Adenovirus-based gene transfer vectors are the most frequently used vector type in gene therapy clinical trials to date, and they play an important role as genetic vaccine candidates during the ongoing SARS-CoV-2 pandemic. Immediately upon delivery, adenovirus-based vectors exhibit multiple complex vector-host interactions and induce innate and adaptive immune responses. This can severely limit their safety and efficacy, particularly after delivery through the blood stream. In this review article we summarize two strategies to modulate Ad vector-induced immune responses: extensive genomic and chemical capsid modifications. Both strategies have shown beneficial effects in a number of preclinical studies while potential synergistic effects warrant further investigations. While viruses have been evolutionary optimized to deliver their genes into host target cells with high specificity and efficiency, the hosts have developed complex defense mechanisms that limit or prevent such infections. To date, the most efficient gene transfer vectors are derived from viruses. As a consequence, to achieve safe and efficacious in vivo gene delivery by virus-based vectors a comprehensive understanding of the molecular mechanisms underlying antiviral immune reactions is mandatory. In addition, tools are needed to modify vectors appropriately with the aim to either prevent or exploit such immune reactions. The immune system is a complex and interwoven network with numerous different cell types and noncellular components being involved. It is divided into an innate and an adaptive arm. The innate immune response relies on a limited number of proteins and reacts within seconds or minutes after an infectious event. Innate immune responses are not antigen-specific and do not result in immunological memory. However, innate immune sensing creates an inflammatory environment that conditions adaptive immune responses. The adaptive parts of the immune system rely on clonal expansion of antigen-specific B- and T-cells and generate an immunological memory. Sensing of viral structures  by the innate immune system may provoke tissue invasion of innate immune cells and release of inflammatory chemokines and cytokines. These events induce an inflammatory  state in the respective tissue, resulting in decreased transduction and potential activation of adaptive immune responses. Activation of dendritic cells (DCs) and subsequent antigen-presentation is a critical step linking innate to adaptive immune responses.12 particles/mL) while meeting clinical good manufacturing practice (GMP) standards. Given these numerous advantages Ad-derived vectors are currently the most commonly used vectors in gene therapy, cancer and vaccine clinical trials worldwide (http://www.abedia.com/wiley/vectors.php (accessed on 10 January 2021)). The first gene therapy/oncolytic products based on Ad were released already eighteen years ago [Adenoviruses (Ad) are non-enveloped, icosahedral viruses with a ~35 kb linear double-stranded (ds) DNA genome ,2. They So-called first-generation (FG) vectors based on human adenovirus type five have a transgene capacity of up to 8 kilobases (kb) due to deletion of the E1 and/or E3 gene regions. These vectors exhibit strong short-term transgene expression in immune-competent individuals due to leaky expression of viral genes that triggers vector-directed immune responses. Viral gene products, and in some cases the transgene itself, influence cellular immune responses leading to extinction of gene expression ,5. There12 vector particles) in human gene therapy trials led to elevated serum level of IL-6 und IL-1 [Systemic Ad vector administration for gene transfer purposes has been shown to result in quick vector sequestration from the blood stream. In mice an almost complete elimination of virus particles from the blood occurs within 30 min, with a half-life of less than 2 min . The remund IL-1 ,21,22. Ound IL-1 . Moreoveund IL-1 . These ound IL-1 . Systemiund IL-1  as well und IL-1 . Of noteund IL-1 . Capsid-Ads and Ad-derived vectors are highly immunogenetic, and because human Ads (hAds) are ubiquitous, the serum prevalence to one or more types is nearly universal . Therefohypoxanthine-guanine phosphoribosyltransferase (HPRT) gene resulted in prolonged gene expression and no induction of a CTL response was detected [The construction of third-generation Ad vectors, also called helper-dependent (HD-Ad) or high-capacity (HC-Ad) Ad vectors, is based on extensive genetic modifications up to the deletion of all viral genes except for cis-acting elements needed for genome replication (ITR) and packaging (ψ). These global deletions offer high cloning capacity  and enable delivery of multiple transgenes, complete genomic loci or at least large regulatory sequences to regulate e.g., tissue-specific transgene expression. Due to the lack of viral gene expression, HD-Ad exhibit prolonged transgene expression and reduced toxicity. For efficient DNA packaging a minimum vector size of 27 kb should be maintained ,47, so ddetected . When chdetected ,50,51. Fdetected ,53. Irredetected .In the following we will exemplarily highlight few promising studies addressing long-term transgene expression and immunogenicity of HD-Ad.apoE cDNA as well as a HD-Ad harboring the genomic apoE locus were compared. After intravenous injection, FG-Ad resulted in transiently (28 days) decreased cholesterol serum level within normal range. In contrast, delivery of the apoE genomic locus using HD-Ad yielded stable reduction of cholesterol serum level for up to two and a half years. In addition, only FG-Ad induced elevated serum level of liver enzymes aspartate transaminase (AST) and alanine transaminase (ALT) indicating hepatoxicity, whereas there was no such evidence for HD-Ad. However, both vectors induced production of Ad-specific antibodies hindering re-administration [For treatment of hypercholesteremia in apolipoprotein E (apoE) deficient mice a first-generation Ad vector (FG-Ad) encoding the stration . Anotherstration .Similar findings were observed in clinically relevant large animal models. Liver-directed, HD-Ad-mediated expression of human α1-antitrypsin (hAAT) in baboons exhibited prolonged hAAT expression for > 1 year in two out of three animals. The third animal, however, had significantly lower hAAT serum levels due to antibody development targeting the hAAT protein. In contrast, when FG-Ad was used for hAAT delivery, transgene expression lasted for only 3–5 month. Here this was not due to antibody formation against hAAT, but development of Ad-specific CD4+ T-cells, resulting in the elimination of all transduced liver cells. A phenomenon that was not observed for HD-Ad . One verAn elegant method to prevent viral vectors from being recognized by both, innate and adaptive immune cells, is to equip the viral capsid with a “magic cap”. Pharmaceutical studies have shown before that polyethylene glycol (PEG) reduces antigenicity and immunogenicity of therapeutic protein compounds ,61. Thuset al. demonstrated that PEGylated vector particles evaded neutralization both, in vitro by purified antihexon antibodies and in pre-immunized mice in vivo [et al., PEGylated Ads mediated prolonged transgene expression and exhibited a decreased induction of Ad-specific CTLs. Analysis of cytokine profile revealed reduced Th1 responses but no difference in Th2 responses. However, Ad vector PEGylation still stimulated development anti-Ad NABs, albeit to a low extent, resulting in reduced transgene expression after sequential vector administration [Due to the hydrophilic nature of covalently attached polymers like PEG and poly-N-(2-hydroxypropyl)methacrylamide (pHPMA), another important shielding moiety , polymer in vivo . Croyle stration . In a costration . These fstration . OutliniAfter coupling, nonreactive ends of monovalent PEGs protrude from the capsid surface, while multivalent pHPMA molecules are linked to numerous reactive sites coupling the polymer to the viral capsid. Capsid shielding using pHPMA prevented vector particles from antibody neutralization  and bindA genetichemical concept for vector shielding overcoming some of these limitations was introduced by Kreppel et al. This concept based on the genetic introduction of cysteines in the capsid at solvent-exposed positions like fiber HI-loop , proteinSo far, these data indicate that shielding of viral capsids is a powerful tool to prevent interactions of the immune system with Ad vectors, circumventing antibody mediated neutralization and mitigating acute toxicity. Chemical capsid modifications of Ad vectors in general need to be performed in a way that does not interfere with intracellular trafficking of the vector particles or prevents particle disassembly after uptake into the target cells ,76,77. TAs described above, Ads are highly immunogenic and can induce both, innate and adaptive immune responses. While this may lead to undesired side-effects in gene replacement strategies, these effects can be exploited for vaccination purposes. A successful vaccination strategy requires the induction of a cytokine profile orchestrating the maturation of B- and cytotoxic T-cells, resulting in a prolonged and boostable immune response. Naturally, Ad vectors cause lytic infections resulting in short antigen presentation time of individually infected cells, thereby potentially favoring a CD4+ immune response. Most Ad vectors currently used for vaccination in clinical and pre-clinical trials are FG-Ad (reviewed for example in ) and benBecause shielding vector capsids with polymers such as PEG has been shown to evade recognition by innate immune cells, prevents vector particles from being trapped by Kupffer cells and macrophages, and protects from neutralizing antibodies ,65,66, iAdenovirus vectors belong to the most immunogenic vectors. Thus, unwanted immune reactions need to be dampened and controlled in order to enable safe Ad vector-mediated gene delivery. Two major strategies have been employed for Ad vectors: (i) genome modifications that delete all viral genes to prevent immune reactions due to leaky virus gene expression from the vectors and (ii) capsid shielding strategies based on chemical capsid modifications. While Ad vectors devoid of all viral coding sequences have shown prolonged gene expression in animal models, chemically “stealthed” vectors exhibited dampened innate immune responses. By combining both approaches with constant results from the discovery of additional rare Ad types and Ad species, it appears very likely that improved gene transfer vectors, Ad-based genetic vaccines and oncolytics will be developed in the near future. Because Ad vector-based vaccines are currently an important tool to fight the COVID-19 pandemic, it might become of even higher relevance to develop novel strategies to circumvent pre-existing antivector immunity.


Clostridium difficileinfection or MDRO acquisition.Copper-impregnated surfaces and linens have been shown to reduceinfections and multidrug-resistant organism (MDRO) acquisition in healthcaresettings. However, retrospective analyses of copper linen deployment at a 40-bedlong-term acute-care hospital demonstrated no significant reduction inincidences of healthcare facility-onset  HO-MDRO acquisitions, defined as a new finding compared to knownstatus, were detected by routine surveillance  or NHSN LabID HAIs.7 Swabs were tested formethicillin-resistant Staphylococcus aureus (MRSA) using Xpert MRSApolymerase chain reaction (PCR) assay  and forvancomycin-resistant enterococci (VRE) using Spectra chromogenic agar .9 Carbapenem-resistant Enterobacteriaceae (CRE)and extensive drug-resistant Acinetobacter baumannii (≥3antibiotic classes) were identified following selective incubation and subculture toColorex agar , followed by modifiedcarbapenem inactivation with Carba-R PCR (Cepheid) or susceptibilities,respectively.12 Facility hand hygiene complianceauditing data were also collected during a combination of 12-hour day and nightshifts.We retrospectively analyzed healthcare facility-onset CDI (HO-CDI) eventsaccording to the National Health and Safety Network (NHSN) laboratory-identified(LabID) definitionsMonthly incidence rates were analyzed over a 27-month control period prior toimplementation (July 2012 through September 2014) followed by a 27-monthintervention period (October 2014 through December 2016), and an additional 10-monthcontrol period (January 2017 through October 2017).Clostridium difficile infection testing wasperformed using real-time PCR of the tcdB gene (Cepheid) during the entire study.Universal contact precautions were employed throughout the study period with good(>92%) audited compliance that did not vary significantly over time.Rates of hygiene compliance were measured through an anonymous auditing program that did not change over thecourse of the study period. An average of 40 hand hygiene opportunities wereassessed per month. P values were obtained using 2-tailedindependent samples t tests for equality of means. In addition,interrupted time-series analyses were performed using quasi-Poisson regression, withpatient days as an offset and hand hygiene compliance as a covariate.Statistical P = .023) and total HO-MDROacquisition (P = .001). In subgroup analysis, differences in totalHO-MDRO acquisition were largely attributable to VRE  and to CRE . Comparisons of the relativelysmall number of HO-MDRO infections during each period revealed no significantdifferences (P = .313). Rates of HO-CDI, HO-MDRO acquisitions, andchanges in infection control–related practices over the study period aredepicted in Overall, 29,342 patient days were observed during the control period and25,243 patient days were observed during the intervention . Copper P = .001); however,HO-CDI was no longer significantly different among groups (P =.081). See the Poorer mean monthly hand hygiene compliance occurred during the interventionperiod (90.9% vs 95.3% for the control period). Quasi-Poisson models demonstrated asimilar effect on total HO-MDRO acquisition , fevers (47% reduction), andbacterial loads measured from sheets with copper linens.3 and studies showing clinical benefit in othersettings.6 The likelihood of the development ofmicrobial resistance to copper is likely small due to the multiple mechanisms ofcopper’s microbicidal action. In the United Kingdom, bacterial resistance hasnot been observed, despite wide deployment of copper.2 However, staff and patients were not blindedto the intervention; thus, we cannot rule out that copper linens may have resultedin degradation of other infection control practices, including hand hygiene,possibly leading to increased pathogen transmission. Similar hypotheses have beenadvanced to explain the negative correlations between glove use and hand hygienecompliance.13 Notably, nochanges to the institutional antibiotic formulary or policies occurred during thestudy period.Although our data demonstrated increased HO-CDI and HO-MDRO acquisitionduring the intervention period, there are several reasons to question a causativerole by copper, including the well-described reduced bioburden by coppermaterials7 There-fore, CDI detection may have beeninflated during the latter portions of the intervention and control periods. Linenswere laundered using standard protocols (according to manufacturer recommendations)and although the manufacturer reports the linens degrade microbes for the life ofthe product, we cannot rule out the possibility that the antimicrobial propertiesmay have waned over time.This study has several limitations. While quasi-experimental studies areoften pragmatic within the field of healthcare epidemiology, time-varying factorsand overlapping interventions  may have14 Second,point-prevalence MDRO surveys were collected on admission and weekly to determinenew colonization events. Most previous studies of copper-containing products onlyreported rates of clinical infections but not rates of new colonization events;predictably, this will underestimate the rate of new total acquisitions.Despite some limitations, our study has several notable aspects. First, theadd-and-remove treatment design strengthens the evaluation of time-varyingfactors.While we did not observe a beneficial effect, copper-impregnated surfaceswith or without copper-impregnated linens may be useful in other settings as anadjunct strategy to existing infection control practices, particularly if incidencesof HAIs and MDRO transmission are high and infection control practices do notdegrade with their use. Trials with more rigorous study designs  and relevant endpoints  are neededto define the efficacy of copper linens and/or surfaces in healthcare settings.Sup001Sup002Sup003


RHE (reversible hydrogen electrode), nearly ideal four-electron pathway and excellent methanol tolerance when compared to commercial 20% Pt/C. The utility of Fe@NCNT-rGO for removal of bisphenol A from contaminated waters is also reported.Till date, waste plastics are either down-cycled to cheap products like fibers or burnt in incinerators to generate heat. In this manuscript, we report a simple and effective technique for microwave induced transformation of waste polyethylene terephthalate (wPET) to carbon nano-tubes (CNT). Iron nano-particles dispersed on graphene substrate acted as catalyst for CNT growth whereas urea served the dual role of de-polymerisation of wPET and also as nitrogen doping agent. Application of our newly synthesized 3-D meso-porous graphene-nitrogen doped carbon nanotube- iron electrode (Fe@NCNT-rGO) as electro-catalyst for oxygen reduction reaction (ORR) shows a positive half-wave potential (E BPA’s exposure to humans is widespread especially by usage of household plastic articles like water bottles, food storage containers , feeding bottles for babies, plastic toys, etc. . BPA conIn summary, we report the microwave synthesis of iron and nitrogen co-doped carbon nanotubes (Fe@NCNT-rGO) from wPET bottles using iron nano-particles anchored on graphene as catalyst, urea as the combined source of nitrogen moieties and de-polymerization agent whereas microwave radiation was external energy source. Morphological structural analysis by SEM and HRTEM reveals that our synthesized Fe@NCNT-rGO hierarchical carbon-nanostructures are composed of millimeter long, bamboo shaped and defect rich carbon nanotubes anchored on reduced graphene oxide substrate with nano-sized iron particles embedded on walls and the interstitial voids inside the carbon nanotubes. Elemental analysis by EDS maps show excellent and uniform distribution of nitrogen and iron moieties in carbon nanotubes which on further analysis by deconvolution of high resolution XPS data shows that the nitrogen moieties are predominantly in Fe-N-C, pyridine and pyrrole structures which boosts the electro-catalytic activity, durability and methanol tolerance of wPET derived Fe@NCNT-rGO when applied as catalyst in ORR reactions. The excellent ability of Fe@NCNT-CF for removal of bisphenol-A from contaminated waters at acidic, basic and neutral pH values is also demonstrated.


Maternal antibodies (MatAbs) passively transferred across the placenta and into breast milk are critical for protection against infectious disease and immune development during the first year of life . PassiveAntibodies contain 2 domains that exert a wide range of effector functions. The antigen-binding fragment (Fab) domain binds foreign antigens and drives antibody diversity , whereasThe IgG Fc domain mediates considerable heterogeneity of its effector functions depending on the subclass and glycan profile. For example, each IgG subclass (IgG1-4) has one N-glycosylation site in each CH2 domain, an important binding site for FcγRs . InteresIgA antibodies bind their own unique Fc receptors that facilitate epithelial cell transcytosis and innate immune cell activation. dIgA antibodies are composed of 2 monomers, linked by a 15-kDa J-chain. Transport of dIgA into breast milk is dependent on C-terminal binding of the J-chain to a portion of pIgR, known as the secretory component, on the basolateral surface of MG epithelial cells . WithoutConsidering breast milk sIgA protects from pathogenic insult and facilitates maturation of the microbiota in early life , understDespite the well-documented benefits of MatAbs on early life immunity , a mountMultiple mechanisms have been proposed to describe both Fab- and Fc-mediated MatAb interference. These include live virus vaccine neutralization, inhibition of B-cell responses by epitope masking , and IgG+ T-cell responses are mostly unaffected by MatAb interference [FH) cells in the germinal center (GC) [FH cells resulted in the reduction or prevention of plasma cell and memory B-cell generation in a MatAb- and antigen dose–dependent manner [Although infant CD4rference ,39, recerference  and limit manner  and incrt manner  in a Fc t manner ,54. HowePassive transfer of MatAbs is central to pathogen protection and immune system development in early life. However, MatAb-mediated interference dampens antibody responses to vaccinations, leaving children more susceptible to infections while increasing transmission rates to unvaccinated cohorts. Recent work demonstrated that maternal IgG antibodies are differentially transferred across the placenta in an Fc glycan–dependent manner ,17. Cons


Background: Ovarian cancer (OC) is the most lethal malignancy in the female reproductive system. Growing evidences demonstrates that competing endogenous RNA (ceRNA) network play crucial roles in the occurrence and progression of tumors. Therefore, we aimed to explore and identify novel mRNA-miRNA-lncRNA ceRNA networks associated with prognosis of OC.Methods: The differentially expressed gene (DEGs) of four expression profiles datasets  were collected from Gene Expression Omnibus (GEO) database and analyzed with NetworkAnalyst. Intersection of DEGs were further employed for Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis. Protein-protein interaction (PPI) network and hub genes of DEGs were also identified. The expression levels and survival analysis of the hub genes in OC and their upstream miRNAs and lncRNAs were performed by various bioinformatics databases. More importantly, ceRNA networks were constructed based on mRNA-miRNA-lncRNA in OC.Results: A total of 178 DEGs including 38 upregulated and 140 downregulated genes from intersected DEGs of four expression profiles were identified in OC. Functional enrichment analysis suggested that the commonly DEGs were enriched in regulating enzyme inhibitor activity, glycosaminoglycan and G protein-coupled receptor binding, cell morphogenesis, and involved in pathways including metabolic process, proteoglycans in cancer. Top 10 hub genes with higher connectivity degree were selected for subsequent expression and prognosis analysis. After take expression levels and prognostic roles of hub genes and their upstream miRNAs and lncRNAs in OC into consideration, 2 mRNAs (TACC3 and CXCR4), 2 miRNAs (hsa-miR-425-5p and hsa-miR-146a-5p) and 3 lncRNAs  were significantly associated with the poor prognosis of OC. The mRNA-miRNA-lncRNA networks (TACC3-hsa-miR-425-5p-FUT8-AS1 and CXCR4-hsa-miR-146a-5p-LINC00665/LINC01535) were eventually constructed in OC based on ceRNA mechanism.Conclusion: We successfully constructed novel ceRNA network associated with the prognosis of ovarian cancer, which may provide a new strategy for early diagnosis and therapeutic intervention of OC. Ovarian cancer is one of the most common malignant tumors in gynecology worldwide Accumulating evidences have demonstrated that noncoding ribonucleic acids (ncRNA) play key roles in the occurrence and development of multiple tumors, including microRNAs (miRNAs), long noncoding RNA (lncRNAs) and circular RNA (circRNA) In this present study, we collected four original expression profiles by array  of ovarian cancer from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between ovarian cancer and normal samples were further identified with various bioinformatics approaches. Additionally, the intersection of DEGs of these four datasets were further employed for functional enrichment analysis, protein-protein interaction (PPI) network construction and top-ranked hub genes identification with online tools. After comprehensive evaluation of expression levels and prognostic roles of hub genes in ovarian cancer, 4 upregulated DEGs and 1 downregulated DEGs were eventually identified for predicting their upstream miRNAs with the miRTarBase online database. Furthermore, the upstream miRNAs which analyzed by expression levels and prognostic values were chosen for predicting the upstream lncRNAs by miRNet database. Similarly, the expression levels and prognostic values of these upstream lncRNAs were also analyzed with online database. As a consequence, an mRNA-miRNA-lncRNA regulatory network associated with the prognosis of patients with ovarian cancer was successfully constructed. This study may provide new insights into exploring and identifying novel diagnostic biomarkers or potential targets for therapeutic intervention of ovarian cancer.www.ncbi.nlm.nih.gov/geo/) online database Four expression profiles by array  of ovarian cancer were collected from the Gene Expression Omnibus (GEO)  P < 0.05 and log2 fold change (log2 FC) ≥1.NetworkAnalyst 3.0 (http://bioinformatics.psb.ugent.be/webtools/Venn/) was employed to explore the intersection genes of the four datasets by venn diagrams. To further explore the potential functions and mechanisms of above commonly DEGs in ovarian cancer, Metascape (http://metascape.org) P <0.05, a minimum count of 3, enrichment factor >1.5 were considered to be statistically significant.All the DEGs of the four datasets identified by NetworkAnalyst 3.0 were further divided into upregulated DEGs and downregulated DEGs, respectively. The commonly DEGs in all of the various datasets were subjected to subsequent analysis. An online tool-Draw Venn Diagram (http://string-db.org) To explore the hub genes correlated with ovarian cancer, PPI networks of commonly DEGs identified (upregulated and downregulated DEGs) were constructed separately with the Search Tool for the Retrieval of Interacting Genes (STRING) (http://gepia.cancer-pku.cn/) P-value < 0.05, |Log2FC| >2. Human Protein Atlas (HPA) (https://www.proteinatlas.org/) http://kmplot.com)  P-values were also explored online. The filter conditions were as follows: cancer: ovarian cancer; survival: progression free survival (PFS); follow-up threshold: 120 months, log-rank P value < 0.05 was regarded as statistically significant difference.The Gene Expression Profiling Interactive Analysis (GEPIA) (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) https://www.picb.ac.cn/dbDEMC/) miRTarBase (https://www.mirnet.ca/) miRNet (https://LncLocatorwww.csbio.sjtu.edu.cn/bioinf/lncLocator/) https://lncipedia.org) LncLocator (P<0.05 and |log2 FC| >1), as shown in the heatmaps and volcano plots, a total of 1420 DEGs (756 upregulated genes and 664 downregulated genes) were identified between ovarian cancer samples and normal samples from GSE54388 dataset , and there were no significant differences in the expression of RNASEH2A, RNASEH2B and RNASEH2C between ovarian cancer samples and normal samples  , hsa-miR-146a-5p  and hsa-miR-150-5p  (the upstream miRNA of CXCR4) were correlated with poor overall survival (OS) of patients with ovarian cancer , and a total of 20 upstream miRNAs were eventually identified to be correlated with 3 upregulated hub genes  according to the powerful evidence , both LINC00665  and LINC01535  were positively correlated with CXCR4 , an online database miRNet was used to predict upstream lncRNA. The research showed that a total of 139 lncRNAs were detected in the database for three downregulated miRNAs   in ovarian cancer and normal samples that were shared by four expression profiles datasets . Then GO and KEGG enrichment analysis of 178 intersected DEGs were explored with Metascape, the GO enrichment analysis showed that these DEGs were mainly located in extracellular matrix and plasma membrane, and involved in biological functions such as regulating enzyme inhibitor activity, glycosaminoglycan and G protein-coupled receptor binding. KEGG pathway enrichment analysis suggested that DEGs were significantly enriched in signaling pathways such as energy metabolism, proteoglycans in cancer. The above biological behavior and signaling pathways played crucial role in the progression and prognosis of ovarian cancer In order to identify the hub genes, two separate PPI networks were investigated with STRING database, and the hub genes were filtered out based on the connectivity degree calculated by Cytoscape software. The top ten upregulated DEGs and top ten downregulated DEGs were further employed for expression validation and survival evaluation. Only high expression of UBE2C, TK1, TACC3 and CXCR4 and low expression of MAOB were proved to be associated with poor prognosis of patients with ovarian cancer, suggesting the five genes were considered to be the key genes in ovarian cancer. Numerous studies have shown that UBE2C overexpression was correlated with poor prognosis and regulated the malignant biological process of various tumors, including endometrial cancer, breast cancer and ovarian cancer  et al. demonstrated that miR-146a-5p could function as a tumor-suppressive miRNA in lung cancer through targeting EGFR and regulating various metabolic and signaling pathways We further explored the upstream miRNAs associated with the five hub genes by the online database-miRTarBase based on the potential ceRNA hypothesis. After taking the expression levels and survival exploration into consideration, only three upstream miRNAs were considered as key miRNAs , all of which were downregulated and associated with poor prognosis in ovarian cancer. Many studies have discovered that these miRNAs functioned as oncogene or tumor suppressor in the tumorigenesis and progression of different tumors. Researchers showed that miR-425-5p inhibited the expression of MALAT1 and TUG1 through inactivating the Wnt/β-catenin signaling pathway and further suppressed the progression of osteosarcoma Numerous studies have shown that lncRNAs could function as miRNAs sponge to regulate downstream genes based on the ceRNA mechanism, with the upstream lncRNAs binding to the miRNAs further identified with miRNet. After comprehensive evaluation of the expression levels, prognosis values and cellular locations, only high expression of three lncRNAs  were significantly associated with poor prognosis of ovarian cancer, which were finally proved to be key upstream lncRNAs of the ceRNA network, suggesting that these lncRNAs play crucial in the occurrence and progression of ovarian cancer. Studies focused on FUT8-AS1 were extremely limited. Some studies showed that LINC00665 contributed to the progression and biological behaviors via regulating downstream miRNAs and targeted genes in many malignancies, such as lung cancer, hepatocellular carcinoma, breast cancer in vivo and vitro remains to be carried out to detect the functional mechanisms of ceRNA networks in the future.In consequence, on the basis of ceRNA mechanism, we successfully constructed novel mRNA-miRNA-lncRNA regulatory networks (TACC3-hsa-miR-425-5p-FUT8-AS1 and CXCR4-hsa-miR-146a-5p-LINC00665/LINC01535) associated with prognosis of ovarian cancer. Although existing research may not be perfect, it is valuable to make a conclusion that the ceRNA networks observed in our study could exert profound influences on the predictive accuracy for ovarian cancer, and additional experimental exploration In summary, with a series of integrated bioinformatics databases, we systematically explored and identified DEGs, miRNAs and lncRNAs associated with the prognosis of ovarian cancer. Based on the ceRNA hypothesis, novel mRNA-miRNA-lncRNA regulatory networks (TACC3-hsa-miR-425-5p-FUT8-AS1 and CXCR4-hsa-miR-146a-5p-LINC00665/LINC01535) in ovarian cancer were successfully constructed. The ceRNA networks observed in our study may provide new insights into exploring potential biomarkers for early diagnosis and targeted therapy of ovarian cancer, and further experimental exploration remains to be carried out in the future.Supplementary figures and tables.Click here for additional data file.


Understanding the prevalence and symptoms of electronic cigarette (e-cigarette) dependence and its association with future e-cigarette use among youth may help to guide pediatric clinical services and health policy.To examine the cross-sectional prevalence and symptom presentation of e-cigarette dependence and to determine whether e-cigarette dependence is associated with subsequent e-cigarette use patterns 6 months later among youth with baseline past-year e-cigarette use.This prospective cohort study used baseline and 6-month follow-up surveys among students in the 12th grade during the 2016 to 2017 school year who reported any past-year e-cigarette use. Surveys were conducted on site in 10 high schools in Los Angeles, California. Data were analyzed from March 2019 to December 2019.Self-reported checklist of 10 tobacco product dependence symptoms reflecting loss of control over use, craving or urge, or withdrawal symptoms while abstinent, completed at baseline and administered separately for e-cigarettes and combustible cigarettes. Reporting 1 or more symptoms indicated a positive screen for dependence. Vaping continuation, defined as any past 6-month vaping, and past 30-day nicotine vaping days , sessions per vaping day , and puffs per session  at 6-month follow-up were assessed.P < .001) and among youth who used e-cigarettes with nicotine than among those who used e-cigarettes without nicotine . After adjusting for baseline vaping and e-cigarette dependence risk propensity scores, baseline e-cigarette dependence symptom status was associated with vaping continuation  and past 30-day number of nicotine vaping days , vaping sessions per day , and puffs per session  at 6-month follow-up.Among 3168 twelfth-grade students who completed the baseline survey, 444 youths  reported past-year e-cigarette use. Among these, 52 youths (11.7%) reported at least 1 e-cigarette dependence symptom. Among youth who reported past-year dual e-cigarette and combustible cigarette use, combustible cigarette dependence, reported by 43 youths (29.7%), was more prevalent than e-cigarette dependence, which was reported by 24 youths (16.4%). The most common symptoms, craving, urge, and need to use, and least common symptoms, abstinence-related concentration and emotional problems, were similar in both combustible and e-cigarette dependence. The prevalence of e-cigarette dependence was higher among youth who reported vaping in the past month than among those who did not (41 youths [17.6%] vs 11 youths [5.2%]; These findings suggest that e-cigarette dependence may be an expression of tobacco use disorder associated with future use persistence and escalation among youth. Electronic cigarette dependence may be a behavioral health consequence of adolescent vaping that warrants consideration in pediatric patient care and public health policy. Specifically, we examined similarities and differences in prevalence and symptom expression between e-cigarette dependence and combustible cigarette dependence among youth, the prevalence of e-cigarette dependence symptoms stratified by subgroups presumed to be at elevated risk , and associations of baseline e-cigarette dependence with subsequent vaping continuation, frequency, and intensity patterns 6 months later. This study aims to provide foundational descriptive evidence on the expression and progression of e-cigarette dependence as a potential presentation of tobacco use disorder among youth. Such evidence could help establish whether e-cigarette dependence is a health outcome of e-cigarette use that should be considered in federal regulatory decisions that weigh the relative harms and benefits of e-cigarettes.10 a prospective cohort study of behavioral health. All ninth-grade students in 10 participating public high schools in Los Angeles County, California, in 2013 were eligible. Semiannual in-classroom assessments were administered from 2013 to 2017. Students who were not in class on survey days completed abbreviated surveys, which excluded dependence measures. Data on e-cigarette dependence were first collected in the fall 12th-grade survey in 2016, which was considered baseline; follow-up data were collected in the spring of 12th grade, approximately 6 months later, in 2017.Data were drawn from the Happiness & Health Study,The University of Southern California Institutional Review Board approved this study. Participants provided active assent and a parent or legal guardian provided written or verbal informed consent prior to study enrollment. This study is reported according to Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.11 which was originally developed to measure combustible cigarette dependence and has demonstrated adequate psychometric properties. Students reported whether they had ever experienced each of 10 dependence symptoms for e-cigarettes and combustible cigarettes separately. Electronic cigarette and combustible cigarette dependence items were identically worded, except for substitution of e-cigarette and vaping terms for cigarette and smoking. Endorsing 1 or more symptoms indicated that the participant screened positive for dependence.11 Students presenting 2 or more or 3 or more total symptoms of e-cigarette or combustible cigarette dependence symptoms were also classified.At baseline, tobacco product dependence symptoms were measured using the Hooked on Nicotine Checklist,12 Affirmative responses to either or both questions over the past year were used to classify past-year any e-cigarette use (yes or no), which was necessary for sample inclusion. Past-month vaping (yes or no) and past-year vaping of e-cigarettes that contained nicotine (yes or no) were also assessed. We assessed baseline past-year combustible cigarette smoking (yes or no) using items from previously derived epidemiologic surveys.12At baseline, use of e-cigarettes with nicotine and e-cigarettes without nicotine or cannabis oil were measured as yes-or-no questions.13Vaping continuation was operationalized as any past 6-month use of e-cigarettes (with or without nicotine) at follow-up. Additional survey items assessed past 30-day nicotine vaping frequency  and intensity, including number of nicotine vaping sessions per vaping day  and puffs per nicotine vaping session  .13We collected several additional measures to describe the sample and assess risk factors for e-cigarette dependence to be included in an e-cigarette propensity score covariate. These variables may have also influenced e-cigarette use progression patterns and therefore confounded associations between e-cigarette dependence and future use.14 Participants reported ever use of alcohol, combustible cannabis, vaporized cannabis, or other drugs (yes or no) using questions derived from previously validated items.15Participants reported age (in years) at e-cigarette use initiation. The following tobacco product use characteristics were measured: past 30-day number of days smoked cigarettes , cigarettes smoked per day on smoking days , and ever (lifetime) use of cigars, hookah, or smokeless tobacco (yes or no).19 Additionally, age, sex , self-reported race/ethnicity  and highest level of parental education  were surveyed, per past work.10To assess a potential association of mental health with e-cigarette dependence and future use, we measured symptoms of major depressive disorder, generalized anxiety disorder, social phobia, panic disorder, obsessive-compulsive disorder, manic symptoms, attention-deficit/hyperactivity disorder, and conduct disorder symptoms .16–19 Ad2 tests. For descriptive data, prevalence of meeting 2 or more (yes or no) or 3 or more (yes or no) symptom thresholds were also reported. The prospective association of baseline e-cigarette dependence symptom status with nicotine vaping status at follow-up was tested using binary logistic regression. Prospective associations of baseline e-cigarette dependence symptom status with nicotine vaping frequency and intensity at follow-up were tested using negative binomial regression models. All regressions included baseline status of each respective outcome as a covariate. Adjusted analyses included an e-cigarette dependence propensity score covariate calculated from a prediction model of e-cigarette dependence status regressed on 25 baseline variables, as detailed in the 20 were applied to control studywise false-discovery rate at 0.05, based on 2-tailed corrected P values. Data were analyzed from March 2019 to December 2019.After descriptive analyses, we reported prevalence of e-cigarette dependence (report of ≥1 symptom) and prevalence of the 10 specific dependence symptoms among all past-year e-cigarette users and by combustible cigarette use status. Among past-year e-cigarette and combustible cigarette dual users, McNemar tests for within-participant comparisons were used to conduct cross–tobacco product comparisons of e-cigarette dependence, combustible cigarette dependence, and individual symptoms. Prevalence of e-cigarette dependence symptoms was compared across binary subclassifications of past-year vaping of e-cigarettes with (vs without) nicotine, past-month vaping (yes or no), and past-year combustible cigarette and e-cigarette dual use (vs e-cigarette use only) using χAmong 4100 eligible students enrolled in ninth grade in the Fall 2013 semester, 3396 students enrolled in the cohort, and 3168 students completed the fall 12th-grade baseline survey in 2016. Of these students, 460 reported past-year use of e-cigarettes with or without nicotine and had baseline data on key measures. Sixteen students lacked follow-up data and were excluded from the analytic sample . The finP < .001) and intensity , were more likely to report use of other tobacco products , smoke more combustible cigarettes per day , vape cannabis , and experience higher mean (SD) symptom levels of major depressive disorder , mania , generalized anxiety disorder , panic disorder , and conduct problems  (P < .001) and reported higher mean (SD) past-month number of nicotine vaping days , nicotine vaping sessions , and puffs per nicotine session   age of vaping initiation was 15.31 (1.58) years . Of past = .008) . Descrip < .001) .The profile of prevalence rates for each of the 10 different tobacco product dependence symptoms for e-cigarettes and combustible cigarettes are reported in P < .001) and were more likely to meet 2 or more  or 3 or more  dependence symptom thresholds for combustible cigarette dependence than for e-cigarette dependence (P = .002), feeling the need to use , and strong need or urge to use when abstinent   . For desP = .04), users of e-cigarettes with nicotine compared with those who used e-cigarettes without nicotine , and youth who used e-cigarettes in the past month compared with those who did not  (P = .004) or 3 or more symptoms ; youth with vaping use in the past 30 days, compared with youth without past 30 days vaping use, were more likely to report 2 or more symptoms ; and youth who used e-cigarettes with nicotine, compared with those who use e-cigarettes without nicotine, were more likely to report 2 or more symptoms  or 3 or more symptoms . Among 146 past-year cigarette and e-cigarette dual users, 145 youths had age of tobacco product use onset data. Among these 145 dual-users, 77 youths (53.1%) reported earlier e-cigarette use onset than cigarette use onset; 32 youths (22.1%) reported same age at onset for both products, and 36 youths (24.8%) reported earlier combustible cigarette use onset than e-cigarette use onset. No association was found of combustible and e-cigarette use onset sequence with e-cigarette dependence symptom status.The prevalence of e-cigarette dependence symptoms was higher in baseline past-year dual users of combustible and e-cigarettes compared with e-cigarette–only users  . The preP = .005) and more frequent and intense past-month nicotine vaping, including more nicotine vaping days , sessions per vaping day , and puffs per session  6 months later in models adjusted for baseline vaping characteristics (P = .02) and more nicotine vaping days , sessions , and puffs per session   .Sensitivity analyses of cross-tobacco product comparisons of dependence prevalence, severity, and symptom patterns that adjusted for age at onset and past 30-day use frequency of the 2 respective products yielded results that were similar to the primary results . AdditioFor descriptive purposes to examine whether results generalized across sex and nonnicotine substance use, tests of differences in cross-sectional and prospective analyses stratified by sex and number of nonnicotine substances used are reported in P = .01); this association was amplified by the number of other substances used  (P < .001) and more cigarettes smoked per day   . Additio < .001) .8 To our knowledge, this is also the first prospective longitudinal investigation of the association of e-cigarette dependence symptoms with subsequent vaping patterns. Our results suggest that e-cigarette dependence symptoms may be associated with future vaping patterns.This cohort study provides some of the most detailed evidence to date on the prevalence and symptom presentation of e-cigarette dependence and its association with future e-cigarette use among youth, to our knowledge. Previous studies have provided less comprehensive characterizations of e-cigarette dependence, without comparisons with combustible cigarette dependence.7 primarily characterized by cravings and a perceived need to vape. Although prevalence and severity of dependence symptoms were approximately 2-fold as large for combustible cigarettes as e-cigarettes in users of both products, the most common  and least common  symptoms were similar across the 2 products, as were the qualitative profiles across all 10 symptoms. Importantly, the cross-product comparisons were conducted within-persons among dual users, eliminating confounding differences between smokers and vapers. Unlike combustible cigarettes, e-cigarettes and e-liquids vary in nicotine content and delivery.3 Few youth in this study likely used the now-popular pod mod–style e-cigarette products  that deliver large amounts of nicotine efficiently.21 Considering the elevated dependence symptoms reported among youth who vaped nicotine in this study despite low probability of pod mod use, these results suggest that e-cigarette dependence may be of notable clinical and public health significance.In this study, the prevalence of e-cigarette dependence symptoms was relatively low (reported by 11.7% of past-year e-cigarette users and 17.6% of past-month users) and, consistent with prior research,22 found that dual users had higher levels of nicotine biomarkers than users of e-cigarettes only. To our knowledge, this is among the first investigations to find that e-cigarette dependence symptoms were experienced even among youth who reported using only e-cigarettes without nicotine—a sizeable proportion of youth e-cigarette users.5 Nicotine-containing products may have been mislabeled, or youth with histories of nicotine vaping in early adolescence who later switched to only nicotine-free vaping over the past year may have experienced cravings triggered by cues associated with the act of vaping.Electronic cigarette dependence symptoms were elevated in certain subgroups expected to be at higher risk, including youth who vaped recently, used e-cigarettes that contained nicotine, or used both e-cigarettes and combustible cigarettes. A 2018 study23 that suggested that vaping continuation, with escalation of use frequency and dependence symptoms, is common. Our study further suggests that youth with dependence symptoms are at elevated risk for continuation and escalation. Symptoms of e-cigarette dependence  may directly increase motivation to use, and increased use may recapitulate a cycle of worsening dependence. Although our observational study design precludes such causal inferences, findings suggest that e-cigarette dependence may be associated with subsequent vaping patterns even after adjusting for dependence propensity defined by numerous potentially confounding influences.Youth who used e-cigarettes and reported at least 1 e-cigarette dependence symptom were more likely to continue vaping and to vape more frequently and intensely 6 months later than their peers who did not report any dependence symptoms. This finding is consistent with a 2019 study2 and our findings suggest that dependence symptoms associated with nicotine exposure via e-cigarettes are associated with greater risk for escalation of vaping behavior. The development of dependence in youth is an important public health consequence that should not be overlooked.In clinical settings, e-cigarette dependence symptom screening questions may identify youth at risk for vaping progression who may benefit from intervention. In regulatory decision-making, dependence is a potential health consequence of e-cigarette use that should be considered. Adolescents are particularly vulnerable to nicotine exposure,24 Third, all measures were self-reported and did not include clinical diagnosis of nicotine dependence. While the measure of dependence was selected for its presumed applicability to both tobacco products and its ability to capture key features of the dependence syndrome ,25 other measures of e-cigarette dependence that are correlated with nicotine exposure merit inclusion in future research to address varying aspects of the dependence syndrome.6 Fourth, the follow-up period was limited to 6 months, leaving unclear the long-term association of e-cigarette dependence with future use.This study has some limitations. First, all participants were recruited from high schools in Los Angeles, California; therefore, extension to different regions would be informative. Second, data were collected in the 2016 to 2017 school year, before high-nicotine e-cigarettes  became popular among youth.In this cohort of youth who used e-cigarettes in the past year, e-cigarette dependence symptoms were similar in nature to combustible cigarette dependence but less prevalent and severe. Symptoms were elevated in subgroups presumed to be at increased risk, including youth who recently vaped, vaped nicotine, and also smoked, and were associated with increased risk of future vaping. These findings suggest that youth who report vaping may benefit from screening for e-cigarette dependence symptoms, such as cravings and perceived need to vape—symptoms that may be associated with risk of future vaping persistence and progression. Dependence is an important health effect of e-cigarette use that should be considered in federal regulatory decisions that weigh the relative harms and benefits of e-cigarettes.SM


Heterogeneity in cell populations poses a significant challenge for understanding complex cell biological processes. The analysis of cells at the single-cell level, especially single-cell RNA sequencing (scRNA-seq), has made it possible to comprehensively dissect cellular heterogeneity and access unobtainable biological information from bulk analysis. Recent efforts have combined scRNA-seq profiles with genomic or proteomic data, and show added value in describing complex cellular heterogeneity than transcriptome measurements alone. With the rising demand for scRNA-seq for biomedical and clinical applications, there is a strong need for a timely and comprehensive review on the scRNA-seq technologies and their potential biomedical applications. In this review, we first discuss the latest state of development by detailing each scRNA-seq technology, including both conventional and microfluidic technologies. We then summarize their advantages and limitations along with their biomedical applications. The efforts of integrating the transcriptome profile with highly multiplexed proteomic and genomic data are thoroughly reviewed with results showing the integrated data being more informative than transcriptome data alone. Lastly, the latest progress toward commercialization, the remaining challenges, and future perspectives on the development of scRNA-seq technologies are briefly discussed. Single cells are the basic structural, biological, and functional unit of organisms ,2. TradiConventional scRNA-seq technologies initially involved manual isolation of cells using mouth pipettes , micropiReview articles on the introduction of scRNA-seq technologies are readily available ,28,29,30Numerous conventional methods exist to isolate single cells for scRNA-seq , and theSwitching Mechanism at 5′ End of RNA Template (Smart-seq) has been introduced to address the limitations of existing technologies such as limited throughput and read coverage across transcripts . Briefly2 significantly improves the cDNA yield. Adding deoxyribonucleoside triphosphates (dNTP) before RNA denaturation increases the pre-amplified cDNA average length possibly due to the stabilization of RNA-oligo(dT) primer hybridization. Utilizing KAPA HiFi Hotstart DNA polymerase during preamplification provides a good amplification efficacy and greater cDNA length . Hence, in addition to stability enhancement, Smart-seq 2 has improved both the length and yield of cDNA libraries generated from single cells as well as the coverage, bias, and accuracy of detection. Time-consuming cell isolation processes using the micropipette and a low number of cells are known as the limitations of Smart-seq.Smart-seq 2 has been introduced to address the challenges of low-yield, coverage, and poor sensitivity in smart-seq 1 . It imprSingle Cell RNA Barcoding and Sequencing (SCRB-seq) is introduced to profile mRNAs from a large number of cells using a minimal amount of reagents and sequencing reads per cell . This meC. elegans embryos and demonstrated the possibility of distinguishing cell types even in the presence of only subtle biological differences.Cell Expression by Linear Amplification and Sequencing (CEL-seq) mainly relies on linear amplification of CDNA by in vitro transcription. This protocol allows pooling of barcoded samples. Therefore, this dramatically improves the amplification efficiency . Single Essentially, CEL-seq, which involves 3′ end cDNA coverage, gives a more sensitive and reproducible outcome than full length cDNA coverage. Compared to Smart-seq, CEL-seq adds the barcode at an earlier stage, which specifically identifies each single cell. Hence, this reduces the hands-on work. However, this technology can only be used for 3′-end sequencing, which provides less transcriptomic information than full length transcript sequencing.CEL-seq 2, which is a modified method of CEL-Seq, adds a 5-base pair UMI upstream of the barcode to identify PCR duplicates in scRNA-seq , which sMassively Parallel RNA Single-Cell Sequencing (MARS-seq) was introduced following a CEL-seq protocol as an automated workflow to analyze transcriptomes of thousands of single cells while minimizing amplification biases and labeling errors . Single MARS-seq 2 is a modified method of MARS-seq that incorporates indexed FACS sorting to enrich cells of interest. This key feature is vital for identification of rare cell subpopulations via scRNA-seq , such asQuartz-seq is a simple and highly-quantitative scRNA-seq approach based on homopolymer tailing-based PCR . BesidesA key feature of Quartz-seq 2 is its high capability of analyzing single cell transcriptome with a limited number of sequence reads . Single 15N6). With the use of ExoSAP-IT, the unreacted primers are removed. The poly(A) tail is added to the 3′ end of the first-stand cDNA using dATP doped with 1% ddATP. Poly(T) primers with a different anchor sequence (AnchorY-T24) are then used to synthesize second-strand cDNA, which is followed by PCR using AnchorY-T24 and AnchorX-T15 primers prior to deep sequencing. This technology was used to study the roles of circular RNAs in mammalian early embryonic development and demonstrated its capability of detecting both circular and polyadenylated RNAs in the mouse preimplantation embryo . A. A19]. AA microfluidic hydrodynamic trap array was developed to enable off-chip transcriptomic sequencing of single cells after multi-generational lineage tracking under the control of culture conditions . The arrMicrofluidic diffusion-based RNA-seq (MID-RNA-seq) was introduced for performing scRNA-seq with a diffusion-based reagent swapping scheme B 51]. T. T51]. TThe single-cell suspension was introduced into the device via the sample inlet. Through operating valves, single cells were trapped in chambers. The chambers were rinsed with phosphate buffered saline (PBS) and then with lysis buffer. The lysis buffer was diffused, which moved the trapped cells into the reaction chamber for the lysis reaction. RT and PCR were performed based on the similar diffusion processes, and the chambers were washed with elution buffer to collect cDNA for library preparation and sequencing. However, unlike most conventional technologies, it enables automated processing and multiplexing. The result obtained by this technology was comparable to that of the conventional scRNA-seq technologies. Like the above-mentioned valve-based microfluidic technologies, this technology requires complex device fabrication processes.Hydrodynamic scRNA-seq (Hydro-seq) was introduced to address the challenges of low-throughput and poor cell capture efficiency exhibited by the existing scRNA-seq technologies C 52]. H. H52]. HDroplet microfluidic technologies were introduced and typically involve the steps of encapsulating single cells in droplets in an inert carrier oil. The use of carrier oil allows the droplets to be moved, merged, split, heated, or stored. These technologies are fast and have high-throughput. Additionally, compartmentalization of up to thousands of cells can be performed in seconds, which is ideal for large-scale applications.The first droplet-based microfluidic technology developed for scRNA-seq was High-Throughput Single-Cell Labeling (Hi-SCL) . This teSimilar to Hi-SCL, reactions of indexing droplets (In-Drop) are carried out in droplets, which allows the indexing of thousands of cells for RNA-seq A 24]. T. T24]. TDrop-seq shares some similarities with In-Drop, which also involves a droplet that encapsulates each single cell with a barcode B 23]. U. U23]. UThe cells are lysed after being isolated in droplets, and the poly(A) tail of mRNA molecules are hybridized to the oligo(dT) tail on the beads, which forms Single-cell Transcriptomes Attached to MicroParticles (STAMPs). The droplets are then broken and subjected to RT in a single tube. PCR and fragmentation are subsequently performed using the Nextera XT kit. This technology enables high throughput analysis  in a cheaper and faster way when compared to the previously mentioned technologies. The cell capture efficiency is ~12.8%, which is higher than that of In-Drop. However, similar to In-Drop, only the 3′ most terminal fragments can be used for sequencing.As mentioned, droplet-based technologies have enabled rapid processing thousands of cells simultaneously, but current technologies has medium throughput, which requires the utilization of custom reagents C 54]. T. T54]. TGel bead in Emulsion (GEM) is the core of this technology that is integrated into a droplet-based microfluidic device that efficiently captures approximately 50% of cells loaded. Each gel bead is functionalized with a barcoded oligonucleotide that consists of illumina adapters, 10x barcodes, UMI, and oligo(dT) that prime RT of polyadenylated RNAs. Cell lysis starts immediately after cell encapsulation. Then gel beads dissolve and release the oligonucleotides for RT. RT is performed inside droplets and transferred to a tube where amplification of cDNAs occurs. The amplicons that have Illumina adapters and sample indices allow pooling and sequencing of multiple libraries simultaneously. The sequencing data can be rapidly processed using an analysis pipeline. This technology was utilized to profile 68k peripheral blood mononuclear cells (PBMCs), which demonstrates its ability to dissect large immune populations . SimilarMultiplexing using lipid-tagged indices for single-cell RNA sequencing (MULTI-seq) allows localization of the DNA barcode to a single cell within an emulsion droplet for scRNA-seq . Single More recently, Nanowell technologies were developed, which offer several benefits over droplet-based devices for single cell analysis including a short cell-loading period, low reagent and sample volumes, and enhanced compatibility with optical imaging . The capGene expression cytometry (Cytoseq) enables massively parallel, stochastic barcoding of RNA from single cells in bead-containing Nanowells, which allows for simultaneous gene expression profiling of thousands of single cells . Cells aTo perform more comprehensive transcriptomic analysis of cell populations, microwell-seq utilized agarose-constructed Nanowells to profile thousands of single cells A 26]. T. T26]. TMore recently, Seq-well was developed, which leveraged the advantage of Nanowell arrays to achieve massively parallel scRNA-seq B 25]. T. T25]. TMycobacterium tuberculosis. It is able to process small amounts of samples and is compatible with on-array imaging cytometry for identifying cell phenotypes from complex biological samples.The array’s three-layer functionalized surface consists of an amino-silane base crosslinked to a bifunctional poly(glutamate)–chitosan through a p-phenylene diisothiocyanate intermediate. Chitosan on the array’s top surface allows efficient sealing to the membrane while poly(glutamate) on the array’s inner surface inhibits nonspecific mRNA binding. Following cell lysis, the arrays are transferred to new dishes and inverted so that the PDMS surfaces is in contact with the dishes to allow beads to be collected after centrifugation. The library preparation is performed based on a Drop-seq protocol. This technology was used to sequence thousands of primary human macrophages exposed to Single cell optical phenotyping and expression sequencing (SCOPE-seq) was developed, which enables identification of each individual cell based on their phenotypic profile and link phenotypic information to scRNA-seq data C 57]. O. O57]. OTo further simplify user steps, single-cell freeze-thaw lysis directly toward 3′ mRNA sequencing (scFTD-seq) of a microchip was developed to perform scRNA-seq using cells that have undergone freeze-thaw lysis . SimilarAs previously mentioned, the high throughput scRNA-seq approaches have proven to be valuable for describing complex cell populations. However, the existing approaches do not provide proteomic information such as expression of cell surface proteins. Paired RNA and protein analyses reveal the information of genetic expression and the cell phenotype by providing a more detailed cell subpopulation classification . Some spCellular indexing of transcriptomes and epitopes by sequencing (Cite-seq) was introduced to simultaneously analyze transcriptomes alongside cell surface protein abundance at the single cell level  Figure A. The meSimilar to Cite-seq, RNA expression and protein sequencing (Reap-seq) enables parallel quantification of mRNAs and protein at a single cell resolution  Figure B. It enaBoth Cite-seq and Reap-seq used a similar approach, generating a protein readout to be sequenced alongside a single cell transcriptome. Unlike the detection of cell surface protein by flow cytometry, these technologies use DNA barcodes to label surface antibodies. DNA barcoding enables the combination of various antibodies by targeting distinct epitopes in a single assay that could be resolved through sequencing. This study has enabled a more comprehensive analysis of single cells, which allows fine discrimination between cell types. This was unobtainable with mRNA data alone. It could also be potentially used in exploring post-transcriptional gene regulation. To date, extending the technology to detect intracellular proteins alongside transcriptomes remains a challenge, particularly due to the requirement of cell permeabilization, which may cause RNA degradation.To detect both transcriptome and secretome from the same cells, one study developed a PDMS-Nanowell technology to study cytokine secretion, which is followed by downstream transcriptomic analysis . The PDMThe existing scRNA-seq approaches do not provide genotypic information such as gDNA copy number, chromosome structure, and number. In fact, paired transcriptomic and DNA analysis reveals the effects of changes in the DNA structure and copy number on gene expression. This would directly link the cell genotype (wild-type or mutant) to its functional states (cell types and states) . For insTo integrate DNA analysis and the transcriptome measurement into an efficient, single cell readout, several technologies have been developed, which are described below. These technologies are summarized in gDNA-mRNA sequencing (DR-Seq) permits simultaneous transcriptomic and DNA analysis of the same single cell using a quasilinear amplification strategy without physically separating mRNA from gDNA A 65]. A. A65]. ASimilar to DR-seq, genome and transcriptome sequencing (G and T-seq) enables simultaneous single cell DNA and transcriptomic analysis to evaluate the effects of genetic variation on gene expression ,69. A siIn addition to the above-mentioned technologies, simultaneous isolation and parallel sequencing of gDNA and total RNA (SIDR-seq) was also introduced to simultaneous sequence gDNA and RNA B 67]. B. B67]. BConcurrent sequencing of the transcriptome and targeted genomic regions (CORTAD-seq) allows simultaneous evaluation of the transcriptome and genome within the same single cell in an automated, high-throughput microfluidic platform (Fluidigm C1) . The FluBesides genome and transcriptome sequencing, single-cell triple omics sequencing (scTrio-seq) also analyzes epigenome or DNA methylome from the same cell C 68]. I. I68]. ITo date, there are a few commercial scRNA-seq sample preparation technologies, including droplet-based and Nanowell-based technologies . DropletTo address these drawbacks, some producers utilize Nanowell-based technologies such as a BD Rhapsody single cell analysis system (BD) D 73], I, I73], ITo easily isolate single cells of interest for downstream processes, the puncher platform (Vycap) was introduced . The maiIn summary, recent development of scRNA-seq technologies such as valve-based, droplet-based, and Nanowell-based scRNA-seq technologies have enabled highly-sensitive, accurate, and high throughput transcriptomic analysis of individual cells. Combining single-cell transcriptomic data with proteomic data enables understanding of how transcriptomic cellular states translate into functional phenotypic states. In addition, it may reveal phenotypic cell states not obtainable from scRNA-seq data alone due to the fact that heterogeneity may present in both post-transcriptional and post-translational processes. Integrating transcriptomic data with genomic data allows detection of a gene mutation alongside transcriptomes. This integration could gain insights into cancer evolution and help address medical challenges not obtainable from RNA sequencing alone, such as dissecting complex cellular immune responses or determining intra-tumor heterogeneity.One area of future research will be improving efficiency of current technologies including sensitivity, multiplexing, throughput, and cost-effectiveness. Specifically, given the requirement of analyzing more cells, the cost of consumables, labor, and sequencing remains high, which poses a barrier for widespread implementation of most scRNA-seq technologies. Therefore, reducing total cost might be a crucial step. Developing high-throughput technologies  with a high-sample processing capacity and low reaction volume along with easily fabricated and operated processes could achieve efficient scRNA-seq at a minimal cost. The automation of devices would reduce assay time and minimize human intervention . This wiFuture progress could explore multi-omics to gain a more comprehensive outlook of cell types and cell states. For example, DNA sequencing and DNA methylation could be detected alongside transcriptomes using long-read sequencing to obtain more cellular information . MeanwhiBesides technological improvements, computational analytical methods are also improving. The sources of review articles on scRNA-seq bioinformatics are extensive and readily available ,81,82. O


Cognitive impairments, including delirium, are common after coronary artery bypass grafting (CABG). Improving cognition pre- and post-operatively using computerised cognitive training (CCT) may be an effective approach to improve cognitive outcomes in CABG patients.Investigate the effect of remotely supervised CCT on cognitive outcomes, including delirium, in older adults undergoing CABG surgery.Thirty-six participants, were analysed in a single-blinded randomised controlled trial . CCT was completed by the intervention group pre-operatively  and post-operatively, beginning 1-month post-CABG (3 x 45–60-minute sessions/week for 12-weeks), while the control group maintained usual care plus weekly phone calls. Cognitive assessments were conducted pre- and post-operatively at multiple follow-ups . Post-operative delirium incidence was assessed daily until discharge. Cognitive change data were calculated at each follow-up for each cognitive test .Adherence to the CCT intervention (completion of three pre-operative or 66% of post-operative sessions) was achieved in 68% of pre-CABG and 59% of post-CABG participants. There were no statistically significant effects of CCT on any cognitive outcome, including delirium incidence.Adherence to the CCT program was comparatively higher than previous feasibility studies, possibly due to the level of supervision and support provided . Implementing CCT interventions both pre- and post-operatively is feasible in those undergoing CABG. No statistically significant benefits from the CCT interventions were identified for delirium or cognitive function post-CABG, likely due to the sample size available (study recruitment greatly impacted by COVID-19). It also may be the case that multimodal intervention would be more effective. Improving cognition through computerised cognitive training (CCT) is an attractive target for pre-operative intervention as it is a scalable, adaptable, relatively inexpensive and safe approach, known to be effective following relatively short interventions .15Potentially modifiable risk factors for post-CABG delirium and cognitive decline exist including pre-operative depression, diabetes, hypertension and cognitive impairment.18 and attention,18 as well as reductions in depression and anxiety.17 In pre-operative settings (non-cardiac patients), cognitive training has significantly reduced cognitive decline (1-week post-operatively)19 and delirium incidence20 when compared with controls. Other studies reported no significant benefits in cardiac and non-cardiac patients,22 albeit with small sample sizes and sub-optimal intervention designs .23 Beneficial effects may be plausible with a more optimally designed CCT program including supervision and less regular and longer sessions,23 which have been implemented in the current study.Post-operative cognitive training in cardiac and noncardiac patients has elicited improvements in trained domains of memory (including working memory)The current paper investigates the effect of remotely supervised CCT on cognitive outcomes following CABG surgery in older adults. Specifically, our primary aims are to determine the effect of 1) pre-CABG CCT on cognitive function at discharge and the incidence and severity of post-operative delirium and 2) pre- and post-CABG CCT on cognitive function at 4, and 6- months.24 In brief, the study was a single-blinded, randomised controlled trial with longitudinal follow-up comparing pre- and post-operative CCT (intervention group) with usual care (control group). Persons scheduled to undergo CABG or CABG plus a concomitant surgery, with at least one-week lead-up time before surgery, who were aged ≥65 years, spoke English proficiently and lived within a 1-hour drive from the Adelaide  central business district were approached. As detailed in the published protocol paper,24 the target sample size was n=120. However, recruitment was severely impacted due to COVID-19 restrictions beginning in 2020, with the cancellation of elective procedures within the South Australian hospital system, resulting in scheduled surgeries of metropolitan patients being completed in private hospitals .A detailed description of the study protocol, including the timing of all study measurements, has been published.24) and consented (note participants received a group-specific information sheet and consent form), cognitive function and depression were measured before surgery (baseline), at discharge, as well as post-operatively at 4- (end of intervention) and 6-months. The presence of delirium was measured post-operatively; in-hospital (daily) and at discharge  interview26 (which informed the scoring of the CAM27) were conducted daily when participants were in the ICU and ward respectively. In addition, chart reviews were utilised to ascertain delirium presence28 over the weekend. In the case of a positive delirium diagnosis  the participant was coded as having delirium in those 24-hours. Delirium severity was quantified using the CAM-ICU 729 (when in the ICU) and the MDAS (when in the ward).The intensive care unit (ICU) Confusion Assessment Method (CAM-ICU)30 Cognition was assessed with a computerised battery  Connect).31 Specific CANTAB tests included Reaction Time (RTI), Paired Associates Learning (PAL), Spatial Working Memory (SWM) and One Touch Stockings of Cambridge (OTS). For each CANTAB test, at least two outcome measures  were extracted for analyses; see 32Global cognitive status was measured utilising the Addenbrookes Cognitive Examination (ACE-III).36 For the intervention group, CCT sessions lasting 45-60 minutes were completed every other day pre-operatively, and three times a week post-operatively . During intervention periods, participants were offered at least one completely supervised session weekly, face-to-face or remotely (via TeamViewer), with a trained researcher (trained by AL). In addition, participants were contacted weekly via phone . Control participants did not complete any CCT but were contacted weekly via phone . Adherence, identified through app metrics, was considered acceptable if three pre-operative sessions were completed, or 66% of the scheduled post-operative sessions were completed. Those who did not adhere pre-operatively could still be considered adherers for the 4- and 6-month follow-ups. Only data from adherers were used in analyses.CCT was delivered through the HappyNeuron Pro platform (on a laptop), with selected exercises targeting cognitive domains most affected in heart failure: psychomotor speed, attention, memory and executive function.37 using the Rstatix package.38 As described in the published protocol paper, a per-protocol approach was utilised,24 due to the importance of investigating the efficacy of the intervention as intended . Due to the limited sample size, effect sizes were produced for each analysis to interpret potential findings, rather than focusing on statistical significance.All analyses were performed using R (version 4.2.0)A logistic regression was performed to assess the effect of CCT  on delirium , with age and sex as covariates. An additional sensitivity analysis was run including baseline cognition (ACE-III) and depression (GDS) as covariates.ACE-III raw scores at the three follow-ups were subtracted from baseline scores to index change in global cognition over time. Independent samples t-tests were conducted assessing the effect of CCT on the change in test performance at each follow-up.39CANTAB test scores were used to assess domain-specific cognition. Z-scores were calculated for each CANTAB test outcome measure see  at baselA linear regression was performed to assess the effect of CCT on delirium duration (number of days delirious), with age and sex as covariates. This included participants who did not experience delirium (0 days).Two separate linear regressions were performed assessing the effect of CCT on delirium severity; 1) delirium severity in the ICU (CAM ICU-7), 2) delirium severity in the ward (MDAS). Both regressions included age and sex as covariates. Unstandardised beta values (B) were used to indicate effect size and direction. Distributions of the residuals of each regression indicated that model assumptions were not violated.See CONSORT diagram  for partp=0.76), covarying for age and sex. Age and sex also did not significantly associate with incident delirium. Model 2, which included baseline global cognition and depression, did not yield any significant effects  at discharge, 4- and 6-month follow-ups. There were also no significant effects of CCT on the change in performance on any cognitive domains (CANTAB) at discharge, 4- and 6-month follow-ups .Pre-operative CCT did not significantly associate with the duration of post-operative delirium , while covarying for age and sex. Pre-operative CCT did not predict the severity of delirium in the ICU  or on the ward , covarying for age and sex. Age significantly predicted delirium severity in the ICU , with older participants experiencing more severe delirium. Sex did not associate with delirium severity in the ICU. Age and sex also did not significantly predict delirium duration or severity on the ward, .29We saw no meaningful effects of CCT on incident delirium and cognitive function in CABG patients. Notably, our study is underpowered, due to substantial difficulties in recruiting CABG patients during the COVID-19 pandemic. Exploratory analyses revealed age as a predictor of increased delirium severity in the ICU, yet, this is a known effect.22 These trials prescribed short (15-20 minutes), frequent (up to twice daily), unsupervised and home-based CCT sessions. This may have driven their lower adherence rates, with participants reporting feeling over-whelmed.22 In contrast, with our approach  participants in a qualitative sub-study reported “it’s manageable but with support”.40 This highlights that support from our research staff (starting sometimes with how to use a computer) was essential in overcoming the perceived barriers of completing CCT, and is likely a driving factor in the adherence rate disparity to the previous feasibility trials.The findings indicate that pre- and post-operative CCT interventions, which include participant-tailored support , are feasible for CABG patients. In the CCT group, adherence was seen in 68% of participants pre-operatively. This rate is comparatively higher than those reported in recent feasibility trials (ranging from 17 to 39%) investigating the effect of pre-operative CCT on post-operative delirium and cognitive decline.22 However, a recent large randomised controlled trial  found that pre-operative CCT (utilising the Lumosity platform) significantly lowered post-operative delirium risk  in cognitively healthy, non-cardiac surgical patients, who were at least minimally compliant to the CCT intervention.20 Although minimum lead-in times to surgery were similar between Neurobics and the current study (one week), CCT was prescribed more frequently in the former . Therefore, hours of completed CCT were likely higher in the Neurobics trial.20 Although, it is known that CCT delivered less frequently (≤ 3 sessions a week) is more effective than more frequent sessions across weeks to months of an intervention.23Although adherence was considerably more favourable compared to previous trials there were no statistically significant differences between study groups concerning the incidence, severity, and duration of delirium post-operatively. These findings are similar to previous feasibility tri-als.41 This change  at a group level only occurred in the control group at 6-month follow-up (as compared to baseline) in a task targeting executive function (SWM). However, this result is likely driven by low baseline scores in the control group in this task. The CCT intervention focused on executive function . However, recent meta-analyses revealed CCT brings about only negligible-to-small improvements in this domain , which do not reach statistical significance, for both healthy older adults23 and those with mild cognitive im-pairments.42There were no statistically significant differences between CCT and control groups, relative to change in performance in cognitive domains  or global cognition (ACE-III), from baseline to discharge, 4- or 6-month follow-ups. Within the literature, meaningful cognitive change for an individual (commonly investigated as decline) is characterised by a 1 standard deviation change at a post-operative follow-up compared to pre-operative performance.43 This same pattern can be seen in our data , due to an increased withdrawal rate in those who experienced delirium. The current study investigated at a group level, comparing control with CCT participants. With more statistical power (participant numbers), it would have been ideal to investigate how CCT affected known post-operative cognitive trajectories: decline, no change, and improvement. It may be that the pattern of general cognitive improvement is masking different patterns and combinations of cognitive trajectories relative to group.A meta-analysis, containing data of over 2,000 CABG patients, reported a trend towards improvement in overall cognitive function across early (3-6 months) and late (6-12 months) follow-ups compared to pre-operative performance.data see , where g23 These analyses were conducted utilising data from CABG patients recruited from a single-centre hospital based in metropolitan South Australia, limiting the ability to generalise results.Due to the small sample size available for analyses, the study is not sufficiently powered. Notably, we did not see any patterns of effect sizes  across outcomes that supported our original hypotheses. The current study was limited by a two-group design. Consequently, the independent effects of pre-CABG and post-CABG CCT could not be investigated. In addition to this, no sham control was utilised within this study. However, this is not a considerable limitation due to; 1) the use of separate consent forms for study groups , and 2) a meta-analysis investigating the effect modifiers of CCT in cognitively healthy older adults found no significant effect of control group (passive versus active).20 further research (within larger samples) is warranted. This study focused on a single-mode intervention (CCT). Multicomponent interventions, where multiple risk factors are targeted concurrently within the one intervention, are another viable method of pre-operative intervention shown to be beneficial in improving (or maintaining) cognition in those at risk of dementia and reducing incident delirium during hospitalisation.47This study provides support for the feasibility of implementing CCT interventions pre- and post-operatively in cardiac surgical settings. Given the reported null effects, it is possible CCT may not be an effective method for reducing post-CABG delirium and cognitive decline; or null effects could be due to our small sample. As there are reported small protective effects of CCT on delirium incidence,Supplementary Material


Elevated levels of serum ferritin (SF) are observed in several types of cancer; however, little is known on the association between ferritin and glioma, the most frequent type of human primary brain tumour. Here we report that GBM patients show significantly increased pre-surgical SF levels (i.e. ferritinaemia) within the SF reference range and a marked ferritin immunoreactivity of resected tumour tissue. Our findings account for an indirect association between ferritin synthesis in glioma-tissue and altered SF levels, which limits the clinical value of SF as a tumour marker in glioma. Importantly, we show for the first time that GBM-derived glioma cells release ferritin in vitro, which exerts an apoptosis-stimulating activity. Albeit the pathophysiologic context of apoptosis induction by a tumour-derived ferritin remains to be defined, our findings account for a distinct growth-regulatory role of these ferritin species in tumour biology. Only three studies have assessed ferritinaemia in gliomas by routine laboratory diagnostics comprising a total number of less than 60 patients,6 the biological background of glioma-associated ferritinaemia remaining elusive. Here, we provide evidence for an upregulated ferritin synthesis in glioma tissue, which is not directly associated with elevated SF levels. In addition, we show for the first time that tumour-derived ferritins are capable of stimulating apoptosis.Ferritin, a 450-kDa multimeric iron-storage protein, built from 24 heavy (FTH) and light (FTL) chains, is essential to cellular iron homoeostasis by regulating the intracellularlabileiron pool via its ferroxidase activity conferred by the FTH chain.7 as well as newborn mouse astrocytes, was investigated under serum-free conditions using a primary rat hepatocyte assay defined for assessing ferritin cytotoxicity.9 Statistical significance was examined by applying non-parametric tests  and Student’s double-sided t-test for independent samples (apoptosis induction) using SPSS version 24.The study was approved by the local research ethics committee Salzburg, Austria (415-EP/33/3-2008). Written informed consent was obtained from all patients. The study was performed in accordance with the Declaration of Helsinki. Pre-surgical serum samples and tumour tissue specimens from 18 GBM patients and 16 meningioma (WHO I) patients were investigated. Serum ferritin (SF) was quantified by employing the Tina-Quant-Ferritin assay  on a Hitachi 917 automatic analyser. Immunohistochemical analysis of formalin-fixed paraffin-embedded specimens of resected tumour tissue followed standard procedures by employing anti-human FTL-specific (LSBio LS-B4383) and FTH-specific (LSBio LS-C105404) antibodies. Determination of the ferritin-labelling index is outlined in Supplementary Fig. p < 0.005) elevated in GBM and meningioma patients, in 22.2% (GBM) and 37.5% (meningioma) of the patients (ExtR cohort) exceeding the 95th percentile of the SF reference range  higher compared with a healthy reference population,3 as well as with the RefR cohort of meningioma patients  higher labelling indices  apoptosis-stimulating activity that was suppressed by neutralising anti-ferritin antibody Ab rH02  collected from a human glioblastoma-derived cell line Fig. . The GBMH02 Fig. . In contH02 Fig. . This de6 which is causal to GBM-associated ferritinaemia. Moreover, the enhanced immunoreactivity of resected GBM tissue for the FTL subunit accounts for the synthesis of a FTL-type isoform, which corresponds well with the reported expression of FTL-type isoferritins in cultured glioblastoma-derived cells,10 and with similar findings in glioblastoma stem-like cells.11 Strikingly, we demonstrate for the first time an apoptosis-stimulating activity of GBM-derived isoferritins. Since this activity is not seen for the isoferritin released from cultured newborn astrocytes, it is conceivable that astrocyte transformation towards a malignant phenotype comes along with the synthesis and secretion of a different ferritin isoform that exerts a pro-apoptotic activity. Albeit the pathophysiological significance of this novel finding is elusive, earlier research has demonstrated that iron-mediated oxidative stress and lipid peroxidation plays a pivotal role in ferritin-mediated apoptosis.12 On the other hand, high cytosolic ferritin levels may confer cytoprotection by enhanced iron sequestration, and affecting stress-associated targets such as the GADD45A/JNK pathway.10 Notably, elevated iron requirements have been demonstrated for glioblastoma stem-like cells where an increased ferritin expression supposedly confers stable intracellular iron buffering.11Our findings strongly support the assumption that glial tumours synthesise and secrete ferritin,13 and vice versa, it may also antagonise the exit of high-molecular-weight ferritin to the circulation. However, evidence exists that glioma cells themselves release soluble factors, such as vascular endothelial growth factor and hepatocyte growth factor, which promote BBB degradation.14 It is tempting to speculate on a similar effect conferred by the pro-apoptotic properties of glioma cell-derived isoferritins contributing to ferritinaemia in advanced GBM. The markedly high SF levels seen in the ExtR cohort of GBM patients could hypothetically reflect such a possibility. Similar contexts, although of less-severe outcome, may also apply to benign brain tumours, such as meningioma, which, as shown here, can also be accompanied by increased serum ferritin levels.Interestingly, no significant correlation between tumour ferritin immunoreactivity and SF levels was found in the investigated GBM patients. Hence, the tumour-based ferritin release may not be directly connected with the circulating SF pool in GBM, which limits the use of serum ferritin as a useful tumour marker for gliomas. With respect to this, it is noteworthy that the blood–brain barrier (BBB) can persist in GBM tissue, which hinders the accessibility of chemotherapeutics to the tumour cells,In conclusion, our findings account for a dual role of altered ferritin expression in glioma development: protecting the tumour cells by solid intracellular iron buffering, and upon release, acting as effector molecule in the tumour microenvironment.Supplemental File


Family caregivers are essential care providers helping to ensure the sometimes complicated recovery of recently hospitalized COVID-19 patients. COVID-19 caregivers face pandemic-specific challenges such as not being at patient bedside throughout the hospital stay and managing social distancing post-discharge. The current study aims to explore the unique experiences of family caregivers of Intensive Care Unit (ICU) COVID-19 patients. In-depth qualitative interviews were conducted by web conference with 13 dyads of adults who were in an ICU for COVID-19 between March and August 2020 and their primary caregiver (n=26). Participants were interviewed about the care recipient’s hospitalization and recovery journey, supports received, challenges experienced, and gaps in the system of care. Thematic qualitative analysis was conducted utilizing Watkins’ (2017) rigorous and accelerated data reduction (RADaR) technique. Caregivers played a critical role in patient admission, discharge, and recovery. Themes of caregiving challenges included self-management of COVID-19 infection, knowledge deficits of available resources and post-discharge care needs, post-infection stigma, separation guilt, deprioritized self-care, financial challenges, and lengthy recoveries with some ongoing health needs. While receipt of emotional support was considered an advantage, some caregivers expressed contact fatigue. Understanding how COVID caregivers experience illness management across the recovery journey can aid our understanding of the COVID caregiving process and identify intervention targets to improve overall health and well-being of the care dyad.