Dual-Signal Feature Spaces Map Protein Subcellular Locations Based on Immunohistochemistry Image and Protein Sequence.

Protein is one of the primary biochemical macromolecular regulators in the compartmental cellular structure, and the subcellular locations of proteins can therefore provide information on the function of subcellular structures and physiological environments. Recently, data-driven systems have been developed to predict the subcellular location of proteins based on protein sequence, immunohistochemistry (IHC) images, or immunofluorescence (IF) images. However, the research on the fusion of multiple protein signals has received little attention. In this study, we developed a dual-signal computational protocol by incorporating IHC images into protein sequences to learn protein subcellular localization. Three major steps can be summarized as follows in this protocol: first, a benchmark database that includes 281 proteins sorted out from 4722 proteins of the Human Protein Atlas (HPA) and Swiss-Prot database, which is involved in the endoplasmic reticulum (ER), Golgi apparatus, cytosol, and nucleoplasm; second, discriminative feature operators were first employed to quantitate protein image-sequence samples that include IHC images and protein sequence; finally, the feature subspace of different protein signals is absorbed to construct multiple sub-classifiers via dimensionality reduction and binary relevance (BR), and multiple confidence derived from multiple sub-classifiers is adopted to decide subcellular location by the centralized voting mechanism at the decision layer. The experimental results indicated that the dual-signal model embedded IHC images and protein sequences outperformed the single-signal models with accuracy, precision, and recall of 75.41%, 80.38%, and 74.38%, respectively. It is enlightening for further research on protein subcellular location prediction under multi-signal fusion of protein.

Bioaccumulation, Detoxification, and Biological Macromolecular Damage of Benzo[a]pyrene in Exposure in Tissues and Subcellular Fractions of Scallop Chlamys farreri.

Because of the persistence and high toxicity of benzo[a]pyrene (B[a]P), the bioaccumulation and detoxification mechanisms of B[a]P have been studied extensively at the tissue level; but the data at the subcellular level in bivalves have not been reported. The present study was conducted to investigate the effects of B[a]P exposure on bioaccumulation, detoxification, and biomacromolecular damage in gills, digestive glands, and their subcellular fractions of the scallop Chlamys farreri. The subcellular fraction contains cytoplasm, mitochondria, microsome, nucleus, cell membrane, and overall organelle. The results demonstrated that B[a]P accumulation showed a clear time-dose effect. Based on the time-dependent accumulation of B[a]P in subcellular fractions, we speculated that the intracellular migration order of B[a]P was cell membrane, organelle, and nucleus in turn. Considering the difference of B[a]P accumulation may be related to B[a]P metabolism, we have further confirmed that the activities of B[a]P metabolizing enzymes in scallop tissues and subcellular fractions were significantly tempted by B[a]P (p < 0.05), including 7-ethoxyresorufin O-deethylase (increased), glutathione-S-transferase (GST; decreased), and superoxide dismutase (increased). First, GST was detected in bivalve cytoplasm and microsome. Second, B[a]P exposure also caused biomacromolecules damage. The results demonstrated that mitochondria and microsome were more vulnerable to lipid peroxidation than cell membrane and nucleus. Taken together, the present study fills some of the gaps in our knowledge of the bioaccumulation and detoxification mechanisms of C. farreri exposed to B[a]P in subcellular fractions and deeply explores the transportation and the main metabolic and damage sites of polycyclic aromatic hydrocarbons (PAHs) in cells, which helped us to comprehensively understand the toxic mechanism of PAHs on bivalves. Environ Toxicol Chem 2022;41:2353-2364. © 2022 SETAC.

A Method to Estimate the Distribution of Proteins across Multiple Compartments Using Data from Quantitative Proteomics Subcellular Fractionation Experiments.

Knowledge of cellular location is key to understanding the biological function of proteins. One commonly used large-scale method to assign cellular locations is subcellular fractionation, followed by quantitative mass spectrometry to identify proteins and estimate their relative distribution among centrifugation fractions. In most of such subcellular proteomics studies, each protein is assigned to a single cellular location by comparing its distribution to those of a set of single-compartment reference proteins. However, in many cases, proteins reside in multiple compartments. To accurately determine the localization of such proteins, we previously introduced constrained proportional assignment (CPA), a method that assigns each protein a fractional residence over all reference compartments (Jadot Mol. Cell Proteomics 2017, 16(2), 194-212. 10.1074/mcp.M116.064527). In this Article, we describe the principles underlying CPA, as well as data transformations to improve accuracy of assignment of proteins and protein isoforms, and a suite of R-based programs to implement CPA and related procedures for analysis of subcellular proteomics data. We include a demonstration data set that used isobaric-labeling mass spectrometry to analyze rat liver fractions. In addition, we describe how these programs can be readily modified by users to accommodate a wide variety of experimental designs and methods for protein quantitation.

RNALocate v2.0: an updated resource for RNA subcellular localization with increased coverage and annotation.

Resolving the spatial distribution of the transcriptome at a subcellular level can increase our understanding of biology and diseases. To facilitate studies of biological functions and molecular mechanisms in the transcriptome, we updated RNALocate, a resource for RNA subcellular localization analysis that is freely accessible at http://www.rnalocate.org/ or http://www.rna-society.org/rnalocate/. Compared to RNALocate v1.0, the new features in version 2.0 include (i) expansion of the data sources and the coverage of species; (ii) incorporation and integration of RNA-seq datasets containing information about subcellular localization; (iii) addition and reorganization of RNA information (RNA subcellular localization conditions and descriptive figures for method, RNA homology information, RNA interaction and ncRNA disease information) and (iv) three additional prediction tools: DM3Loc, iLoc-lncRNA and iLoc-mRNA. Overall, RNALocate v2.0 provides a comprehensive RNA subcellular localization resource for researchers to deconvolute the highly complex architecture of the cell.

Transmission Electron Microscopy as a Powerful Tool to Investigate the Interaction of Nanoparticles with Subcellular Structures.

Nanomedical research necessarily involves the study of the interactions between nanoparticulates and the biological environment. Transmission electron microscopy has proven to be a powerful tool in providing information about nanoparticle uptake, biodistribution and relationships with cell and tissue components, thanks to its high resolution. This article aims to overview the transmission electron microscopy techniques used to explore the impact of nanoconstructs on biological systems, highlighting the functional value of ultrastructural morphology, histochemistry and microanalysis as well as their fundamental contribution to the advancement of nanomedicine.

Long Non-coding RNA PVT1 Inhibits miR-30c-5p to Upregulate Rock2 to Modulate Cerebral Ischemia/Reperfusion Injury Through MAPK Signaling Pathway Activation.

Long non-coding RNAs (lncRNAs) play a key role in a variety of disease processes. Plasmacytoma variant translocation 1 (PVT1), a lncRNA, is known to regulate cell functions and play a key role in the pathogenesis of many malignant tumors. The function and molecular mechanisms of lncRNA-PVT1 in cerebral ischemia remain unknown. Real-time PCR (qRT-PCR) was used to detect lncRNA-PVT1 and microRNA-30c-5p (miR-30c-5p) expression in the brain tissues of mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reperfusion (OGD/R)-treated mouse primary brain neurons. Gain- or loss-of-function approaches were used to manipulate PVT1, miR-30c-5p, and Rho-associated protein kinase 2 (Rock2). The mechanism of PVT1 in ischemic stroke was evaluated both in vivo and in vitro via bioinformatics analysis, CCK-8, flow cytometry, TUNEL staining, luciferase activity assay, RNA FISH, and Western blot. PVT1 was upregulated in the brain tissues of mice treated with MCAO/R and primary cerebral cortex neurons of mice treated with OGD/R. Mechanistically, PVT1 knockdown resulted in a lower infarct volume and ameliorated neurobehavior in MCAO mice. Consistent with in vivo results, PVT1 upregulation significantly decreased the viability and induced apoptosis of neurons cultured in OGD/R. Moreover, we demonstrated that PVT1 acts as a competitive endogenous RNA (ceRNA) that competes with miR-30c-5p, thereby negatively regulating its endogenous target Rock2. Overexpression of miR-30c-5p significantly promoted cell proliferation and inhibited apoptosis. Meanwhile, PVT1 was confirmed to target miR-30c-5p, thus activating Rock2 expression, which finally led to the activation of MAPK signaling. We demonstrated that PVT1, as a ceRNA of miR-30c-5p, could target and regulate the level of Rock2, which aggravates cerebral I/R injury via activation of the MAPK pathway. These findings reveal a new function of PVT1, which helps to broadly understand cerebral ischemic stroke and provide a new treatment strategy for this disease.

Methods to Study Translated Pseudogenes: Recombinant Expression and Complementation, Targeted Proteomics, and RNA Profiling.

The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow one to exogenously express the pseudogenic protein of interest, either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homolog. An alternative approach is to detect the endogenous protein using targeted proteomics analysis and to assess the full range of endogenous RNA isoforms, in order to consider additional coding and noncoding RNA functionality.

Sex Differences in Insulin Sensitivity are Related to Muscle Tissue Acylcarnitine But Not Subcellular Lipid Distribution.

OBJECTIVE: Sex differences in insulin sensitivity are present throughout the life-span, with men having a higher prevalence of insulin resistance and diabetes compared with women. Differences in lean mass, fat mass, and fat distribution-particularly ectopic fat-have all been postulated to contribute to the sexual dimorphism in diabetes risk. Emerging data suggest ectopic lipid composition and subcellular localization are most relevant; however, it is not known whether they explain sex differences in obesity-induced insulin resistance. METHODS: To address this gap, this study evaluated insulin sensitivity and subcellular localization of intramuscular triacylglycerol, diacylglycerol, and sphingolipids as well as muscle acylcarnitines and serum lipidomics in people with obesity. RESULTS: Insulin sensitivity was significantly lower in men (P < 0.05); however, no sex differences were found in localization of intramuscular triacylglycerol, diacylglycerol, or sphingolipids in skeletal muscle. In contrast, men had higher total muscle acylcarnitine (P < 0.05) and long-chain muscle acylcarnitine (P < 0.05), which were related to lower insulin sensitivity (r = -0.42, P < 0.05). Men also displayed higher serum ceramide (P = 0.05) and lysophosphatidylcholine (P < 0.01). CONCLUSIONS: These data reveal novel sex-specific associations between lipid species involved in the coupling of mitochondrial fatty acid transport, ß-oxidation, and tricarboxylic acid cycle flux that may provide therapeutic targets to improve insulin sensitivity.

Ca2SiO4 chemigation reduces cadmium localization in the subcellular leaf fractions of spinach (Spinacia oleracea L.) under cadmium stress.

Heavy metal like cadmium (Cd) is inessential and highly toxic and is posing serious environmental problems for agriculture worldwide. Presence of Cd gives rise to several physiological and structural disorders that leads to reduction in growth and performance of agricultural plants. Evidence related to subcellular distribution and accumulation of Cd is still enigmatic. Experiment was conducted using hydroponic culture to examine the subcellular accumulation of Cd in Spinacia oleracea L. leaves under Cd stress (50 µM and 100 µM); moreover, the Cd toxicity alleviation using 5 mM silicon (Si) was investigated. Our findings suggest that fresh and dry biomass, shoot and root length, leaf area and length of leaf declined when exposed to Cd stress (50 µM and 100 µM); however, an increase was noticed when Cd treated plants were supplied with Si (5 mM). The content of Ca2+, Mg2+ and Fe2+ in apoplastic washing fluid and symplasm were found to be lower in plants treated with alone Cd, when compared to control. Higher Cd2+:Ca2+, Cd2+:Fe2+ and Cd2+:Mg2+ ratios were detected under cadmium stress in both apoplast and symplast of leaves which were lowered by the addition of 5 mM Si. The novelty of the current study is the detection of increased apoplastic and symplastic Cd concentration in aerial part (i.e., spinach leaves) under alone Cd treatment which was considerably reduced when supplied with Si. Moreover, a noticeable increase in spinach growth and beneficial ionic concentrations suggest that Si can ameliorate the Cd stress in crop plants.

High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS).

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

1Selenium supply alters the subcellular distribution and chemical forms of cadmium and the expression of transporter genes involved in cadmium uptake and translocation in winter wheat (Triticum aestivum).

BACKGROUND: Cadmium (Cd) accumulation in crops affects the yield and quality of crops and harms human health. The application of selenium (Se) can reduce the absorption and transport of Cd in winter wheat. RESULTS: The results showed that increasing Se supply significantly decreased Cd concentration and accumulation in the shoot and root of winter wheat and the root-to-shoot translocation of Cd. Se application increased the root length, surface area and root volume but decreased the average root diameter. Increasing Se supply significantly decreased Cd concentration in the cell wall, soluble fraction and cell organelles in root and shoot. An increase in Se supply inhibited Cd distribution in the organelles of shoot and root but enhanced Cd distribution in the soluble fraction of shoot and the cell wall of root. The Se supply also decreased the proportion of active Cd (ethanol-extractable (FE) Cd and deionized water-extractable (FW) Cd) in root. In addition, the expression of TaNramp5-a, TaNramp5-b, TaHMA3-a, TaHMA3-b and TaHMA2 significantly increased with increasing Cd concentration in root, and the expression of TaNramp5-a, TaNramp5-b and TaHMA2 in root was downregulated by increasing Se supply, regardless of Se supply or Cd stress. The expression of TaHMA3-b in root was significantly downregulated by 10 µM Se at both the 5 µM and 25 µM Cd level but upregulated by 5 µM Se at the 25 µM Cd level. The expression of TaNramp5-a, TaNramp5-b, TaHMA3-a, TaHMA3-b and TaHMA2 in shoot was downregulated by increasing Se supply at 5 µM Cd level, and 5 µM Se upregulated the expression of those genes in shoot at 25 µM Cd level. CONCLUSIONS: The results confirm that Se application limits Cd accumulation in wheat by regulating the subcellular distribution and chemical forms of Cd in winter wheat tissues, as well as the expression of TaNramp5-a, TaNramp5-b and TaHMA2 in root.

Principles and innovative technologies for decrypting noncoding RNAs: from discovery and functional prediction to clinical application.

Noncoding RNAs (ncRNAs) are a large segment of the transcriptome that do not have apparent protein-coding roles, but they have been verified to play important roles in diverse biological processes, including disease pathogenesis. With the development of innovative technologies, an increasing number of novel ncRNAs have been uncovered; information about their prominent tissue-specific expression patterns, various interaction networks, and subcellular locations will undoubtedly enhance our understanding of their potential functions. Here, we summarized the principles and innovative methods for identifications of novel ncRNAs that have potential functional roles in cancer biology. Moreover, this review also provides alternative ncRNA databases based on high-throughput sequencing or experimental validation, and it briefly describes the current strategy for the clinical translation of cancer-associated ncRNAs to be used in diagnosis.

Significance of Autoantibodies in Autoimmune Encephalitis in Relation to Antigen Localization: An Outline of Frequently Reported Autoantibodies with a Non-Systematic Review.

Autoantibodies related to central nervous system (CNS) diseases propel research on paraneoplastic neurological syndrome (PNS). This syndrome develops autoantibodies in combination with certain neurological syndromes and cancers, such as anti-HuD antibodies in encephalomyelitis with small cell lung cancer and anti-Yo antibodies in cerebellar degeneration with gynecological cancer. These autoantibodies have roles in the diagnosis of neurological diseases and early detection of cancers that are usually occult. Most of these autoantibodies have no pathogenic roles in neuronal dysfunction directly. Instead, antigen-specific cytotoxic T lymphocytes are thought to have direct roles in neuronal damage. The recent discoveries of autoantibodies against neuronal synaptic receptors/channels produced in patients with autoimmune encephalomyelitis have highlighted insights into our understanding of the variable neurological symptoms in this disease. It has also improved our understanding of intractable epilepsy, atypical psychosis, and some demyelinating diseases that are ameliorated with immune therapies. The production and motility of these antibodies through the blood-brain barrier into the CNS remains unknown. Most of these recently identified autoantibodies bind to neuronal and glial cell surface synaptic receptors, potentially altering the synaptic signaling process. The clinical features differ among pathologies based on antibody targets. The investigation of these antibodies provides a deeper understanding of the background of neurological symptoms in addition to novel insights into their basic neuroscience.

Dengue Virus Capsid Protein Dynamics Reveals Spatially Heterogeneous Motion in Live-Infected-Cells.

Dengue is the single most important human viral infection transmitted by insects. The function of the viral proteins andtheir interactions with the host cell is under exhaustive investigation with the aim of identifying antiviral strategies. Here,using recombinant full-length dengue virus genomes, carrying a fluorescent mCherry fused to capsid, we studied biophysicalproperties of the viral protein during one infectious cycle in living cells. Dengue virus capsid protein associates to differentcellular compartments but its function in these locations is largely unknown. We evaluated the diffusion of capsid inside the celland determined a higher effective diffusion coefficient in the cytoplasm than in the nucleus. Using advanced fluorescencecorrelation methods, including the recently developed two-dimensional pair correlation analysis, we constructed for the first timehigh resolution maps of capsid mobility in an infected cell. We observed that the motion of capsid in the nucleoplasm-nucleolusinterface was highly organized, indicating an obstacle in this interface. Although nucleoli are membraneless structures, theydisplayed liquid-liquid phase separation. Once inside nucleoli, the protein showed isotropic mobility, indicating free diffusion orimmobilized capsid inside these structures. This is the first study presenting spatial and temporal dynamics of the dengue viruscapsid protein during infection.

Intratumor δ-catenin heterogeneity driven by genomic rearrangement dictates growth factor dependent prostate cancer progression.

Only a small number of genes are bona fide oncogenes and tumor suppressors such as Ras, Myc, ß-catenin, p53, and APC. However, targeting these cancer drivers frequently fail to demonstrate sustained cancer remission. Tumor heterogeneity and evolution contribute to cancer resistance and pose challenges for cancer therapy due to differential genomic rearrangement and expression driving distinct tumor responses to treatments. Here we report that intratumor heterogeneity of Wnt/ß-catenin modulator δ-catenin controls individual cell behavior to promote cancer. The differential intratumor subcellular localization of δ-catenin mirrors its compartmentalization in prostate cancer xenograft cultures as result of mutation-rendered δ-catenin truncations. Wild-type and δ-catenin mutants displayed distinct protein interactomes that highlight rewiring of signal networks. Localization specific δ-catenin mutants influenced p120ctn-dependent Rho GTPase phosphorylation and shifted cells towards differential bFGF-responsive growth and motility, a known signal to bypass androgen receptor dependence. Mutant δ-catenin promoted Myc-induced prostate tumorigenesis while increasing bFGF-p38 MAP kinase signaling, ß-catenin-HIF-1α expression, and the nuclear size. Therefore, intratumor δ-catenin heterogeneity originated from genetic remodeling promotes prostate cancer expansion towards androgen independent signaling, supporting a neomorphism model paradigm for targeting tumor progression.

Visualization of cell-type dependent effects of anti-E2 antibody and interferon-gamma treatments on localization and expression of Broccoli aptamer-tagged alphavirus RNAs.

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7-8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Differences in root surface adsorption, root uptake, subcellular distribution, and chemical forms of Cd between low- and high-Cd-accumulating wheat cultivars.

The differences in the mechanism of cadmium (Cd) accumulation in the grains of different wheat (Triticum aestivum L.) cultivars remain unclear. Thus, we conducted a hydroponic experiment in a greenhouse to compare root surface adsorption, root uptake, subcellular distribution, and chemical forms of Cd between low- and high-Cd-accumulating wheat cultivars at seedling stage, to improve our understanding of the differences between cultivars. The results showed that Cd adsorbed on the root surface was mainly in a complexed form, and the total amount of Cd on the Yaomai16 (YM, high-Cd-accumulating genotypes) root surface was higher (p < 0.05) than that on Xinmai9817 (XM, low-Cd-accumulating genotypes). A large amount of Cd ions adsorbed on root surface would cause plant damage and inhibit growth. Comparing the root-to-shoot translocation factors of Cd, the transfer coefficients of YM were 1.017, 1.446, 1.464, and 1.030 times higher than those of XM under 5, 10, 50, and 100 µmol L-1 Cd treatments, respectively. The subcellular distribution of Cd under Cd exposure is mainly in the cell wall and soluble fraction. The proportions of Cd in YM shoot soluble fraction were higher than those in XM, which was the main detoxification mechanism limiting the activity of Cd and may be responsible for low Cd accumulation in grains, while the effects of the chemical forms of Cd on migration and detoxification were not found to be related to Cd accumulation in the kernels.

Disruption of thin- and thick-wall microalgae using high pressure gases: Effects of gas species, pressure and treatment duration on the extraction of proteins and carotenoids.

Industrial scale microalgal cell disruption requires low cost, high efficiency and structural conservation of biomolecules for biorefinery. Many cultivated microalgae have thick walls and these walls are barriers for efficient cell disruption. Until recently, despite the high biodiversity of microalgae, little attention has been paid to thin-wall microalgal species in the natural environment for the production and recovery of valuable biomolecules. Instead of developing high power cell disruption devices, utilization of thin-wall species would be a better approach. The present paper describes a simple device that was assembled to evaluate the viability and effectiveness of biomolecule extraction from both thin- and thick-wall species as a proof of concept. This device was tested with high-pressure gases including N2, CO2 plus N2, and air as the disruption force. The highest nitrogen pressure, 110 bar, was not able to disrupt the thick-wall microalgal cells. On the other hand, the thin-wall species was disrupted to different degrees using different pressures and treatment durations. In the same treatment duration, higher nitrogen pressure gave better cell disruption efficiency than the lower pressure. However, in the same pressure, longer treatment duration did not give better efficiency than the shorter duration. High pressure CO2 treatments resulted in low soluble protein levels in the media. The best conditions to disrupt the thin-wall microalgal cells were 110 bar N2 or air for 1 min among these tests. In these conditions, not only were the disruption efficiencies high, but also the biomolecules were well preserved.

Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems.

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles.

Membraneless organelles are liquid compartments within cells with different solvent properties than the surrounding environment. This difference in solvent properties is thought to result in function-related selective partitioning of proteins. Proteins have also been shown to accumulate in polyelectrolyte complexes, but whether the uptake in these complexes is selective has not been ascertained yet. Here, we show the selective partitioning of two structurally similar but oppositely charged proteins into polyelectrolyte complexes. We demonstrate that these proteins can be separated from a mixture by altering the polyelectrolyte complex composition and released from the complex by lowering the pH. Combined, we demonstrate that polyelectrolyte complexes can separate proteins from a mixture based on protein charge. Besides providing deeper insight into the selective partitioning in membraneless organelles, potential applications for selective biomolecule partitioning in polyelectrolyte complexes include drug delivery or extraction processes.