Segmentation-based analysis of single-cell immunoblots.

From genomics to transcriptomics to proteomics, microfluidic tools underpin recent advances in single-cell biology. Detection of specific proteoforms-with single-cell resolution-presents challenges in detection specificity and sensitivity. Miniaturization of protein immunoblots to single-cell resolution mitigates these challenges. For example, in microfluidic western blotting, protein targets are separated by electrophoresis and subsequently detected using fluorescently labeled antibody probes. To quantify the expression level of each protein target, the fluorescent protein bands are fit to Gaussians; yet, this method is difficult to use with noisy, low-abundance, or low-SNR protein bands, and with significant band skew or dispersion. In this study, we investigate segmentation-based approaches to robustly quantify protein bands from single-cell protein immunoblots. As compared to a Gaussian fitting pipeline, the segmentation pipeline detects >1.5× more protein bands for downstream quantification as well as more of the low-abundance protein bands (i.e., with SNR ∼3). Utilizing deep learning-based segmentation approaches increases the recovery of low-SNR protein bands by an additional 50%. However, we find that segmentation-based approaches are less robust at quantifying poorly resolved protein bands (separation resolution, Rs < 0.6). With burgeoning needs for more single-cell protein analysis tools, we see microfluidic separations as benefitting substantially from segmentation-based analysis approaches.

Multimodal detection of protein isoforms and nucleic acids from low starting cell numbers.

Protein isoforms play a key role in disease progression and arise from mechanisms involving multiple molecular subtypes, including DNA, mRNA and protein. Recently introduced multimodal assays successfully link genomes and transcriptomes to protein expression landscapes. However, the specificity of the protein measurement relies on antibodies alone, leading to major challenges when measuring different isoforms of the same protein. Here we utilize microfluidic design to perform same-cell profiling of DNA, mRNA and protein isoforms (triBlot) on low starting cell numbers (1-100 s of cells). After fractionation lysis, cytoplasmic proteins are resolved by molecular mass during polyacrylamide gel electrophoresis (PAGE), adding a degree of specificity to the protein measurement, while nuclei are excised from the device in sections termed "gel pallets" for subsequent off-chip nucleic acid analysis. By assaying TurboGFP-transduced glioblastoma cells, we observe a strong correlation between protein expression prior to lysis and immunoprobed protein. We measure both mRNA and DNA from retrieved nuclei, and find that mRNA levels correlate with protein abundance in TurboGFP-expressing cells. Furthermore, we detect the presence of TurboGFP isoforms differing by an estimated <1 kDa in molecular mass, demonstrating the ability to discern different proteoforms with the same antibody probe. By directly relating nucleic acid modifications to protein isoform expression in 1-100 s of cells, the triBlot assay holds potential as a screening tool for novel biomarkers in diseases driven by protein isoform expression.

Quantitative UV-C dose validation with photochromic indicators for informed N95 emergency decontamination.

With COVID-19 N95 shortages, frontline medical personnel are forced to reuse this disposable-but sophisticated-multilayer respirator. Widely used to decontaminate nonporous surfaces, UV-C light has demonstrated germicidal efficacy on porous, non-planar N95 respirators when all surfaces receive ≥1.0 J/cm2 dose. Of utmost importance across disciplines, translation of empirical evidence to implementation relies upon UV-C measurements frequently confounded by radiometer complexities. To enable rigorous on-respirator measurements, we introduce a photochromic indicator dose quantification technique for: (1) UV-C treatment design and (2) in-process UV-C dose validation. While addressing outstanding indicator limitations of qualitative readout and insufficient dynamic range, our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%) required for UV-C dose measurements. In a measurement infeasible with radiometers, we observe a striking ~20× dose variation over N95s within one decontamination system. Furthermore, we adapt consumer electronics for accessible quantitative readout and use optical attenuators to extend indicator dynamic range >10× to quantify doses relevant for N95 decontamination. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical considerations for both photochromic indicators themselves and UV-C decontamination processes.

Current Understanding of Ultraviolet-C Decontamination of N95 Filtering Facepiece Respirators.

Introduction: The COVID-19 pandemic has led to critical shortages of single-use N95 filtering facepiece respirators. The US Centers for Disease Control and Prevention has identified ultraviolet-C (UV-C) irradiation as one of the most promising decontamination methods during crisis-capacity surges; however, understanding the mechanism of pathogen inactivation and post-treatment respirator performance is central to effective UV-C decontamination. Objective: We summarize the UV-C N95 decontamination evidence and identify key metrics. Methods: We evaluate the peer-reviewed literature on UV-C decontamination to inactivate SARS-CoV-2, viral analogues, and other microorganisms inoculated on N95s, as well as the resulting effect on respirator fit and filtration. Where peer-reviewed studies are absent, we discuss outstanding questions and ongoing work. Key Findings: Evidence supports that UV-C exposure of ≥1.0 J/cm2 inactivates SARS-CoV-2 analogues (≥3-log reduction) on the majority of tested N95 models. The literature cautions that (1) viral inactivation is N95 model-dependent and impeded by shadowing, (2) N95 straps require secondary decontamination, (3) higher doses may be necessary to inactivate other pathogens (e.g., some bacterial spores), and (4) while N95 fit and filtration appear to be preserved for 10-20 cycles of 1.0 J/cm2, donning and doffing may degrade fit to unacceptable levels within fewer cycles. Results and Discussion: Effective N95 UV-C treatment for emergency reuse requires both (1) inactivation of the SARS-CoV-2 virus, achieved through application of UV-C irradiation at an appropriate wavelength and effective dose, and (2) maintenance of the fit and filtration efficiency of the N95. Conclusions: UV-C treatment is a risk-mitigation process that should be implemented only under crisis-capacity conditions and with proper engineering, industrial hygiene, and biosafety controls.

Best Practices for Germicidal Ultraviolet-C Dose Measurement for N95 Respirator Decontamination.

Ultraviolet-C (UV-C) decontamination holds promise in combating the coronavirus disease 2019 pandemic, particularly with its potential to mitigate the N95 respirator shortage. Safe, effective, and reproducible decontamination depends critically on UV-C dose, yet dose is frequently measured and reported incorrectly, which results in misleading and potentially harmful protocols. Understanding best practices in UV-C dose measurement for N95 respirator decontamination is essential to the safety of medical professionals, researchers, and the public. Here, we outline the fundamental optical principles governing UV-C irradiation and detection, as well as the key metrics of UV-C wavelength and dose. In particular, we discuss the technical and regulatory distinctions between UV-C N95 respirator decontamination and other applications of germicidal UV-C, and we highlight the unique considerations required for UV-C N95 respirator decontamination. Together, this discussion will inform best practices for UV-C dose measurement for N95 respirator decontamination during crisis-capacity conditions.

UV-C decontamination for N95 emergency reuse: Quantitative dose validation with photochromic indicators.

With COVID-19 N95 respirator shortages, frontline medical personnel are forced to reuse this disposable - but sophisticated - multilayer textile respirator. Widely used for decontamination of nonporous surfaces, UV-C light has germicidal efficacy on porous, non-planar N95 respirators when ≥1.0 J/cm^2 dose is applied across all surfaces. Here, we address outstanding limitations of photochromic indicators (qualitative readout and insufficient dynamic range) and introduce a photochromic UV-C dose quantification technique for: (1) design of UV-C treatments and (2) in-process UV-C dose validation. Our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%). Furthermore, we adapt consumer electronics for accessible quantitative readout and extend the dynamic range >10× using optical attenuators. In a measurement infeasible with radiometers, we observe striking 20× dose variation over 3D N95 facepieces. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical design considerations for both photochromic indicators and UV-C decontamination.

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.

Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels.

From whole tissues to single-cell lysate, heterogeneous immunoassays are widely utilized for analysis of protein targets in complex biospecimens. Recently, benzophenone-functionalized hydrogel scaffolds have been used to immobilize target protein for immunoassay detection with fluorescent antibody probes. In benzophenone-functionalized hydrogels, multiplex target detection occurs via serial rounds of chemical stripping (incubation with sodium-dodecyl-sulfate (SDS) and ß-mercaptoethanol at 50-60 °C for ≥1 h), followed by reprobing (interrogation with additional antibody probes). Although benzophenone facilitates covalent immobilization of proteins to the hydrogel, we observe 50% immunoassay signal loss of immobilized protein targets during stripping rounds. Here, we identify and characterize signal loss mechanisms during stripping and reprobing. We posit that loss of immobilized target is responsible for ≥50% of immunoassay signal loss, and that target loss is attributable to disruption of protein immobilization by denaturing detergents (SDS) and incubation at elevated temperatures. Furthermore, our study suggests that protein losses under non-denaturing conditions are more sensitive to protein structure (i.e., hydrodynamic radius), than to molecular mass (size). We formulate design guidance for multiplexed in-gel immunoassays, including that low-abundance proteins be immunoprobed first, even when targets are covalently immobilized to the gel. We also recommend careful scrutiny of the order of proteins targets detected via multiple immunoprobing cycles, based on the protein immobilization buffer composition.

SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets.

While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific 'network wiring'. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib-a tyrosine kinase inhibitor (TKI)-in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI.

Critical appraisal of tocilizumab in the treatment of moderate to severe rheumatoid arthritis.

Recent advances in our understanding of the role of interleukin (IL)-6 in autoimmunity and in particular rheumatoid arthritis (RA) have brought about important changes in the way we think about autoimmune diseases. Encouraging data from several phase III clinical trials of tocilizumab, a humanized monoclonal antibody against IL-6R, have led to its approval in Europe for the treatment of moderate to severe RA. Data on clinical efficacy, patient-reported outcomes, safety, and cost-effectiveness with the use of tocilizumab in patients with RA will be summarized in this review, with particular emphasis on phase III clinical trials. Furthermore, adverse events associated with the use of tocilizumab will be reviewed. Future clinical trials will evaluate the role of tocilizumab in other autoimmune diseases. The goal of this review is to describe the current understanding of the role of IL-6 in mediating the inflammatory response in RA, as well as the role of tocilizumab in the treatment of RA and the evolving role of this agent in other autoimmune diseases.

AID-mediated diversification within the IgL locus of chicken DT40 cells is restricted to the transcribed IgL gene.

Somatic hypermutation (SHM) and gene conversion (GCV) are closely related processes that increase the diversity the primary immunoglobulin repertoire. In both processes the activation-induced cytidine deaminase (AID) converts cytosine residues to uracils within the DNA of the immunoglobulin (Ig) genes in a transcription-dependent manner, and subsequent error-prone repair processes lead to changes in the antigen recognition site of the encoded receptors. This activity is specifically recruited to the Ig loci by unknown mechanisms. Our analyses of the chicken B-cell line DT40, and derivatives thereof, now revealed that even the closest neighbors of the Ig light chain (IgL) gene are protected from AID activity, albeit being transcribed and thus acting as potential targets of AID. Our findings are in support of a model in which cis-acting DNA boundary elements restrict AID activity to the IgL locus and guard the genome in the vicinity of the IgL gene from deleterious mutations.

Somatosensory cortical barrel dendritic abnormalities in a mouse model of the fragile X mental retardation syndrome.

The Fragile X mental retardation syndrome is the largest source of inherited mental retardation. The syndrome usually results from the transcriptional silencing of the fragile X mental retardation gene (FMR1). To date the most prominent reported neuronal abnormalities for the fragile X mental retardation syndrome include a higher density of long thin spines similar to those found in sensory deprived and developing tissue, suggesting a possible deficit in pruning of immature spines. Dendrites on spiny stellate cells in the inner 1/3 of the barrel wall in layer IV of the rodent somatosensory cortex have been shown to exhibit developmental pruning similar to that affecting spines. To determine if FMRP plays a role in dendritic development, these neurons were examined in two strains of adult FMRP knockout (FraX) mice. FraX mice in both strains exhibited a greater amount of septa-oriented dendritic material, a morphology consistent with pre-pruning status early in development. This observation suggests that FMRP could be necessary for normal developmentally regulated dendritic pruning.