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Tissue diagnosis is important during surgical excision of solid tumors for margin evaluation. Conventional histopathologic methods rely heavily on image-based visual diagnosis by specialized pathologists, which can be time-consuming and subjective. We report a three-dimensional (3D) histological electrophoresis system for rapid labeling and separation of the proteins within tissue sections, providing a more precise assessment of tumor-positive margin in surgically resected tissues. The 3D histological electrophoresis system uses a tumor-seeking dye labeling strategy to visualize the distribution of tumor-specific proteins within sections and a tumor finder that automatically predicts the tumor contour. We successfully demonstrated the system's capability to predict the tumor contours from five murine xenograft models and distinguish the tumor-invaded region of sentinel lymph nodes. Specifically, we used the system to accurately assess tumor-positive margins from 14 patients with cancer. Our 3D histological electrophoresis system serves as an intraoperative tissue assessment technology for more accurate and automatic pathologic diagnosis.

The article presents a study which explores the ultrasound-activatable platinum prodrug used for sonosensitized chemotherapy. It also highlights the practical utility of ultrasound to precisely activate Pt(IV) anticancer prodrugs for the eradication of deep tumor lesions and broadens the biomedical uses of Pt coordination complexes.

The article presents a study which reported the cryo-electron microscopy structures of human APOBEC3G (A3G) bound to HIV-1 Vif in complex with T cell transcription cofactor core binding factor beta (CBF-ß) and multiple components of the Cullin-5 (CUL5) RING E3 ubiquitin ligase. Topics discussed include reconstitution of functional A3G-Vif complex in vitro, RNA fork recognition and interdomain configuration of A3G, and architecture of the A3G-Vif-CUL5 E3 ligase complex.

The article presents a study on the protein tau associated with microtubules to maintain neuronal health. It describes the structure of the microtubule-binding domain of tau using the solid-state nuclear magnetic resonance (NMR) technique. It also discusses how posttranslational modifications of tau interfere with this binding, leading to tau aggregation in neurodegenerative disorders.

The article looks at a study which report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. It mentions structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 amino-terminal region can help nucleate interactions between the amino termini of the syntaxin-1 and synaptobrevin SNARE motifs. It also mentions SNARE complex assembly experiments, and fusion assays with reconstituted proteoliposomes.

The article discusses a study that solves the electron cryo-microscopy structure of the F1F0-adenosine 5'-triphosphate (ATP) synthase from Acinetobacter baumannii. It notes that infections from the bacteria is causing the global spread of multidrug resistance. It concludes that the absence of features of the nucleotide-converting F1 subcomplex in mitochrondrial ATP synthases promises the development of next-generation drugs.

The article focuses on processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex. Topics include the Lon protease is the prototype of a family of proteolytic machines with adenosine triphosphatase modules built into a substrate degradation chamber, and the Lon is known to degrade protein substrates in a processive fashion, cutting a protein chain processively into small peptides before commencing cleavages of another protein chain.

The article presents a study which reported complete three-dimensional (3D) structures of the Lon protease from Meiothermus taiwanensis (MtaLon), in two substrate-bound states, determined to 2.4-Å resolution by cryo-electron microscopy (cryo-EM). Topics discussed include the assembly of interlocked helix triangle on the top of the hexameric core, implication of the tensegrity helix triangle on the assembly process, and a triangular platform for substrate binding, selection, and unfolding.

Extensive effort has been devoted to the discovery, development, and validation of biomarkers for early disease diagnosis and prognosis as well as rapid evaluation of the response to therapeutic interventions. Genomic and transcriptomic profiling are well-established means to identify disease-associated biomarkers. However, analysis of disease-associated peptidomes can also identify novel peptide biomarkers or signatures that provide sensitive and specific diagnostic and prognostic information for specific malignant, chronic, and infectious diseases. Growing evidence also suggests that peptidomic changes in liquid biopsies may more effectively detect changes in disease pathophysiology than other molecular methods. Knowledge gained from peptide-based diagnostic, therapeutic, and imaging approaches has led to promising new theranostic applications that can increase their bioavailability in target tissues at reduced doses to decrease side effects and improve treatment responses. However, despite major advances, multiple factors can still affect the utility of peptidomic data. This review summarizes several remaining challenges that affect peptide biomarker discovery and their use as diagnostics, with a focus on technological advances that can improve the detection, identification, and monitoring of peptide biomarkers for personalized medicine., Competing Interests: Competing interes ts: The authors declare that they have no competing interests., (Copyright © 2023 Lin Li et al.)

Despite extensive knowledge on phage resistance at bacterium level, the resistance of bacterial communities is still not well-understood. Given its ubiquity, it is essential to understand resistance at the community level. We performed quantitative investigations on the dynamics of phage infection in Klebsiella pneumoniae biofilms. We found that the biofilms quickly developed resistance and resumed growth. Instead of mutations, the resistance was caused by unassembled phage tail fibers released by the phage-lysed bacteria. The tail fibers degraded the bacterial capsule essential for infection and induced spreading of capsule loss in the biofilm, and tuning tail fiber and capsule levels altered the resistance. Latent infections sustained in the biofilm despite resistance, allowing stable phage-bacteria coexistence. Last, we showed that the resistance exposed vulnerabilities in the biofilm. Our findings indicate that phage lysate plays important roles in shaping phage-biofilm interactions and open more dimensions for the rational design of strategies to counter bacteria with phage.

Acoustic tweezers have gained substantial interest in biology, engineering, and materials science for their label-free, precise, contactless, and programmable manipulation of small objects. However, acoustic tweezers cannot independently manipulate multiple microparticles simultaneously. This study introduces acousto-dielectric tweezers capable of independently manipulating multiple microparticles and precise control over intercellular distances and cyclical cell pairing and separation for detailed cell-cell interaction analysis. Our acousto-dielectric tweezers leverage the competition between acoustic radiation forces, generated by standing surface acoustic waves (SAWs), and dielectrophoretic (DEP) forces, induced by gradient electric fields. Modulating these fields allows for the precise positioning of individual microparticles at points where acoustic radiation and DEP forces are in equilibrium. This mechanism enables the simultaneous movement of multiple microparticles along specified paths as well as cyclical cell pairing and separation. We anticipate our acousto-dielectric tweezers to have enormous potential in colloidal assembly, cell-cell interaction studies, disease diagnostics, and tissue engineering.

The nanoscale morphology of the photoactive layer notably impacts the performance of organic solar cells (OSCs). Conventional methods to tune the morphology are typically chemical approaches that adjust the properties (such as solubility and miscibility) of the active components including donor, acceptor, and/or additive. Here, we demonstrate a completely different approach by applying an external electric field (EEF) on the active layer during the wet coating. The EEF-coating method is perfectly compatible with an ambient blade coating using environmentally friendly solvents, which are essential requirements for industrial production of OSCs. A record 18.6% efficiency is achieved using the EEF coating, which is the best value for open-air, blade-coated OSCs to date. Our findings suggest broad material applicability and attribute-enhanced performance to EEF-induced fiber formation and long-range ordering of microstructures of acceptor domains. This technique offers an effective method for producing high-performance OSCs, especially suited for industry OSC production based on open-air printing.

Once considered as a "metabolic waste," lactate is now recognized as a major fuel for tricarboxylic acid (TCA) cycle. Our metabolic flux analysis reveals that skeletal muscle mainly uses lactate to fuel TCA cycle. Lactate is transported through the cell membrane via monocarboxylate transporters (MCTs) in which MCT1 is highly expressed in the muscle. We analyzed how MCT1 affects muscle functions using mice with specific deletion of MCT1 in skeletal muscle. MCT1 deletion enhances running performance, increases oxidative fibers while decreasing glycolytic fibers, and enhances flux of glucose to TCA cycle. MCT1 deficiency increases the expression of mitochondrial proteins, augments cell respiration rate, and elevates mitochondrial activity in the muscle. Mechanistically, the protein level of PGC-1α, a master regulator of mitochondrial biogenesis, is elevated upon loss of MCT1 via increases in cellular NAD + level and SIRT1 activity. Collectively, these results demonstrate that MCT1-mediated lactate shuttle plays a key role in regulating muscle functions by modulating mitochondrial biogenesis and TCA flux.

Procalcitonin (PCT) serves as a crucial biomarker utilized in diverse clinical contexts, including sepsis diagnosis and emergency departments. Its applications extend to identifying pathogens, assessing infection severity, guiding drug administration, and implementing theranostic strategies. However, current clinical deployed methods cannot meet the needs for accurate or real-time quantitative monitoring of PCT. This review aims to introduce these emerging PCT immunoassay technologies, focusing on analyzing their advantages in improving detection performances, such as easy operation and high precision. The fundamental principles and characteristics of state-of-the-art methods are first introduced, including chemiluminescence, immunofluorescence, latex-enhanced turbidity, enzyme-linked immunosorbent, colloidal gold immunochromatography, and radioimmunoassay. Then, improved methods using new materials and new technologies are briefly described, for instance, the combination with responsive nanomaterials, Raman spectroscopy, and digital microfluidics. Finally, the detection performance parameters of these methods and the clinical importance of PCT detection are also discussed., Competing Interests: Competing interests: The authors declare that they have no competing interests., (Copyright © 2024 Jiayue Huang et al.)

The Golgi-localized golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23. The amino-terminal domain of TBC1D23 binds to the golgins, and the carboxyl-terminal domain of TBC1D23 captures the vesicles, but how it recognizes specific vesicles was unclear. A search for binding partners of the carboxyl-terminal domain unexpectedly revealed direct binding to carboxypeptidase D and syntaxin-16, known cargo proteins of the captured vesicles. Binding is via a threonine-leucine-tyrosine (TLY) sequence present in both proteins next to an acidic cluster. A crystal structure reveals how this acidic TLY motif binds to TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the carboxyl-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle.

The grain size structure of phytoplankton has great influence on shellfish culture. The present study aimed to assess the spatial and temporal variation in the phytoplankton community structure in the Yalu River Estuary and to explore the relationship between the phytoplankton community structure and various environmental parameters in 2020. High-throughput sequencing was used in this study. The results showed that nanophytoplankton, especially Karlodinium veneficum, dominated the estuary throughout the year. The biomass ratio of picophytoplankton, nanophytoplankton, and microphytoplankton were 20:63:17 in spring, 30:44:26 in summer, 1:38:61 in autumn, and 2:45:53 in winter, respectively. Meanwhile, Dinophyta had the greatest biomass throughout the year, followed by Bacillariophyta. On the spatial dimension (Station average), COD, T, SST had a positive impact on total phytoplankton communities, and Dep had a negative impact. In the time dimension (Monthly average), the environmental factor that significantly controlled the phytoplankton community structure were NO2 and SST. [ABSTRACT FROM AUTHOR]

The article presents the triggered formation of membrane tubes in response to specific DNA signals that actuate membrane-bound DNA clamps from an open state to various predefined closed states, releasing prestored energy to activate membrane deformation. Topics discussed include effect of actuating membrane-bound DNA clamps on vesicles, possible vesiculation by removing DNA clamps from membrane tubes, and design and assembly of DNA origami structures.

The article presents a study which demonstrated that toxin-coregulated pilus (TCP)F interacts with the minor pilin, TcpB, of TCP and elucidated the crystal structures of TcpB alone and in complex with TcpF. Topics discussed include crystal structures of TcpB in complex with a TcpF (1-15) peptide, TcpB-TcpF complex association in solution, and structural model of the TcpF-TCP filament complex.

The article highlights a study to visualize a Polyketide synthases (PKS) bimodule core using cryo–electron microscopy and resolve a two-dimensional meshwork of the bimodule core formed by homotypic interactions between modules. It discusses that the sheet-like organization provides the framework for efficient substrate transfer and for sequestration of transacting enzymes required for polyketide production.

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. We grew Brachypodium distachyon hydroponically in fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (nitrate, ammonium, and ammonium nitrate), followed by nitrogen starvation. Analyses of exudates with liquid chromatography-tandem mass spectrometry, biomass, medium pH, and nitrogen uptake showed EcoFAB 2.0's low intratreatment data variability. Furthermore, the three inorganic nitrogen forms caused differential exudation, generalized by abundant amino acids-peptides and alkaloids. Comparatively, nitrogen deficiency decreased nitrogen-containing compounds but increased shikimates-phenylpropanoids. Subsequent bioassays with two shikimates-phenylpropanoids (shikimic and p -coumaric acids) on soil bacteria or Brachypodium seedlings revealed their distinct capacity to regulate both bacterial and plant growth. Our results suggest that (i) Brachypodium alters exudation in response to nitrogen status, which can affect rhizobacterial growth, and (ii) EcoFAB 2.0 is a valuable standardized plant research tool.

Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases. Conventional methods for sEV isolation from blood are incompatible with routine clinical workflows, significantly hampering the utilization of blood-derived sEVs in clinical settings. Here, we present a simple, viscoelastic-based microfluidic platform for label-free isolation of sEVs from human blood. The separation performance of the device is assessed by isolating fluorescent sEVs from whole blood, demonstrating purities and recovery rates of over 97 and 87%, respectively. Significantly, our viscoelastic-based microfluidic method also provides for a remarkable increase in sEV yield compared to gold-standard ultracentrifugation, with proteomic profiles of blood-derived sEVs purified by both methods showing similar protein compositions. To demonstrate the clinical utility of the approach, we isolate sEVs from blood samples of 20 patients with cancer and 20 healthy donors, demonstrating that elevated sEV concentrations can be observed in blood derived from patients with cancer.

The article focuses on structure of the hexameric fungal plasma membrane proton pump in its autoinhibited state. Topics include the fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients, and the autoinhibited Pma1 hexamers in the plasma membrane of starving fungi are activated by glucose signaling and subsequent phosphorylation of the autoinhibitory domain.

Shotgun proteomics has been widely used to identify histone marks. Conventional database search methods rely on the "target-decoy" strategy to calculate the false discovery rate (FDR) and distinguish true peptide-spectrum matches (PSMs) from false ones. This strategy has a caveat of inaccurate FDR caused by the small data size of histone marks. To address this challenge, we developed a tailored database search strategy, named "Comprehensive Histone Mark Analysis (CHiMA)." Instead of target-decoy-based FDR, this method uses "50% matched fragment ions" as the key criterion to identify high-confidence PSMs. CHiMA identified twice as many histone modification sites as the conventional method in benchmark datasets. Reanalysis of our previous proteomics data using CHiMA led to the identification of 113 new histone marks for four types of lysine acylations, almost doubling the number of previously reported marks. This tool not only offers a valuable approach for identifying histone modifications but also greatly expands the repertoire of histone marks.

To compete in certain low-light environments, some cyanobacteria express a paralog of the light-harvesting phycobiliprotein, allophycocyanin (AP), that strongly absorbs far-red light (FRL). Using cryo-electron microscopy and time-resolved absorption spectroscopy, we reveal the structure-function relationship of this FRL-absorbing AP complex (FRL-AP) that is expressed during acclimation to low light and that likely associates with chlorophyll a-containing photosystem I. FRL-AP assembles as helical nanotubes rather than typical toroids due to alterations of the domain geometry within each subunit. Spectroscopic characterization suggests that FRL-AP nanotubes are somewhat inefficient antenna; however, the enhanced ability to harvest FRL when visible light is severely attenuated represents a beneficial trade-off. The results expand the known diversity of light-harvesting proteins in nature and exemplify how biological plasticity is achieved by balancing resource accessibility with efficiency.

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate-dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo-electron microscopy structure of the Thermus thermophilus RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The β-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.

Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma , commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, nonmotile minimal bacterium, JCVI-syn3B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, combinations of Spiroplasma MreB4-MreB5 and MreB1-MreB5 produced a helical cell shape and swimming. These results suggest that the swimming originated from the differentiation and coupling of bacterial actins, and we obtained a minimal system for motility of the synthetic bacterium.

Mitochondrial complex I is a redox-driven proton pump that generates proton-motive force across the inner mitochondrial membrane, powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic fungus Chaetomium thermophilum , determined by cryoEM up to 2.4-Å resolution. We show that the complex undergoes a transition between two conformations, which we refer to as state 1 and state 2. The conformational switch is manifest in a twisting movement of the peripheral arm relative to the membrane arm, but most notably in substantial rearrangements of the Q-binding cavity and the E-channel, resulting in a continuous aqueous passage from the E-channel to subunit ND5 at the far end of the membrane arm. The conformational changes in the complex interior resemble those reported for mammalian complex I, suggesting a highly conserved, universal mechanism of coupling electron transport to proton pumping.

Pancreatic β cell failure is a hallmark of diabetes. However, the causes of β cell failure remain incomplete. Here, we report the identification of tetranectin (TN), an adipose tissue-enriched secretory molecule, as a negative regulator of insulin secretion in β cells in diabetes. TN expression is stimulated by high glucose in adipocytes via the p38 MAPK/TXNIP/thioredoxin/OCT4 signaling pathway, and elevated serum TN levels are associated with diabetes. TN treatment greatly exacerbates hyperglycemia in mice and suppresses glucose-stimulated insulin secretion in islets. Conversely, knockout of TN or neutralization of TN function notably improves insulin secretion and glucose tolerance in high-fat diet-fed mice. Mechanistically, TN binds with high selectivity to β cells and inhibits insulin secretion by blocking L-type Ca 2+ channels. Our study uncovers an adipocyte-β cell cross-talk that contributes to β cell dysfunction in diabetes and suggests that neutralization of TN levels may provide a new treatment strategy for type 2 diabetes.

Although gamete fusion represents the central event in sexual reproduction, the required protein machinery is poorly defined. In sperm cells, Izumo1 and several Izumo1-associated proteins play an essential role for this process. However, so far, the mechanisms underlying transport and maturation of Izumo1 and its incorporation into high molecular weight complexes are incompletely defined. Here, we provide a detailed characterization of the C11orf94 protein, which we rename Frey, which provides a platform for the assembly of Izumo1 complexes. By retaining Izumo1 in the endoplasmic reticulum, Frey facilitates its incorporation into high molecular weight complexes. To fulfill its function, the unstable Frey protein is stabilized within the catalytic center of an intramembrane protease. Loss of Frey results in reduced assembly of Izumo1 complexes and male infertility due to impaired gamete fusion. Collectively, these findings provide mechanistic insights into the early biogenesis and functional relevance of Izumo1 complexes.

We analyze the structure of the cytoplasm by performing single-molecule displacement mapping on a diverse set of native cytoplasmic proteins in exponentially growing Escherichia coli . We evaluate the method for application in small compartments and find that confining effects of the cell membrane affect the diffusion maps. Our analysis reveals that protein diffusion at the poles is consistently slower than in the center of the cell, i.e., to an extent greater than the confining effect of the cell membrane. We also show that the diffusion coefficient scales with the mass of the used probes, taking into account the oligomeric state of the proteins, while parameters such as native protein abundance or the number of protein-protein interactions do not correlate with the mobility of the proteins. We argue that our data paint the prokaryotic cytoplasm as a compartment with subdomains in which the diffusion of macromolecules changes with the perceived viscosity.

Dissolved organic matter (DOM) is a distinct component of Earth's hydrosphere and provides a link between the biogeochemical cycles of carbon, nutrients, and trace metals (TMs). Binding of TMs to DOM is thought to result in a TM pool with DOM-like biogeochemistry. Here, we determined elemental stoichiometries of aluminum, iron, copper, nickel, zinc, cobalt, and manganese associated with a fraction of the DOM pool isolated by solid-phase extraction at ambient pH (DOM SPE-amb ) from the Amazon plume. We found that the rank order of TM stoichiometry within the DOM SPE-amb fraction was underpinned by the chemical periodicity of the TM. Furthermore, the removal of the TM SPE-amb pool at low salinity was related to the chemical hardness of the TM ion. Thus, the biogeochemistry of TMs bound to the DOM SPE-amb component in the Amazon plume was determined by the chemical nature of the TM and not by that of the DOM SPE-amb .

The heterogeneous pathophysiology of traumatic brain injury (TBI) is a barrier to advancing diagnostics and therapeutics, including targeted drug delivery. We used a unique discovery pipeline to identify novel targeting motifs that recognize specific temporal phases of TBI pathology. This pipeline combined in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis. We identified targeting motifs based on the complementarity-determining region 3 structure of dAbs for acute (1 day post-injury) and subacute (7 days post-injury) post-injury time points in a preclinical TBI model (controlled cortical impact). Bioreactivity and temporal sensitivity of the targeting motifs were validated via immunohistochemistry. Immunoprecipitation-mass spectrometry indicated that the acute TBI targeting motif recognized targets associated with metabolic and mitochondrial dysfunction, whereas the subacute TBI motif was largely associated with neurodegenerative processes. This pipeline successfully discovered temporally specific TBI targeting motif/epitope pairs that will serve as the foundation for the next-generation targeted TBI therapeutics and diagnostics.

Recent studies demonstrate that α cells contribute to glucose-stimulated insulin secretion (GSIS). Glucagon-like peptide-1 receptor (GLP-1R) agonists potently potentiate GSIS, making these drugs useful for diabetes treatment. However, the role of α and β cell paracrine interactions in the effects of GLP-1R agonists is undefined. We previously found that increased β cell GLP-1R signaling activates α cell GLP-1 expression. Here, we characterized the bidirectional paracrine cross-talk by which α and β cells communicate to mediate the effects of the GLP-1R agonist, liraglutide. We find that the effect of liraglutide to enhance GSIS is blunted by α cell ablation in male mice. Furthermore, the effect of β cell GLP-1R signaling to activate α cell GLP-1 is mediated by a secreted protein factor that is regulated by the signaling protein, 14-3-3-zeta, in mouse and human islets. These data refine our understanding of GLP-1 pharmacology and identify 14-3-3-zeta as a potential target to enhance α cell GLP-1 production.

The emergence of large-scale collective phenomena from simple interactions between individual units is a hallmark of active matter systems. Active colloids with alignment-dominated interparticle interactions tend to develop orientational order and form motile coherent states, such as flocks and swarms. Alternatively, a combination of self-propulsion and excluded-volume interactions results in self-trapping and active phase separation into dense clusters. Here, we reveal unconventional arrested-motility states in ensembles of active discoidal particles powered by induced-charge electrophoresis. Combining experiments and computational modeling, we demonstrate that the shape asymmetry of the particles promotes the hydrodynamically assisted formation of active particles' bound states in a certain range of excitation parameters, ultimately leading to a spontaneous collective state with arrested motility. Unlike the jammed clusters obtained through self-trapping, the arrested-motility phase remains sparse, dynamic, and reconfigurable. The demonstrated mechanism of phase separation seeded by bound state formation in ensembles of oblate active particles is generic and should be applicable to other active colloidal systems.

The convective transport rate of polymers through confined geometries depends on their size, allowing for size-based separation of polymer mixtures (chromatography). Here, we investigate whether mixtures of active polymers can be separated in a similar manner based on their activity. We use thin, living Tubifex tubifex worms as a model system for active polymers and study the transport of these worms by an imposed flow through a channel filled with a hexagonal pillar array. The transport rate through the channel depends strongly on the degree of activity, an effect that we assign to the different distribution of conformations sampled by the worms depending on their activity. Our results demonstrate a unique way to sort mixtures of active polymers based on their activity and provide a versatile and convenient experimental system to investigate the hydrodynamics of active polymers.

Protein SUMOylation plays an essential role in maintaining cellular homeostasis when cells are under stress. However, precisely how SUMOylation is regulated, and a molecular mechanism linking cellular stress to SUMOylation, remains elusive. Here, we report that cAMP, a major stress-response second messenger, acts through Epac1 as a regulator of cellular SUMOylation. The Epac1-associated proteome is highly enriched with components of the SUMOylation pathway. Activation of Epac1 by intracellular cAMP triggers phase separation and the formation of nuclear condensates containing Epac1 and general components of the SUMOylation machinery to promote cellular SUMOylation. Furthermore, genetic knockout of Epac1 obliterates oxidized low-density lipoprotein-induced cellular SUMOylation in macrophages, leading to suppression of foam cell formation. These results provide a direct nexus connecting two major cellular stress responses to define a molecular mechanism in which cAMP regulates the dynamics of cellular condensates to modulate protein SUMOylation.

Engineered molecular circuits that process information in biological systems could address emerging human health and biomanufacturing needs. However, such circuits can be difficult to rationally design and scale. DNA-based strand displacement reactions have demonstrated the largest and most computationally powerful molecular circuits to date but are limited in biological systems due to the difficulty in genetically encoding components. Here, we develop scalable cotranscriptionally encoded RNA strand displacement (ctRSD) circuits that are rationally programmed via base pairing interactions. ctRSD circuits address the limitations of DNA-based strand displacement circuits by isothermally producing circuit components via transcription. We demonstrate circuit programmability in vitro by implementing logic and amplification elements, as well as multilayer cascades. Furthermore, we show that circuit kinetics are accurately predicted by a simple model of coupled transcription and strand displacement, enabling model-driven design. We envision ctRSD circuits will enable the rational design of powerful molecular circuits that operate in biological systems, including living cells.

Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that appear to silence gene expression. One nucleoid-associated silencing factor is the conserved protein Hfq. Although seemingly nonspecific in its DNA binding properties, Hfq is strongly enriched at AT-rich DNA regions, characteristic of prophages and mobile genetic elements. Here, we demonstrate that polyphosphate (polyP), an ancient and highly conserved polyanion, is essential for the site-specific DNA binding properties of Hfq in bacteria. Absence of polyP markedly alters the DNA binding profile of Hfq, causes unsolicited prophage and transposon mobilization, and increases mutagenesis rates and DNA damage–induced cell death. In vitro reconstitution of the system revealed that Hfq and polyP interact with AT-rich DNA sequences and form phase-separated condensates, a process that is mediated by the intrinsically disordered C-terminal extensions of Hfq. We propose that polyP serves as a newly identified driver of heterochromatin formation in bacteria.

Lateral flow assays (LFAs) use capillary flow of liquids for simple detection of analytes. While useful for spontaneously wicking samples, the capillary flow inherently limits performing complex reactions that require timely application of multiple solutions. Here, we introduce a technique to control capillary flow on paper by imprinting roadblocks on the flow path with water-insoluble ink and using the gradual formation of a void between a wetted paper and a sheath polymer tape to create timers. Timers are drawn at strategic nodes to hold the capillary flow for a desired period and thereby enable multiple liquids to be introduced into multistep chemical reactions following a programmed sequence. Using our technique, we developed (i) an LFA with built-in signal amplification to detect human chorionic gonadotropin with an order of magnitude higher sensitivity than the conventional assay and (ii) a device to extract DNA from bodily fluids without relying on laboratory instruments.

Enzymatic DNA synthesis (EDS) is a promising benchtop and user-friendly method of nucleic acid synthesis that, instead of solvents and phosphoramidites, uses mild aqueous conditions and enzymes. For applications such as protein engineering and spatial transcriptomics that require either oligo pools or arrays with high sequence diversity, the EDS method needs to be adapted and certain steps in the synthesis process spatially decoupled. Here, we have used a synthesis cycle comprising a first step of site-specific silicon microelectromechanical system inkjet dispensing of terminal deoxynucleotidyl transferase enzyme and 3' blocked nucleotide, and a second step of bulk slide washing to remove the 3' blocking group. By repeating the cycle on a substrate with an immobilized DNA primer, we show that microscale spatial control of nucleic acid sequence and length is possible, which, here, are assayed by hybridization and gel electrophoresis. This work is distinctive for enzymatically synthesizing DNA in a highly parallel manner with single base control.

The telomerase reverse transcriptase elongates telomeres to prevent replicative senescence. This process requires exposure of the 3'-end, which is thought to occur when two sister telomeres are generated at replication completion. Using two-dimensional agarose gel electrophoresis (2D-gels) and electron microscopy, we found that telomeric repeats are hotspots for replication fork reversal. Fork reversal generates 3' telomeric ends before replication completion. To verify whether these ends are elongated by telomerase, we probed de novo telomeric synthesis in situ and at replication intermediates by reconstituting mutant telomerase that adds a variant telomere sequence. We found variant telomeric repeats overlapping with telomeric reversed forks in 2D-gels, but not with normal forks, nontelomeric reversed forks, or telomeric reversed forks with a C-rich 3'-end. Our results define reversed telomeric forks as a substrate of telomerase during replication.

The human oral microbiome correlates with numerous diseases, including lung cancer. Identifying the functional changes by metaproteomics helps understand the disease-related dysbiosis, yet characterizing low-abundant bacteria is challenging. Here, we developed a free-flow isoelectric focusing electrophoresis-mass spectrometry- (FFIEF-MS-) based metaproteomics strategy to reduce host interferences and enrich low-abundant bacteria for in-depth interpretation of the oral microbiome. With our method, the number of interfering peptides decreased by 52.87%, whereas the bacterial peptides and species increased by 94.97% and 44.90%, respectively, compared to the conventional metaproteomics approach. We identified 3647 bacterial proteins, which is the most comprehensive oral metaproteomics study to date. Lung cancer-associated bacteria were validated among an independent cohort. The imbalanced Fusobacterium nucleatum and Prevotella histicola and their dysregulated functions in inhibiting immune response and maintaining cell redox homeostasis were revealed. The FFIEF-MS may serve as a valuable strategy to study the mechanisms between human diseases and microbiomes with broader applications., Competing Interests: The authors declare no competing interests., (Copyright © 2022 Xiaoteng Jiang et al.)