LKB1 . Their s(CXCL10) . Survivi(CXCL10) .The activation of the cGAS-STING pathway plays a crucial role in both tumor cells and immune cells as an innate immune sensor, which could regulate multiple steps in cancer-immunity cycle. This cytosolic DNA sensing has been well-characterized, which can induce IFN production and arouse host immune responses mediated by infiltration of immune cells such as T cells and natural killer (NK) cells , 25. Act+ T cell cross-priming via Batf3-lineage dendritic cells (DCs) [+ DC survival and antigen retention, which enhances DC’s cross-presentation [Pre-clinical studies have demonstrated the activation of STING pathway in host immune cells is vital for IFN-β production , and in ls (DCs)   , or tumols (DCs) , therebyentation . Besidesentation . Throughentation , 36. In entation . Further+ T cell responses, and this ability could be further enhanced when both PD-1 and CTLA-4 were blocked. Their further studies showed this immune response depended on the production of type I IFNs from endothelial cells in TME, indicating the strategy of targeting tumor endothelial cells for melanoma immunotherapy. Another study enhanced STING activation of the tumor and lymph nodes by using the cGAMP-based nanoparticles, which enhanced the cytosolic delivery of cGAMP by promoting its endosomal escape and triggered the formation of a “hot” type TME with enriched T cell infiltration [Due to the correlation between deficiencies in cGAS-STING pathway and surviving cancer cells, as well as the importance of cGAS-STING in the regulation of cancer-immunity cycle, STING agonists were developed to mimic this activation to enhance anti-cancer effects Table . CDNs haltration . This noltration . Theses n = 26), physician or patient decision (n = 8), and death (n = 1) [Beyond naturally derived CDNs, synthetic CDNs with better properties were developed. The anti-tumor compound dithio CDN  showed a high binding affinity to hSTING alleles . This CD (n = 1) . Dose-liA growing body of evidence showed the pharmacological function of non-CDN agents in cGAS-STING activation. The initiated agonist targeting the STING pathway is dimethyloxoxanthenyl acetic acid (DMXAA) . ActuallThe design of agent amidobenzimidazole (ABZI) represented a new breakthrough of STING agonist in immune-modifying cancer treatment . This noAppropriate adjuvants play an essential role in tolerance overcome and tumor-specific immunity enhancement, and innate immunity activation is able to boost antigen-presenting cell (APC) activation, which facilities the immunogenicity of tumor-associated antigens (TAAs) . STINGVAα+ DCs [+, CD8+, GZMB+, and CD45RO+ cells) represent favorable prognosis and positive clinical results for cancer patients [Several STING agonists have been used as an anti-cancer therapy in clinical trials, and the STING agonists/ICIs combinations were also developed Table . cGAS-STα+ DCs , which ppatients . Cytotoxpatients . In addipatients . Indeed,patients .The threshold of T lymphocyte activation can be reduced by the application of anti-CTLA-4 therapy . EvidencThe cGAMP/antigenic peptide nanosatellite vaccine SatVax, combined with anti-PD-L1 therapy in the xenograft model, showed elevated E7-specific CD8+ CTLs, and decreased ratio of CD8+ Tim3+ and CD8+ PD-1+ T cells . This suOncolytic viruses have been regarded as a versatile platform to treat cancer. They are viral vectors that can kill tumor cells selectively and also have the potential to amplify the immune response and enhance anti-tumor effects . The oncEngineered T cell has the ability to recognize the targeted antigen of tumor cells with the single-chain variable fragment domain, through transferring gene encoding chimeric antigen receptor (CAR) . The CARUndoubtedly, STING agonists showed impressive potential in antitumor immunity. However, emerging evidence suggested the pro-tumor roles of the cGAS-STING pathway, from tumor initiation and development, to metastasis –72 Fig., which mBeyond the pro-tumor chronic inflammation and CIN, the ER stress response as well as autophagy also serve as a barrier to the anti-tumor effect of STING agonists. They play a part in the advanced tumor progression by making tumor cells survive under stressful conditions . The ER Another major challenge is how to carefully select patients to enhance clinical response to STING agonists. The preliminary clinical phase 1 trial of STING agonist (MK-1454) in solid tumors and lymphomas has shown only a modest clinical response using single-agent treatment with no marked activity seen, and the co-administration of it with ICIs in advanced cancers showed only partial responses . TherefoIn addition, there may have technical limitations of the bulk RNA sequencing since the relationship between patient outcomes and cGAS-STING RNA levels is incongruent across different tumors. For example, poor patient survival was revealed with downregulated cGAS-STING signaling in a subset of human tumors , 72. WhiA comprehensive mechanistic understanding of the biologic STING signaling may greatly help to develop agents to potently activate it while reducing its immune-suppressive effects. For example, several feedback loops were found to attenuate STING activation including autophagy induction, AIM2 inflammasome activation, and autocrine IFN signaling , 83. Com−/− mice lost the antitumor effect, indicating the importance of host immune cell priming [Another implication for selectively promoting desired outputs is to increase the local concentration of type I IFNs. One method is the tumor-targeted monoclonal antibodies combined with IFN-β. For example, a method of coupling either anti-EGFR or anti-Her2 monoclonal antibodies to IFN-β can result in tumor regression in tumor-bearing mice . In addi priming . Thus, tIt is also critical to understanding the STING-based TME changes in pre-clinical experiments and clinical trials, so that less side effects may be produced in cancer treatment. Most STING agonists have not encountered pro-tumor effects because just a few doses of treatment could result in a burst of type I IFN production to activate anti-tumor immune system . TherefoThe burgeoning interest in the STING pathway, using patients’ own immunity to eradicate tumors, is extremely appealing and several STING agonists were developed for cancer treatment with promising pre-clinical results. As a critical immune sensor, the STING pathway plays an important role in tumor control through tumor-derived DNA sensing and T cell priming. This induces T cell presentation in tumors, making the STING pathway a promising strategy combined with ICI therapy. STING agonists can elevate the efficacy of therapies from cancer vaccine, to ICIs to CAR-T immunotherapies. However, emerging pro-tumor roles have been described and a greater understanding of STING-associated TME and biologic mechanism is needed. We believe cGAS-STING pathway manipulation might become a promising strategy combined with cancer immunotherapy.


Despite the exponential growth in research following the rapid spread of SARS-CoV-2 and subsequent COVID-19 pandemic, the underlying pathophysiological mechanisms in COVID-19 patients remain poorly understood. The increased incidence of cardiovascular and thromboembolic complications, immune cell deactivation and sepsis-like multiple organ failure suggests the involvement of multiple pathways. Accordingly, recent studies have proposed that virus-induced endothelial dysfunction and damage, resulting in impaired vascular blood flow, coagulation and leakage, may partially explain the development of organ dysfunction and oedema [Whilst numerous pro-inflammatory cytokines and blood biomarkers have already been compared in patients with different severities of COVID-19 - to date - no study, report or editorial has described the potential role of adrenomedullin (ADM) during the host response to COVID-19. This is surprising, since ADM has been shown to play a key role in reducing vascular (hyper) permeability and promoting endothelial stability and integrity following severe infection . Thus, AFurthermore, recent clinical studies on sepsis patients upon emergency department (ED) presentation and during intensive care (ICU) treatment using the stable protein surrogate, mid-regional proadrenomedullin (MR-proADM), found that its assessment could accurately identify disease progression in patients with non-severe clinical signs and symptoms, safely increase out-patient treatment with decreased readmission rates and no subsequent mortalities , and ideThe assessment of MR-proADM in future COVID-19 studies may therefore provide important information into the pathophysiological mechanisms underlying endotheliitis and subsequent organ dysfunction. The early identification of patients likely to develop severe clinical symptoms requiring subsequent hospitalisation, as well as the safe discharge of those already hospitalised, may be of particular importance in regions where healthcare systems are used to full capacity.


Ebola virus (EBOV) is a zoonotic pathogen causing severe hemorrhagic fevers in humans and non-human primates with high case fatality rates. In recent years, the number and extent of outbreaks has increased, highlighting the importance of better understanding the molecular aspects of EBOV infection and host cell interactions to control this virus more efficiently. Many viruses, including EBOV, have been shown to recruit host proteins for different viral processes. Based on a genome-wide siRNA screen, we recently identified the cellular host factor carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as being involved in EBOV RNA synthesis. However, mechanistic details of how this host factor plays a role in the EBOV life cycle remain elusive. In this study, we analyzed the functional and molecular interactions between EBOV and CAD. To this end, we used siRNA knockdowns in combination with various reverse genetics-based life cycle modelling systems and additionally performed co-immunoprecipitation and co-immunofluorescence assays to investigate the influence of CAD on individual aspects of the EBOV life cycle and to characterize the interactions of CAD with viral proteins. Following this approach, we could demonstrate that CAD directly interacts with the EBOV nucleoprotein NP, and that NP is sufficient to recruit CAD into inclusion bodies dependent on the glutaminase (GLN) domain of CAD. Further, siRNA knockdown experiments indicated that CAD is important for both viral genome replication and transcription, while substrate rescue experiments showed that the function of CAD in pyrimidine synthesis is indeed required for those processes. Together, this suggests that NP recruits CAD into inclusion bodies via its GLN domain in order to provide pyrimidines for EBOV genome replication and transcription. These results define a novel mechanism by which EBOV hijacks host cell pathways in order to facilitate genome replication and transcription and provide a further basis for the development of host-directed broad-spectrum antivirals. Ebolavirus within the order Filoviridae, and is the causative agent of severe hemorrhagic fevers in humans and non-human primates with high case fatality rates , and the anti-c-myc antibody used for IFA analysis was obtained from Thermo Fisher Scientific [A-21281]. Primary antibodies against NP , GAPDH (mouse anti-GAPDH clone 0411), and CAD (rabbit anti-CAD clone EP710Y) were ordered from IBT Bioservices , Santa Cruz , or Abcam . Secondary antibodies used for IFA analysis against mouse (Alexa Fluor 488 anti-mouse [A-11029]), rabbit (Alexa Fluor 568 anti-rabbit [A-11036]), and chicken IgY (Alexa Fluor 647 anti-chicken [A-21449]) were obtained from Thermo Fisher Scientific. For Western blotting, secondary antibodies against mouse (IRDye 680RD anti-mouse [926-68070]) and rabbit IgG (IRDye 800CW anti-rabbit [926-68071]) were purchased from Li-COR , while anti-mouse IgG (Kappa light chain) Alexa Fluor 680 [115-625-174] used for coIP analyses was ordered from Dianova .Zaire ebolavirus rec/COD/1976/Mayinga-rgEBOV (GenBank accession number KF827427.1), which is identical in sequence to the EBOV Mayinga isolate with the exception of four silent mutations as genetic markers , was use100mM uridine or cytidine (both Sigma-Aldrich) stock solutions were prepared in dimethyl sulfoxide (DMSO) and further diluted in cell culture medium. Diluted pyrimidines or DMSO corresponding to 1% of the supernatant volume in 12-well plates was added to the cells at the time of transfection and after medium changes. All concentrations indicated in the figures are final concentrations.For siRNA knockdown of endogenous CAD, 293T cells were reverse transfected  with 12 pmol pre-designed silencer select siRNAs  or a self-designed EBOV-anti-L siRNA [5′-UUU AUA UAC AGC UUC GUA CUU-3′] ordered from Eurofins Genomics . Transfection was performed in 12-well plates using Lipofectamine RNAiMax (Thermo Fisher Scientific) following the manufacturer’s instructions. 48 h post-siRNA transfection, the cells were transfected using Transit-LT1  with all minigenome assay components, i.e., pCAGGS-based expression plasmids for NP (62.5 ng), VP35 (62.5 ng), VP30 (37.5 ng), L (500 ng), codon-optimized T7-polymerase (125 ng), firefly luciferase , and the T7-driven monocistronic minigenome . For analyses of vRNA and mRNA levels the control firefly luciferase was replaced with GFP (200 ng), and for the replication-deficient minigenome assay a Pol-II-driven replication-deficient minigenome (pCAGGS-EBOV-1cis-vRNA-nLuc-RdM) was used. Transfections were performed using Transit LT1 as previously described [RNA isolation from minigenome cell lysates was performed following the manufacturer’s instructions using a NucleoSpin RNA kit . After RNA purification, all samples were treated with DNase  following the manufacturer’s instructions to avoid plasmid contamination. For cDNA generation, RNA samples were incubated with an oligo(dT)-primer for mRNA quantification or with a strand-specific primer (5′-AGT GTG AGC TTC TAA AGC AAC C-3′) for vRNA quantification using the RevertAid Reverse Transcriptase (Thermo Fisher Scientific) following the manufacturer’s instructions. The subsequent qPCR was performed using a PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) with 1 µL of cDNA and primers targeting either the reporter gene , or GFP as a control . Values for vRNA and mRNA levels were normalized to control GFP mRNA levels.++ (PBS with 0.9M Ca2+ and 0.5M Mg2+)) for 10 min. Then, cells were permeabilized with 0.1% Triton X-100 in PBS for another 10 min and incubated with 10% fetal calf serum (FCS) in PBS for 45 min. Primary antibodies  were diluted in PBS with 10% FCS and cells were incubated for 1 h at room temperature with the prepared antibody solutions. Secondary antibodies  were prepared as described for the primary antibodies. After 45 min of staining, cells were washed with PBS and water before mounting with ProLong Diamond Antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific). Slides were analyzed by confocal laser scanning microscopy using a Leica SP5.Huh7 cells, which are more suitable for IFA than 293T cells, were seeded on coverslips in 12-well plates and transfected 24 h later with 500 ng pCAGGS-EBOV-NP and 500 ng pCAGGS-FLAG-HA-CAD (or CAD mutants) and, in selected experiments, additionally with 500 ng pCAGGS-myc-VP35 as indicated. For a mock control, cells were transfected with pCAGGS. Transfection was performed using polyethylenimine (Sigma-Aldrich) following the manufacturer’s instructions. 48 h post-transfection, cells were fixed using 4% paraformaldehyde  in DMEM for 20 min and then treated with 1 M glycine  and transfected as described above  with 500 ng pCAGGS-FLAG/HA-CAD. At 48 h post-transfection, the transfected cells were infected with EBOV at an MOI of 1, and the samples were fixed 16 h post-infection in 10% formalin twice overnight prior to removal from the BSL4 facility and immunofluorescence analysis.CoIPs were performed as previously described . BrieflyFor validation of CAD knockdown efficiency and analyses of coIP input and lysates, samples were subjected to SDS-PAGE and Western blotting as previously described . FLAG-taOne-way ANOVA with a Dunnett’s multiple comparisons test was performed using the GraphPad Prism 8.1.0 software.Using a genome-wide siRNA screen, we previously identified CAD to be important for EBOV RNA synthesis and/or viral protein expression . HoweverNext, we performed a classical minigenome assay A in connTo further dissect the influences of CAD on viral genome replication, mRNA transcription, and later steps of viral protein expression, we performed classical minigenome assays in the context of an siRNA knockdown of CAD and measured vRNA and mRNA levels in cell lysates using RT-qPCR. For this, we used either an oligo-dT primer for reverse transcription of mRNAs, or a strand-specific primer for reverse transcription of vRNA, followed by qPCR against the reporter gene. CAD siRNA-treated cells showed a strong reduction in both vRNA and mRNA levels in comparison to the control cells, demonstrating that CAD is important for both EBOV transcription and viral genome replication D,E.As CAD is an important component for pyrimidine synthesis , we wantSimilar to other negative-sense RNA viruses, EBOV and in particular its nucleoprotein NP is known to induce the formation of cytoplasmic inclusion bodies, which are sites of viral genome replication and transcription ,9. SinceTo assess the contribution of individual domains of CAD in its recruitment into NP-induced inclusion bodies, we focused on the GLN and the CPS domains. When we expressed deletion mutants lacking these domains, they showed a similar intracellular distribution compared to wild-type CAD when expressed alone in cells. During coexpression of NP and CAD-ΔCPS, we observed recruitment of this mutant into NP-driven inclusion bodies , indicating that the CPS domain of CAD is not required for its accumulation in inclusion bodies . In starAs NP recruits CAD into EBOV inclusion bodies, we next assessed whether CAD interacts with NP. To this end, we performed coIP assays using FLAG-CAD expressed in the presence of NP by precipitating CAD with an anti-FLAG antibody and then detecting NP by Western blotting. We could readily co-precipitate NP with CAD, indicating that CAD is able to interact with NP . Becausel-aspartate (PALA), which transiently inhibits the aspartate transcarbamylase activity of CAD, were effective in vitro against various viruses, including vaccinia virus and arenaviruses [In this work, we identified CAD, an essential component of the de novo pyrimidine synthesis pathway, to be important for both EBOV genome replication and transcription, and demonstrated that the function of CAD in pyrimidine synthesis is responsible for this effect. Knockdown of CAD was also shown to affect replication and transcription of other viruses, e.g., hepatitis C viruses . Furtheraviruses ,40. The aviruses , will haOur results are consistent with the fact that several pyrimidine synthesis inhibitors are effective against EBOV in vitro, underlining the importance of the pyrimidine pathway for these viruses ,42. ExamWe were further able to show that CAD is recruited to EBOV inclusion bodies, which represent the site of EBOV replication and transcription ,9. SinceIn the context of its cellular function, CAD has been shown to localize primarily in the cytoplasm, although small amounts can also be detected in the nucleus of dividing cells. Redistribution of CAD into nuclear compartments during cell growth and proliferation is believed to be in response to phosphorylation by MAP kinases at position Thr-456, which results in upregulation of the enzymatic activity of CAD . Since NOverall, we have shown that CAD is recruited into NP-induced and virus-induced inclusion bodies to provide sufficient amounts of pyrimidines for EBOV genome replication and transcription. Furthermore, we demonstrated that the GLN domain of CAD is required for recruitment into inclusion bodies. These findings increase our understanding of EBOV and its host cell interactions, and provide a basis for future identification of molecular targets for the development of novel therapies against this virus.


Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new type of coronavirus that causes the Coronavirus Disease 2019 (COVID-19), which has been the most challenging pandemic in this century. Considering its high mortality and rapid spread, an effective vaccine is urgently needed to control this pandemic. As a result, the academia, industry, and government sectors are working tightly together to develop and test a variety of vaccines at an unprecedented pace. In this review, we outline the essential coronavirus biological characteristics that are important for vaccine design. In addition, we summarize key takeaways from previous vaccination studies of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), highlighting the pros and cons of each immunization strategy. Finally, based on these prior vaccination experiences, we discuss recent progress and potential challenges of COVID-19 vaccine development. Coronaviruses (CoVs) are a group of related viruses that can cause respiratory tract infection in humans ranging from mild symptoms to lethal outcomes. Until now, there are seven genera of CoVs that are known to infect humans . Four ofCoronaviruses, whose name derives from their characteristic crown-like appearance under the electron microscope, are enveloped RNA viruses with a diameter of approximately 80–160 nm , 10. TheMany viral proteins are essential for the life cycle of CoVs. For entering target cells, S protein first binds to cellular receptors through its receptor-binding domain (RBD), and the receptor-virus complex is subsequently translocated to endosomes Fig.  15]. Bo. Bo15]. The S protein is particularly important for virus-cell receptor binding and virus-cell membrane fusion, suggesting that it can be an effective target for CoV vaccine design . In factThere are also immunopathological complications associated with the SARS-CoV and MERS-CoV vaccines that require addressing and further optimization. One adverse effect is the induction of antibody-dependent enhancement (ADE) effect, which is usually caused by vaccine-induced suboptimal antibodies that facilitates viral entry into host cells , 31. A sVarious forms of vaccines targeting SARS-CoV and MERS-CoV have been developed and tested in preclinical models. However, only a few of them entered clinical trials and none of them have been FDA approved. These approaches include protein subunit vaccines, virus-like particle vaccines, DNA vaccines, viral vector vaccines, whole-inactivated vaccines and live-attenuated vaccines. The following sections outline the principles of various forms of SARS-CoV and MERS-CoV vaccine development Table , and theProtein subunit vaccines consist of viral antigenic fragments produced by recombinant protein techniques. They are easy to produce, and relatively safe and well-tolerated compared to whole virus vaccines and viral vector vaccines. The drawback of protein subunit vaccines is their low immunogenicity. Therefore, adjuvants and fusion with immunostimulatory molecules are usually used together with subunit vaccines to overcome this challenge.The development of SARS-CoV protein subunit vaccines was initially surrounding full-length S protein-based vaccines and then later focused on S protein RBD-based vaccines. None of the SARS-CoV protein subunit vaccines entered clinical trials, but they induced potent antibody responses and protective effects in preclinical models , 40–44. Guided by previous SARS-CoV experiences, most proteins subunit vaccines of MERS-CoV are focused on RBD-based vaccines. RBD-based MERS-CoV vaccines generally show great immunogenicity and elicit potent neutralizing antibodies, cell-mediated immunity, and protective effect against MERS-CoV infection , 26. OneE. coli, which is probably due to the native conformation and post-translational modification maintained in mammalian cellular system [Apart from antigen design, several other factors also affect the efficacy of protein subunit vaccines . The expr system . In addir system . Their dr system , 51. TheVirus-like particles (VLPs) are self-assembled viral structural proteins that mimic the conformation of native viruses but lack the viral genome. Compared with protein subunit vaccines, VLP vaccines present epitope in conformation that is more similar to the native virus, leading to better immunization responses. In addition, compared to whole virus vaccines, the production of VLP vaccines does not involve live virus or inactivation steps, which makes them safer vaccine candidates. The highly repetitive antigenic surface of VLP vaccines also help induce stronger antibody response by efficiently cross-linking B-cell surface receptors. Up to now, VLP vaccines have been commercialized for the protection against human papillomavirus (Cervarix™ and Gardasil®) and hepatitis B virus (Engerix® and Recombivax HB®) .Few SARS-CoV and MERS-CoV VLP vaccines have been reported so far. For SARS-CoV, Lokugamage et al. have demonstrated that chimeric VLPs composed of SARS-CoV S protein and mouse hepatitis virus E, M and N proteins can induce neutralizing antibody responses and reduce SARS-CoV virus titer in mice lung after viral challenge . In addiDNA vaccines contain genes encoding viral antigenic components that are expressed by plasmid vectors and delivered into cells through electroporation. Compared with other vaccine technologies, DNA vaccines offer a fast and flexible platform for vaccine development and production, making it an attractive technology to combat emerging epidemics like SARS-CoV-2. In addition, antigen production of DNA vaccines happens in the target cells, which helps recapitulate the native conformation and post-translational modification of viral antigens. However, an important drawback of DNA vaccines is their limited immunogenicity due to their inability to spread and amplify in vivo. Therefore, it is important to consider strategies that can enhance the potency of DNA vaccines, such as adding adjuvant or using a prime-boost regimen. Besides, the genomic integration of DNA vaccines into the host chromosome is another biosafety concern, which may lead to mutagenesis and oncogenesis . Even thE. coli expressed recombinant S protein booster can also induce higher neutralization titers than DNA or protein subunit vaccine alone [Several DNA vaccine candidates have been reported for SARS-CoV, including the S-, M-, and N protein-based vaccines –64. Althne alone .Similar to SARS-CoV, several studies on MERS-CoV DNA vaccines have demonstrated optimistic results. Muthumani et al. reported that a full-length S protein-based MERS-CoV DNA vaccine can induce potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels, and macaques vaccinated with this DNA vaccine were protected against MERS-CoV challenge without demonstrating any clinical or radiographic signs of pneumonia . BuildinTaken together, DNA vaccines encoding full-length S or S1 protein have demonstrated encouraging results to fight against SARS-CoV and MERS-CoV. The same strategy is likely to be generalizable to SARS-CoV-2 DNA vaccine considering the biological similarity.Viral vector vaccines are recombinant viruses that encode antigens of interest in an unrelated modified virus. They deliver antigen into the cells mimicking natural infection, so they induce strong antigen-specific cellular and humoral immune responses per se, thereby obviating the need for additional adjuvants. In addition, viral vectors are able to accept large insertions in their genome, providing a flexible platform for antigen design. Despite these advantages, there are several drawbacks. The manufacturing process for viral vector vaccines is more complicated than other approaches, including the optimization of cellular systems and the exclusion of contaminants, which can greatly affect the efficiency of viral vectors . MoreoveSimilar to DNA and protein subunit vaccines, most viral vector coronavirus vaccines target the S antigen. Numerous viral vectors have been used to develop SARS-CoV and MERS-CoV vaccines, which have been described in detail in previous review articles , 75. In Adenovirus is a popular viral vector vaccine that has been tested in clinical trials for a wide variety of diseases, and several studies have also examined the efficacy of adenovirus-based SARS-CoV vaccine. The feasibility of SARS adenovirus vector vaccine was first demonstrated in two study by Gao et al. and Liu et al. , 77. TheModified vaccinia virus Ankara (MVA) is another well-established vaccine platform to combat emerging infectious diseases . Bisht eFor Venezuelan equine encephalitis (VEE) virus-based SARS-CoV vaccine, Deming et al. has reported that VEE virus replicon particles (VRP) expressing S protein provides complete short- and long-term protection against homologous strain challenge in young and senescent mice . To furtSeveral additional viral vectors have also shown promising results for SARS-CoV vaccines. Two studies from Buchholz et al. and Bukreyev et al. have used an attenuated parainfluenza virus as vector to express SARS-CoV S protein, showing that parainfluenza-based vaccine can induce neutralizing antibody responses and protective effect against SARS-CoV challenge in hamsters and monkeys , 87. BesSeveral adenovirus-based MERS-CoV vaccines have been developed. Human adenovirus type 5 (Ad5) and type 41 (Ad41) expressing MERS-CoV S or S1 protein have been shown to induce neutralizing antibodies in mice , 90. HowBesides, chimpanzee adenovirus has been employed as viral vector with an aim to overcome the pre-existing immunity problem of human adenoviruses. A MERS-CoV S protein vaccine based on a chimpanzee adenoviral vector (ChAdOx1) was shown to induce high levels of neutralizing antibodies and cell-mediated immune in mice, and to protect hDPP4-transduced mice from lethal MERS-CoV challenge , 95. In The vaccine of modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV S protein has been reported to induce not only virus-neutralizing antibody responses and MERS-CoV-specific CD8+ T cell response, but also provide protective effect against MERS-CoV in DPP4-transduced mice . FurtherMERS-CoV vaccines dependent on Venezuelan equine encephalitis (VEE) virus have also been studied. Agnihothram et al. have demonstrated that VEE virus replicon particles (VRP) expressing MERS-CoV S protein can induce neutralizing antibodies in young and aged mice . AnotherSeveral other vaccine platforms have been applied for the development of MERS-CoV vaccine. Measle virus- and rabies virus-based MERS-CoV S protein vaccines have been shown to induce neutralizing antibodies and provide protective effect against MERS-CoV in hDDP4-transduced mice , 105. NeIn summary, SARS-CoV and MERS-CoV vaccines based on many viral vectors, including adenovirus, modified vaccinia virus Ankara, Venezuelan equine encephalitis virus, parainfluenza virus, vesicular stomatitis virus, Measle virus, and rabies virus, have been shown to elicit protective immunity against viral challenges. Some of these viral vectors has already become promising candidate platforms for the development of SARS-CoV-2 vaccine.Whole inactivated vaccines are composed of chemically or radiationally inactivated virions. They contain a full repertoire of immunogenic components of the original virus, and compared with attenuated viruses, they carry no risk of viral reactivation if properly inactivated. Although safer than live attenuated vaccines, the immunogenic epitopes of inactivated viruses may be structurally deformed during the inactivation process, which can undermine the protection they may provide. Moreover, both SARS-CoV and MERS-CoV whole inactivated vaccines have been reported to induce eosinophil-related lung pathology , 37. TheDuring the early development of SARS-CoV vaccines, inactivated whole virus was once a leading strategy. Studies have shown that UV- and formaldehyde-inactivated SARS-CoV can induce neutralizing antibody response, and a phase I clinical trial using β-propiolactone-inactivated SARS-CoV vaccine demonstrated that it is safe, well-tolerated, and can elicit SARS-CoV-specific neutralizing antibodies –110. HowLive attenuated vaccines are live viruses weakened by deleting or mutating the pathogenic component of the viral genome. Similar to whole inactivated vaccines, they possess nearly the full immunogenic components of the original virus. Furthermore, they preserve the native conformation of viral antigens and present antigens to the immune system as in natural infections. Therefore, live attenuated vaccines are the most immunogenic kind of vaccine and have a long history of success in controlling a variety of infectious diseases . HoweverO-methyltransferase that is required for 5′ capping of viral RNA [Although a few SARS-CoV and MERS-CoV live attenuated vaccines have demonstrated efficacy in animal models, none of them have proceeded to clinical trials –117. Theiral RNA . This meiral RNA . Both SAiral RNA , 116. Moiral RNA . The losiral RNA . Graham Compared with SARS and MERS, which tended to resolve spontaneously after regional outbreak, the worldwide magnitude of the COVID-19 pandemic has made development of vaccine an unprecedented urgency. This urgent need has led to many different approaches in vaccine development considerations. First of all, unconventional vaccine platforms, such as nucleic acid vaccines and viral vector vaccines, are becoming the leading players in the race of COVID-19 vaccine development due to their ability to be developed using sequence information alone . These nIn this section, we will discuss the latest preclinical and clinical development of COVID-19 vaccines . We will highlight representative COVID-19 vaccines from each major vaccine platform that have published clinical data  vaccine . BNT162bCurrently, there are 12 viral vector vaccines in clinical trials, and an additional 36 viral vector vaccines under preclinical development . Many vin = 2,741) [n = 8,895) [11 and 5 × 1010 viral particles, and showed that the 5 × 1010 dose causes less severe adverse reactions without compromising the immunogenicity [AZD1222 is a chimpanzee adenovirus-based viral vector vaccine (ChAdOx1) expressing SARS-CoV-2 spike protein . This Ch= 2,741) . On the = 8,895) . Due to = 8,895) . They co= 8,895) . In both= 8,895) . Their r= 8,895) . Both va= 8,895) . Now thi= 8,895) . Their r= 8,895) . They al= 8,895) . There w= 8,895) . These p= 8,895) , 93. In Currently, there are 7 whole inactivated COVID-19 vaccine in clinical trials . From thThere are also several other COVID-19 vaccine candidates using different technologies other than the platforms mentioned above. Virus-like particle-based vaccine, which has been demonstrated to induce humoral and cell-mediated immunity in SARS-CoV and MERS-CoV preclinical models, has one candidate COVID-19 vaccine in phase I clinical trial (NCT04450004) and 14 vaccine candidates under preclinical development . HoweverIn addition to these traditional platforms, scientists have also developed COVID-19 vaccines using unconventional approaches. Aivita Biomedical, Inc. has developed AV-COVID-19, which is an autologous dendritic cells vaccine loaded with SARS-CoV-2 antigens . AV-COVIFinally, people have been testing existing licensed vaccines and trying to repurpose them to combat COVID-19. It has been shown that tuberculosis vaccine bacillus Calmette–Guérin (BCG) can train innate immunity and induce nonspecific host defensive reaction against viral pathogens, including respiratory syncytial virus (RSV), influenza A virus and herpes simplex virus type 2 (HSV2) –162. AddGiven the rapid transmission and asymptomatic spread of COVID-19, it is clear that an effective vaccine with global immunization coverage is required to bring people’s lives back to normalcy. However, even when an effective SARS-CoV-2 vaccine becomes available, the duration of vaccine-induced immunity is still largely unknown. Previous SARS studies have shown that SARS-specific IgG and neutralizing antibodies were only maintained for approximately 2 years in patients who recovered from SARS-CoV infection , 165. AsSince the discovery of human coronaviruses in 1960s, new types of coronaviruses have kept emerging and have gradually become a serious threat to global public health. Even though there have been almost two decades since the first coronavirus outbreak, the scientific and medical community are not well prepared with effective weapons to combat these pathogens. One lesson we learned from this is that the financial and regulatory mechanism of current pharmaceutical market does not provide enough incentive to encourage vaccine development before a deadly outbreak happens. To make up for this, now academic institutions and companies all over the world are developing an explosive numbers of vaccine candidates with highly compressed clinical trial schedules. Fortunately, the biological and clinical lessons we learned from SARS-CoV and MERS-CoV researches, together with the vaccine development experience we gained from other diseases, have already guided us to come up with multiple promising candidate solutions. Besides, multiple therapeutic candidates targeting molecules in SARS-CoV-2 life cycle and human immune response against COVID-19 have also been rapidly explored, with Remdesivir and Dexamethasone being the two leading drugs that showed promising clinical evidences in shortening the time to recovery and decreasing mortality rates , 172. Th


Coronavirus disease 2019 (COVID-19), a respiratory syndrome, is a global pandemic. Therefore, there is an urgent need to explore mechanisms implicated in the pathogenesis of the disease. Clinical and autopsy studies show a complex chain of events preceding COVID-19-related death. The disease is characterized by endothelial dysfunction, platelet activation, thrombosis, coagulopathy, and multiple organ failure. Globally, millions of patients with coronary heart disease undergo percutaneous coronary intervention (PCI) each year. These patients undergo high-intensity antithrombotic therapy during hospitalization and dual antiplatelet therapy (DAPT) for at least 6 months post PCI. COVID-19 is characterized by changes in platelet counts. Treatment of ischemic events that occur during stent implantation is associated with bleeding complications in patients following PCI complicated by COVID-19. This review summarizes recent progress in activation status and levels of COVID-19-related platelet changes. These findings will provide information on the effectiveness of antithrombotic therapy for the management of platelet changes in COVID-19 patients. Coronavirus disease 2019 (COVID-19), a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is a worldwide pandemic. In November 2020, the World Health Organization reported over 50 million confirmed cases of COVID-19 and 1.3 million deaths globally  on the surface of host cells , 7, in aA recent study reports significant changes in platelet gene expression and function in COVID-19 patients. These changes result in platelet activation and aggregation, which are potential novel mechanisms for management of COVID-19-associated thrombosis and coagulopathy . NotablyA previous clinical trial reports that the prevalence of hypertension, diabetes, and coronary heart disease among COVID-19 patients in the first 2 months of the outbreak was 15, 7.4, and 2.5%, respectively . PrevaleSARS-CoV-2 is a new coronavirus strain that belongs to the same class with SARS reported in 2003 . ClinicaViral pneumonia accounts for one third of adult community-acquired pneumonia. Most viral pneumonia cases are caused by influenza, rhinovirus, and coronavirus infections . Viral pDuring 2002 and 2003, the SARS-CoV caused severe respiratory infection in more than 8,000 people and led to 774 deaths, with a mortality rate of 9.6% . The typThe early pulmonary pathological changes in SARS-CoV2-infected lungs included edema, proteinaceous exudate, and focal reactive hyperplasia of pneumocytes with patchy inflammatory cellular infiltration, whereas hyaline membranes were not prominent . The keyThere have been accumulating pathologic findings of COVID-19 outside China , 31–34. It should be noted that the above pathological findings are mainly the direct and indirect consequences of lung tissue destruction induced by intracellular viral proliferation. In the rapid progressive and life-threatening form of viral pneumonia, the underlying pathological process is often diffuse alveolar injury, coagulopathy, and MOF . CollectReplication and dissemination of SARS-CoV-2 in systemic circulation lead to extrapulmonary manifestations, which play key roles in disease progression , 36. A pEndothelial dysfunction induces inflammation and vascular remodeling , which aSARS-CoV-2 enters host cells by binding to ACE-2 on pulmonary epithelial cells resulting in lung damage , 7. MoreA previous study reports that lungs have a high hematopoietic potential, thus they contribute to terminal platelet production (nearly 50%) . TherefoAt the early phase of infection, SARS-CoV-2 invades the lung tissue of the host, which may activate platelets through changes in gene expression , 42. Plain situ activation of lung megakaryocyte-derived platelets or have a direct impact on lung megakaryocytes. This may lead to changes in gene expression profile of platelets as observed in COVID-19 patients  , high coDuring the COVID-19 global pandemic lockdown period, the number of patients presenting with acute coronary syndrome (ACS) and emergency coronary procedures reduced significantly in Europe –58, the Early diagnosis and timely management are critical in reducing morbidity and mortality related to ACS. Ischemic time duration is a major determinant of infarct size in patients with STEMI. Current delays in COVID-19 testing, termed as “COVID-19-related delay,” may contribute to total ischemia time . Tam et Recent studies showed that patients with STEMI presenting with concurrent COVID-19 present with unique findings during coronary angiography , 76. A sWhile primary PCI remains the treatment of choice for STEMI, the balance between exposure risk of medical staff and benefit of patient from thrombolysis should be considered in certain circumstances. Strategic Reperfusion Early After Myocardial Infarction study demonstrated that even a single hour of delay may affect the effectiveness of primary PCI compared with thrombolysis .In China and Iran, thrombolytic therapy is recommended over primary PCI for STEMI management if COVID-19 was confirmed or could not be excluded within a short time. On the other hand, SARS-CoV-2 infection is excluded first for non-STEMI and unstable angina pectoris approaches –69. ConvA high risk of thrombotic complications in patients with COVID-19 complicates the dosage of anticoagulation in hospitalized patients with COVID-19 . AnticoaThe 2018 European Society of Cardiology guideline does not give guidelines on routine post-procedural anticoagulant therapy after primary PCI . STEMI pin situ platelet activation in lung vasculature   . A recen= 0.002) . TherefoCurrently, the long-term impact of COVID-19-related endothelial activation, hypercoagulability, microvascular thrombosis, and myocardial injury is not well-known . PreviouZhang et al.  reportedFindings from clinical observations and autopsy studies show a complex chain of events preceding COVID-19-related death. The adverse event chain starts with viral infection and proliferation, followed by endothelial dysfunction induced by local and systemic viral dissemination. Further, platelet activation, thrombosis, and platelet and coagulation factor depletion occur leading to MOF and life-threatening bleeding. Patients treated with PCI and patients on antithrombotic treatment should undergo post-procedural anticoagulation and prolonged anticoagulation following intravenous thrombolysis and standard DAPT treatment to reduce the risk of thrombotic complications during early-to-mid stages of COVID-19 progression. Pros and cons of these antithrombotic treatment regimens should be evaluated in an individualized manner in cases of clinical thrombocytopenia (induced either by platelet consumption or by heparin) and/or bleeding complications.HL and ZW performed the manuscript writing and illustration drawing. HS performed part of literature collecting. TT and YL provided critical scientific input and discussions. XZ and QY conceived, designed, and supervised all studies and the drafting and editing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent epidemiologic, virologic, and modeling reports support the possibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission from persons who are presymptomatic (SARS-CoV-2 detected before symptom onset) or asymptomatic (SARS-CoV-2 detected but symptoms never develop). SARS-CoV-2 transmission in the absence of symptoms reinforces the value of measures that prevent the spread of SARS-CoV-2 by infected persons who may not exhibit illness despite being infectious. Critical knowledge gaps include the relative incidence of asymptomatic and symptomatic SARS-CoV-2 infection, the public health interventions that prevent asymptomatic transmission, and the question of whether asymptomatic SARS-CoV-2 infection confers protective immunity. As the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) unfolds, an increasing number of reports have indicated that some infected persons may not exhibit signs or symptoms of illness, including persons who are presymptomatic (SARS-CoV-2 RNA is detectable before symptom onset) or asymptomatic (SARS-CoV-2 RNA is detectable but symptoms never develop) . We classified studies as reporting epidemiologic, virologic, or modeling evidence for presymptomatic or asymptomatic transmission of SARS-CoV-2.Most reports of presymptomatic . Presymp>2 to >9 days.However, cases of presymptomatic transmission have been reported from other countries before widespread community transmission occurred. A report from Germany documented infection of a German businessman after exposure to a mildly symptomatic colleague visiting from China (t) values. The RT-PCR Ct value represents the number of PCR cycles required to detect SARS-CoV-2 RNA; lower values indicate higher viral load and imply higher infectiousness  or by viral culture and demonstration of cytopathic effect   and ability to isolate infectious SARS-CoV-2 provide plausible virologic evidence for SARS-CoV-2 transmission by persons not demonstrating symptoms.Four reports documented the presence of SARS-CoV-2 RNA with lower Ceveloped . Two repTwo studies used models to estimate the serial interval (time between symptom onset in a primary patient and the secondary patient) . They esTwo models attempted to estimate the number of infections caused by asymptomatic, presymptomatic, or mildly symptomatic infected persons (Each of the epidemiologic, virologic, and modeling studies described has limitations. However, in the aggregate, these diverse studies suggest that SARS-CoV-2 can be transmitted by persons with presymptomatic or asymptomatic infection, which may meaningfully contribute to the propagation of the COVID-19 pandemic. This literature summation was conducted to support changes in CDC recommendations to reduce the risk for asymptomatic transmission and was not a systematic review. These conclusions are drawn from the literature available at the time and may change, given the rapidly evolving nature of the evidence base for asymptomatic transmission.The existence of persons with asymptomatic SARS-CoV-2 infection who are capable of transmitting the virus to others has several implications. First, the case-fatality rate for COVID-19 may be lower than currently estimated ratios if asymptomatic SARS-CoV-2 infections are included (The existence of SARS-CoV-2 transmission while infected persons are presymptomatic and asymptomatic raises 3 key questions that need to be answered to inform public health action. First, the incidence of asymptomatic compared with symptomatic SARS-CoV-2 infection needs to be determined. The extent of presymptomatic or asymptomatic SARS-CoV-2 infection may be clarified by studies using serial virologic data, serologic data, or a combination of both in observational cohorts or surveillance systems. If a substantial proportion of infections are asymptomatic, enhanced testing strategies may be needed to detect these persons. Second, given that a large proportion of infections probably result from transmission from asymptomatic or presymptomatic persons (


Evidence suggests that ectopic fat deposition interferes with skeletal muscle structure and function, but few studies have examined underlying morphological and contractile properties at the single fiber level. Healthy older (65-75 y) men (n=9) and women (n=9) underwent dynamometry for assessment of knee extensor maximal torque, water-fat magnetic resonance imaging to quantify quadriceps muscle cross-sectional area (CSA) and fat fraction (FF), and vastus lateralis biopsies to determine morphology and function of type I and II muscle fibers. Despite similar body mass indices  and daily moderate-to-vigorous physical activity , women had greater FF  than men, indicating increased adipose tissue deposition in skeletal muscle. Women also had smaller quadriceps CSA , specific torque  and type II fiber CSA  than men. Type I CSA did not differ by sex . In older women, FF was inversely associated with single fiber CSA in type I  and II  fibers, and tended to be associated with slower myosin-actin cross-bridge kinetics (longer myosin attachment time) in type I fibers . These relationships were not observed in men. Overall, healthy older women have greater intramuscular fat than men, which may contribute to sex-specific effects on knee extensor specific torque through differences in muscle fiber size and cross-bridge kinetics.


Rational: Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. While effective medical interventions are available for some kinds of heart failure, one age-related impairment, diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is lacking a clinically effective treatment. Methods and Results: Using the pH indicator cpYFP in the model of naturally aging mice and rats, we show direct evidence of increased mitochondrial proton leak in aged heart mitochondria following a pH gradient stress. Furthermore, we identified Adenine Nucleotide Translocator 1 (ANT1) as mediating the increased proton permeability of old cardiomyocytes. Most importantly, acute (2 hours) in vitro treatment with the tetra-peptide drug SS-31 (elamipretide) reverses age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore (mPTP) opening and rejuvenates mitochondrial function. Moreover, we show that SS-31 benefits the old mitochondria by direct association with ANT1 and stabilization of the mitochondrial ATP synthasome, leading to substantial reversal of diastolic dysfunction. Conclusion: Our results uncover excessive mitochondrial proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 is able to reverse this clinically important complication of cardiac aging.