Life sciences discovery and technology highlights

Abstract

Towards high throughput and high information coverage: advanced single-cell mass spectrometric techniques. Mass spectrometry (MS) is attractive for single-cell analysis because of its high sensitivity, rich information, and large dynamic ranges, especially for the single-cell metabolome and proteome analysis. Efforts have been made to deal with the throughput and information coverage problems in typical manual single-cell MS techniques. In this review, advanced techniques to improve the automation and throughput for single-cell sampling and single-cell metabolome and proteome MS detection have been discussed. Furthermore, representative MS-based strategies that can increase the in-depth cellular information coverage and achieve the more comprehensive single-cell multiomics information during high throughput detection have been highlighted, providing an ongoing perspective of the MS performance for the single-cell research (Xu, S. et al., Anal Bioanal Chem, 2022, 414(1), 219-233). Background: Profiling the plant root architecture is vital for selecting resilient crops that can efficiently take up water and nutrients. The high-performance imaging tools available to study root-growth dynamics with the optimal resolution are costly and stationary. In addition, performing nondestructive high-throughput phenotyping to extract the structural and morphological features of roots remains challenging. Results: Lube et al developed the MultipleXLab: a modular, mobile, and cost-effective setup to tackle these limitations. The system can continuously monitor thousands of seeds from germination to root development based on a conventional camera attached to a motorized multiaxis-rotational stage and custom-built 3D-printed plate holder with integrated light-emitting diode lighting. The authors also developed an image segmentation model based on deep learning that allows the users to analyze the data automatically. The authors tested the MultipleXLab to monitor seed germination and root growth of Arabidopsis developmental, cell cycle, and auxin transport mutants non-invasively at high-throughput and showed that the system provides robust data and allows precise evaluation of germination index and hourly growth rate between mutants. Conclusion: MultipleXLab provides a flexible and user-friendly root phenotyping platform that is an attractive mobile alternative to high-end imaging platforms and stationary growth chambers. It can be used in numerous applications by plant biologists, the seed industry, crop scientists, and breeding companies (Lube, V. et al, Plant Methods, 2022, 18(1), 38). Experiences From Developing Software for Large X-Ray Crystallography-Driven Protein-Ligand Studies. The throughput of macromolecular X-ray crystallography experiments has surged over the last decade. This remarkable gain in efficiency has been facilitated by increases in the availability of high-intensity X-ray beams, (ultra)fast detectors and high degrees of automation. These developments have in turn spurred the development of several dedicated centers for crystal-based fragment screening which enable the preparation and collection of hundreds of single-crystal diffraction datasets per day. Crystal structures of target proteins in complex with small-molecule ligands are of immense importance for structure-based drug design (SBDD) and their rapid turnover is a prerequisite for accelerated development cycles. While the experimental part of the process is well defined and has by now been established at several synchrotron sites, it is noticeable that software and algorithmic aspects have received far less attention, as well as the implications of new methodologies on established paradigms for structure determination, analysis, and visualization. Pearce et al will review three key areas of development of large-scale protein-ligand studies. First, the authors looked into new software developments for batch data processing, followed by a discussion of the methodological changes in the analysis, modeling, refinement and deposition of structures for SBDD, and the changes in mindset that these new methods require, both on the side of depositors and users of macromolecular models. Finally, the authors highlight key new developments for the presentation and analysis of the collections of structures that these experiments produce, and provide an outlook for future developments (Pearce, N. M. et al. Front. Mol. Biosci., 2022, 9, 861491). Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening. Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential (Diego, B. et al., J Enzyme Inhib Med Chem, 2022, 37(1), 912-929). Thermoplastic polymers are besides glass the material of choice for the industrialization of microfluidic and organ-on-chip applications. In most cases, however, such devices are developed on the basis standard lithographic clean room technologies and subsequent casting into PDMS. This results in comparably fast progress in the development of functional designs but important aspects with respect to later industrialization are thereby largely neglected. For that reason, it is advisable to switch at a rather early stage of development from PDMS to a thermoplastic polymer such as, for instance, cyclo-olefin (co)polymer (COC, COP). By making this step, additional challenges related to the anticipated manufacturing process can be identified, which is particularly important when aiming at industrialization. The authors present herein a standard process sequence for mastering of microfluidic devices by two-photon polymerization and final transfer into COC films by hot embossing. In addition, the authors describe the laser micromanufacturing of polymeric mold inserts and subsequent prototype injection molding of small series of COP samples (Kristiansen, P.M et al, Methods. Mol. Biol., 2022, 2373, 39-55). This review focuses on experimental work on nonlinear phenomena in microfluidics, which for the most part are phenomena for which the velocity of a fluid flowing through a microfluidic channel does not scale proportionately with the pressure drop. Examples include oscillations, flow-switching behaviors, and bifurcations. These phenomena are qualitatively distinct from laminar, diffusion-limited flows that are often associated with microfluidics. Battat et al explore the nonlinear behaviors of bubbles or droplets when they travel alone or in trains through a microfluidic network or when they assemble into either one- or two-dimensional crystals. The authors consider the nonlinearities that can be induced by the geometry of channels, such as their curvature or the bas-relief patterning of their base. By casting posts, barriers, or membranes situated inside channels from stimuli-responsive or flexible materials, the shape, size, or configuration of these elements can be altered by flowing fluids, which may enable autonomous flow control. The authors also highlight some of the nonlinearities that arise from operating devices at intermediate Reynolds numbers or from using non-Newtonian fluids or liquid metals. The authors include a brief discussion of relevant practical applications, including flow gating, mixing, and particle separations (Battat, S. et al, Chem. Rev., 2022, 122(7), 6921-6937). Simultaneous detection of different biomarkers from a single specimen in a single test, allowing more rapid, efficient, and low-cost analysis, is of great significance for accurate diagnosis of disease and efficient monitoring of therapy. Recently, developments in microfabrication and nanotechnology have advanced the integration of nanomaterials in microfluidic devices toward multiplex assays of biomarkers, combining both the advantages of microfluidics and the unique properties of nanomaterials. In this review, Wang et al focus on the state of the art in multiplexed detection of biomarkers based on nanomaterial-assisted microfluidics. Following an overview of the typical microfluidic analytical techniques and the most commonly used nanomaterials for biochemistry analysis, the authors highlight in detail the nanomaterial-assisted microfluidic strategies for different biomarkers. These highly integrated platforms with minimum sample consumption, high sensitivity and specificity, low detection limit, enhanced signals, and reduced detection time have been extensively applied in various domains and show great potential in future point-of-care testing and clinical diagnostics (Wang, Y. et al, Mikrochim Acta, 2022, 189(4), 139). Traditional diagnostic strategies for infectious disease detection require benchtop instruments that are inappropriate for point-of-care testing (POCT). Emerging microfluidics, a highly miniaturized, automatic, and integrated technology, are a potential substitute for traditional methods in performing rapid, low-cost, accurate, and on-site diagnoses. Molecular diagnostics are widely used in microfluidic devices as the most effective approaches for pathogen detection. This review summarizes the latest advances in microfluidics-based molecular diagnostics for infectious diseases from academic perspectives and industrial outlooks. First, Wang et al introduce the typical on-chip nucleic acid processes, including sample preprocessing, amplification, and signal read-out. Then, four categories of microfluidic platforms are compared with respect to features, merits, and demerits. The authors further discuss application of the digital assay in absolute nucleic acid quantification. Both the classic and recent microfluidics-based commercial molecular diagnostic devices are summarized as proof of the current market status. Finally, the authors propose future directions for microfluidics-based infectious disease diagnosis (Wang, X. et al, Mil. Med. Res., 2022, 9(1), 11). Electrochemical energy conversion is an important supplement for storage and on-demand use of renewable energy. In this regard, microfluidics offers prospects to raise the efficiency and rate of electrochemical energy conversion through enhanced mass transport, flexible cell design, and ability to eliminate the physical ion-exchange membrane, an essential yet costly element in conventional electrochemical cells. Since the 2002 invention of the microfluidic fuel cell, the research field of microfluidics for electrochemical energy conversion has expanded into a great variety of cell designs, fabrication techniques, and device functions with a wide range of utility and applications. The present review aims to comprehensively synthesize the best practices in this field over the past 20 years. The underlying fundamentals and research methods are first summarized, followed by a complete assessment of all research contributions wherein microfluidics was proactively utilized to facilitate energy conversion in conjunction with electrochemical cells, such as fuel cells, flow batteries, electrolysis cells, hybrid cells, and photoelectrochemical cells. Moreover, emerging technologies and analytical tools enabled by microfluidics are also discussed. Lastly, opportunities for future research directions and technology advances are proposed (Ibrahim, O. A. et al, Chem. Rev., 2022, 122(7), 7236-7266). Research Techniques Made Simple: Spatial Transcriptomics. Transcriptome profiling of tissues and single cells facilitates interrogation of gene expression changes within diverse biological contexts. However, spatial information is often lost during tissue homogenization or dissociation. Recent advances in transcriptome profiling preserve the in situ spatial contexts of RNA molecules and together comprise a group of techniques known as spatial transcriptomics (ST), enabling localization of cell types and their associated gene expression within intact tissues. In this paper, Pineiro et al review ST methods; summarize data analysis approaches, including integration with single-cell transcriptomics data; and discuss their applications in dermatologic research. These tools offer a promising avenue toward improving our understanding of niche patterning and cell‒cell interactions within heterogeneous tissues that encompass skin homeostasis and disease (Pineiro, A. et al. J Invest Dermatol, 2022, 142(4), 993-1001.e1). The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here the authors present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, the authors provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. The authors then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. The authors also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. The authors demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis (Guilliams, M. et al, Cell, 185(2), 379-396.e38).