Alzheimer’s disease (AD) is the most common form of dementia, characterized by progressive cognitive impairment and neurodegeneration. Extensive clinical and genomic studies have revealed biomarkers, risk factors, pathways, and targets of AD in the past decade. However, the exact molecular basis of AD development and progression remains elusive. The emerging single-cell sequencing technology can potentially provide cell-level insights into the disease. Here we systematically review the state-of-the-art bioinformatics approaches to analyze single-cell sequencing data and their applications to AD in 14 major directions, including 1) quality control and normalization, 2) dimension reduction and feature extraction, 3) cell clustering analysis, 4) cell type inference and annotation, 5) differential expression, 6) trajectory inference, 7) copy number variation analysis, 8) integration of single-cell multi-omics, 9) epigenomic analysis, 10) gene network inference, 11) prioritization of cell subpopulations, 12) integrative analysis of human and mouse sc-RNA-seq data, 13) spatial transcriptomics, and 14) comparison of single cell AD mouse model studies and single cell human AD studies. We also address challenges in using human postmortem and mouse tissues and outline future developments in single cell sequencing data analysis. Importantly, we have implemented our recommended workflow for each major analytic direction and applied them to a large single nucleus RNA-sequencing (snRNA-seq) dataset in AD. Key analytic results are reported while the scripts and the data are shared with the research community through  GitHub. In summary, this comprehensive review provides insights into various approaches to analyze single cell sequencing data and offers specific guidelines for study design and a variety of analytic directions. The review and the accompanied software tools will serve as a valuable resource for studying cellular and molecular mechanisms of AD, other diseases, or biological systems at the single cell level.The online version contains supplementary material available at 10.1186/s13024-022-00517-z. Alzheimer’s disease (AD) is one of the most devastating forms of dementia common in the elderly, estimated to affect over 6.2 million individuals in the United States and 24 million worldwide , 2. Clin, rare familial AD, sporadic early-onset (EOAD), and late-onset (LOAD) . The two samples were normalized and integrated into a dataset Fig. A. Gene aGene regulatory network (GRN) inference hypothesizes that the etiology of a complex genetic disease is driven by complex signaling cascades  and aimsTo this end, scRNA-seq has recently emerged to uncover cell-level signaling pathways associated with neurodegenerative diseases and their markers , 194, anGRN inference in scRNA-seq is presented with challenges to construct robust and reproducible network models. Drop-out reads present false zero-expressions in addition to the sparse cell-wise transcriptome. These noises in scRNA-seq underscored near-random performances of gene-gene similarity measures to uncover meaningful gene interactions in benchmark scRNA-seq data . Model-bEstablished bulk-based GRN inference tools have been applied in scRNA-seq as these tools constructed robust network models in complex diseases and are immediately accessible. While there are several excellent reviews on bulk-based GRN inference methods –320, we Co-expression networks identify gene interactions by gene pairwise association measures such as correlations or information-theoretic measures . WeighteSeveral statistical frameworks infer directed interactions between causal and effector genes in contrast to undirected interactions. Bayesian Network (BN) inference provides a flexible framework for identifying directed interactions in causal cascades and integrating upstream regulations such as genetic variants as prior network , 328. WeApplications in scRNA-seq have discovered key pathways and markers of heterogeneous cell populations underlying human disease tissues. WGCNA has been applied to identify pathways to activate dormant neural stem cells , regulatOn the other hand, naïve applications of bulk-based GRN methods involve several shortcomings. They show lower retrieval of known functional links than those inferred from bulk RNA-seq data  and primf, which formulates the state of a target gene based on the states of some other genes through clauses consisting of only Boolean operators AND (∧), OR (∨) and NOT (¬) [The Boolean network model simplifies the complex biological pathways into a switch-like process that transits the network change from one state to another . In Bool NOT (¬) . Several NOT (¬) , 342 is  NOT (¬) . Unlike  NOT (¬)  does not NOT (¬) .x is a time-labeled vector of C single-cell transcriptomic profiles, x1, x2, …, xC ∈ ℝG in which xi represents the expression, for the ith cell, of G genes, and A is a square matrix that characterizes the regulatory network among the genes [GRN inference from time-stamped scRNA-seq data can also be facilitated by ordinary differential equation (ODE) models. In these models, a set of ODEs from potential regulators describe temporal changes in the target genes. This model can be expressed in the form of he genes , 346. Onhe genes . It firsA BN inference approach, AR1MA1-VBEM  , uses a scRNA-seq data provides an opportunity to estimate the cell-level temporal dynamics by assuming gradual changes in the cellwise transcriptome occurs over time and constitute a trajectory. While cell trajectory inference is an active research area in the scRNA-seq domain , 194, thDifferent statistical frameworks have also been adopted to evaluate gene-gene relationships across different time windows. LEAP (Lag-based Expression Association for Pseudotime-series) utilizes Pearson’s correlation of normalized expressions at a time window with those expressions from lagged time windows to establish time-lagged associations between the genes . SCENIC In contrast to other model-based methods, association-based networks objectively evaluate the likelihood of gene-gene interactions by ‘guilt-by-association’ . SeveralA popular strategy involves a series of data transformation or modeling steps to handle sparsity and dropout expressions and evaluate significant association by correlations or information-theoretic measures. In bigSCale, gene-expressions are grouped into cell clusters , and difCell-cell communication is vital for multicellular organisms to coordinate functionally unique cell populations in response to internal and external stimuli. Such communication is primarily mediated by ligand (L)-receptor (R) interactions and can be visualized by networks where each node is a cell type and the edges are L-R interactions . SeveraliTalk relies on a built-in curated database of 2648 non-redundant and known L-R pairs to infer communications across or within distinct cell types . It modeACTION attributes functional similarity to genes with relatively weak but preferential expression in specific cell types. It identifies cell clusters, termed archetypes, by low-dimensional geometric constructs in the functional space. Further, it infers cell-type-specific TF regulatory networks (TRNs) by assessing significant TFs with their targeted top-ranked cell type markers. Thus, ACTION provides functional annotation and subsequently the phenotype associated with each cell type . SCINET The confidence of the inferred interactions is often crucial in identifying key mechanisms and putative regulators, and ultimately, formulating robust and testable biological hypotheses. One approach is to examine the congruence between the established regulatory pathways or relationships and the inferred networks. Gene perturbation databases in established disease models such as Library of Integrated Network-based Cellular Signatures (LINCS)  or CREEDOn the other hand, simulation studies provide the full controls over the model and noise parameters to generate tailored data, whose underlying reference network is known for objective comparison with inferred networks . GeneNetInference of robust and accurate network models in single-cell transcriptome requires appropriate modeling of data noises specific to scRNA-seq. These involve low per-cell coverage, doublets, dropout reads as the main sources of noises and should be handled as illustrated in QC guidelines and impute the dropout reads with model-based methods. The model-based imputations infer cellwise dropout rates to facilitate the selection of cells robustly expressing each gene to improve the performance of inferred gene-gene interactions . In addiDepending on the type of an inferred GRN, the expression data should be adequately normalized to optimize the accuracy of inferred gene interactions. The denoised counts should be log-transformed to closely follow Gaussian distributions for constructing regression-based or association-based GRNs. On the contrary, kinetic-based methods such as Boolean or ODE-based networks may perform better in the gene count space.However, GRN inference with scRNA-seq data is still in its infancy and requires an objective performance evaluation. BEELINE  and GeneTo illustrate the application of network analysis in AD single-cell data, we constructed gene co-expression and Bayesian networks for an excitatory neuron cluster identified in the ROSMAP snRNA-seq data using two well-established bulk-tissue gene network inference tools. Figure scRNA-seq enables accurate classification of individual cells by gene expression profiles, and cluster-based differential gene expression analysis of scRNA-seq data can help resolve brain-region specific changes in heterogeneous cell populations in AD brains. As loss of neurons and increased microglia activation are characteristic of typical AD brains , 369, anFor example, Olah et al. used scRNA-seq to characterize differences in the distribution of distinct microglia subpopulations in human cerebral cortex specimens . The stuIn another single-nucleus transcriptomic study of human prefrontal cortex specimens, Mathys et al. identified transcriptionally distinct sub-populations across six major brain cell types, with many of the top DEGs recurring across multiple cell types . Interesdeconvolving’ the cell compositions. Early deconvolution approaches rely on cell markers and gene expressions from cell sorting experiments. For example, CIBERSORT applies support vector regression to characterize the cell composition of complex tissues from their gene expression profiles [In the past decade, bulk RNA-seq datasets have been massively accumulated. However, cell type compositions in the bulk tissues represent a significant confounding factor influencing sample comparisons. scRNA-seq can be utilized to estimate cell fractions of the bulk RNA-seq datasets, thus ‘profiles , and Celprofiles . More reIn contrast to traditional methods that mainly use marker genes from cell sorting experiments, single-cell-based deconvolution methods utilize sparse gene expression matrix from scRNA-seq and models gene/sample specific variations unique to single-cell experiments. For example, DeconvSeq utilizes a generalized linear model in feature quantification to construct a projection matrix and resolves cell type fractions by a sequential quadratic programming based solver ; MuSiC aLeveraging the ROSMAP AD cohort with both bulk RNA-seq data for over 600 subjects and snRNA-seq data for a subset of the samples, we performed a mini-benchmarking of the performance of several deconvolution methods. Here we aggregated the cells of each individual in the snRNA-seq data by major cell types to calculate a cell type proportion estimate. As shown in Fig. The cell-type proportions estimated from deconvolution methods can be used for a number of downstream analyses for AD. For example, in contrast to traditional DE analysis confounded by cell type proportions, deconvolution results can provide new insights into the cell-intrinsic DEGs by adjusting cell type proportions . The frap-value or fold enrichment) [Established strategies for prioritizing co-expressed modules and key causal drivers from bulk transcriptomic data can be directly applied to cell cluster-based coexpression and causal networks , 381–383ichment) . Similarichment) . Such keichment) , 386–390ichment) .Cellular spatial location is not well retained in the single-cell transcriptomics methods described above. Given that cellular gene expression patterns change in response to environmental cues maintaining the neighborhood environment allows for a deeper understanding of tissue-wide dynamics and biological function of genes and cells. Many of the current spatial technologies were derived from RNA in situ hybridization (ISH) and/or principles of laser capture microdissection. While ISH, a commonly used technique to histologically visualize mRNA localization in tissues on microscope slides, allows for maintained tissue integrity and entire tissue analyses, a major limitation of traditional ISH is the number of targets that can be analyzed concurrently. On the other hand, laser capture microdissection, developed in the 1990s, uses low-power infra-red laser beams to microdissect cells of interest to overcome this obstacle and allows the whole transcriptome profiling, but the laser capture microdissection is destructive for the cells.Several advanced approaches have been developed to profile whole transcriptomes while preserving spatial information in the past few years. These include fluorescence in situ hybridization (FISH) –396, in These technologies open an unprecedented opportunity to dissect the cellular complexity and characterize the tissue microenvironment for identifying inter-cellular interactions and signaling pathways in complex diseases such as cancer , 403, amNL-G-F mice. They identified two co-expression networks, one of which  is particularly interesting since their activities are strongly associated with the Aβ plaque. We applied Giotto [NL-G-F) mice at the 18-month-old age. By using Leiden clustering, we identified 8 distinct clusters , with aCell clustering in scRNA-seq is mostly performed within widely adopted scRNA-seq workflows such as Scater , Seurat While several dimension reduction methods such as PCA, t-SNE , DiffusiThe surge in trajectory inference method development urgently needs appropriate scenario-specific methods. A growing effort has been put into this direction , 434. A Even though single-cell based CNV and sc-eQTL analyses have been primarily performed in cancer researches , 212 andSingle-cell sequencing can identify key molecular pathways targets from distinct cell types in AD and resolve the mixed signals from bulk tissues. Particularly, cell-type-specific signatures from scRNA-seq can be useful for repositioning FDA-approved drugs for treating AD. Through reversal of the cell-type-specific signatures, Connectivity map (CMap) and LINCS have been used to predict candidate FDA-approved drugs for several diseases, including pulmonary arterial hypertension (PAH) , COVID-1As high-throughput sequencing techniques produce numerous data and hypotheses, additional experimental validation is often required to confirm the findings. Several scRNA-seq studies have revealed and experimentally validated AD-associated gene regulation and cell subpopulations with a primary emphasis on the glial cells , 57. Co-In conclusion, we comprehensively reviewed the state-of-the-art bioinformatics approaches to analyze single-cell sequencing data and their applications to AD in 14 major directions. The basic analyses include data quality control and normalization, cell cluster identification and cell subpopulation characterization and differential expression while more advanced analyses involve trajectory inference, copy number variation, eQTL identification, and integrative gene network inference. We also reviewed the recent progress on analyzing scATAC-seq and spatial transcriptomics data and integrating single-cell multi-Omics data. We summarized their advantages and disadvantages for multiple methods in each direction to help users select the most appropriate approach for specific applications. More importantly, we have implemented the recommended workflow for each major analytic direction and applied it to an snRNA-seq dataset in AD while the scripts and the data are shared with the research community. We further discussed the potential future development of bioinformatics of single-cell sequencing data. We expect both less experienced and advanced single cell data analysts would be greatly benefited from the review and the accompanied software tools. As such, this review not only provides insights into various methods to analyze scRNA-seq data and guidelines for analyzing AD scRNA-seq data but also serves as an invaluable resource for the AD research community and the single cell sequencing community in general.Additional file 1: Supplementary Table S1. The chr19 amplification region inferred in the Cluster 6 of late-pathology AD cases. Supplementary Table S2. Genes in the chr19 amplification region in the Cluster 6 of late pathology ADs.


In this review, we discuss the use of the alkylating agent temozolomide (TMZ) in the treatment of IDH-mutant gliomas. We describe the challenges associated with TMZ in clinical (drug resistance and tumor recurrence) and preclinical settings (variabilities associated with in vitro models) in treating IDH-mutant glioma. Lastly, we summarize the emerging therapeutic targets that can potentially be used in combination with TMZ. Gliomas are the most common primary malignant tumors in the central nervous system. Grade 2 and 3 gliomas are referred to as lower grade gliomas (LGG) and harbor mutations in the isocitrate dehydrogenase (IDH) gene . IDH-mutR132H) at the catalytic site of the enzyme. IDH mutation produces a neomorphic enzyme that converts α-KG to D-(R)-2-hydroxyglutarate (2-HG), leading to the accumulation of 2-HG in the tumor [The IDH gene encodes the enzyme isocitrate dehydrogenase, which converts isocitrate to α-ketoglutarate (α-KG). α-KG an intermediate of the tricarboxylic acid (TCA) cycle that contributes to the production of NADPH. NADPH is necessary to reduce oxidized glutathione to glutathione, which directly neutralizes free radicals and reactive oxygen species (ROS). Overall, 65% of total NADPH in glioblastoma (GBM) is driven by the enzymatic activity of IDH, which is reduced to 38% when IDH is mutated . There ahe tumor . The onche tumor ,13. Thishe tumor  and altehe tumor   or temozolomide (TMZ). Here, we focus on the use of TMZ in IDH-mutant LGGs. First, we will present the effect of IDH mutation on cellular metabolism, epigenetic modifications, and the targeted therapies associated with these alterations. Second, we will discuss the use of TMZ in the treatment of IDH-mutant gliomas, including its toxicity, TMZ-associated molecular signature in tumor recurrence, and drug resistance, and discuss the synthetic lethality opportunities that emerge with TMZ treatment of IDH-mutant gliomas. Third, we will discuss the challenges of using TMZ to treat IDH-mutant gliomas in the preclinical setting, including non-consensus TMZ dosage and regimen, variable methods in measuring cell viability, and difficulties in culturing IDH-mutant glioma cell lines. To conclude the review, we will discuss targeted vaccine therapy that may facilitate the treatment of IDH-mutant gliomas. Redox balance. IDH is a central enzyme of the TCA cycle; therefore, many studies have sought to understand the unique metabolic profiles reprogrammed by mutant IDH1 to therapeutically exploit these metabolic vulnerabilities  Figure . IDH actGlutamate. Metabolomic analysis using glioma cell lines and surgical specimens indicated significantly reduced levels of glutamate in IDH-mutant gliomas ,20,21,22Lipid synthesis. Altered lipid biosynthesis is another significant vulnerability of IDH-mutant gliomas. Biopsies from glioma patients showed lower phospholipid levels in IDH-mutant tumors compared with IDH-wt tumors. This effect was mediated by autophagic degradation of the endoplasmic reticulum (ER), termed ER-phagy . Since ENAD. Another extreme metabolic vulnerability is the low NAD+ levels in IDH-mutant cells, making further depletion of NAD+ a good therapeutic target . BiosyntEpigenetic modifications. IDH-mutant gliomas exhibit a cytosine-phosphate-guanine (CpG) island methylator phenotype (G-CIMP)  characteStandard-of-care treatment for gliomas includes maximal surgical resection, possibly followed by radiotherapy (RT) and chemotherapy with PCV or TMZ. Several randomized clinical trials  investigCurrently, there are no mature data comparing TMZ and PCV or their combination with radiation for LGGs. The ongoing clinical trial ALLIANCE-N0577-CODEL comparing RT + TMZ with RT + PCV for anaplastic oligodendrogliomas with 1p/19q co-deletion could potentially provide a more definitive comparison between the two regimens . Both PC2 for 5 out of 28 days for 6–12 cycles [7-MeG), 10–20% of the methyl groups are added at adenine (N3-MeA), and 10% of methyl groups at guanine (O6-MeG) [7-MeG and N3-MeA, are readily removed by the rapid and efficient base excision repair (BER) system before replication. Therefore, the key toxic insult of TMZ is attributed to the O6-meG lesions [TMZ is administered orally in capsules at a dose of 150–200 mg/m2 cycles     instead of cytosine (C) during DNA replication (6-meG. The mismatch repair (MMR) system can detect and repair these mismatches through the MutS and MutL complexes. The MutS recognition complex, including MutSα (an MSH2/MSH6 heterodimer) and MutSb (MSH2/ MSH3 heterodimer), identifies base–base mismatches and binds the O6-meG: T mismatch. Upon binding to the mismatch, the MutS complex recruits MutL (MLH1/PMS2 dimer) to the site of DNA damage. Together, these proteins excise a stretch of single-stranded DNA (ssDNA) containing the mispaired T, creating a gap in the DNA, while leaving the O6-meG adduct on the template strand intact [6-meG, triggering another round of MMR which leads to repeated attempts to repair the same base T. This futile MMR cycling and accumulation of ssDNA gaps lead to successively longer DNA reinsertion and excision, which generates double strand breaks (DSBs) in subsequent rounds of replication and induce cell cycle arrest in G2/M phase, apoptosis and autophagy [Olication c. Duringd intact . DNA polutophagy . Thus, iutophagy   through covalent transfer, a process that effectively repairs the alteration prior to replication  is a potent inhibitor of the repair protein O6–alkylguanine–DNA alkyltransferase (AGT) that effectively inhibits MGMT activity by suicide inactivation. O6-BG binds and inactivates AGT, and until new AGT protein is synthesized, the cells have increased sensitivity to TMZ [6-BG and TMZ [6-BG plus TMZ restored TMZ sensitivity in patients with TMZ-resistant anaplastic IDH-mutant gliomas [MGMT. Oy to TMZ ,67,68, l and TMZ ,71,72,73MMR. MMR is regulated by multiple signaling pathways and responds to many stimuli . MutatioBER. Many efforts have been made to target the downstream ssDNA repair and HR system to maintain TMZ sensitivity. One target from the base-excision repair (BER) pathway is poly (ADP-ribose) polymerase 1 (PARP1), which facilitates DNA repair by binding to single-strand breaks and recruiting DNA repair proteins to the site of damage . PARP inHR. One target from the HR pathway is the homologous recombinase RAD51. RAD51 is involved in DNA strand exchange between homologous DNA sequences. Exogenously expressed mutant IDH1 increases RAD51-driven HR and leads to increased TMZ resistance, and RAD51 knockdown increases the sensitivity of glioma cells to TMZ . SeveralTumor Microtubes. Recently, there has been increasing evidence that tumor microtubes (TM) are an important mechanism of therapy resistance in gliomas. Gliomas interconnect and communicate through a network of TMs . TM-connThe first report investigating the effect of TMZ in the treatment of LGGs  showed t6-meG lesions. In the absence of MGMT-mediated repair in conjunction with deficient MMR, long-term TMZ treatment causes cells to accumulate G:C>A:T transitions throughout the genome, resulting in a hypermutator phenotype in recurrent tumors [TMZ resistance can be acquired either by elevated MGMT levels ,103 or bt tumors ,109  from primary IDH-mutant and IDH-wt gliomas of all grades revealed amplification of cyclins and cyclin-dependent kinase genes in IDH-mutant gliomas  Table 1Table 1. R132H mutation was not lost during progression and remained clonal in all progressed tumors [R132H vaccines for targeted therapies.However, a study by the GLASS consortium comparing 23 pairs of untreated primary and TMZ-treated recurrent IDH-mutant gliomas  did not d tumors , providiCRISPR-based screening enables sensitive detection of drug-gene interactions directly in human cells. Although no genome-wide CRISPR-Cas9 screen has been performed in IDH-mutant glioma models, results from GBM patient-derived lines  and GBM Pathway analysis from RNA-seq data obtained from preclinical GBM models showed that epithelial–mesenchymal transition, Wnt signaling, and immune response were the most significantly activated pathways in TMZ-resistant cell lines . In addiDNA methylation. Preclinical studies have shown that high TMZ concentration leads to a short-term increase in total 5-methylcytosine (hypermethylation), while repeated low TMZ doses lead to DNA hypomethylation . This inHistone methylation. An inhibitor of histone methyltransferase (HMT) G9a, BIX01294, also exerted a synergistic effect with TMZ in GBM , possiblHistone acetylation. Several histone deacetylase (HDAC) inhibitors synergize with TMZ. For example, vorinostat  is well Here, we summarize the current epigenetic drugs with TMZ combination therapy in Glutamate. Previous studies have indicated that long-term TMZ treatment leads to changes in amino acid metabolism in preclinical models of oligodendroglioma . Other rPhospholipid. The late-stage autophagy inhibitors chloroquine (CQ) and bafilomycin A1 (BAF) restore phospholipid levels and inhibit clonogenicity of IDH-mutant glioma cells. CQ enhances the cytotoxic effects of TMZ in GBM , and itsNAD+. TMZ treatment leads to NAD+ consumption driven by PARP activation, as NAD+ is a known PARP cofactor. In IDH1-mutant cells with already low basal NAD+ levels, this surge in consumption leads to a further reduction in NAD+. Importantly, this metabolic imbalance introduces a window of hypervulnerability to NAD+ biosynthesis inhibitors . Indeed,Glucose. TMZ treatment has been shown to increase the expression of glucose transporters (GLUTs) ,162, whiA triple therapy combination with TMZ, CQ, and rapamycin decreased mitochondrial function and induced lysosome-dependent apoptotic cell death , suggestA recent review summarizes the four main limitations associated with the use of TMZ in preclinical models of GBM : (1) themax) and the integrated area under the plasma concentration-time curve (AUC), typically calculated from time zero to infinity, associated with the highest recommended dose of the drug [Human pharmacokinetic (PK) data determined exposure defined by the maximum plasma concentration  cell proliferation assay monitors cell division by evaluating DNA synthesis through thymidine insertion. BrdU is a thymidine analog and can be incorporated into the newly synthesized DNA of replicating cells. BrdU levels can be detected by flow cytometry using a BrdU antibody conjugated with fluorochrome. Particular care must be taken with the incubation time for BrdU labeling as it is cell cycle dependent.Soft agar colony formation assay measures the ability of single cells to form colonies in semi-solid soft agar medium in an anchorage-independent manner, which is closer to in vivo conditions. Depending on the cell lines and the number of cells seeded, the colony formation can take several weeks to months.There is no perfect method to assess cell viability. While some assays can differentiate between dead and live cells, and others can just assess the relative performance of cells. To increase reliability of the results, researchers should use more than one assay method to better assess cell proliferation .The IDH-mutant glioma cell lines used in preclinical research include four types: (1) genetically engineered lines overexpressing mutant IDH1 such as immortalized human astrocytes (IHA) ,36,181, Patient-derived IDH-mutant glioma cell lines are difficult to establish and propagate in vitro. One reason is the low incidence of IDH-mutant LGG patients at a given medical center, and another reason is that IDH-mutant tumor cells are difficult to culture  or to grDue to rapid culturing and retention of the original genetic aberrations, some of the glioma cell lines have been used in several in vitro drug screens . HoweverRecently, 3D cultures of organoids have proven to be effective models. Organoids can be grown from patient-derived normal and tumor tissues  and evenHere, we present the challenges and opportunities for treating IDH-mutant gliomas with the chemotherapy drug TMZ. We summarize the genetic, epigenetic, metabolic vulnerabilities that arise from IDH mutation and that could be exacerbated by treatment with TMZ. In the future, the fast-developing technologies, such as single-cell RNA-seq, or in vivo/in vitro shRNA and CRISPR screenings, will help us to identify more therapeutic targets in each specific molecular context, accelerating personalized medicine treatment in IDH-mutant gliomas. The recent success of the IDH1 mutation-specific vaccine in treating IDH1-mutated gliomas  may open


The use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) as first-line treatment in patients with lung adenocarcinoma (LUAD) harboring EGFR-activating mutations has resulted in a dramatic improvement in the management of the disease. However, the long-term clinical benefit is inevitably compromised by multiple resistance mechanisms. Accumulating evidence suggests that metabolic landscape remodeling is one of the mechanisms that EGFR-mutant LUAD cells activate, thus acquiring higher plasticity, tolerating EGFR TKI-mediated cytotoxic stress, and sustaining their oncogenic phenotype. Several metabolic pathways are upregulated in EGFR TKI-resistant models modulating the levels of numerous metabolites such as lipids, carbohydrates, and metabolic enzymes which have been suggested as potential mediators of resistance to EGFR TKIs. Moreover, metabolites have been shown to carry signals and stimulate oncogenic pathways and tumor microenvironment (TME) components such as fibroblasts, facilitating resistance to EGFR TKIs in various ways. Interestingly, metabolic signatures could function as predictive biomarkers of EGFR TKI efficacy, accurately classifying patients with EGFR-mutant LUAD. In this review, we present the identified metabolic rewiring mechanisms and how these act either independently or in concert with epigenetic or TME elements to orchestrate EGFR TKI resistance. Moreover, we discuss potential nutrient dependencies that emerge, highlighting them as candidate druggable metabolic vulnerabilities with already approved drugs which, in combination with EGFR TKIs, might counteract the solid challenge of resistance, hopefully prolonging the clinical benefit. EGFR mutations occur in nearly 50% of Asian and approximately 15% of Caucasian patients with LUAD [EGFR-sensitizing mutations. Several phase 3 clinical trials have consistently shown the superior efficacy of first-generation  and second-generation  EGFR tyrosine kinase inhibitors (TKIs) in comparison with standard first-line platinum-based chemotherapy in patients with LUAD harboring these EGFR-activating mutations [EGFR-dependent and EGFR-independent genetic alterations are known to be associated with resistance to EGFR TKIs. Among them, the secondary “gatekeeper” mutation EGFR T790M in exon 20 is the dominant resistance mechanism occurring in around 50% of patients treated with first- or second-generation TKIs [EGFR-activating mutations and the EGFR T790 mutation. In the FLAURA trial, osimertinib resulted in improved survival outcomes compared to first-generation EGFR TKIs, and consequently entered in clinical practice as standard therapy for treatment-naïve patients [EGFR C797S mutation in exon 20 are reported in only 7% of patients treated with first-line osimertinib [KRAS mutations, MET amplification, small-cell transformation, and gene fusions have been observed in a considerable percentage of patients [Lung cancer remains one of the most frequent and deadly types of cancer for both genders, while predictions about the future do not suggest dramatic improvements in incidence and mortality rates ,2. Thereith LUAD . Specifiutations ,7,8,9. Dion TKIs ,11. Osimpatients ,13. Unfomertinib ,15. In cpatients ,17. The EGFR-mutant LUAD cells overcome EGFR TKI-mediated cytotoxicity through parallel resistance mechanisms, stemming from different multi-omics sources [EGFR-mutant LUAD and how alterations in the levels of oncometabolites could induce resistance to therapy. Moreover, we describe promising metabolism-modulating interventions that might enhance EGFR TKI efficacy and combat the emergence of resistance.Beyond genomics, accumulating evidence suggests that  sources ,19. Canc sources . In the  sources . In thisEGFR-mutant LUAD, the aberrant overactivation of EGFR signaling axis leads to increased glucose uptake, excessive lactate formation, upregulation of the pentose phosphate pathway (PPP), and de novo pyrimidine biosynthesis in EGFR-mutant in vitro models [EGFR-mutant LUAD has been demonstrated by treating gefitinib/erlotinib-sensitive  and -resistant (H1975) EGFR-mutant LUAD cells. In particular, the treatment abrogated the activity of crucial metabolic pathways including glycolysis and PPP, downregulating effectively intermediate metabolites such as fructose 1,6-bisphosphate, lactate, and dihydroxyacetone phosphate in EGFR TKI-sensitive cells compared to resistant cells. Interestingly, quantification of these oncometabolites and establishment of cut-offs in the clinic might serve as auxiliary prediction biomarkers to EGFR TKI efficacy in patients with EGFR-mutant LUAD [Physiologically, EGFR activation stimulates different signaling pathways such as PI3K/Akt and MEK/Erk that are tightly linked downstream with the regulation of a highly diverse set of metabolic pathways ,23,24. Io models . Moreoveant LUAD .EGFR-mutant LUAD cells, enhanced mitochondria-mediated pyruvate oxidation, thus decreasing lactate production. Intriguingly, decreased mitochondrial function has been correlated with an epithelial–mesenchymal transition (EMT) phenotype, a well-known resistance mechanism to EGFR TKIs [Glycolysis is a multi-level metabolic process assigned to numerous enzymes. Pyruvate dehydrogenase complex (PDH) is a multienzyme complex that catalyzes the oxidative phosphorylation of pyruvate towards acetyl coenzyme A (Acetyl-CoA). However, phosphorylation of PDH by pyruvate dehydrogenase kinase 1 (PDHK1) inactivates it, resulting in the overproduction of lactate. Interestingly, mRNA levels of PDHK1 along with genes of multiple glycolytic enzymes have been found to be significantly higher in NSCLC compared to normal tissue . Today, GFR TKIs ,27. CombGFR TKIs .EGFR exon 19 deletion-containing LUAD HCC827 cell line sensitive to EGFR TKIs. Interestingly, derived cell lines from these models were not resistant to erlotinib in vitro. The resistant phenotype was rescued when CAF-enriched conditioned media was cocultured. Importantly, monocarboxylate transporter 4 (MCT4), a lactate transporter and well-known readout of the Warburg effect, was found to be increased in resistant HCC827 cells compared to parental cells, confirming the metabolic rewiring towards aerobic glycolysis. Moreover, stromal HGF and MCT4 were upregulated in NSCLC samples progressed after gefitinib/erlotinib treatment, suggesting potential clinical relevance of the findings. Collectively, the implication of lactate metabolism and MET signaling in EGFR TKI resistance warrants additional research exploring the efficacy of combinatorial treatments with lactate/MET inhibitors and EGFR TKIs [The tumor microenvironment (TME) is a mixture of active and dynamic components, including a plethora of metabolites that all interact to promote cancer cell survival and proliferation. In particular, the lactate secreted by EGFR TKI-resistant cells is engulfed by cancer-associated fibroblasts (CAFs), triggering the overproduction of hepatocyte growth factor (HGF) and the subsequent activation of MET signaling, suggesting the presence of a non-cell-autonomous metabolism-based mechanism of resistance to EGFR TKIs . ErlotinGFR TKIs .EGFR-mutant HCC827 and PC9 cells to osimertinib and gefitinib respectively induced rapid cell death. However, a subpopulation of treated cells evaded the EGFR TKI-induced cytotoxic effect using an EGFR-independent resistance mechanism. Expression profile analysis of gefitinib-resistant PC9 clones identified the downregulation of genes related with glutamine metabolism and the tricarboxylic acid (TCA) cycle [In another study, the exposure of A) cycle . FurtherA) cycle .EGFR-sensitizing mutations does not guarantee an equal survival benefit in all patients with EGFR-mutant LUAD. Therefore, and besides the inevitable resistance mechanisms, the high variability of response rate must be taken into consideration, demonstrating the urgent need for identifying the factors responsible for this reality. To that end, one study analyzed the serum metabolic profile of 44 patients with EGFR-mutant LUAD treated with icotinib and divided them into equal groups of good and poor responders, considering progression-free survival longer or shorter than 11 months [In the clinical setting, the presence of 1 months . Among 81 months .EGFR-mutant sensitive  and resistant (H1975) LUAD cell lines, respectively. The use of statins such as atorvastatin (ATV) has been demonstrated to abrogate the function of caveolin-1 (CAV-1), a factor that regulates cellular cholesterol homeostasis. Indeed, ATV abrogated the expression of CAV-1 and decreased the survival of EGFR-mutant LUAD cells by upregulating apoptosis. Interestingly, a combination of ATV with gefitinib significantly enhanced cell death compared to ATV alone. However, the addition of mevalonate, an intermediate metabolite of cholesterol formation, restricted the tumor cell suppression and reinduced the CAV-1 expression in ATV-treated cells, demonstrating the tight link between cholesterol and CAV-1 [Many non-oncological drugs have been repurposed for anti-cancer effects . For exand CAV-1 . Glucosend CAV-1 , and GLUnd CAV-1 . CAV-1 und CAV-1 .EGFR-mutant LUAD cells compared to wild type (wt). Combinatorial treatment of ATV and EGFR TKIs resulted in higher inhibition than single treatment, highlighting the importance of lipid biosynthesis in EGFR TKI resistance [EGFR-mutant LUAD cells by significantly reducing the presence of lipid droplets. Moreover, the lipidomic profile of osimertinib-treated cells has revealed decreased concentrations of lipid classes such as diacylglycerol, triacylglycerol, ceramides, and phosphatidylethanolamine [Another study identified a higher expression of low-density lipoprotein receptor (LDLR) mediated by sterol regulatory-element-binding protein 1 (SREBP1) in sistance . Generalsistance ,45. Morenolamine . Mechaninolamine . Similarnolamine . Moreovenolamine .EGFR-mutant and EGFR-wt patients with LUAD was able to discriminate the presence of EGFR mutation based on a seven-lipid profile including mainly 20 and 22 carbon chain length polyunsaturated fatty acids (PUFAs) and phospholipids [In clinical routine, limited availability of tissue frequently does not allow molecular analysis or leads to inconclusive histological diagnosis . Therefoholipids .EGFR-mutant LUAD enhances glycolysis to support cell survival. At a specific stage of glycolysis, LUAD cells exert an activating transcription factor 4-mediated metabolic rewiring, shifting glycolysis towards serine synthesis as demonstrated by upregulation of serine synthesis pathway metabolic enzymes such as phosphoserine phosphatase and phosphoglycerate dehydrogenase. Serine is a precursor of cystine which leads to glutathione (GSH) formation, which acts as an antioxidant and participates in ROS scavenging, inhibiting cell death [The cells during physiological processes such as oxidative metabolism or response to cytokines or pathogen infection produce by-products of so-called reactive oxygen species (ROS). When ROS are accumulated without clearance, they induce cell death . Moreovell death . Anotherll death . Transcrll death .EGFR-mutant HCC827 cells [BCAT1 promoter was gradually decreased during resistance acquisition, indicating a possible negative correlation between them. Aberrant BCAT1 activation led to a metabolic rewiring involving the redox pathway and increasing GSH generation, resulting in ROS scavenging and EGFR TKI resistance. Interestingly, BCAT1 and H3K9 were found to be negatively correlated in EGFR-mutant LUAD samples at baseline and relapse. Moreover, BCAT1 was higher in the relapse group and was correlated with poor response to treatment. The use of ROS-inducing agents such as piperlongumine or auranofin as well as buthionine sulfoximine, a GSH synthesis inhibitor, managed to partially overcome the gefitinib resistance both in vitro and in vivo [EGFR-mutant LUAD. Specifically, PE-derived metabolites were analyzed between naïve and first-generation EGFR TKI-treated groups. Principal component analysis identified 34 metabolites that were differentially expressed between the groups, and specifically higher in the treatment group. Further classification shared these metabolites into six metabolic pathways associated with amino acid metabolism  and biosynthesis  [An example of multi-omics interconnection in the EGFR TKI resistance involves branched-chain amino acid (BCAA) metabolism. BCAAs are a group of three essential amino acids: leucine, isoleucine, and valine. Alterations in BCAA metabolism in response to erlotinib treatment have been demonstrated in 27 cells . Branche in vivo . Interes in vivo ,52. A suptophan) .EGFR-mutant LUAD cells develop to tolerate EGFR TKI treatment [The acquisition of a stem-cell-like phenotype is among the resistance mechanisms that reatment . Indeed,reatment . Of notereatment . Similarreatment . Moreovereatment .EGFR-mutant LUAD are primarily based on tumor genetic profiles. However, patients with identical EGFR TKI-sensitizing alterations present various response rates and relapse-free survival. Therefore, the effective stratification of patients with EGFR-mutant LUAD towards a long-term clinical benefit remains an unmet challenge and suggests that additional mechanisms beyond genetic alterations might exist which co-determine patients’ treatment efficacy. Metabolic reprogramming is inevitably triggered by constitutively active EGFR signaling pathways, empowering cancer initiation and progression. As reviewed here, EGFR TKIs acutely affect numerous metabolic processes, predominantly glucose metabolism, fatty acid biogenesis, and redox homeostasis, increasing cell death and tumor reduction. However, it seems that these oncometabolites rapidly recover their levels, reactivating EGFR signaling and establishing a resistant phenotype. Here, we discussed different comprehensive studies which have used a wide range of metabolic-modulating agents with remarkable efficacy against EGFR TKI resistance. We also discussed metabolic vulnerabilities that could be addressed by already FDA-approved drugs used for other diseases such as hyperlipidemia and diabetes, thus accelerating their introduction into clinical use for EGFR TKI-treated patients. A serious concern about metabolism-modulating drugs is the systemic toxicity that might be triggered when they are delivered to unselected cancer patients lacking indication of metabolic dependency. However, experiments in EGFR-mutant LUAD in vivo models with metabolic agents summarized in this review showed a high safety profile without affecting vital points of the in vivo models such as body weight.Today, the clinical treatment decisions in EGFR Mutation Test v2 as a companion diagnostic test for EGFR TKI-eligible NSCLC patients. Although the findings are limited, biofluids such as serum and PE of patients with EGFR-mutant LUAD could be used as sources of metabolites and provide information regarding treatment efficacy. The regular diet of patients naturally shapes the availability of nutrients. Although no dietary patterns such as an alkaline diet have been introduced in the treatment management of patients with lung cancer, future clinical trials investigating whether patient diet affects EGFR TKI response would be of high interest. Moreover, cancer-related cachexia is a catabolic syndrome that affects an important percentage of patients with NSCLC, and has been associated with patient survival and treatment efficacy. Therefore, future approvals of metabolism-targeted therapies should take into serious consideration this aspect, since drugs that block different anabolic processes might further aggravate the disease.Liquid biopsy represents a promising alternative solution to traditional tissue analysis. Interestingly, one of the few FDA-approved applications of liquid biopsy is the Cobas EGFR-sensitizing alterations, metabolomics and epigenomics collaborate to promote EGFR TKI resistance in EGFR-mutant LUAD. Although many -omics regarding EGFR TKI resistance in EGFR-mutant LUAD are still understudied, preliminary insights indicate that resistance occurrence is mediated at a multi-omics level and evaluation of EGFR TKI treatment should be performed with a more holistic approach. Collectively, we highly encourage the continuation of metabolism-oriented studies in EGFR-mutant LUAD for the identification of predictive and prognostic metabolic biomarkers. Moreover, future research should include clinical studies enrolling patients with EGFR-mutant LUAD and exploring the potential clinical value of translational insights from laboratory-based findings regarding the metabolism-targeted therapies in combination with EGFR TKIs.Finally, as presented here, in the presence of


COVID-19 is an on-going pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent evidence suggests that SARS-CoV-2 may be associated with various neuropsychiatric symptoms, including mania. We present a case of a middle aged man presenting with acute mania with psychotic symptoms 20 days post COVID infection in the absence of prior psychiatric illness. This report highlights the need for rigorous neuropsychiatric assessment in patient with symptoms of SARS-CoV-2 infection.A 52-year-old man of West African origin with past history of hypertension and no previous history of mental health illness presented with acute manic symptoms on background of two weeks of high fever, diarrhoea, mild headache, dry cough and anosmia. He was tested positive for SARS-CoV-2 infection on COVID PCR test. He was under self-isolation along with his family members who exhibited mild symptoms of SARS-CoV-2, none of them required hospital admission. He was initially fearful to seek medical attention but was brought in by family after exhibiting behaviour changes, obsession with toilet cleaning, reckless spending and getting aggressive approximately two weeks after the onset of acute upper respiratory symptoms. He presented elated in mood with pressure of speech and grandiose ideas. Investigations like neuroimaging and bloods were unremarkable. Initial psychiatric assessment found symptoms consistent with acute mania and he was detained under the Mental Health Act. During admission, he was sexually disinhibited and agitated on the ward requiring IM antipsychotics. He was treated with high dose of Olanzapine and Sodium valproate and his symptoms subsided within two weeks.This case emphasises the manifestation of neuropsychiatric illness post COVID-19 without a background of psychiatric illness, hypoxemia and cerebral infarction.Based on the CORONERVE Programme and latest retrospective Lancet cohort studies, the period between 14 and 90 days after diagnosis, 5.8% COVID-19 survivors had their first recorded diagnosis of psychiatric illness.It is also important to consider other organic disease given the simultaneous diagnosis of COVID-19. Although it is not yet possible to confirm here due to the lack of a validated CSF-PCR assay, previous reports have implicated SARS-CoV-2 in the development of viral encephalitis, and this remains an important differential.Clinicians should be alert to the possibility of patients with COVID-19 developing neuropsychiatric complications post SARS-CoV-2 infection, mandating the need for vigilant initial neuropsychiatric assessment and possibly follow-up care in 3 months.


Nanotechnology enables new ways to diagnose and treat cancer. Nanomedicines can increase payload concentration at the disease site, reduce toxicity, and enhance therapeutic effects compared to drugs in their “free” form. Spherical Nucleic Acids (SNAs) emerged as a new class of oligonucleotide nanotherapeutics that are currently being explored as gene-regulatory and immunostimulatory architectures to overcome drug resistance and immunosuppression in solid tumors. This review highlights seminal studies that identified SNAs as a powerful platform for gene regulation, the activation of innate immunity, and the development of next-generation cancer vaccines, discusses recent efforts to translate fundamental discovery from the laboratory into the clinic, and provides an outlook on future research aimed at harnessing the full therapeutic potential of the SNA platform.Spherical Nucleic Acids (SNAs) emerged as a new class of nanotherapeutics consisting of a nanoparticle core densely functionalized with a shell of radially oriented synthetic oligonucleotides. The unique three-dimensional architecture of SNAs protects the oligonucleotides from nuclease-mediated degradation, increases oligonucleotide bioavailability, and in the absence of auxiliary transfection agents, enables robust uptake into tumor and immune cells through polyvalent association with cell surface pattern recognition receptors. When composed of gene-regulatory small interfering (si)RNA or immunostimulatory DNA or RNA oligonucleotides, SNAs silence gene expression and induce immune responses superior to those raised by the oligonucleotides in their “free” form. Early phase clinical trials of gene-regulatory siRNA-based SNAs in glioblastoma (NCT03020017) and immunostimulatory Toll-like receptor 9 (TLR9)-agonistic SNAs carrying unmethylated CpG-rich oligonucleotides in solid tumors (NCT03086278) have shown that SNAs represent a safe, brain-penetrant therapy for inhibiting oncogene expression and stimulating immune responses against tumors. This review focuses on the application of SNAs as precision cancer therapeutics, summarizes the findings from first-in-human clinical trials of SNAs in solid tumors, describes the most recent preclinical efforts to rationally design next-generation multimodal SNA architectures, and provides an outlook on future efforts to maximize the anti-neoplastic activity of the SNA platform. Oligonucleotide-based therapeutics for gene regulation and immunostimulation have merged as a powerful and novel concept of cancer therapy that can address many of the challenges of conventional drug development ,2,3,4. TInvented by Chad Mirkin and colleagues , SNAs ar3O4) [2) [2) [First-generation SNA nanoconstructs were composed of gold nanoparticle templates modified with a densely packed, highly oriented radial shell of DNA oligonucleotides via thiol adsorption. Research over the past decade has shown that SNAs are highly tailorable structures, which can be composed of various types of oligonucleotides and core materials. Types of oligonucleotides include DNA oligonucleotides , locked 3O4) ,24, quan3O4) [2) , core–sh [2) [2) , liposom [2) [2) , lipid n [2) [2) , poly(la [2) [2) , micelle [2) [2) , protein [2) [2) , and T8  [2) [2) .The ability to fine-tune core material and oligonucleotide identity has been critical for developing SNAs as cancer therapeutics and diagnostic tools, with many SNA architectures commercialized or enrolled in clinical testing. SNA-based Verigene™  and SmartFlare™ systems  use SNA technology to detect pathogens or disease-specific markers and enable RNA detection and quantification in live cells, respectively, with a degree of sensitivity and selectivity exceeding that of conventional diagnostic tools . In addiSNA-mediated gene regulation represents a powerful therapeutic approach for treating cancer and other diseases with a defined genetic basis. Here, SNA target specificity is controlled by the sequence of the gene-regulatory oligonucleotide. Different SNA therapeutics can be generated by modifying the oligonucleotide sequence using digital drug design to target a broad spectrum of oncogenes. Early studies of gene-regulatory SNAs, i.e., gold nanoparticles cores functionalized with antisense oligonucleotides or small interfering RNAs (siRNAs), demonstrated potent knockdowns of model targets . These iC-terminal 14 amino acid sequence with significant homology to the BH (Bcl-2 Homology) 2 domain found in several members of the Bcl-2 family [Bcl2L12 is a potential prognostic factor, as glioblastoma patients with high-level overexpression of Bcl2L12 mRNA have shorter progression-free survival than patients with low or underexpression of Bcl2L12 [Building upon these studies, Stegh, Mirkin, and colleagues began evaluating SNAs as gene-regulatory modalities for delivering siRNAs and miRNAs to glioblastoma, the most prevalent and aggressive form of brain cancer. Functional genomics investigations have identified numerous genetic events involved in glioblastoma development. An incomplete understanding of how these genetic aberrations influence tumor response to therapy, combined with the lack of effective delivery of drugs across the blood–brain and blood–tumor barriers (BBBs/BTBs) to the central nervous system, have contributed to making glioblastoma one of the most challenging cancers for which to achieve improved treatment outcomes . Althoug2 family ,40,41,422 family ,40,41,42 Bcl2L12 ,44,45. M Bcl2L12 . Given tIII)-conjugated SNAs, and by quantification of gold content in resected tumor tissue using Inductively Coupled Plasmaresonance Mass Spectrometry (ICP-MS) [To neutralize Bcl2L12 expression in an established glioma, Jensen et al.  and Kour(ICP-MS) ,18. Accu(ICP-MS) ,18.A recent study developed an innovative methodology to optimize SNA gene regulatory activity in vivo. Sita et al.  used a g−3 fg of Au per μm3, with gold also accumulating in tumor-associated macrophages and the tumor-associated neo-vasculature [Building upon these preclinical studies, a first-in-human, window-of-opportunity phase 0 clinical trial of siBcl2L12-carrying SNAs (drug moniker: NU-0129) in patients with recurrent glioblastoma (NCT03020017) was conducted to assess SNA safety, pharmacokinetics, biodistribution, and target engagement . This clculature . SNA uptculature . These rImmunotherapy has rapidly become the fourth pillar of cancer treatment, alongside surgery, radiation, and chemotherapy . It has Immunostimulatory SNAs are well-defined nanostructures that can present multiple immunostimulatory cues. The prototypic immune-stimulatory SNA architecture consists of a liposomal core conjugated with a radial surface shell of synthetic TLR-agonistic oligonucleotides at a high density . TLRs arLike TLR-9 activating constructs, SNAs carrying TLR7/8-activating RNA, which is attached to hydrophobic cholesterol moieties of the liposomal core template, can engage TLR7/8, trigger NF-κΒ activation in primary immune cells, promote pro-inflammatory cytokine production, and induce the expression of co-stimulatory molecules more effectively than cationic lipid-transfected RNA of the same sequence . In addi+ effector T cells: a non-cleavable linker  succinimide ester, BMPS); a cleavable linker (succinimidyl 3-(2-pyridyldithio)propionate, SPDP), which upon cleavage under reducing conditions within the endosome, leaves a molecular pendant group (3-mercaptopropionamide) attached to the antigen; and a traceless linker (4-nitrophenyl 2-(2-pyridyldithio)ethyl carbonate, NDEC), which upon reduction, engages in intramolecular cyclization followed by the release of the antigen in an unmodified form. All SNA architectures robustly co-delivered the adjuvant and antigen to APCs and effectively activated the APCs, suggesting that the linker chemistry does not impact SNA uptake endosomal TLR9 activation within APCs. Using T cell receptor transgenic CD8+ T cells that specifically recognize gp100, SNAs with a traceless linker most effectively induced effector T cell expansion and activation by promoting IFN-γ, TNF-α, granzyme-B, and IL-6 production [Immunostimulatory SNAs can be co-functionalized with peptide antigens in various structural arrangements to activate APCs and tumor-targeting effector T cells . Here, Soduction . Furtheroduction . Taken tAs a modular and chemically well-defined structure assembled from synthetic components, SNA-based vaccines allow for the systematic variation of the vaccine architecture to optimize the timing of APC activation through the induction of cell surface co-stimulatory molecules and the intracellular processing of the peptide antigen. Using a ‘rational vaccinology’ approach, Wang et al.  began tosn-glycero-3-phosphocholine (DOPC), loaded with tumor cell lysates derived from triple-negative breast cancer (TNBC) cell lines, and decorated with a shell of radially oriented TLR9-agonistic CpG oligonucleotides antigen-derived peptides represent a rational approach for tumor types, which ubiquitously and homogenously express known tumor-associated antigens. For tumors with less well-defined and heterogeneously expressed antigens, the use of cell lysates derived from a patient’s tumor represents an alternative approach that addresses some of the limitations of using a finite set of well-defined antigens. These include restricted epitope expression by only one of the major histocompatibility complexes (MHCs), inter- and intratumoral heterogeneous expression of antigen, and loss of antigen expression during tumor progression or recurrence. However, the suboptimal cell uptake and bioavailability of tumor cell lysates have limited therapeutic efficacy . To enhaleotides . Immune l memory . These rl memory .TM macrophages were engineered to secrete NF-κB-inducible embryonic alkaline phosphatase (SEAP). NF-κB is a major transcription factor that is activated by innate immune sensing involving TLRs and other DNA sensors [The seminal studies described above evaluated the impact of a finite number of structural features on SNA vaccine performance. Now, high-throughput methods for synthesizing and evaluating SNA vaccines combined with machine learning approaches allow for the systematic, large-scale screening of structural features that contribute to in vitro potency. Yamankurt et al.  develope sensors ,76. Macr sensors ,78,79. Ssn-glycero-3-phosphoethanolamine (DOPE), CpG DNA oligonucleotides, and a peptide derived from ovalbumin as a model antigen. Results from this screen revealed that the lipid composition of the core and surface chemistries for oligonucleotide anchoring to liposomal cores, together with oligonucleotide concentration, orientation, and backbone, influenced SNA immune-stimulatory activity [An analysis of SNA structure-activity relationships provided insights into the importance of individual SNA design components and how combinations of these components impact immune activation . The immactivity . Multifaactivity , as SNAsactivity ,82, indiIn addition to SNA vaccines, the most well-defined class of multimodal SNA nanotherapeutics, a series of SNA architectures have been synthesized and evaluated preclinically. In addition to oligonucleotide payloads, these SNA conjugates co-deliver chemotherapeutic drugs ,84, anti50 values than the free drug. These data provided the first proof-of-concept that SNAs can be used as delivery vehicles for chemotherapeutic drugs to enhance efficacy and reduce adverse side effects [Platinum (Pt) compounds, such as cisplatin or carboplatin, effectively treat a broad spectrum of cancer types, including testicular and ovarian cancers ,88. To d effects . A simil effects . Here, g effects .III complexes. Song et al. [III-conjugated SNAs as an MRI agent, enabling high-level GdIII loading, efficient cellular uptake, and excellent contrast enhancement. GdIII was conjugated to poly DNA thymine (poly dT) oligonucleotides and linked to gold nanoparticle cores via thiol adsorption using copper-catalyzed click chemistry. GdIII-SNAs showed high-level cell uptake and brighter MRI signal compared to non-SNA-conjugated GdIII. When intratumorally administered to glioblastoma-bearing mice, these architectures were used to define SNA pervasive infiltration of intracranial tumors using ex vivo MRI on resected tumor-bearing mouse brain [III loading, relaxivity, and cell uptake.In addition to increasing chemotherapeutic drug solubilities and efficacies, SNAs can deliver bioactivated contrast agents, including paramagnetic Gdg et al.  developese brain . In sum,SNAs architectures, i.e., nanoparticle core templates densely packed with highly oriented oligonucleotides, represent emerging and potentially paradigm-shifting oligonucleotide-based therapeutics. Comprehensive preclinical characterizations revealed that SNAs are recognized and endocytosed by scavenger receptors; potently activate endosomal TLRs; deliver peptides to antigen-presenting cells; and following endosomal escape, can potently and persistently neutralize gene expression due to increased resistance toward nuclease-driven degradation. These preclinical studies have now been translated into the clinic, where early phase clinical trials demonstrate efficacy of gene-regulatory and immunostimulatory SNAs in solid tumors.What is next? The development of high-throughput screening and machine learning approaches to investigate SNA structure-activity-relationships and define and predict critical design parameters to optimize SNA activity demonstrated that the systematic variations in SNA structure can lead to significant improvements in SNA performance. Enabled by their modular synthesis and chemically well-defined building blocks, SNAs will allow for the development of potent gene-regulatory conjugates or vaccines with well-defined presentation and stoichiometry of components.For vaccine development, SNAs must be systematically innovated for optimal antigen-specific T cell activation by incorporating multiple peptide antigens that engage both MHC-I and MHC-II, by improving tumor cell lysate loading into hollow nanoparticle cores, by fine-tuning SNA surface and linker chemistries for the most robust co-delivery of immunostimulatory cues to antigen-presenting cells, and by developing oligonucleotides that target multiple innate immune receptors or intracellular DNA sensors. In addition to developing the next generation of SNA vaccines for clinical development, this line of research will also help us better understand the structural and mechanistic basis for vaccine function through systematic iterations in SNA synthesis and design .As concerns the optimization of gene-regulatory SNAs, we must understand, on a fundamental level, how SNAs architectures can escape the endosome upon SR-A-dependent internalization, how to increase cell uptake and cytosolic localization, and augment siRNA loading on nanoparticle templates to engage the RNAi machinery more effectively. A recent study exemplified the need to further optimize gene-regulatory SNAs. Vasher et al.  developeFurthermore, we must begin to develop innovative treatment regimens that combine the power of SNA-enabled gene regulation to neutralize virtually any oncogenic lesion and the proven efficacy of chemo- and targeted therapeutics. The concept is to design and comprehensively characterize multimodal SNA architectures carrying both chemotherapeutic drugs and gene-regulatory oligonucleotides to target critical factors that drive chemo- and targeted therapy resistance .Besides optimizing the oligonucleotide shell for the activation of innate immunity, vaccine development, and gene regulation, future studies must focus on the nanoparticle template as a therapeutic cue. For example, gold nanoparticle cores were shown to act as a radiosensitizers . RadioseRecent studies have shown that proteins can be used as SNA templates, as the ability of SNAs to enter cells and tissue is mediated by the unique 3D architecture of the oligonucleotide shell and is independent of the type of core material. By densely functionalizing a protein’s surface with a shell of radially oriented oligonucleotides, protein-based SNAs (ProSNAs) can delivery cell-impermeable bioactive proteins to cells and tissue, particularly when conjugated with G-quadruplex-containing oligonucleotides ,96,97. STaken together, these future preclinical and clinical studies are poised to establish gene-regulatory and immunostimulatory SNAs as a customizable, on-demand therapeutic platform that can rapidly be developed to combat a broad spectrum of cancer types.A.H.S. is the inventor on patent US20150031745A1, which describes SNA nanoconjugates to cross the blood–brain barrier.


Platinum-based drugs cisplatin, carboplatin, and oxaliplatin are widely used for chemotherapeutic eradication of cancer. However, the side effects of platinum drugs, such as lack of selectivity, high systemic toxicity, and drug resistance, seriously limit their clinical application. With advancements in nanotechnology and chemical synthesis, Pt-based anti-cancer drugs have made great progress in cancer therapy in recent years. Many strategies relied on the anti-cancer mechanism similar to cisplatin and achieved some success by modifying existing platinum drugs. Pt-based nanodrugs, such as platinum nanoclusters, have novel anti-cancer mechanisms and great potential in tumor-targeted therapy and have shown promising results in clinical application. In this review, we systematically explored the development of first-line platinum chemotherapy drugs in the clinic and their anti-cancer mechanisms. We also summarize the progress of Pt-based anti-cancer drug application in cancer therapy, emphasizing their modification to enhance the anti-tumor effect. Finally, we address challenges faced by platinum chemotherapy drugs, especially Pt nanocluster-based nanodrugs, in cancer treatment. The new platinum drugs and their targeted modifications undoubtedly provide a promising prospect for improving the current anti-cancer treatments. Chemotherapy is an effective method of anti-tumor treatment The platinum-based anti-cancer drugs, including cisplatin in vitro, and low systemic toxicity and biocompatibility in vivoMany reformed and new anti-cancer platinum drugs have been formulated and reported recently. Among the diverse anti-cancer platinum drugs, multifunctional high-performance platinum nanocluster-based (Pt NC-based) nanodrugs, fabricated using different biocompatible materials leading to flexible designs, have attracted much attention for cancer-specific therapy and drug delivery In this review, we summarize recent scientific advances in Pt-based drugs designed for anti-cancer treatment and address other relevant issues: (1) investigation of molecular mechanisms of platinum drugs in clinical use; (2) summary of the strategies developed to avoid systemic toxicity and improve bioavailability of platinum drugs; (3) development of novel Pt NC-based nanodrugs designed for anti-cancer treatment; (4) challenges of platinum chemotherapy drugs, especially Pt NC-based nanodrugs, for the anti-cancer treatment.Cisplatin, carboplatin, and oxaliplatin are clinically approved worldwide and are the first choice for malignant tumor treatment 3)2Cl(OH2)]+ and cis-[Pt(NH3)2(OH2)2]2+As Pt-based anti-cancer treatment, cisplatin, carboplatin, oxaliplatin, and other drugs are widely used in the clinic with obvious therapeutic effects and a clear mechanism of action It is generally accepted that the principal mechanism of cisplatin anti-cancer action is platinum binding to DNA by forming intra-stranded and inter-stranded crosslinks Numerous studies have addressed the limitations of first-line platinum chemotherapy drugs due to potential toxicity and side effects and developed strategies to improve anti-cancer efficiency while reducing systemic toxicity As stated earlier, the efficacy of first-line Pt drugs is limited due to the occurrence of severe side effects  together with the ability of cancer cells to limit drug accumulation Extensive research indicated that the killing effect of Pt-based drugs on cancer cells could be improved by integrating the cancer cell-targeting moiety into Pt(IV) prodrugs. Notably, peptide-based drug delivery systems can enhance drug targeting properties and significantly reduce side effects . The tripeptide arginine-glycine-aspartic (abbreviated as RGD) recognizes integrin αvβ3 overexpressed on tumor cells vβ3-overexpressing cancer cells, an apoptosis sensor composed of tetraphenylsilole (TPS) fluorophore with aggregation-induced emission (AIE) characteristics, and a caspase-3 enzyme-specific Asp-Glu-Val-Asp (DEVD) peptide. The targeted Pt(IV) prodrug could selectively bind to integrin αvβ3 overexpressed on cancer cells to facilitate cellular uptake. Furthermore, the Pt(IV) prodrug was reduced to active Pt(II) drug in cells, releasing the apoptosis sensor TPS-DEVD. The reduced Pt(II) drug could induce cell apoptosis and activate the caspase-3 enzyme to cleave the DEVD peptide sequence. Due to the free rotation of the phenylene rings, TPS-DEVD was nonemissive in aqueous media. The specific cleavage of DEVD by caspase-3 generated the hydrophobic TPS residue, which aggregated, resulting in restriction of intramolecular rotations of the phenyl rings and ultimately leading to fluorescence enhancement  conjugate  can also be internalized by αvβ5 integrin opens up the door to delivering promising anti-cancer metallodrugs to tumors overexpressing αvβ5 integrin or to tumors coexpressing αvβ3 and αvβ5 integrins. The multi-integrin targeting approach would provide new metal-based anti-cancer strategies and benefit a broader range of patients by increasing the types of tumors which can be targeted.Similarly, Gandioso et al. reported an anti-cancer agent based on the conjugation of a photoactivatable Pt(IV) prodrug to a cyclic RGD-containing peptide Furthermore, cell-penetrating peptides (CPP), such as HIV-1 TAT, which can penetrate proteins and oligoarginine, are valuable tools for transporting therapeutic macromolecules into tumor cells in vitro and in vivo data suggested that Pt-Mal-LHRH elicits tumor-targeted drug delivery with increased potency, efficacy, and a possible reduction in chemotherapeutic side effects allowing the use of a high dose of chemotherapy in patients compared to other platinum drugs. Also, in vitro scratch assay data demonstrated a reduction in migration of tumor cells. Importantly, in vivo metastasis was investigated since the major cause of mortality in breast cancer patients is metastasis to distant sites, including the lungs. The in vivo data supported the in vitro data, as a significant decrease was observed in tumor volume and lung tumor colonization by Pt-Mal-LHRH treatment. Thus, the Pt-Mal-LHRH conjugate was selective for tumors overexpressing LHRH receptors while avoiding systemic distribution.The luteinizing hormone-releasing hormone (LHRH) peptide also acts as a targeting moiety, whose receptors are overexpressed in several types of cancer cells, such as breast, ovarian, prostate, lung, and liver Interestingly, conjugating non-functional small molecules with platinum drugs improves the targeting ability of Pt drugs. For example, Muhammad et al. reported the design and biological properties of two Pt(IV) complexes, Pt-Bio-I and Pt-Bio-II, carrying one or two biotin moieties in the axial positions of the Pt center Figure D 84. TetNanocarrier-based platinum drug delivery systems are promising alternatives to avoid the disadvantages of conventional platinum drugs 3)2Cl2(OH)(O2CCH2CH2CO2H)]) (MDDP), and then functionalized with a targeting ligand folic acid (FA) Utilizing the unique biological effects of GNCs, Zhou et al. employed BSA-protected GNCs as a dual-functional nanoplatform for drug delivery and fluorescence imaging of the tumor Figure  18. The Brown et al. tethered the active component of the anti-cancer drug oxaliplatin to gold nanoparticles (AuNPs) for improved drug delivery Superparamagnetic iron oxide nanoparticles (SPIONs) are usually employed as targeted delivery regents due to their advantages of low toxicity, biocompatibility, biodegradability, and well water dispersion. SPIONs can bind to drugs and be directed to the tissues of interest or tumors using an external magnetic field 3)2]2+ was attached via the terminal carboxylate groups  showed no cytotoxicity at concentrations up to 2 μM. However, in this study, the cisplatin Pt@Au@FeNPs, despite having activity at nanomolar concentrations, were cross-resistant with cisplatin in A2780/cp70 cells  to improve their delivery to tumors and increase efficacy s Figure A. The sus Figure B. The cys Figure C.in vitro anti-cancer activity comparable to free cisplatin at the same drug concentrations. In addition, they exhibited an enhanced anti-cancer effect in vivo on a cisplatin-resistant HT-29 tumor model in mice, particularly when a magnetic field was applied to the tumor area -graft-poly(ethyleneglycol methacrylate) (p(MAA-g-EGMA)) by radical copolymerization of methoxy-PEG-methacrylate with methacrylic acid Figure A. The cia Figure C-D. Duria Figure E, indicaa Figure F were idBesides SPIONs and GNCs, other nanomaterials, such as mesoporous silica, have multiple potential applications as drug carriers Glutathione (GSH) is one of the most abundant non-protein thiols in tumor cells, with its intracellular content of about 0.5-10 mM in vitro and in vivo. After efficient internalization of SFN-CDDP-NPs by tumor cells, the rapidly released SFN could notably decrease the GSH content and thus significantly increase DNA-bound Pt, resulting in severe DNA damage and cellular apoptosis. Due to the improved chemosensitivity and preferential tumor accumulation, SFN-CDDP-NPs greatly inhibited orthotopic breast cancer progression with reduced toxic side effects.Sulforaphane (SFN) has been reported to deplete GSH by directly binding with GSH to form the GSH-SFN complex, which can be exported out of the cell. Recently, Xu et al. proposed that the therapeutic efficacy of SFN-CDDP-NPs could be significantly improved by SFN-mediated GSH depletion in vivo efficacy and safety results showed that NPs effectively inhibited the growth of cisplatin-resistant xenograft tumors with an inhibition rate of 83.32% while alleviating serious side effects associated with cisplatin. GSH-scavenging polymeric NP technology reported herein could provide a unique strategy for improving the therapeutic efficacy of current Pt drugs  (Cys-PDSA) polymers (Cys-8E polymer) that readily react with GSH via disulfide-mediated reduction and their combination with a series of Pt(IV) prodrugs with tunable hydrophobicity. Optimized polymers rapidly disassembled and released Pt drugs in response to intracellular GSH while simultaneously consuming GSH to restore Pt sensitivity in cisplatin-resistant tumor cells s Figure B. Liang s Figure C.Pt NCs, similar to noble metal clusters such as GNCs, are relatively stable molecular aggregates composed of up to hundreds of metal atoms Although multiple studies of the Pt(II) complex and modified Pt(IV) prodrugs have attempted to improve the anti-cancer efficiency of Pt drugs, these strategies relied on anti-cancer mechanisms similar to cisplatin and were not very successful 2+ ions leaching under low pH conditions, such as in cell endosomes, to induce DNA damage 2 yolk-shell nanoclusters exhibited an IC50 of 35.5 ng/mL (4.7 ng/mL of Pt) in HeLa cells that was much lower than cisplatin (230 ng/mL of Pt). The FePt nanoclusters were oxidized to generate Pt2+ and Fe3+ ions, especially in intracellular late lysosome with a low pH (pH < 5.5) environment, as hollow nanospheres were found in mitochondria of cancer cells, implying breakdown of the FePt core. Thus, after cellular uptake, FePt cores disintegrated to generate metal ions inside the acidic environment of secondary lysosomes. Subsequently, these metal ions could escape from endosomes and enter the cell nucleus to bind DNA, forming DNA-Pt adducts and eventually leading to tumor cell apoptosis  cells. Subsequently, its metabolites entered nuclei to interact with DNA, and finally, the DNA-Pt complex triggered TNBC cell apoptosis via the p53 pathway. These data revealed that Pt6GS4 was comparable to carboplatin for cancer cell uptake, nuclear localization, and cancer cell proliferation inhibition. More significantly, compared with carboplatin, Pt6GS4 was non-toxic for the liver and kidneys, and Pt6GS4-treated mice lived longer. Our study opened a new avenue to explore polynuclear Pt compounds with accurate architecture for enhancing therapeutic effects and reducing systemic toxicity.Recently, our research group explored a facile approach to develop an endogenous GSH-chelated Pt molecule containing multiple Pt atoms for efficient cancer treatment Figure  121. TheIn vitro experiments showed that CytcApt-Pt NCs could kill 4T1 tumor cells in a pH-dependent manner but did not affect normal 293T cells. These results showed good therapeutic efficacy and excellent biosafety of CytcApt-Pt NCs, indicating their great potential for tumor treatment and reducing systemic toxicity Targeting peptides have widely been used to synthesize Pt NCs Xia et al. synthesized a Pt nanocluster assembly (Pt-NA) composed of assembled Pt NCs incorporating a pH-sensitive polymer and hepatocellular carcinoma (HCC)-targeting peptide Figure  125. Pt-in vivo. Results indicated that this chemotherapeutic had efficacy comparable to cisplatin.Another example is of a first-generation dendrimer-caged Pt nanocluster (CPN) with the size of an atomic level (0.93 ± 0.22 nm in diameter). CPN was endowed with targeting function by conjugating with the iRGD peptide Figure A 126. EsTraditional tumor chemotherapy employs chemo-drugs, which usually have strong side effects for normal cells and tissues The challenges accompanying these advances provide us with future directions and efforts for designing and constructing more effective Pt-based drugs for possible clinical applications:(1) For cisplatin and other platinum anti-cancer drugs, systemic toxicity is still the most challenging problem. Platinum drugs are modified by many methods, such as linking target molecules and adding drug delivery carriers, with the ultimate goal of reducing their toxicity.in vivo and their interaction with biomolecules to treat malignant tumors and other diseases (2) Many new platinum nanodrugs, such as Pt NCs, have been developed. Pt NCs generate platinum ions in cells and induce irreversible DNA damage (3) As a new type of platinum anti-cancer drug, the molecular composition of Pt NCs needs to be further improved. Mass spectrometric techniques, such as MALDI-TOF-MS and ESI-MS, would help characterize the molecular formula of clusters or the metal to ligand ratio in clusters, but the precise structural characterization methods of cluster molecules need to be further explored (4) Combining Pt-based nanodrugs with other therapeutic methods, such as synergistic chemo-electrodynamic therapy, can maximize the bio-function of Pt NCs and strengthen their anti-tumor effect


The use of face masks or respirators (N95/KN95) is recommended to reduce transmission of SARS-CoV-2, the virus that causes COVID-19  or negative (control-participants) SARS-CoV-2 test result, from among all California residents, without age restriction, who received a molecular test result for SARS-CoV-2 during February 18–December 1, 2021  in the 14 days preceding testing and whether they wore a face mask or respirator all, most, some, or none of the time in those settings. Interviewers recorded participants’ responses regarding COVID-19 vaccination status, sociodemographic characteristics, and history of exposure to anyone known or suspected to have been infected with SARS-CoV-2 in the 14 days before participants were tested. Participants enrolled during September 9–December 1, 2021, (534) were also asked to indicate the type of face covering typically worn  in indoor public settings.The primary analysis compared self-reported face mask or respirator use in indoor public settings 14 days before SARS-CoV-2 testing between case- (652) and control-  participants. Secondary analyses accounted for consistency of face mask or respirator use all, most, some, or none of the time. To understand the effects of masking on community transmission, the analysis included the subset of participants who, during the 14 days before they were tested, reported visiting indoor public settings and who reported no known exposure to persons known or suspected to have been infected with SARS-CoV-2. An additional analysis assessed differences in protection against SARS-CoV-2 infection by the type of face covering worn, and was limited to a subset of participants enrolled after September 9, 2021, who were asked to indicate the type of face covering they typically wore; participants who indicated typically wearing multiple different mask types were categorized as wearing either a cloth mask (if they reported cloth mask use) or a surgical mask (if they did not report cloth mask use). Adjusted odds ratios comparing history of mask-wearing among case- and control-participants were calculated using conditional logistic regression. Match strata were defined by participants’ week of SARS-CoV-2 testing and by county-level SARS-CoV-2 risk tiers as defined under California’s Blueprint for a Safer Economy reopening scheme.A total of 652 case- and 1,176 control-participants were enrolled in the study equally across nine multi-county regions in California . The majWearing an N95/KN95 respirator  or wearing a surgical mask  was associated with lower adjusted odds of a positive test result compared with not wearing a mask . WearingDuring February–December 2021, using a face mask or respirator in indoor public settings was associated with lower odds of acquiring SARS-CoV-2 infection, with protection being highest among those who reported wearing a face mask or respirator all of the time. Although consistent use of any face mask or respirator indoors was protective, the adjusted odds of infection were lowest among persons who reported typically wearing an N95/KN95 respirator, followed by wearing a surgical mask. These data from real-world settings reinforce the importance of consistently wearing face masks or respirators to reduce the risk of acquisition of SARS-CoV-2 infection among the general public in indoor community settings.These findings are consistent with existing research demonstrating that face masks or respirators effectively filter viruses in laboratory settings and with ecological studies showing reductions in SARS-CoV-2 incidence associated with community-level masking requirements  variant, which is more transmissible than earlier variants. Sixth, face mask or respirator use was self-reported, which could introduce social desirability bias. Seventh, small strata limited the ability to account for reasons for testing in the adjusted analysis, which may be correlated with face mask or respirator use. Finally, this analysis does not account for potential differences in the intensity of exposures, which could vary by duration, ventilation system, and activity in each of the various indoor public settings visited.The findings of this report reinforce that in addition to being up to date with recommended COVID-19 vaccinations, consistently wearing face masks or respirators while in indoor public settings protects against the acquisition of SARS-CoV-2 infection (Face masks or respirators (N95/KN95s) effectively filter virus-sized particles in laboratory settings. The real-world effectiveness of face coverings to prevent acquisition of SARS-CoV-2 infection has not been widely studied.Consistent use of a face mask or respirator in indoor public settings was associated with lower odds of a positive SARS-CoV-2 test result (adjusted odds ratio = 0.44). Use of respirators with higher filtration capacity was associated with the most protection, compared with no mask use.In addition to being up to date with recommended COVID-19 vaccinations, consistently wearing a comfortable, well-fitting face mask or respirator in indoor public settings protects against acquisition of SARS-CoV-2 infection; a respirator offers the best protection.


The extra-pulmonary computed tomography (CT) findings of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or coronavirus disease 2019 (COVID-19) have been described in the literature, including the neurologic, cardiac, and abdominal manifestations. Ischemic and inflammatory bowel changes were the most frequent abdominal CT findings. Acute pancreatitis was seldom found all over the world outside of China and described in case reports, but not in Egypt.A 44-years-old female patient presented with severe abdominal pain, vomiting, and mild fever for six days. No respiratory symptoms were encountered till the time of the radiological investigation. Abdominal CT examination revealed interstitial edematous pancreatitis with acute peri-pancreatic fluid collection according to the revised Atlanta classification. No pancreatic necrosis or vascular complications were depicted. Secondary pyloro-duodenitis was noticed. The modified Balthazar CT-severity index was moderate (6/10). Basal chest scans showed bilateral variable-sized bronchocentric and sub-pleural consolidative pneumonic patches with mild bilateral pleural effusion. The patient was admitted to the intensive-care unit (ICU) for two weeks. The serum amylase and lipase titers were elevated and the polymerase chain reaction (PCR) test for COVID-19 was positive. She received pancreatic, circulatory, and pulmonary medical support, then she was discharged after stabilization of her condition.The authors provided this case report for the association between COVID-19 infection and acute pancreatitis, which is mostly the first in Egypt. It documented their Egyptian experience to be added to the international literature. It radiologically described their chest and abdominal CT findings in detail using the COVID-19 Reporting and Data System (CO-RADS) and the revised Atlanta classification with modified Balthazar CT-severity index (CTSI). This could eventually enrich the radiological point of view in addition to the previously published clinical case reports. Several extra-pulmonary manifestations of SARS-CoV-2 COVID-19) have been described, including the neurologic, cardiac, and abdominal manifestations 9 have be–6. On thScientists attributed the pancreatic injury that accompanies COVID-19 infection to either primary direct viral attack or secondary angiotensin receptor-converting-enzyme 2 abnormalities by the viral proteins . The excThe authors provide this case report for the association between COVID-19 infection and acute pancreatitis, which is mostly the first in Egypt.Different from the majority of the previous case reports in the literature, acute pancreatitis in the current case report was the initial clinical presentation that preceded the respiratory symptoms of COVID-19 infection. The authors hence believed that COVID-19 infection should be always concerned in patients presenting with acute pancreatitis without signs of cholelithiasis or a history of metabolic diseases or alcohol consumption.Additionally, the authors were keen to radiologically describe their chest and abdominal CT findings in detail using the CO-RADS and the revised Atlanta classification with modified Balthazar CT-severity index (CTSI).A 44-years-old Egyptian female patient presented to our radiology department in June 2021 with a clinical request for abdominal CT imaging. The referring clinician suspected pancreatitis after performing an ultrasound examination for the patient in the out-patient clinic.Clinically, the patient was complaining of acute abdominal pain and vomiting for six days. The patient was feverish at 38 degrees celsius (38 °C). She did not have any respiratory complaints till the date of the radiological investigation. The past history was negative regarding diabetes mellitus, hyperlipidemia, hypertension, and auto-immune diseases. She had a surgical history of cholecystectomy five years ago.Radiologically, the abdominal scans showed:A large peri-pancreatic and left anterior pararenal homogenous and hypo-attenuated fluid collections measuring 8 Hounsfield units (8 Hu) with a mild stranding of the nearby fat planes. It extends to the lesser sac with nearby mural thickening and sub-mucosal edematous changes of the pyloro-duodenal junction, denoting secondary gastro-duodenitis.The pancreas itself retained a normal size and homogenous tissue matrix without non-enhancing necrotic areas. The main pancreatic duct (MPD) was not dilated.The superior mesenteric vessels as well as the portal vein, superior mesenteric vein, and splenic vein were patent and adequately opacified without vascular thrombosis.The spleen was mildly enlarged, reaching 14 × 10.6 × 6 cm in maximum cranio-caudal (CC), anteroposterior (AP), and side to side (at the level of splenic hilum) dimensions (with 542 ml splenic volume and 882 splenic index). It showed a normal homogenous tissue matrix and absent focal lesions.No ascites were detected elsewhere. Clear cholecystectomy surgical bed with an average caliber of the common bile duct (CBD) and intrahepatic biliary radicles.Mild bilateral pleural collection was noted.The fore-mentioned CT findings were suggestive of interstitial edematous pancreatitis (IEP) with acute peri-pancreatic fluid collection (APFC) according to the revised Atlanta Classification. The modified Balthazar CT-severity index (CTSI) was 6/10 (moderate).The basal chest CT cuts additionally showed bilateral variable-sized bronchocentric and sub-pleural consolidative patches, they were suggestive of basal pneumonic changes. The viral origin was highly suspicious radiologically (CO-RADS 5), hence clinico-laboratory correlation was requested. This was attributed to the current pandemic status, in addition to the multi-centricity and characteristics appearance of the pulmonary parenchymal involvement. . The C-reactive protein (CRP) was elevated at 77.5 mg/L. The serum ferritin and D-dimer were elevated also (338 and 443 ng/mL respectively). The vital signs and laboratory tests results of the patients are detailed in (Table The patient was urgently admitted to the intensive care unit (ICU). At the time of ICU admission, the patient had mild tachypnea (20 breaths/min) and mild tachycardia (92 beats/min). His blood pressure was 130/80. The serum amylase and lipase titers were significantly increased (672 and 1619 U/L respectively) and this confirmed the diagnosis of acute pancreatitis. The PCR test for COVID-19 was performed and proved positive. The white cell count was not elevated (9.5 × 109/L). The C-reactive protein (CRP) also decreased (24 mg/L). The serum amylase and lipase titers had significantly decreased (77 and 34 U/L respectively). The renal functions had improved.The patient stayed in the intensive care unit (ICU) for two weeks and received pancreatic and pulmonary medical support. This included off-oral intake with IV-fluids replacement, high-flow nasal oxygen, prophylactic heparin anticoagulation, intravenous prophylactic antibiotics, and dexamethasone. She was discharged later after stabilization of her condition. The fever had subsided. The leukocytic count decreased . These CT findings predominantly included the presence of peripheral located mixed ground glass and consolidative patches in both lung fields in addition to the bulky pancreas with stranding of the surrounding fat planes and multi-locular peri-pancreatic fluid collections.