In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow ...molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30 μm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6 h, and thus this method provides several advantages over traditional western blotting.
Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell ...immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (xy) for cell isolation and lysis. Single-cell lysate in each microwell is "electrophoretically projected" into the 3
dimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is > 2.5 cells/s (70× faster than published serial sampling), with 25 immunoblots/mm
device area (>10× increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cells. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.
This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of the Lewy bodies and Lewy neurites found in Parkinson's disease brains and ...other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addition of preformed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds the recruitment of endogenous α-syn into aggregates characterized by detergent insolubility and hyperphosphorylation. Aggregate formation follows a lag phase of 2-3 d, followed by formation in axons by days 4-7, spread to somatodendritic compartments by days 7-10 and neuron death ~14 d after PFF addition. Here we provide methods and highlight the crucial steps for PFF formation, PFF addition to cultured hippocampal neurons and confirmation of aggregate formation. Neurons derived from various brain regions from nontransgenic and genetically engineered mice and rats can be used, allowing interrogation of the effect of specific genes on aggregate formation.
Senescent cells (SnCs) accumulate in many vertebrate tissues with age and contribute to age-related pathologies, presumably through their secretion of factors contributing to the ...senescence-associated secretory phenotype (SASP). Removal of SnCs delays several pathologies and increases healthy lifespan. Aging and trauma are risk factors for the development of osteoarthritis (OA), a chronic disease characterized by degeneration of articular cartilage leading to pain and physical disability. Senescent chondrocytes are found in cartilage tissue isolated from patients undergoing joint replacement surgery, yet their role in disease pathogenesis is unknown. To test the idea that SnCs might play a causative role in OA, we used the p16-3MR transgenic mouse, which harbors a p16
(Cdkn2a) promoter driving the expression of a fusion protein containing synthetic Renilla luciferase and monomeric red fluorescent protein domains, as well as a truncated form of herpes simplex virus 1 thymidine kinase (HSV-TK). This mouse strain allowed us to selectively follow and remove SnCs after anterior cruciate ligament transection (ACLT). We found that SnCs accumulated in the articular cartilage and synovium after ACLT, and selective elimination of these cells attenuated the development of post-traumatic OA, reduced pain and increased cartilage development. Intra-articular injection of a senolytic molecule that selectively killed SnCs validated these results in transgenic, non-transgenic and aged mice. Selective removal of the SnCs from in vitro cultures of chondrocytes isolated from patients with OA undergoing total knee replacement decreased expression of senescent and inflammatory markers while also increasing expression of cartilage tissue extracellular matrix proteins. Collectively, these findings support the use of SnCs as a therapeutic target for treating degenerative joint disease.
Neutrophils are a vital component of immune protection, yet in cancer they may promote tumour progression, partly by generating reactive oxygen species (ROS) that disrupts lymphocyte functions. ...Metabolically, neutrophils are often discounted as purely glycolytic. Here we show that immature, c-Kit
neutrophils subsets can engage in oxidative mitochondrial metabolism. With limited glucose supply, oxidative neutrophils use mitochondrial fatty acid oxidation to support NADPH oxidase-dependent ROS production. In 4T1 tumour-bearing mice, mitochondrial fitness is enhanced in splenic neutrophils and is driven by c-Kit signalling. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and T cell suppression when glucose utilisation is restricted. Consistent with these findings, peripheral blood neutrophils from patients with cancer also display increased immaturity, mitochondrial content and oxidative phosphorylation. Together, our data suggest that the glucose-restricted tumour microenvironment induces metabolically adapted, oxidative neutrophils to maintain local immune suppression.
The importance of autophagy in the generation of memory CD8(+) T cells in vivo is not well defined. We report here that autophagy was dynamically regulated in virus-specific CD8(+) T cells during ...acute infection of mice with lymphocytic choriomeningitis virus. In contrast to the current paradigm, autophagy decreased in activated proliferating effector CD8(+) T cells and was then upregulated when the cells stopped dividing just before the contraction phase. Consistent with those findings, deletion of the gene encoding either of the autophagy-related molecules Atg5 or Atg7 had little to no effect on the proliferation and function of effector cells, but these autophagy-deficient effector cells had survival defects that resulted in compromised formation of memory T cells. Our studies define when autophagy is needed during effector and memory differentiation and warrant reexamination of the relationship between T cell activation and autophagy.
Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3'-splice site (3'ss) recognition during RNA splicing; however, the molecular mechanisms ...of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1(R625/K666) mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3'ss. Modelling the differential junctions in SF3B1(WT) and SF3B1(R625/K666) cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1(WT) knockdown or overexpression do not reproduce the SF3B1(R625/K666) splice pattern, qualifying SF3B1(R625/K666) as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1(R625/K666)-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.
ELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary ...serologic assay for COVID-19. The Jess
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Simple Western system, an automated capillary-based assay, was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as the source of antigen, and total immunoglobulins (IgG, IgM, IgA) detection. In total, 602 sera were tested including 223 from RT-PCR-confirmed COVID-19 patients, 76 from patients diagnosed with seasonal HCoVs and 303 from coronavirus-negative control sera. We also compared this assay with the EUROIMMUN® SARS-CoV-2 IgG ELISA kit. Among 223 sera obtained from RT-PCR-confirmed COVID-19 patients, 180/223 (81%) exhibited reactivity against the nucleocapsid and 70/223 (31%) against the spike protein. Nucleocapsid reactivity was further detected in 9/76 (14%) samples collected from patients diagnosed with seasonal HCoVs and in 15/303 (5%) coronavirus-negative control samples. In the subset of sera collected more than 2 weeks after the onset of symptoms, the sensitivity was 94% and the specificity 93%, the latter value probably reflecting cross-reactivity of SARS-CoV-2 with other coronaviruses. The automated Western immunoblotting presented a substantial agreement (90%) with the compared ELISA (Cohen’s Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposure of people to HCoVs including SARS-CoV-2.
Inflammation and subsequent fibrotic encapsulation that occur after implantation of biomaterials are issues that fostered efforts in designing novel biocompatible materials to modulate the immune ...response. In this study, glycosaminoglycans (GAG) like hyaluronic acid (HA) and heparin (Hep) that possess anti‐inflammatory activity were covalently bound to NH2‐modified surfaces using EDC/NHS cross‐linking chemistry. Immobilization and physical surface properties were characterized by atomic forces microscopy, water contact angle studies and streaming potential measurements demonstrating the presence of GAG on the surfaces that became more hydrophilic and negatively charged compared to NH2‐modified. THP‐1 derived macrophages were used here to study the mechanism of action of GAG to affect the inflammatory responses illuminated by studying macrophage adhesion, the formation of multinucleated giant cells (MNGCs) and IL‐1β release that were reduced on GAG‐modified surfaces. Detailed investigation of the signal transduction processes related to macrophage activation was performed by immunofluorescence staining of NF‐κB (p65 subunit) together with immunoblotting. We studied also association and translocation of FITC‐labeled GAG. The results show a significant decrease in NF‐κB level as well as the ability of macrophages to associate with and take up HA and Hep. These results illustrate that the anti‐inflammatory activity of GAG is not only related to making surfaces more hydrophilic, but also their active involvement in signal transduction processes related to inflammatory reactions, which may pave the way to design new anti‐inflammatory surface coatings for implantable biomedical devices.
The anti‐inflammatory activity of GAG is not only related to making surfaces more hydrophilic and negatively charged which was determined by the physiochemical investigations, but also their active involvement in signal transduction processes related to inflammatory reactions which was shown by the significant decrease in NF‐κB level as well as the ability of macrophages to associate with and uptake HA and Hep, respectively. HA and Hep may have a significant impact in reducing the inflammatory response after implantation, which may increase functionality and lifetime of sensors and other implanted medical devices. (Part of the graphics were obtained as single images from the website https://smart.servier.com/ while the design and other needed drawings were self‐made).
Activated natural killer (NK) cells engage in a robust metabolic response that is required for normal effector function. Using genetic, pharmacological and metabolic analyses, we demonstrated an ...essential role for Srebp transcription factors in cytokine-induced metabolic reprogramming of NK cells that was independent of their conventional role in the control of lipid synthesis. Srebp was required for elevated glycolysis and oxidative phosphorylation and promoted a distinct metabolic pathway configuration in which glucose was metabolized to cytosolic citrate via the citrate-malate shuttle. Preventing the activation of Srebp or direct inhibition of the citrate-malate shuttle inhibited production of interferon-γ and NK cell cytotoxicity. Thus, Srebp controls glucose metabolism in NK cells, and this Srebp-dependent regulation is critical for NK cell effector function.
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