Dysmotility and postoperative ileus (POI) are frequent major clinical problems post-abdominal surgery. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine that promotes recovery of the intestine in various injury models. While EPO receptors (EPOR) are present in vagal Schwann cells, the role of EPOR in POI recovery is unknown because of the lack of EPOR antagonists or Schwann-cell specific EPOR knockout animals. This study was designed to explore the effect of EPO via EPOR in vagal nerve Schwann cells in a mouse model of POI.flox/flox / Mpz-EPOR-KO) confirmed using PCR and qRT-PCR techniques. We then measured the intestinal transit time (ITT) at baseline and after induction of POI with and without EPO treatment. Although we have previously shown that EPO accelerates functional recovery in POI in wild type mice, EPO treatment did not improve functional recovery of ITT in POI of Mpz-EPOR-KO mice.The structural features of EPOR and its activation by EPO-mediated dimerization were understood using structural analysis. Later, using the Cre-loxP system, we developed a myelin protein zero (Mpz) promoter-driven knockout mouse model of Schwann cell EPOR (MpzCre-EPORTo the best of our knowledge, this is the first pre-clinical study to demonstrate a novel mouse model of EPOR specific knock out on Schwan cells with an effect in the gut. We also showed novel beneficial effects of EPO through vagus nerve Schwann cell-EPOR in intestinal dysmotility. Our findings suggest that EPO-EPOR signaling in the vagus nerve after POI is important for the functional recovery of ITT.The online version contains supplementary material available at 10.1186/s12896-023-00781-x. Postoperative ileus (POI) is a common clinical complication after an abdominal surgery that involves inhibition of intestinal motility, prolonged parenteral nutrition, abdominal discomfort , and increased morbidity , 2. The Erythropoietin (EPO) is a U.S. Food and Drug Administration (FDA) approved drug for the treatment of anemia associated with chronic kidney disease. The functional role of EPO via EPO receptors (EPOR) has been reported in many other tissues including the intestine –10. Our Thus, we aimed to understand the obligatory role of VN Schwann-cell specific EPOR in EPO-induced intestinal functional recovery. While EPOR is present in Schwann cells of the VN, the role of EPOR in POI recovery is unknown because of the lack of EPOR antagonists or Schwann cell specific EPOR knockout animals. This study was designed to address these questions by testing a clinically relevant systemic dosage of EPO using the Mpz-EPOR-KO-in vivo murine model after inducing POI.To understand the structural features of EPOR and EPOR activation by EPO mediated dimerization, we performed a 3-dimensional bioinformatics analysis. The X-ray crystal structures of EPOR bound to EPO and antagonist bound EPOR were used  for thisfor missing atoms or unknown units. In addition, all the heteroatoms including water molecules and any co-crystallized solvent were removed from the PDB file to simplify the visualization.Furthermore, a schematic 2-dimensional representation of the protein-protein complexes DIMPLOT-plots interactions across selected protein or domain interfaces was studied  Upon comparing the binding map of both chains of EPOR with EPO there is surprisingly a 44% overlap spatially even though interaction amino acid identity is only 14% and biochemical conservancy of 29%. This is an example of binding convergence where two peptides bind to the same site, but the interesting thing is both of those distinct peptides are present in a single EPO hormone molecule. The most critical amino acid side chain residues were Ser91 and His153 on EPOR  they were involved in both dimerization and binding of agonists/ antagonists. The distance between both residue side chains is 66Å which is much larger for a drug target site  also there is the absence of a possible small molecule binding cavity at the site.The unique 120-degree angular relationship of the two EPOR sub-units as previously reported was clear from visualizing the dimer . This asflox/flox homozygous and MpzCre hemizygous mice [flox/flox homozygous mice with floxed is 390 and, 428 bp respectively  using three markers  by IF staining Fig. A. The geflox/flox served as EPOR controls. This was also true in all three segments of the intestine  , which are associated with the cell bodies and processes of enteric neurons throughout the digestive tract . To verium) Fig. C. In a nWe aimed to determine the effect of EPO treatment on the gastrointestinal motility of Mpz-EPOR-KO mice after IM. ITT, a method to assess gastrointestinal transit, was evaluated in controls (no manipulation) and intestine-manipulated animals 24 h after surgery, as mentioned in our established protocol . Figure We have generated a novel Schwann cell-specific conditional knockout EPOR mouse model. Our results provided direct evidence that vagus nerve Schwann cells specific EPOR plays an obligatory role in EPO-induced improvement in intestinal motility following POI. Here, EPO treatment has not shown a beneficial effect on ITT compared to the saline group. However, our previous study has shown a significant increase in ITT following EPO treatment in the setting of the POI-wildtype mouse model, where EPOR was intact in VN Schwann cells . EPO treNext, we set a confirmatory experiment to see the status of the EPOR in cultured SCs (S100/p75NTR/Mpz positive) of Mpz-EPOR-KO mice. In vitro, SCs studies are applicable to elucidate EPO molecular signaling under different translationally relevant stressor conditions. Our result shows the absolute knockout of EPOR in passage zero, VNSCs. Interestingly, we have observed partial deletion of EPOR in passage one of the VNSCs, which might be due to a shift in the myeline status of the cells. Thus, our data precisely confirms EPOR deletion was absolute with myelinated Schwann cells but not with the non-myeline cells that include neuronal fibroblasts, endothelial cells, and immune cells.In parallel, we have shown our further interest in characterizing Mpz-EPOR-KO mice, where we aimed to determine the status of EPOR-KO in the intestine that is innervated and regulated by VN. Genotyping data shows no deletion of EPOR in all the intestinal segments. Inquisitively, this was also true with EGCs. In summary, the development of the EPOR-KO condition mice was specific to neuronal-glial cells (VNSCs) rather than intestinal glial cells (EGCs). The absence of the functional improvement in POI with EPO treatment in the KO mice, speaks for the role of vagus nerve EPOR in mediating intestinal recovery.In conclusion, using vagus nerve Schwann cells-specific Mpz-EPOR-KO mice, our preliminary findings indicate that EPO-EPOR coupling plays an obligatory role in exogenous EPO-induced functional recovery following POI. Further characterization of this novel Mpz-EPOR-KO mouse model and investigations of EPO-induced downstream signaling pathways concerning EPOR expression, paracellular communications, the severity of POI, EPO dosages, and timing of EPO administration could provide remarkable insights into clinical translation use.http://www.ebi.ac.uk/thorntonsrv/software/LigPlus/). DIMPLOT produced a schematic 2-D representation of the protein-protein complexes DIMPLOT-plots interactions across selected protein or domain interfaces [UCSF chimera software was used to create a schematic visualization of EPOR activation by EPO-mediated dimerization . The X-rterfaces . The diaterfaces . In Fig.The experimental design and animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at The Pennsylvania State University College of Medicine, Hershey, PA. Twelve- to 16-week-old male mice weighing 25 to 30 g were used.flox/flox, synonym with Mpz-EPOR-KO) was generated by cross-mating floxed EPOR (EPORflox/flox) mice with myelin protein zero (Mpz) promoter-driven Cre recombinase (MpzCre) mice from Jackson Laboratories as described in our publication [The specific knockout of EPOR in Schwann cells  of isoflurane in a sterile condition of drop jar method followed by cervical dislocation, where the depth of the anesthesia was confirmed using respiration status and the pedal reflex.flox/flox and EPORflox/flox (control) mice were harvested and the total RNA was extracted using miRNeasy mini kit  [To assess EPOR gene expression, the vagus nerve of the MpzCre-EPOR Qiagen) . Next, RMouse vagus nerve-derived Schwann-cell (VNSC) isolation and ex vivo expansion was performed as described in the previous studies , 28. BriMouse enteric glial cell isolation and ex vivo expansion was performed as previously described with slight modification . BrieflyEighteen mice were randomly allocated into three groups. Group 1 animals were normal with no surgical manipulation (n = 4). Groups 2 and 3 animals underwent intestinal manipulation (IM) and these mice received intraperitoneal injections of either normal saline  or EPO  immediately after surgery. IM was carried out as described in our previous publication . No maniTwenty-four hours after IM, in vivo gastrointestinal transit study was performed as described in our protocol . BrieflyAll data were analyzed using GraphPad Prism Version 8.4.3 . Comparisons between three groups with n ≥ 4 were performed via ordinary one-way analysis of variance (ANOVA), and Tukey’s multiple comparisons test after confirmation of normally distributed data. For comparisons of two groups, (n per group ≥ 3), two-tailed, unpaired t-tests were used. All values are presented as means ± SEM. Significance levels  were documented using standard symbols .Below is the link to the electronic supplementary material.Supplementary Material 1


Dear Editor:Like many countries, Canada faced a triple threat of respiratory illness  in the fall of 2022, after all COVID-19 measures had been lifted, placing significant pressure on the healthcare system Tanne, . AlthougThe International COVID-19 Awareness and Responses Evaluation (iCARE) Study is an ongoing multi-wave cross-sectional survey of population attitudes and behaviours surrounding COVID-19 . Further, risk assessment education may be needed for those who may be underestimating their risk of contracting COVID-19 after recovering from previous infections.Findings have implications for government policy planning. Public health measures should be determined based on the pandemic situation and


The aim of this systematic review was to identify the correlations between spectrum sensing, clustering algorithms, and energy-harvesting technology for cognitive-radio-based internet of things (IoT) networks in terms of deep-learning-based, nonorthogonal, multiple-access techniques. The search results and screening procedures were configured with the use of a web-based Shiny app in the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) flow design. AMSTAR, DistillerSR, Eppi-Reviewer, PICO Portal, Rayyan, and ROBIS were the review software systems harnessed for screening and quality assessment, while bibliometric mapping (dimensions) and layout algorithms (VOSviewer) configured data visualization and analysis. Cognitive radio is pivotal in the utilization of an adequate radio spectrum source, with spectrum sensing optimizing cognitive radio network operations, opportunistic spectrum access and sensing able to boost the efficiency of cognitive radio networks, and cooperative spectrum sharing together with simultaneous wireless information and power transfer able increase spectrum and energy efficiency in 6G wireless communication networks and across IoT devices for efficient data exchange. Cognitive radio has been developed due to spectrum scarcity and diminished exploitation  of allocCognitive radio technology can dynamically distribute the unlicensed spectrum  for IoT-IoT-based large-scale wireless connections can bring about a serious spectrum scarcity issue , and thuThe search results and screening procedures were configured by use of a web-based Shiny app in the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) flow design. In July 2023, a quantitative literature review of major scholarly databases  was carried out, and the search terms were: “cognitive radio-based IoT networks” + “spectrum sensing”, “clustering algorithms”, and “energy harvesting technology”, with the analyzed research being published between 2019 and 2023; only 347 sources qualified for initial inclusion . A totalAs increasing demands for significant data rates overload standard radio frequency technologies , millimeCognitive radio networks can articulate multipurpose spectrum management  by enablCognitive radio can optimize wireless communication spectral efficiency  by enablThe traffic behavior modeling of impermanent spectrum use for IoT applications across collective bands  can shapCognitive radio networks reach a compromise  between A cognitive radio network comprises primary and secondary users : the latCognitive-radio-based IoT systems  develop Spectrum sensing is decisive  in cogniDeep-learning-based cognitive radio technology can be harnessed throughout wireless communication systems , increasThe design and advancement of energy- and spectrum-efficient proposals, such as cognitive radio sensor networks , articulClustering and data aggregation are decisive in IoT-based wireless communication , while eSpectrum dynamics and energy use can be assimilated in network-stability-aware clustering  that cohClustering arranges nodes into groups  so as toA coherent and green machine-learning-based dynamic clustering mechanism integrating power demand and data volume can assist cognitive IoT networks in terms of intelligent processing, secure delivery, and far-reaching awareness , leadingCognitive wireless sensor networks can harness the inactive authorized frequency band to find a solution to the spectrum resource scarcity issue : by leveCognitive-radio-network-based real-time high-speed communication systems  require Metaheuristic algorithms and deep-neural-network-based clustering techniques  can imprEnergy harvesting and cognitive radio technologies can assist wireless sensor networks , extendiAs IoT sensor and devices use a massive volume of power in data transmission , radio fAvailability and ultrareliability demands, together with energy-harvesting technology and dynamic spectrum access, impose specific performance compromises , typifyiA deep-Q-learning based algorithm can be deployed across energy-harvested cognitive radio networks with the aim of optimal resource distribution : primaryThe spectral and energy efficiency of device-to-device communication can be enhanced by employing cognitive radio systems and radio frequency energy-harvesting technologies  while stThe massive increase in smart devices articulating content-centric data traffic amplifies cellular-network-based group communication services : multicaNonorthogonal multiple access can enhance 5G cellular network throughput and spectrum efficiency and facilitate ultrareliable and low-latency communications , articulEnergy-harvesting-powered cognitive machine-to-machine networks can mitigate the intensifying deficient spectrum, as a result of large-scale smart devices and simultaneous access demand that bring about operational deterioration and massive energy use , by ensuCognitive radio technology and nonorthogonal multiple-access techniques  can solvData-driven and wireless communication technologies, by integrating a large volume of end users, have led to a crowded radio spectrum , while gWireless sensor networks assist IoT devices  in physiMultiple-access schemes that can increase the volume of users transmitting over a medium, together with cognitive radio networks that enable harnessing vacant frequency bands of a spectrum in a dynamic or opportunistic manner , can harOur systematic review clarifies that cognitive radio is pivotal in the utilization of an adequate radio spectrum source , with spCognitive radio and nonorthogonal multiple access  are pivoAs we performed research based only on ProQuest, Scopus, and Web of Science sources and in the past few years, when we identified the most robust correlations, we possibly ignored some valuable research on optimizing spectrum sensing, clustering algorithms, and energy-harvesting technology for cognitive-radio-based IoT networks in terms of deep-learning-based nonorthogonal multiple-access techniques. Distinct review software systems and bibliometric network construction and visualization tools would also generate different outcomes. Future research should investigate the use of extended reality technologies and artificial-intelligence-based spectrum sensing, clustering, and energy harvesting in digital-twin-based virtual factories supported by cognitive-radio-based IoT networks.Long-range wide-area networks can assist IoT applications  by integ


Oral immune tolerance is a physiological process to achieve tolerance against autoimmunity by oral ingestion of self-antigen(s) or other therapeutics. At the cellular level, oral tolerance suppresses autoimmune diseases by activating FoxP-positive and -negative regulatory T cells (Tregs) and/or causing clonal anergy or deletion of autoreactive T cells, affecting B cell tolerance. However, oral delivery of antigens/biologics is challenging due to their instability in the harsh environment of the gastrointestinal (GI) tract. Several antigen/drug delivery tools and approaches, including micro/nanoparticles and transgenic plant-based delivery systems, have been explored to demonstrate oral immune tolerance for different autoimmune diseases successfully. However, despite the effectiveness, variation in results, dose optimization, and undesirable immune system activation are the limitations of the oral approach to further advancement. From this perspective, the current review discusses the oral tolerance phenomenon, cellular mechanisms, antigen delivery tools and strategies, and its challenges. Autoimmune diseases, comprised of 80 different autoimmunity-dependent disorders, affect millions worldwide. Their prevalence is around 3–5% of the world population, with autoimmune type 1 diabetes being one of the most common diseases worldwide .Autoimmune diseases are chronic, painful, and sometimes display life-threatening clinical manifestations that significantly affect the quality of life, causing enormous economic burdens to society. Despite intensive efforts at the preclinical and clinical levels, curing these diseases has yet to be feasible. However, new-generation biologics such as TNF-α inhibitors and B and T lymphocyte-targeted inhibitors  are currently available to tackle the autoimmune symptoms and effects temporarily. These biologicals considerably suppress chronic inflammation by blocking the synthesis/secretion of inflammatory cytokines or activating B and T cell receptors. However, the patients need potential drugs for treatment in milligram quantities at specific time intervals for a long duration, manifesting severe side effects . TherefoSimilarly, antigen-specific immunotherapy targets autoimmune deviations by repeatedly exposing the immune system to autoantigen(s). In contrast to an allergic immune response biased towards Th2-type immune responses, an autoimmune response usually allied with Th1 and Th17 cell responses was developed against self-tissues . Therefo+ T cells were shown to contribute to defects in B cell tolerance [Immunological tolerance is the healthy state of the immune system discriminating self- and non-self-antigens, thereby protecting the host from external threats, for example, microbes. Failure of self-tolerance leads to the activation of autoimmune responses. Both central and peripheral tolerance mechanisms activate the regulation of self-reactive T cells, which will also affect B cell tolerance outcomes. For example, in an experimental autoimmune disease (Myasthenia gravis), IL-17-producing CD4olerance . For a dolerance . Generalolerance . An aberolerance , allows Antigen-specific immune tolerance can be directed towards effector T cells through various mechanisms, including clonal deletion, anergy, or Treg activation. These promote the secretion of several anti-inflammatory cytokines to regulate the autoreactive T cells ,13 Figu. Anergy +T cells from the lymphoid organs after peptide exposure was dependent on apoptosis and/or Fas/Fas ligand-mediated cellular death pathways [Interestingly, a decline in the number of T cell clones was negligible after intravenous administration of the same peptide . Similarpathways ,18.+CD25+CD4+) Tregs are essential for maintaining the self-tolerance and homeostasis of immune system [+FoxP3lo resting Tregs, CD45RA−FoxP3hi activated Tregs, which were suppressive under in vitro condition, and cytokine-secreting CD45RA−FoxP3lo non-suppressive T cells [The Forkhead box protein 3-positive . A low amount of antigen induces the activation of Tregs. At the same time, a high dose favors the induction of clonal anergy or the deletion of T cells to suppress the autoimmunity ,44. For The bystander effect refers to suppressing autoreactive immune cells by ingesting a specific antigen that can stop the immune responses generated by another antigen. Generally, fed antigens structurally mimic another antigen, yielding this effect . Under iThe success of oral immune tolerance depends on the nature, dosage, and frequency of the administered antigen, the innate background of the recipient, and the extent of antigen uptake by APCs of the gut-associated lymphoid tissue. In the harsh environment of the GI tract, the amount of antigen delivered to the intestinal site is minimal, probably due to the low bioavailability of the antigen at the target site because of possible degradation in the GI environment . Therefoco-glycolide) (PLGA) nanoparticles (NPs) suppressed autoimmunity in collagen-induced arthritis (CIA), and synthesis of NPs was performed through the double-emulsion method. Mice administrated with a single dose of CII-containing PLGA particles reduced the clinical symptoms of experimental arthritis and systemic CII-specific antibodies [The particulate systems in nano and micron size ranges were explored to deliver different antigens to achieve peripheral immune tolerance. For instance, collagen type II (CII)-encapsulated poly was tested against autoimmune arthritis. Oral administration of Hesperidin-loaded lecithin-folic acid-based silver nanoparticles (Ag-NPs) after emulsification in CFA in an induced rat arthritis model led to anti-rheumatic activities. In addition, the oral treatment showed promising effects on relieving arthritis symptoms, such as the alleviation of joint inflammation, cartilage depletion, and bone erosion, by downregulating the secretion of anti-inflammatory cytokines  while upregulating of the anti-inflammatory cytokine TGF-β.Moreover, a low level of M1 macrophage activation and low production of RANKL, a receptor activator of the nuclear factor kappa beta ligand, and matrix metalloproteinase (MMP)-2/9 are associated with Ag-NPs-based oral immunotherapy . In thisArabidopsis thaliana transgenic plant expressing a collagen-based fusion protein, CTA1(R7K)-COL-DD, prevented arthritis development in the same strain of mice with mild arthritic symptoms and a low CD4+T cell response, compared to the control group [Upon ingestion, the transgenic plant-based systems expressing autoantigens, such as insulin and glutamic acid decarboxylase 65 (GAD), delivered intact proteins to the gut lymphoid tissue ,58. For ol group .Apart from antigens, other therapeutic molecules, including hormones, antibodies, and natural products, have been demonstrated after oral administration to suppress or prevent autoimmune symptoms.+Foxp3+ Tregs in EAE mice [+ and γδ IL-17 cells in the central nervous system [Hormones, especially polypeptide hormones secreted from the pituitary gland, can control autoimmune responses. In a recent study, the therapeutic efficacy of adrenocorticotropic hormone (ACTH) was demonstrated after oral administration in a mouse model to alleviate the clinical symptoms of MS disease. Orally administrated ACTH at a specific frequency decreased the expression of IL-17-positive T cells in the gut lamina propria and spleen by activating the CD4EAE mice . A diffes system .®) was proven using C57BL/6 (B6) mice. TCZ-fed mice inhibited EAE onset and CNS inflammation by upregulating Th2 cytokines while concomitantly downregulating the Th1-type cytokines [®), a drug for psoriasis, was tested against EAE development in B6 mice. Mice orally fed with the UTZ drug suppressed the ongoing inflammatory responses.Commercially available monoclonal antibodies are promising in ameliorating disease symptoms after repeated oral feeding to the mice. For instance, the oral impact of tocilizumab  peptide 35–55 by suppressing the synthesis of proinflammatory cytokines while increasing the production of Th2 cytokines . RecentlBifidobacterium animalis PTCC 1631 and Lactobacillus plantarum in B6 mice, with a daily dose, reduced the induction of EAE. In addition, mice fed with probiotics had a delay in disease onset and showed mild clinical signs. Moreover, a high percentage of Tregs was observed in the mouse spleen and lymph nodes, which received an oral probiotics treatment [Bacillus amyloliquefaciens supplement in camel milk fed to mice significantly reduced the disease symptoms by increasing the intestinal flora and short-chain fatty acids production in a MOG protein-induced EAE model in B6 mice.Recently, probiotics have shown promising results in suppressing or blocking autoimmune responses. For example, the therapeutic effects after oral administration of probiotics in the form of a mixture of reatment . In a diLactobacillus reuteri-based oral system was tested against rheumatoid arthritis. The Lactobacillus reuteri secretes the kv1.3 potassium blocker component Shk-235 (Lrs235) to control the ion channel activity, which blocks the proliferation of human T-effector memory lymphocytes. Interestingly, a single-time oral gavage of the engineered system significantly suppressed the inflammation in a delayed-type hypersensitivity reaction occurring in the atopic dermatitis animal model. Probiotic-fed mice had regulated levels of proinflammatory cytokines and myeloperoxidase . InteresLactococcus lactis in preventing and controlling EAE development in B6 mice. In both regimens, everyday oral administration of the HSP-producing bacterial strain reduced the MS-associated clinical symptoms by activating the LAP+CD4+Foxp3+ T cells in the spleen and the inguinal lymph nodes. Moreover, treated mice showed reduced leukocyte adherence in the venule structure of the spinal cord [Moreover, a daily oral dose of the engineered probiotic reduced the clinical symptoms of arthritis and joint inflammation in arthritis rats . Anothernal cord .+-effector cells (CD44hiCD62Llow), CXCR6+ CD4, CD8, and Th17 cells in the EAE mice [Plant-based products have shown promising results in preclinical studies in achieving successful immune tolerance. For example, oral administration of the plant-derived “Baicalein” considerably reduced EAE clinical symptoms by blocking IL-17A production and suppressing MOG-specific antibodies. Moreover, Baicalein-based oral treatment reduced the pathogenic Th17 cells, CD4EAE mice . In anot+/F4/80+ immune cells in the pancreatic lymph nodes, which secreted anti-inflammatory proteins such as IL-10 and PD-L1 and controlled the inflammatory autoimmune responses [Concanavalin A injection was used to develop autoimmune hepatitis disease. In a dose-dependent manner, orally fed Koumine reduced the serum liver-associated inflammatory markers, such as alanine aminotransferase, aspartate aminotransferase, TNF-α, and IL-6, leading to the blocking of the development of hepatitis in mice. Mechanistically, Koumine regulates the expression of antioxidation factors such as Ho-1 and Nrf2 proteins by activating the Nrf-2 cell signaling pathway and altering the gut microbiota . Differeesponses .The oral route is a safe, robust, and more sustainable model to achieve tolerance by deleting the autoreactive T cells or generating T cell clonal anergy or Tregs. Though oral immune tolerance has shown promising results at the preclinical level, different challenges, including proper selection of antigen(s), dose optimization, and inappropriate immune system activation, hamper its progress to the next level. In the future, a more appropriate therapeutic target with fine-tuned dose optimization might be beneficial to achieve a successful immune tolerance strategy for ameliorating autoimmune disorders.


The Cyclin-dependent kinases (CDKs) play crucial roles in a range of essential cellular processes. While the classical two-step activation mechanism is generally applicable to cell cycle-related CDKs, both CDK7 and CDK8, involved in transcriptional regulation, adopt distinct mechanisms for kinase activation. In both cases, binding to their respective cyclin partners results in only partial activity, while their full activation requires the presence of an additional subunit. Recent structural studies of these two noncanonical kinases have provided unprecedented insights into their activation mechanisms, enabling us to understand how the third subunit coordinates the T-loop stabilization and enhances kinase activity. In this review, we summarize the structure and function of CDK7 and CDK8 within their respective functional complexes, while also describing their noncanonical activation mechanisms. These insights open new avenues for targeted drug discovery and potential therapeutic interventions in various diseases related to CDK7 and CDK8. Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that primarily phosphorylate and regulate key proteins involved in cell cycle progression, gene transcription, and other cellular processes . The memIn principle, CDK monomers exhibit only minimal detectable kinase activity . For canß-sheet-rich N-terminal lobe and a compact C-terminal lobe, which encloses a catalytic cleft responsible for ATP binding and substrate recognition. The T-loop in monomeric CDK2 adopts an inactive conformation, which obstructs the catalytic cleft and renders substrates inaccessible  of CDK, which greatly enhances its kinase activity to a higher level . Among tcessible  , which plays a pivotal role in reversing elongation repression by phosphorylating the Ser2 of RNAPII CTD, the hSPT5 subunit of DSIF and the NELF-E/RD subunit of NELF . NotablyThe eukaryotic Mediator is a multisubunit (25–30 proteins) transcriptional coactivator complex that transmits regulatory signals from activators and repressors to the RNAPII transcription machinery . This coin vitro  in proximity to the T-loop of CDK8. The acidic triad (E33-D34-E35) at the beginning of this activation helix is responsible for positioning it properly for CDK8 activation . This acXenopus as MO15, and its homologues were later identified in mammals and named as CDK7  . In the e T-loop . A consee T-loop . The rolThe inactive T-loop in monomeric CDK adopts a folded conformation, which hinders the catalytic cleft  Bondt e. To actiInterestingly, among the CDKs, CDK8 stands alone in lacking the conserved threonine residue on its T-loop. This absence suggests the utilization of an alternative mechanism to rearrange the Y342 sidechain in order to interact with the conserved arginine residues. In the CKM structure, the N-terminal H1 helix of Med12 interacts with two residues (H341 and T343) of the Y-loop, leading to repositioning of the Y342 sidechain . This adSeveral canonical CDK proteins have been extensively investigated through functional and structural studies, revealing the essential role of both cyclin binding and T-loop phosphorylation in achieving a fully active state . However


Spatial transcriptomic technologies enable measurement of expression levels of genes systematically throughout tissue space, deepening our understanding of cellular organizations and interactions within tissues as well as illuminating biological insights in neuroscience, developmental biology and a range of diseases, including cancer. A variety of spatial technologies have been developed and/or commercialized, differing in spatial resolution, sensitivity, multiplexing capability, throughput and coverage. In this paper, we review key enabling spatial transcriptomic technologies and their applications as well as the perspective of the techniques and new emerging technologies that are developed to address current limitations of spatial methodologies. In addition, we describe how spatial transcriptomics data can be integrated with other omics modalities, complementing other methods in deciphering cellar interactions and phenotypes within tissues as well as providing novel insight into tissue organization. Since the first development of single cell RNA sequencing (scRNAseq) , it has Different spatial transcriptomic methodologies cover various aspects regarding the number of detected genes, resolution and whole transcriptome analysis. These methods are mainly classified as follows: (1) sequencing-based methods; (2) probe-based methods; (3) imaging-based methods; (4) image-guided spatially resolved single cell RNA sequencing methods . The claSequencing or next-generation sequencing (NGS)-based methods A are unb10X Genomics Visium is adapted from the method reported in Stahl et al. 2016 [al. 2016 , 10X Genal. 2016 . This meal. 2016 , cancer al. 2016 .Slide-seq is similar to Visium, Slide-seq [lide-seq , anotherlide-seq , neurosclide-seq ,25,26 anlide-seq . Slide-slide-seq . Moreovelide-seq .Both Visium and Slide-seq methods have been shown to improve cell (sub)typing by integrating the spatial transcriptome data with scRNAseq data . VariousSpatio-temporal enhanced resolution omics sequencing (Stereo-seq), a newly developed NGS-based spatial profiling method, uses randomly barcoded DNA nanoballs deposited in arrays to achieve nanoscale resolution, thereby reaching ~single cell resolution. The method enables large field-of-view (FOV) (cm × cm) spatial transcriptomics at ~cellular resolution and has been applied to mouse organogenesis [ogenesis , plant bogenesis , neuroscogenesis  and deveogenesis .Light-seq, another newly developed NGS-based spatial omics method, uses light-directed DNA barcoding in fixed cells and tissues followed by ex situ NGS sequencing [quencing . The metDespite the advances in NGS-based methods, most of them lack single cell resolution or have relatively low transcript counts, reducing their sensitivity to low abundance transcripts ,15,39,40In contrast to NGS-based spatial transcriptomic methods, oligonucleotide probe-based methods like NanoString’s digital spatial profiling system were designed to capture targeted transcripts in manually selected ROIs ,46. TargTMNanoString’s digital spatial profiling system, GeoMx, uses in situ hybridization probes, consisting of target complimentary oligonucleotide sequences linked to indexing oligonucleotide barcodes via a UV-photocleavable linker  enables quantification and visualization of up to 1000 targeted transcripts and 64 protein analytes via automated, cyclic fluorescence, in situ hybridization [dization . CosMx cMultiplexed error-robust fluorescencehybridization (MERFISH) in situ  combines fluorescence in situ hybridization with combinatorial labels to enable sequential rounds of hybridization and imaging. The serial images are then decoded using error-robust barcodes that are designed to label specific RNA molecules [olecules ,49,50. Tsequential fluorescence in situ hybridization (seqFISH and seqFISH+) is able to detect ~10,000 genes at sub-cellular resolution by combining colors into pseudocolors [Similar to MERFISH, docolors ,51,52, adocolors .Spatially-resolved transcript amplicon readout mapping (STARmap) [STARmap)  integratMost imagining-based methods are featured on single cell resolution, high efficiency, specificity and experimental reproducibility. Even though, pre-selected targeted transcripts need to be determined in advance and the FOV of tissues is limited .Geographical position sequencing (Geo-seq) has combined LCM and scRNAseq to investigate the spatial transcriptome of mouse early embryo, mouse brain and pathological liver and sperm tissues [Zipseq [Despite the great advances in spatial omics, most of the existing methods lack in-depth single cell whole transcriptomic profiling, analyzing tens of thousands of genes per cell. Image-guided or microscopy-based methods have been combined with state-of-the-art scRNAseq to address this limitation. These image-guided spatially resolved scRNAseq methods D can sel tissues . Zipseq  [Zipseq , anotherNICHE-seq [spatially annotated functional single cell sequencing (FUNseq) [NICHE-seq has been applied to identify rare niche-specific immune subpopulations and gene programs in infected B cell follicles and tumors [Spatially annotated FUNseq, developed in our group, has been applied to unravel intratumoral heterogeneity and decipher the tumor invasiveness of cells located in the outermost edge of a cell mass [ICHE-seq  and spat(FUNseq) ,42, on td tumors . Spatialell mass . In addiell mass ,42; (3) ell mass ,42; (5) ell mass  followedImage-guided spatially resolved single cell transcriptomic sequencing enables in-depth single cell whole transcriptomic profiling by integrating state-of-the-art scRNAseq with live cell imaging. Our spatially annotated FUNseq, in particular, allows imaging a large FOV (up to 1.28 cm × 1.28 cm) of live clinical tissue sections while maintaining single cell/subcellular resolution using our ultrawide field-of-view optical (UFO) microscope ,42; it aThese photoactivation-based spatial profiling methods are reproducible, sensitive and specific, but there is room for improvement regarding throughput .Slide-DNA-seq [With the advent of improved resolution and sensitivity of spatial transcriptomic technologies, integration with other omics modalities can provide broader and more comprehensive information on tissue characterization. The combination of spatial genome sequencing with in situ transcriptomic profiling could shed light onto our understanding of how genome organization and function are programmed. With the advent of spatial genomic sequencing in intact tissues , integra-DNA-seq , a metho-DNA-seq .Deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) [Spatial proteomics enable profiling protein expression within individual cells at subcellular resolution, resolving spatiotemporal protein locations as well as protein–protein interactions . Most ofBiT-seq) , an NGS-BiT-seq)  or CITE-BiT-seq) . Using tBiT-seq) . In addiBiT-seq)  and spatBiT-seq) ), showedmass spectrometry imaging (MSI) [multiplexed ion beam imaging (MIBI) [and matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) [Mass spectrometry-based methods, like ng (MSI) ,68, multg (MIBI) ,70 and mry (IMS) ,72,73, aSignificant improvements have been made in MSI and spatially resolved MS-analysis as a whole over the past decade, and many of these have focused on enabling spatial proteomics and metabolomics at a single cell level . MIBI, fco-detection by indexing (CODEX) [tissue cyclic immunofluorescence (t-cyCIF) [In addition to mass spectrometry-based methods, other high-throughput spatial proteomic profiling methods, such as  (CODEX)  and tisst-cyCIF) , providet-cyCIF) , mouse st-cyCIF)  and tumot-cyCIF) .spatial-ATAC-seq) [Spatial-CUT&Tag (cleavage under targets and tagmentation) enables spatially resolving of genome-wide profiling of histone modifications by combining in situ CUT&Tag chemistry, microfluidic deterministic barcoding and next-generation sequencing [Epigenomic MERFISH enables spatially resolved single cell epigenomic profiling and has been demonstrated to map active promoters and putative enhancers in the mouse brain [Spatial epigenomics captures epigenomic modifications, including DNA methylation, histone modifications and chromatin accessibility, in tissue sections. Spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (TAC-seq)  and combTAC-seq) . Spatialquencing . Epigenose brain .Technological advances that enable the integration of different spatial modalities will greatly improve our ability to study tissue complexity and our understanding of biological processes in greater detail.Features of the spatial omics methods mentioned in this review are summarized in Spatial omics can be utilized to deepen our understanding of tissue complexity as the technology provides thorough information of cell (sub)types, cell–cell interactions and heterogeneity of individual cells in intact tissues.The methods have been widely applied in the fields of oncology, cancer immunology and developmental biology. For example, diverse heterogeneity of tumor-associated macrophages (TAMs) and their role in influencing the tumor microenvironmental landscape was better understood via spatial omics profiling ,81. The In developmental biology, Spatial-CUT&Tag has been applied to reveal epigenetic control of cortical layer development and spatial patterning of cell types determined by histone modification in the mouse brain, thereby dissecting the underlying mechanisms of how spatial epigenetic patterns modulate gene expression and tissue development .In cardiac research, spatial omics technologies have been applied to the investigation of heart development and injury . For insIn order to comprehend the intricate molecular mechanisms of cellular interactions within tissues, emerging spatial omics technologies compete in terms of owing higher resolution (single cell or subcellular resolution), faster throughput, better sensitivity, deeper coverage, greater multiplexity, more user-friendly procedures and greater versatility of assayed samples . Owing to the rapid development of these various aspects in the spatial transcriptomics field, a deeper understanding of cellular states and functions within tissues has been greatly noted. The newer invention of spatial genomics, epigenomics, metabolomics and proteomics further complements the findings made by spatial transcriptomics.With future development in the spatial omics field, systematically assaying larger tissue areas for 3D reconstruction as well as deciphering transcriptomic (or other omic) changes over time may be possible. To reach this, NGS-based methods need improved spatial resolution and probe-based and imaging-based methods require an increase in profiling speed and the number of targets that can be profiled. Image-guided spatially resolved methods need to improve either the profiling speed  or the multiplexing capacity . In addition, transcriptomic (or other omic) variations associated with (time-dependent) changes in cellular dynamics or interactions in response to treatments or the surrounding microenvironment may be feasible when technologies for spatial profiling of live tissues  are made more mature. Furthermore, developments in both 3D reconstruction and temporal profiling require more advanced microscopic imaging systems tailored to these applications as well as associated visualization analysis frameworks.In addition to addressing the technological challenges, development of new computational tools and algorithms will be accompanied. Current challenges of spatial omics data analysis include, amongst others, how to properly normalize data matrices and remove low quality data, how to increase the signal-to-noise ratio, how to or whether to smooth data to improve sensitivity and how to remove unwanted technical and biological variations. Furthermore, more and comprehensive integration strategies across different modalities are expected to be developed, for instance, integrating spatial metabolomics with other modalities. The development of algorithms or analysis packages that are robust for each technology as well as compatible with other platforms is crucial to increase the applications of these technologies.Another challenge for the spatial omics field will be to create novel, yet validated, models about how multi-cellular spatial networks communicate and orchestrate biological functions. For example, will traditionally identified cell types be seen as new cell types in different multi-cellular structures or patterns at the tissue level? How do microenvironmental cues reprogram cell or tissue fate? Leveraging on the upcoming big spatial omics data and existing single cell omics profiles, new realization of how cells communicate between each other at the tissue level, how spatial organization and patterns influence biological functions and how environmental cues impact (multi-) cellular development will be made.These deeper biological insights will enable a comprehensive understanding of spatial patterns, cellular interactions and tissue architectures as well as unraveling novel mechanisms of disease formation and organism development.


Brain diseases remain a significant global healthcare burden. Conventional pharmacological therapy for brain diseases encounters huge challenges because of the blood–brain barrier (BBB) limiting the delivery of therapeutics into the brain parenchyma. To address this issue, researchers have explored various types of drug delivery systems. Cells and cell derivatives have attracted increasing interest as “Trojan horse” delivery systems for brain diseases, owing to their superior biocompatibility, low immunogenicity, and BBB penetration properties. This review provided an overview of recent advancements in cell- and cell-derivative-based delivery systems for the diagnosis and treatment of brain diseases. Additionally, it discussed the challenges and potential solutions for clinical translation. Brain diseases, including neurodegenerative disorders, cerebrovascular diseases, and brain cancers, remain a leading cause of disability and mortality worldwide . HoweverThe BBB is mainly comprised of tightly connected brain capillary endothelial cells functionally supported by pericytes and astrocytic perivascular endfeet . The BBBRecently, cell-based delivery systems manufactured from biological materials such as cells, cell components, or cell-derived vesicles have received increasing interest because of their bio-mimic properties . With inIn this review, we provide an overview of the prospects of cell- and cell-derivative-based delivery systems for overcoming the BBB and the research progress on the diagnosis and treatment of brain diseases . FinallyCell-based delivery systems have the advantages of good biocompatibility, low immunogenicity, specific tissue-homing characteristics, and inherent ability to penetrate various biological barriers  . For exaThe candidate cells as carriers should possess good drug-loading potential. The drug-loading process can be based on the physicochemical nature of the target drugs and the biological properties of the candidate cells. General ways for drug loading into carrier cells include cell surface coupling via covalent or non-covalent methods; internalization via endocytosis, phagocytosis, or macropinocytosis; direct cellular entrance through direct translocation, lipid fusion, electroporation, or microinjection; and antibody-antigen recognition ,16.Internalization of drugs into the carrier cells is the most used loading method. For phagocytes , their phagocytic properties can be a convenience for drug loading . For celThe mechanisms involved in the carrier cell migration across the BBB include rolling, activation, arrest, crawling, and diapedesis ,25. Afte3+/64Cu were conducted for glioma delivery and multimodal optical/MR/PET imaging (MSCs are pluripotent stem cells with tumor-homing capacity that can transmigrate the BBB through paracellular and transcellular routes through G-protein coupled receptor and integrin very late antigen-4 and its ligand vascular cell adhesion molecule-1 (VLA-4/VCAM-1) dependent mechanisms ,27. MSCs imaging  28]. Th. Th3+/64 imaging B,C [28]. imaging D, exemplNSCs are multipotent cells originating in the central nervous system with the potential to generate neurons and glial cells. NSCs exhibited targeted migration toward glioblastoma after being injected intravenously . The engADSCs are mesenchymal stem cells obtained from adipose tissue with the capacity of homing to brain cancer via stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 (SDF-1α/CXCR4) signaling axis, thus could serve as therapeutic vehicles for targeted brain tumor delivery . The engThe inflammatory responses occurring in CNS diseases  and the first type of leukocyte recruited to the lesions of inflammation and tumorigenesis, making them prospective in cell-based drug delivery. With the ability to traverse the BBB/BBTB ,35, NEs 3O4 magnetic nanogels showed the navigating capability upon exposure to a rotating magnetic field, with increased accumulation in the diseased regions, as well as facilitated by the chemotaxis-driving BBB penetration of neutrophils for targeted drug delivery [Neutrophils internalizing doxorubicin-loaded magnetic mesoporous silica nanoparticles (ND-MMSNs) have been reported for the therapy of postsurgical glioma  33]. ND. ND33]. delivery .In addition, neutrophil-based vehicles have been explored for glioblastoma multiforme photoacoustic imaging , and cercl PGP-PEG-DGL/CAT-Aco NPs) were internalized by the circulating neutrophils via endocytosis and micropinocytosis and transported across the BBB to the ischemic subregion; the delivery method could enhance the therapeutic efficiency of neuroprotectant in the middle cerebral artery occlusion (MCAO) mice and extend the therapeutic time window in acute cerebral ischemia . s. s75]. sNSCs, derived from the neuroepithelial cells of the neural tube, maintain the capacity for self-renewal and generation of neurons, astrocytes, and oligodendrocytes throughout their whole lives . The NSCNPCs, differentiated from NSCs, can give rise to the glial and neuronal cells that populate the CNS  and are Endometrial stem cells (EnSCs) in the basal and functional layers of the human endometrium involve in the remodeling of the tissue . The EVsEmbryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of blastocysts and characterized by unlimited proliferation, self-renewal, and multidirectional differentiated ability . ESCs caSNCA (siSNCA) for Parkinson’s disease treatment . . 132]. PThere is no simple protocol to generate large numbers of neutrophils for therapeutic purposes because neutrophils have a very short circulatory half-life and are difficult to cultivate and retain their inherited functions. Alternatively, employing the neutrophil membrane for coating nanoparticles can be an option . In a st2O2, which subsequently reacted with CPPO to generate chemical energy for activating the photosensitizer Ce6, thus performing tumor-killing functions [Cancer cell membrane coating has been actively explored for cancer drug delivery by leveraging the homotypic recognition characteristics of cancer cells . Inspireunctions . In anotunctions . Intraveunctions . Moreoveunctions . MPC@siRunctions .Cancer cell membrane coating has also been used for ischemic stroke treatment. The overexpressed CD138 and VCAM-1 on the 4T1 cell membrane can interact with CD31 and VLA-4 on various cerebral cells , thus facilitating the cargos to penetrate the BBB and target the cerebral ischemic lesions [The NSC membrane was engineered to overexpress CXCR4, which substantially interacted with SDF-1 enriched in the ischemic region, and thus enhanced the delivery of the payload to the ischemic area . GlyburiEscherichia coli K1, were used to encapsulate the doxorubicin-loaded PLGA nanoparticles to treat breast cancer brain metastases; the brain-targeted delivery was achieved via the OmpA-gp96 interaction-mediated transcytosis [The Gram-negative bacterial membrane can interact with gp96 on BBB endothelial cells via its outer membrane protein A (OmpA) component and has a potential application for brain-targeted drug delivery . The LPSscytosis .Hybrid membrane frameworks were prepared using various types of cell membranes, including tumor cell-DC , tumor cCell- and cell-derivative-based delivery systems have been demonstrated with promising preclinical results in overcoming the formidable BBB challenge. Yet, continual efforts must be made to promote their translation from bench to bedside.First of all, there is still a bottleneck issue to achieving large-scale production of carrier cells or exosomes. For example, it is difficult to produce large numbers of neutrophils, since these short-lived cells are difficult to cultivate and can only be isolated from peripheral blood ,145. A pSecondly, poor drug-loading efficiency and uncontrolled drug release also limit the therapeutic application of cell- and EV-based drug delivery systems ,38. LoadThirdly, besides improving drug-loading efficiency, minimizing the impact of the drug-loading processes and the loaded drugs on carrier cells is another noteworthy issue. The complex fabrication processes may affect the in vivo kinetic characteristics of the carrier cells, and therefore hamper the delivery efficiency and the therapeutic effect . In addiFourthly, short circulation time and insufficient targeting capability are also typical drawbacks of natural EV-based drug delivery systems. Bioengineering strategies employed to improve these adverse properties include genetic engineering, click chemistry, cloaking, and bio-conjugation ,64. SurfIt should be pointed out that there could be potential side effects of cell- and EV-based drug delivery systems. Given that tumor cell-derived EVs (TDEVs) participate in cancer progression and metastasis , the appOverall, the application of cells or cell derivatives as Trojan delivery systems for the diagnoses and treatments of brain diseases is a new field. Multidisciplinary efforts should be made to achieve large-scale production, develop standardized drug-loading and final product quality control methods, optimize the pharmacokinetics, and demonstrate safety properties to promote clinical translation.


Enveloped viruses co-opt host glycosylation pathwaysto decoratetheir surface proteins. As viruses evolve, emerging strains can modifytheir glycosylation patterns to influence host interactions and subvertimmune recognition. Still, changes in viral glycosylation or theirimpact on antibody protection cannot be predicted from genomic sequencesalone. Using the highly glycosylated SARS-CoV-2 Spike protein as amodel system, we present a lectin fingerprinting method that rapidlyreports on changes in variant glycosylation state, which are linkedto antibody neutralization. In the presence of antibodies or convalescentand vaccinated patient sera, unique lectin fingerprints emerge thatdistinguish neutralizing versus non-neutralizing antibodies. Thisinformation could not be inferred from direct binding interactionsbetween antibodies and the Spike receptor-binding domain (RBD) bindingdata alone. Comparative glycoproteomics of the Spike RBD of wild-type(Wuhan-Hu-1) and Delta (B.1.617.2) variants reveal O-glycosylationdifferences as a key determinant of immune recognition differences.These data underscore the interplay between viral glycosylation andimmune recognition and reveal lectin fingerprinting to be a rapid,sensitive, and high-throughput assay to distinguish the neutralizationpotential of antibodies that target critical viral glycoproteins. Lectin fingerprinting is a high-throughputand facile methodto rapidly profile the neutralization potential of patient antibodiesraised against viral glycoproteins. Many viral pathogensshare a common strategy of employing densely glycosylated surfaceproteins to facilitate viral entry into host cells and modulate hostimmune defenses.2 Because of their abundanceand accessibility, viral glycoproteins often serve as key antigensupon which the host immune system mounts an attack. As the virus evolves,acquired mutations within these glycoproteins, such as the addition3 or remodeling of glycans,5 helpthe virus evade immune detection. Viral genome sequencing readilyreveals amino acid mutations in emerging viral strains, yet alterationsto protein glycosylation cannot be predicted from sequencing informationalone. New tools to rapidly profile changes in viral protein glycosylationand their impact on immune recognition would be valuable assets inmonitoring viral pathogens.6Viruses are the primary cause of respiratorytract infections worldwide,accounting for more than 2 million deaths annually.9  strain  of the Spike glycoprotein and host angiotensin-convertingenzyme 2 (ACE2) receptor, recent studies have demonstrated that Spikeglycans enhance viral entry through interactions with host lectins.12 Thus, modification of N- and O-glycans on Spike could influencehost responses to SARS-CoV-2.Humancoronaviruses are emblematic of viruses that undergo mutation,but the corresponding changes in protein glycosylation tend to beenigmatic. The coronavirus SARS-CoV-2 uses its Spike glycoprotein,which harbors significant glycosite microheterogeneity9 1A, to me) strain 1b.8,9 Al14 still, not all antibodies that bind Spike can neutralizethe virus and prevent viral entry into host cells.16 Moreover, variants with changes proximal to Spike glycosites haveemerged during the pandemic.19 These evolved strains are ofgrowing concern as they exhibit heightened transmissibility and mayreduce the neutralization efficacy of convalescent or vaccinated patientantibodies.22Changes in viral glycosylation pose a risk to globalvaccinationefforts. With SARS-CoV-2, the host humoral response is predominantlydirected against the Spike protein;Two typesof assays are traditionally used to determine the neutralizationcapacity of antibodies to SARS-CoV-2: (1) cellular assays that measurethe ability of antibodies to block viral infection or (2) in vitroassays that screen for the ability of antibodies to directly competewith Ace2 receptor binding. The former requires specialized equipment/facilitiesand is low throughput, and the latter assay does not account for antibodiesthat bind Spike at positions distal to the Ace2 recognition site butare nonetheless capable of neutralizing the virus. As such, complementaryplatforms that rapidly screen patient antibodies for Spike-neutralizingor non-neutralizing capabilities would be advantageous for understandingthe spread of SARS-CoV-2 and informing therapeutic and vaccine design.25 We reasoned that tools to quickly and easily report on changes inSpike glycosylation among viral variants would reveal insights intohow viruses such as SARS-CoV-2 modify their glycans to evade immunesurveillance. Moreover, these glycan reporters could be leveragedto generate recognition patterns, or fingerprints, from neutralizingand non-neutralizing antibodies. Unique fingerprints generated byantibodies with distinct mechanisms of action  could then serve as standardsfor evaluating if patient sera contain antibodies with similar properties.Here we report a lectin fingerprinting strategy to predict the neutralizationpotential of antibodies against WT and Delta SARS-CoV-2 variants andunderstand how viruses utilize glycosylation to subvert host immunedefenses.The ability of enveloped viruses to alter their glycan coat toevade antibody neutralization is well-documented. Spike is the majorglycoprotein present on coronavirus envelopes, and antibody recognitionoccurs on epitopes proximal to or in direct contact with known glycosites.9 Given advances in glycan profiling and diagnostics,28 we reasoned that nonantibody carbohydrate-binding proteins, or lectins,could detect changes in viral glycan microheterogeneity. Because glycansabound on the Spike glycoprotein and its RBD, we reasoned that lectinswould bind differentially and compete with neutralizing antibodies.Thus, we set out to identify a collection of lectins to fingerprintthe Spike RBD.The Spike glycoprotein of SARS-CoV-2 contains overlapping yet distinctglycan compositions at each of its many glycosylation sites.29 to collate the depositedglycan array data sets for 24 plant- and fungal-derived lectins against611 glycans.30 Lectins displaying overlappingspecificities and those containing distinct carbohydrate recognitionproperties were identified using correlation analysis  and publicly available data.31 The glycan-binding specificities of each lectinwere subsequently mapped onto the 46 unique glycoforms reported foreach of the 74 binding sites on the trimeric ectodomain of recombinantfull-length Spike (FL-Spike) to predict which lectins would recognizeSpike glycans (We began by using the Glycan Array Dashboard(GLAD) toolanalysis S1 and puFigure S2). FL-Spike has limited amounts of exposed galactose, N-acetylgalactosamine , and fucose (Fuc). Lectinsrecognizing these carbohydrates  showed minimal binding toFL-Spike.To test the in silico projections, we optimizedan enzyme-linkedlectin assay (ELLA) that would allow us to identify which lectinsfrom the curated suite bind FL-Spike and act as reporters of glycosylation.We immobilized HEK-expressed FL-Spike via passive adsorption to ahydrophilic 96-well plate. Biotinylated lectins were applied to theSpike protein-coated wells, and lectins bound to FL-Spike glycanswere detected with StrepTactin-HRP conjugate , indicating that complexation is specific.Additionally, treating FL-Spike with the N-glycosidase PNGase F reducedlectin binding, confirming that protein–glycan interactionsdrive the observed signal .Several lectins predicted to bind weakly  still gave moderatesignals. The variance between predicted and experimental data mayarise from changes in viral glycan display and minor differences inrecombinant protein preparation. Our data indicate that lectin valencyalso alters Spike recognition. For example, AAL, UEA I, and LTL canall interact with fucose residues, but AAL is the best Spike binder.AAL is a dimer with five fucose binding sites per monomer . In contrast, UEA I is a dimer of monomers with one bindingsite each , and LTL is a tetramer of monomers withone binding site each . Thus, AAL, the lectin predictedto have the most multivalent interactions, exhibited the highest functionalaffinity.Of the 24 plant and fungal lectins tested, 19 boundFL-Spike witha moderate to strong signal. Lectin binding was dose-dependent .32 These data indicatethat the lectin binding is specific and can rapidly report on differencesin glycosylation for SARS-CoV-2 glycoproteins.We next evaluated the generality of lectin fingerprinting and itsability to report on glycosylation of different SARS-CoV-2 domains.We posited that the RBD of Spike would generate a lectin fingerprintdistinct from the FL-Spike. As anticipated, the RBD bound fewer lectinsthan FL-Spike 1E. To de33 Proximalto the ACE2 binding site are two N-glycans, N331 and N343, and thecorresponding glycans in the MERS-CoV and SARS-CoV-1 Spike proteincontact neutralizing antibodies. We therefore postulated that neutralizingantibodies would compete with lectins for Spike occupancy, leadingto competitive displacement of lectins upon the addition of neutralizingantibody.Upon infection with SARS-CoV-2, patients rapidly developa humoral response to viral antigens 2A. In ma34 We initially examined lectin binding to immobilizedcommercial antibodies (1 μg/mL) and found that, with the exceptionof ConA and LCA, minimal interactions were detected relative to thoseobtained using RBD binding .These data indicate that antibody–RBD interactions, and notantibody glycans themselves, should be responsible for competitivefingerprints obtained upon antibody addition.Antibodies themselves carry a single N-glycosylationmark thatcould complicate the interpretation of the lectin fingerprints.Figure S7A). A separate competitivefingerprint was generated for FL-Spike. Displacement patterns forthe lectins jacalin GalNAc, O glycan), SBA ,and STL (GlcNAc) were observed for both Spike and the RBD . A direct comparison . The antibody’s abilityto displace lectins bound to immobilized RBD was assessed in a competitiveELLA assay 2B. CompeTable S1) to expand the detection of glycan microheterogeneity. Wecharacterized the lectin’s unique lectin fingerprints to FL-Spikeand RBD .The data led us to ask whether a panel of lectinsmight distinguishneutralizing from non-neutralizing antibodies. We therefore queriedwhether neutralizing and non-neutralizing antibodies purified fromconvalescent patients would possess unique lectin fingerprints. Inaddition to the 24 plant and fungal lectins, we introduced six commerciallyavailable prokaryotic lectins with defined monosaccharide specificities and two commercial mouse antibodies that werecross-reactive with SARS-CoV-1 RBD but were either known or likelyto be non-neutralizing against SARS-CoV-2 (α-RBD-D002 and α-S1-CR3022). To reduce the high dimensionalityof competitive lectin fingerprints, we employed hierarchical clusteringanalysis with Euclidean distance measure,36 where the similarity between individual lectin data is calculatedcombinatorially. The row and column dendrograms are automaticallygenerated with seriation to find similarities in the lectin fingerprints.37 Application of this protocol indicated thatcommercial neutralizing antibodies cluster separately from patientantibodies, indicating differences in lectin displacement. Withinthe patient antibody cluster, neutralizing antibody SCV2mAb-1 grouped separately fromthe other patient antibodies. Weakly neutralizing antibody SCV2mAb-2 and neutralizing antibody SCV2mAb-3 clustered together,indicating a possible shared mechanism of binding that is distinctfrom SCV2mAb-1.The non-neutralizing antibody, SCV2mAb-4, clusters broadly with patient antibodies whencompared with commercial antibodies, but separately from the otherneutralizing patient antibodies . As indicated by the hierarchical clustering analysis, the profilesof α-S1 cross-reactive SARS-CoV-1 antibodies (α-RBD-D002 and α-S1-CR3022), which engage RBD distal tothe ACE2 binding site, are comparable.38 These results suggest that lectin fingerprinting can distinguishunique or overlapping binding modes of patient antibodies to SpikeRBD.To avoid complicationsfrom FL-Spike structural and conformationalplasticity, we used the RBD for competitive lectin fingerprints frompatient antibodies. We tested four monoclonal antibodies purifiedfrom convalescent patients , two had antibodies that bound WT Spike RBD butwere non-neutralizing by an in vitro infection assay , and two generated antibodiesthat had no apparent Spike binding or neutralization activity  . In each analysis, the clustering patterns separated by sera type.Both samples with neutralizing serum clustered together, while non-neutralizingconvalescent sera showed distinct lectin fingerprints. These initialfindings indicate that competitive lectin fingerprinting could bebenchmarked and used as a diagnostic tool for evaluating sera neutralizingpotential.Rapid serological testsV2ser-6) 2D. CompeV2ser-6) 2E and byTable S2). Collected sera elicited ID50 values between12 and 2053 in a SARS-CoV-2 infection assay with WT and Delta strains between relative SpikeRBD affinity and pseudovirus neutralization  have been generatedfrom mRNA (mRNA)-based vaccination against SARS-CoV-2 Spike protein.We analyzed sera collected from individuals that received either oneor two doses of mRNA-1273 and BNT162b2 mRNA vaccines ( strains3A. The o strains3B.We folization 3C.r2 = 0.892).Viruses are notorious for altering their aminoacid sequences duringevolution. The effects of these changes on neighboring glycosylationsites is poorly understood. The Delta variant has two amino acid changesin its Spike RBD, L452R and T478K 3D, that 19Lectins with differencesin their binding to WT versus Delta includeECL , GSL II (GlcNAc), jacalin , LCA , MAL I , SBA ,STL (GlcNAc), and α-Man. These lectins recognize features ofO-glycans suggesting that WT and Delta strains have major differencesin O-glycosylation 3F. To coWe next asked if lectin fingerprinting could assess the neutralizingpotential of vaccinated patient sera by analyzing samples from patientswho received the BNT162b2 mRNA vaccine 3I. Sera 40 To determinewhether mRNA vaccination strategy changes how vaccinated patient seraengage Spike RBD, we performed lectin fingerprinting on all patientsera (n = 16). Hierarchical clustering analysis oflectin fingerprinting showed dissimilar patterns from the BNT162b2and mRNA-1273 vaccinees, suggesting that differential vaccinationstrategies elicit variations in Spike RBD glycoforms. The result islectin fingerprints that vary with vaccination strategy (Table S1). We therefore conducteda lectin fingerprint correlation analysis . Inthe case of SARS-CoV-2 infections, where glycosylation of the Spikeprotein plays a central role in viral infection and immune evasion,such tools would illuminate how viral protein glycosylation changesover the course of infection and its inevitable evolution betweenhosts. In this study, we developed a lectin fingerprinting methodthat provides a snapshot of glycan microheterogeneity on recombinantFL-Spike and its RBD.As SARS-CoV-2 variants emerge and evolvefor immune escape, changesin glycosite composition as well as the appearance of new glycosites27 Unlike other methods for profiling protein glycosylation, lectin fingerprinting requiresminimal sample processing and uses common laboratory equipment andreagents. Furthermore, this method can be readily scaled up for high-throughputscreening applications and easily formatted for probing any glycoproteinof interest.To generate this lectin fingerprint, weused 24 plant and fungallectins and six prokaryotic lectins to index the accessible glycanson the viral protein surfaces. Lectin fingerprints for FL-Spike andRBD were consistent with mass spectrometric analysis of SARS-CoV-2glycoproteins, demonstrating the accuracy of lectin fingerprintingas a readout of protein glycosylation. Additionally, lectin fingerprintingrapidly reported on differences in glycosylation for SARS-CoV-2 glycoproteinsproduced in different cell types. Together, the data suggest thatthis method may be useful for evaluating alterations to protein glycosylationduring proteostatic stress  or in viralinfections with multitissue tropism that could contribute to viralglycan heterogeneity, depending on the cell type propagating the virus.Although Still, not all antibodies generated againstRBD are neutralizing,recent reports indicate that the majority of anti-SARS-CoV-2 neutralizingantibodies target the RBD. These observations suggest that privilegedepitopes exist on RBD and targeting of these sites by a host may conferincreased immunity against the virus. The assays currently used toexamine binding of convalescent patient antibodies to RBD cannot distinguishwhich sites on RBD are bound, nor which antibodies are neutralizing.However, structural studies on antibody–Spike interactionssuggest that antibodies make contacts with glycans on RBD. Taken together,we reasoned that tools capable of distinguishing antibody engagementof RBD could offer a new route to evaluate convalescent patient antibodiesand their likelihood of conferring protection from SARS-CoV-2 infection.Such reagents could also uncover mechanisms of glycan-mediated immuneevasion.If anti-SARS-CoV-2 antibodies engage epitopes on SpikeRBD proximalto its glycosites, we postulated that antibodies should be able tocompetitively displace bound lectins and perturb the lectin fingerprint.Indeed, we found that commercially available neutralizing antibodiesbound proximally to exposed glycosites and perturbed the lectin fingerprints.Upon binding RBD, non-neutralizing antibodies also perturbed the lectinfingerprint, but in a manner that was distinct from neutralizing antibodies.These data indicate that, in engaging their respective epitopes, antibodiescompetitively displace lectins in a pattern that corresponds to theirneutralization capacity. We employed lectin fingerprinting to evaluatethe neutralization potential of antibodies within prevaccination convalescentpatient sera. The use of patient sera is more relevant for clinicalevaluations but demands the evaluation of antibodies within a morecomplex mixture of serum glycoproteins. We found that convalescentsera yielded distinct lectin fingerprints that corresponded to theirneutralization efficacy.42 Differences in S323/T325 O-glycosylation in the Spike RBD of WTand Delta variants highlight a potentially critical nexus in the evasionof SARS-CoV-2. When O-glycosylation was reduced, as seen in the mutationof T372A from scanned SARS-CoV-2 genomes, infectious virus showedincrease replication42 suggesting thata delicate balance of glycosylation status is critical for maintainingimmune evasion and infectivity. The differences in antibody responseselicited from BNT162b2 and mRNA-1273 that encode different Spike antigensresult in changes in glycosylation. How population heterogeneity selectsfor specific glycan microheterogeneity may influence the evolutionof SARS-CoV-2.Glycan changes have been implicatedin stabilizing Spike proteinstructure and influencing immune selective pressure.Our data suggest that lectin fingerprinting canbe used as a diagnostictool for evaluating antibody efficacy against SARS-CoV-2 antigensand other glycosylated proteins. We anticipate that the method canbe used to profile alterations in SARS-CoV-2 glycosylation that arepresent in rapidly emerging, highly infectious VOCs. Understandinghow changes in glycan structure influence antibody recognition mayreveal new insights into how new viral variants are able to evolveto evade immune responses. More broadly, we believe that lectin fingerprintingcan be used to map protein glycosylation for other proteins in whichglycosylation plays a key functional role.


A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harsh  COL-HA The physical characterization of COL-HA hydrogels revealed that UV-triggered crosslinking resulted in reproducibly form-stable constructs that matched elasticity levels of native tissues, despite the crosslinker-free hydrogel design. Independently of differences in stiffness, COL-HA had larger pores in the lyophilized state than both Gel-HA controls.3.3.6 MSCs/ml were resuspended in each gel formulation prior to photo-crosslinking. Cells remained viable inside the hydrogels for at least a week  at different levels, thereby forming distinct cytokine profiles for the different experimental groups. To evaluate potential biological consequences resulting from these differences, gene ontology (GO) term enrichment analysis for biological processes was performed. Out of the 81 consistently detected cytokines, 56 analytes could be assigned to at least one of the following five categories: angiogenesis, neuro-regeneration, immunomodulation, hemostasis and ECM remodeling  , pro-apoTo validate the protein array data, VEGF and OPG, which showed very distinct regulation, were additionally measured by ELISA . The levThe comparison of paracrine profiles from MSCs exposed to different biomaterial microenvironments suggested complex biomaterial-guided regulation of the cellular secretome. In depth analysis revealed an especially strong secretion of pro-angiogenic factors from COL-HA-Lam encapsulated cells, predicting an increased pro-angiogenic potency of cells encapsulated in this cell carrier material.3.5.To confirm the increased pro-angiogenic potency from cells encapsulated in COL-HA hydrogels, a validated capillary-like structure formation assay was used , 25. PriTo subsequently assess whether the MSC-hydrogel constructs were able to induce the formation of interconnected capillary-like network structures, the number of intact meshes and the total area covered by complete meshes was determined . While CThe biological angiogenesis readout confirmed both the functional relevance of augmented pro-angiogenic factor secretion from COL-HA-encapsulated MSCs and additionally reflected the further enhanced pro-angiogenic potency predicted for COL-HA-Lam.4.The clinical success of MSC therapy is limited by the delivery method, therefore, the aim of this study was to fabricate, characterize and optimize an application-oriented carrier material with improved bio-instructive properties for the encapsulation and delivery of therapeutic MSCs.Two bioactive polymers, collagen and HA, were used to photo-crosslink into a hydrogel with defined, tissue-compatible mechanical properties. Both polymers are used in reconstructive or surgical indications in patients and can therefore be considered safe and clinically applicable –60. In a™ (Gel-HA), was used for comparison. Hystem™ has contributed to important advances in 3D cell culture and organoid development in recent years and exhibits a comparable photo-triggered phase change as COL-HA [To put the properties of COL-HA into the context of commercially available biomaterials, an industry standard hydrogel kit, Hystems COL-HA , 64, 65.s COL-HA , 28. Thes COL-HA , 34. Anos COL-HA . We elimet al achieved a difference in scaffold pore size by varying the lyophilization conditions between experimental groups of chemically identical scaffolds [Interestingly, structural differences were observed between collagen- and gelatin-based biomaterials . Differecaffolds . ConsequEncapsulation of MSCs in COL-HA demonstrated that the cells remained viable and assumed an elongated morphology within the hydrogel in comparison to commercially available hydrogels for cell culture. This observation suggested differences in interactions at the cell–material interface and we hypothesized that these differences would be reflected in the MSCs’ secretory phenotype. While most studies that investigate cell–material interactions focus only on few cell-secreted mediators, we performed a detailed analysis of biomaterial-guided changes in the paracrine profile to validate our hypothesis. This analysis revealed complex regulation patterns and predicted a potent pro-angiogenic profile for MSCs encapsulated in COL-HA. Based on our previous studies, we hypothesized that the loss of cell attachment and spreading within the biomaterial negatively affects MSC’s secretory phenotype . In suppin vivo studies, where the injected material would need to immobilize cells in a bone fracture gap, nerve gap or contracting muscle while complying to surrounding native tissue to avoid scarring and inflammation [The differences in paracrine activity between COL-HA and Gel-HA groups could be the consequence of different cell viabilities, however, 2D TCP cultures, which are known to maintain very high cell viability, showed a limited paracrine activity in our study. Another important influence on the paracrine activity could be the hydrogel stiffness. To test this possibility, the stiff Gel-HA control was included in this study. Although the modulus of this Gel-HA stiff biomaterial matched the COL-HA groups, their secretome profiles had very little similarity. Instead, the Gel-HA stiff biomaterial resulted in the lowest cytokine levels of all experimental groups, demonstrating that higher bulk stiffness alone does not augment the paracrine activity of MSCs. Tissue-specific bulk stiffness is a relevant design parameter for future ammation , 72. Howammation , 73, 74.ammation , 75, 76.As a standard reference we compared the secretome of COL-HA encapsulated cells to 2D MSC culture on polystyrene TCP. Interestingly, the osteoblastic marker OPG was one of the few factors that were secreted at highest levels from MSCs cultured on TCP. MSCs are known to be able to differentiate toward osteoblastic lineages and this result could be carefully interpreted in line with studies that demonstrate stiffness dependent regulation of cell fate , while oEarlier studies have provided evidence that cell–cell and cell–matrix interactions within the 3D hydrogel are crucial for the regenerative function of MSCs , 78. Her5.in vivo characterization of COL-HA-encapsulated MSC in in vivo angiogenic and neuro-regenerative disease models as the next step towards bringing effective MSC therapies to patients.This study demonstrates the fabrication and functionality of a biopolymer hydrogel to modulate the paracrine effects of therapeutic cells. We have combined biologically relevant material cues with important application-oriented features such as injectability and controlled crosslinking to produce a bio-instructive cell carrier for MSC encapsulation and protection. The fast fabrication protocol and on demand crosslinking mechanism are scalable and clinically translatable and were designed for human application, with all individual components of COL-HA or comparable biopolymers having been previously safely implanted into patients in a clinical setting –60. MicrSupplement for Bio-instructive hydrogel expands the paracrine potency of mesenchymal stem cells