Rapid and signal crowdedness-robust in situ sequencing through hybrid block coding.

Spatial transcriptomics technology has revolutionized our understanding of cell types and tissue organization, opening possibilities for researchers to explore transcript distributions at subcellular levels. However, existing methods have limitations in resolution, sensitivity, or speed. To overcome these challenges, we introduce SPRINTseq (Spatially Resolved and signal-diluted Next-generation Targeted sequencing), an innovative in situ sequencing strategy that combines hybrid block coding and molecular dilution strategies. Our method enables fast and sensitive high-resolution data acquisition, as demonstrated by recovering over 142 million transcripts using a 108-gene panel from 453,843 cells from four mouse brain coronal slices in less than 2 d. Using this advanced technology, we uncover the cellular and subcellular molecular architecture of Alzheimer's disease, providing additional information into abnormal cellular behaviors and their subcellular mRNA distribution. This improved spatial transcriptomics technology holds great promise for exploring complex biological processes and disease mechanisms.

Structured learning in time-dependent Cox models.

Cox models with time-dependent coefficients and covariates are widely used in survival analysis. In high-dimensional settings, sparse regularization techniques are employed for variable selection, but existing methods for time-dependent Cox models lack flexibility in enforcing specific sparsity patterns (ie, covariate structures). We propose a flexible framework for variable selection in time-dependent Cox models, accommodating complex selection rules. Our method can adapt to arbitrary grouping structures, including interaction selection, temporal, spatial, tree, and directed acyclic graph structures. It achieves accurate estimation with low false alarm rates. We develop the sox package, implementing a network flow algorithm for efficiently solving models with complex covariate structures. sox offers a user-friendly interface for specifying grouping structures and delivers fast computation. Through examples, including a case study on identifying predictors of time to all-cause death in atrial fibrillation patients, we demonstrate the practical application of our method with specific selection rules.

Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen.

The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which recruit complement component C1q and induce complement activation. Biophysical characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in solution by native mass spectrometry and size-exclusion chromatography. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-saturated IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.

Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation.

Immunoglobulin (Ig) G molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of Staphylococcus aureus, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)6:C1q complexes and prevents downstream complement activation on the surface of S. aureus. Finally, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.

Evaluating hybrid controls methodology in early-phase oncology trials: A simulation study based on the MORPHEUS-UC trial.

Phase Ib/II oncology trials, despite their small sample sizes, aim to provide information for optimal internal company decision-making concerning novel drug development. Hybrid controls (a combination of the current control arm and controls from one or more sources of historical trial data [HTD]) can be used to increase statistical precision. Here we assess combining two sources of Roche HTD to construct a hybrid control in targeted therapy for decision-making via an extensive simulation study. Our simulations are based on the real data of one of the experimental arms and the control arm of the MORPHEUS-UC Phase Ib/II study and two Roche HTD for atezolizumab monotherapy. We consider potential complications such as model misspecification, unmeasured confounding, different sample sizes of current treatment groups, and heterogeneity among the three trials. We evaluate two frequentist methods (with both Cox and Weibull accelerated failure time [AFT] models) and three different commensurate priors in Bayesian dynamic borrowing (with a Weibull AFT model), and modifications within each of those, when estimating the effect of treatment on survival outcomes and measures of effect such as marginal hazard ratios. We assess the performance of these methods in different settings and the potential of generalizations to supplement decisions in early-phase oncology trials. The results show that the proposed joint frequentist methods and noninformative priors within Bayesian dynamic borrowing with no adjustment on covariates are preferred, especially when treatment effects across the three trials are heterogeneous. For generalization of hybrid control methods in such settings, we recommend more simulation studies.

Dealloying-Induced Zeolite-like Metal Framework of AB2 Laves Phase Intermetallic Electrocatalysts.

Exploring an efficient and robust electrocatalyst for hydrogen evolution reaction (HER) at high pH and temperature holds the key to the industrial application of alkaline water electrolysis (AWE). Herein, we design an open tunnel structure by dealloying a series of Laves phase intermetallics, i.e., MCo2 and MRu0.25Co1.75 (M = Sc and Zr). The dealloying process can induce a zeolite-like metal framework for ScCo2 and ScRu0.25Co1.75 by stripping Sc metal from the center of a tunnel structure. This structural engineering significantly lowers their overpotentials at a current density of 500 mA/cm2 (η500) ca. 80 mV in 1.0 M KOH. Through a simple process, ScRu0.25Co1.75 can be easily decorated on a carbon cloth substrate and only requires 132 mV to reach 500 mA/cm2. More importantly it can maintain activity over 1000 h in industrial conditions (6.0 M KOH at 333 K), showing its potential for practical industrial applications.

Regulation of Released Alkali from Gel Polymer Electrolyte in Quasi-Solid State Zn-Air Battery.

Gel polymer electrolyte (GPE) in quasi-solid state Zn-air battery (QSZAB) will release alkali during cycling, resulting in gradual dehydration of GPE, corrosion of Zn electrode, Zn dendrites growth, and therefore inferior performance. Here, hollow Sn microspheres are prepared on Zn substrate by the technique of colloidal self-assembly. The inner surfaces of hollow Sn microspheres are modified by 2-hydroxypropyl-ß-cyclodextrin (hollow Sn-inner HPßCD) to regulate the released alkali at GPE|anode interface. The hollow Sn-inner HPßCD can lessen the leakage of released alkali, make stored alkali diffuse back to GPE during the charging process, and mitigate the loss of soluble Zn(OH)4 2- to suppress Zn dendrites growth. Resultantly, GPE in QSZAB with hollow Sn-inner HPßCD exhibits a high retention capacity for alkaline solution. The cell also exhibits a long cyclic lifespan of 127 h due to the effective regulation of released alkali, which outperforms QSZAB without hollow Sn-inner HPßCD by 7.94 times. This work rivets the regulation of released alkali at GPE|anode interface, providing new insight to improve QSZABs' performance.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions.

In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems.

Microfluidic Platform for Time-Resolved Characterization of Protein Higher-Order Structures and Dynamics Using Top-Down Mass Spectrometry.

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex's architecture and subunits' identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for top-down MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody-antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.

Evaluation of the reliability of MS1-based approach to profile naturally occurring peptides with clinical relevance in urine samples.

RATIONALE: The profiling of natural urinary peptides is a valuable indicator of kidney condition. While front-end separation limits the speed of peptidomic profiling, MS1-based results suffer from limited peptide coverage and specificity. Clinical studies on chronic kidney disease require an effective strategy to balance the trade-off between identification depth and throughput. METHODS: CKD273, a urinary proteome classifier associated with chronic kidney disease, in samples from diabetic nephropathy patients was profiled in parallel using capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography with mass spectrometry (LC-MS), and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Through cross-comparison of results from MS1 of unfractionated peptides and elution-time-resolved MS1 as well as MS/MS in LC- and CE-MS approaches, we evaluated the contribution of false-positive identification to MS1-based identification and quantitation, and analyzed the benefit of front-end separation in terms of accuracy and efficiency. RESULTS: In LC- and CE-MS, although MS1 data resulted in higher number of identifications than MS/MS, elution-time-dependent analysis revealed extensive interference by non-CKD273 peptides, which would contribute up to 50% to quantitation if they are not separated from genuine CKD273 peptides. In the absence of separation, MS1 data resulted in lower numbers of identifications and abundance pattern that significantly deviated from those by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or capillary electrophoresis with tandem mass spectrometry (CE-MS/MS). CE showed higher identification efficiency even when less sample was used or achieved faster separation. CONCLUSIONS: To ensure the reliability of MS1-based urinary peptide profiling, front-end separation should not be omitted, and elution time should be used in addition to intact mass for identification. Including MS/MS in data acquisition does not compromise the speed or identification number, while benefiting data reliability by providing real-time sequence verification.

Releasing Nonperipheral Subunits from Protein Complexes in the Gas Phase.

The quaternary structure is an important feature regulating protein function. Native mass spectrometry contributes to untangling quaternary structures by preserving the integrity of protein complexes in the gas phase. Tandem mass spectrometry by collision-induced dissociation (CID) can then be used to release subunits from these intact complexes, thereby providing structural information on the stoichiometry and topology. Cumulatively, such studies have revealed the preferred release of peripheral subunits during CID. In contrast, here we describe and focus on dissociation pathways that release nonperipheral subunits from hetero-complexes in CID at high collision energies. We find that nonperipheral subunits are ejected with a high propensity, as a consequence of sequential dissociation events, upon initial removal of peripheral subunits. Alternatively, nonperipheral subunits can be released directly from a charge-reduced or an elongated intact complex. As demonstrated here for a range of protein assemblies, releasing nonperipheral subunits under controlled conditions may provide unique structural information on the stoichiometry and topology of protein complexes.

Estimating treatment importance in multidrug-resistant tuberculosis using Targeted Learning: An observational individual patient data network meta-analysis.

Persons with multidrug-resistant tuberculosis (MDR-TB) have a disease resulting from a strain of tuberculosis (TB) that does not respond to at least isoniazid and rifampicin, the two most effective anti-TB drugs. MDR-TB is always treated with multiple antimicrobial agents. Our data consist of individual patient data from 31 international observational studies with varying prescription practices, access to medications, and distributions of antibiotic resistance. In this study, we develop identifiability criteria for the estimation of a global treatment importance metric in the context where not all medications are observed in all studies. With stronger causal assumptions, this treatment importance metric can be interpreted as the effect of adding a medication to the existing treatments. We then use this metric to rank 15 observed antimicrobial agents in terms of their estimated add-on value. Using the concept of transportability, we propose an implementation of targeted maximum likelihood estimation, a doubly robust and locally efficient plug-in estimator, to estimate the treatment importance metric. A clustered sandwich estimator is adopted to compute variance estimates and produce confidence intervals. Simulation studies are conducted to assess the performance of our estimator, verify the double robustness property, and assess the appropriateness of the variance estimation approach.

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces.

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States.

Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted 2H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with 2H during separation based on their disulfide-dependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo- and apo-Mb.

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.

How can native mass spectrometry contribute to characterization of biomacromolecular higher-order structure and interactions?

Native mass spectrometry (MS) is an emerging approach for characterizing biomacromolecular structure and interactions under physiologically relevant conditions. In native MS measurement, intact macromolecules or macromolecular complexes are directly ionized from a non-denaturing solvent, and key noncovalent interactions that hold the complexes together can be preserved for MS analysis in the gas phase. This technique provides unique multi-level structural information such as conformational changes, stoichiometry, topology and dynamics, complementing conventional biophysical techniques. Despite the maturation of native MS and greatly expanded range of applications in recent decades, further dissemination is needed to make the community aware of such a technique. In this review, we attempt to provide an overview of the current body of knowledge regarding major aspects of native MS and explain how such technique contributes to the characterization of biomacromolecular higher-order structure and interactions.

Using Effect Scores to Characterize Heterogeneity of Treatment Effects.

This JAMA Guide to Statistics and Methods article explains effect score analyses, an approach for evaluating the heterogeneity of treatment effects, and examines its use in a study of oxygen-saturation targets in critically ill patients.

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface.

Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF-MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF-MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF-MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights ( Mw) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact Mw values showed ppm level accuracy compared to theoretical predictions.

Multi-step conformational transitions in heat-treated protein therapeutics can be monitored in real time with temperature-controlled electrospray ionization mass spectrometry.

Heat-induced conformational transitions are frequently used to probe the free energy landscapes of proteins. However, the extraction of information from thermal denaturation profiles pertaining to non-native protein conformations remains challenging due to their transient nature and significant conformational heterogeneity. Previously we developed a temperature-controlled electrospray ionization (ESI) source that allowed unfolding and association of biopolymers to be monitored by mass spectrometry (MS) in real time as a function of temperature. The scope of this technique is now extended to systems that undergo multi-step denaturation upon heat stress, as well as relatively small-scale conformational changes that are precursors to protein aggregation. The behavior of two therapeutic proteins (human antithrombin and an IgG1 monoclonal antibody) under heat-stress conditions is monitored in real time, providing evidence that relatively small-scale conformational changes in each system lead to protein oligomerization, followed by aggregation. Temperature-controlled ESI MS is particularly useful for the studies of heat-stressed multi-domain proteins such as IgG, where it allows distinct transitions to be observed. The ability of native temperature-controlled ESI MS to monitor both the conformational changes and oligomerization/degradation with high selectivity complements the classic calorimetric methods, lending itself as a powerful experimental tool for the thermostability studies of protein therapeutics.